Retrospective study of toxoplasmosis prevalence in pregnant women in Benin and its relation with malaria.

BACKGROUND: Globally distributed with variable prevalence depending on geography, toxoplasmosis is a zoonosis caused by an obligate intracellular protozoan parasite, Toxoplasma gondii. This disease is usually benign but poses a risk for immunocompromised people and for newborns of mothers with a primary infection during pregnancy because of the risk of congenital toxoplasmosis (CT). CT can cause severe damage to fetuses-newborns. To our knowledge, no study has been conducted in sub-Saharan Africa on toxoplasmosis seroprevalence, seroconversion and CT in a large longitudinal cohort and furthermore, no observation has been made of potential relationships with malaria.
METHODS: We performed a retrospective toxoplasmosis serological study using available samples from a large cohort of 1,037 pregnant women who were enrolled in a malaria follow-up during the 2008-2010 period in a rural area in Benin. We also used some existing data to investigate potential relationships between the maternal toxoplasmosis serological status and recorded malaria infections.
RESULTS: Toxoplasmosis seroprevalence, seroconversion and CT rates were 52.6%, 3.4% and 0.2%, respectively, reflecting the population situation of toxoplasmosis, without targeted medical intervention. The education level influences the toxoplasmosis serological status of women, with women with little or no formal education have greater immunity than others. Surprisingly, toxoplasmosis seropositive pregnant women tended to present lower malaria infection during pregnancy (number) or at delivery (presence) and to have lower IgG levels to Plasmodium falciparum Apical Membrane Antigen 1, compared to toxoplasmosis seronegative women.
CONCLUSIONS: The high toxoplasmosis seroprevalence indicates that prevention against this parasite remains important to deploy and must be accessible and understandable to and for all individuals (educated and non-educated). A potential protective role against malaria conferred by a preexisting toxoplasmosis infection needs to be explored more precisely to examine the environmental, parasitic and/or immune aspects.

Introduction

Toxoplasmosis is one of the most common parasitic diseases caused by Toxoplasma gondii, an intracellular protozoan parasite belonging to the phylum Apicomplexa. This parasite can infect all warm-blooded animals. Approximately 30% of the human population is infected worldwide, but the prevalence of infection is variable between both areas and communities, according to climate, lifestyle and diet [1]. Toxoplasmosis is the third burden of foodborne illness in Europe for WHO [2], while the American Centers for Disease Control and Prevention (CDC) considers it a neglected disease due to the scant attention paid to it in terms of surveillance, prevention and/or treatment [3, 4].

Toxoplasmosis is usually benign. However, it has adverse consequences in immunocompromised people and fetuses-newborns from women who have contracted toxoplasmosis during their pregnancy. Congenital toxoplasmosis (CT) is the result of mother-to-child vertical transmission of the parasite, and the risk increases with gestational age. The severity of the encountered disorders is inversely related to the pregnancy period at maternal infection. The disorders may range from severe abnormalities (mainly neurological and ophthalmic) or abortion during the first trimester of gestation, to manifestations of variable severity during the second trimester and asymptomatic traces at the third trimester [1, 5, 6]. Other risk factors, including immune and genetic host factors as well as characteristics of the T. gondii strain, are involved in the severity of the disease [6]. In T. gondii non-immune pregnant women, treatment is recommended as soon as the toxoplasmosis contamination is proven in order to reduce the risk of parasite transmission to the fetus and of sequelae in neonates. The contamination can be detected indirectly by the seroconversion of the woman, which is defined by the appearance of specific IgG directed to T. gondii. Treatments are given depending on the stage of pregnancy. For example, in France, spiramycine 9 million units/day is recommended during the first trimester [6, 7] whereas pyrimethamine 50mg/day associated with sulfadiazine 3x1g/day and folinic acid 50mg/week are recommended from the second trimester to the end of pregnancy [6].

The most effective way to detect seroconversion during pregnancy, and then to apply an antiparasitic treatment to prevent CT, is to implement a national serological screening program of pregnant women. Although this practice would be effective and less financially burdensome than monitoring pregnancy [8-10], it is rarely instituted globally [5, 6, 11] and therefore hygiene and diet guidelines remain the most used means of first-line prevention [6, 12]. The lack of systematic serological follow-up of pregnant women limits the informative data on actual seroprevalence and the incidence of seroconversion, especially as the infection is asymptomatic, potentially hiding a considerable number of CT cases. Although many countries lack adequate data, some epidemiological meta-analyses have been performed, leading to global estimates of 33.8% for acquired toxoplasmosis among pregnant women [13], 0.6%–1.1% for the seroconversion rate [14] and 190,100 cases/year (1.5 cases per 1,000 births) for the overall CT incidence [15].

Although data on human toxoplasmosis are less numerous in Africa than in some other countries [13, 16], they show that the seroprevalence rate, recently estimated at a global value of 48.7%, undergoes a great variability between countries [13]. Indeed, the seroprevalence rates recorded during the last decade in West Africa ranged from 20% to 70%, depending on countries [17-20], despite different methodologies used. A downward trend is recorded compared to previous periods, thus the toxoplasmosis seroprevalence decreased from 74% in 2006 [21] to 40% in 2016 [19] in Ghana, and from 54% in 1993 [22] to 49% in 2012 [23] in Cotonou, the economic capital of Benin. A recent meta-analysis in Benin indicated a toxoplasmosis mean seroprevalence of 47% among pregnant women over the 1990–2018 period [24]. Inter-regional variability is also observed in Benin with seroprevalence varying from 30% in 2011 in the Atacora department in the north [25] to 49% in 2012 in the Littoral department in the south including Cotonou [23], and 36% in 2016 in the Atlantic department in the west [26].

As with many other sub-Saharan countries, Benin is also impacted by malaria. This disease is caused by Plasmodium spp., another Apicomplexa parasite. The main species involved is Plasmodium falciparum, known to impair the fetal development in case of pregnancy-associated malaria. The latest WHO report indicated that 11 million pregnant women living in sub-Saharan malaria-endemic areas were exposed in 2018 to malaria infection. This resulted in 16% of the cases of low-birth-weight children recorded in these areas [27]. WHO recommends an intermittent preventive treatment against malaria during pregnancy (IPTp) using a sulfadoxine-pyrimethamine (SP) combination (IPTp-SP), the same molecules as for toxoplasmosis treatment but dosed differently. Since October 2012, IPTp-SP has been recommended as soon as possible from the second trimester of pregnancy, with at least three doses spaced one month apart until delivery [27].

To date, there is no description of toxoplasmosis seroprevalence, seroconversion and CT rates on large cohorts of pregnant women and their infants in West Africa. To our knowledge, only one study performed in Ghana addressed the diagnosis of Plasmodium and Toxoplasma co-infections at delivery among fewer than 100 pregnant women recruited during the third trimester of pregnancy [28].

In this study, we provide additional information on toxoplasmosis during pregnancy in Benin by performing a retrospective serological study using plasmas of pregnant women from a previous study on gestational malaria. This offered a unique opportunity to explore the potential relationships between maternal toxoplasmosis serological status and Plasmodium malaria and IPT-SP in pregnancy.

Material and methods

Population group

The women under study participated in the “Strategy To Prevent Pregnancy-Associated Malaria” (STOPPAM) project, implemented from 2008 to 2010 in southern Benin, where the climate is subtropical [29, 30]. The STOPPAM project was designed to explore the relationships between the timing of malaria infection during pregnancy and clinical and immunological consequences for both mothers and infants.

Women were enrolled in three dispensaries from one semi-rural and two rural sites, in the district of Comé in the Mono province, located 70km west of Cotonou. The main occupations of the population are farming, fishing and trading. The inclusion criteria were: living for more than 6 months within 15 km from the dispensary, having a gestational age under 24 weeks and having planned to deliver at the hospital. A total of 1,037 women were enrolled, and 982 were followed-up through pregnancy, including 891 until delivery [29].

At each visit (e.g., monthly antenatal care (ANC) visit, in case of an emergency and at delivery), clinical and obstetrical data as well as biological samples (including blood samples which were used for this study) were collected.

The STOPPAM project followed WHO recommendations in effect at that time, so women received IPTp, iron and folic acid as per national guidelines. When diagnosed malaria-infected, women were treated with quinine or SP following the recommended WHO process [29].

Design of the serological study on toxoplasmosis

Determination of the toxoplasmosis seroprevalence rate

Serology against T. gondii was carried out on the available ethylenediaminetetraacetic acid (EDTA) frozen plasma samples obtained at each woman’s enrollment into the STOPPAM project (Fig 1), either at the first ANC visit or at the consultation following it, usually after one month. ToRC (Toxoplasmosis, Rubella, Cytomegalovirus) IgG on the BioPlex® 2200 System (Bio-Rad) was assessed according to the manufacturer’s recommendations. It is a multiplex immunoassay for quantitative detection of anti-T. gondii and anti-rubella IgG and for qualitative detection of anti-CMV (cytomegalovirus) IgG in a single reaction from a single serum or plasma sample. Anti-rubella and anti-CMV IgG results are not presented here. A specific software associated with the BioPlex 2200 automated analyzer (Bio-Rad) was used, and T. gondii results were expressed according to the following thresholds: results ≤ 9 IU/mL were negative, those between 10–11 IU/mL were doubtful, and results ≥ 12 IU/mL were positive. In case of positive results at the inclusion, the woman was considered as immunized and no further IgG assay was performed.

Fig 1

Flow chart of the serological study on toxoplasmosis.

*: PS = plasma sample. a: available plasma samples at inclusion, or during the first antenatal care visit or the next visit, not exceeding one month after the first one. b: diluted and undiluted plasma samples (see Materials and Methods: “Methodological constraints”). c: poor quality plasma samples with too much cell debris. d: serological toxoplasmosis results positive (Toxo POS) or negative (Toxo NEG). e: available plasma samples corresponding to the end of the study, usually at delivery or during a previous visit not exceeding one month before delivery. **: CT = congenital toxoplasmosis.

Determination of the toxoplasmosis seroconversion rate during pregnancy

For toxoplasmosis seronegative women at inclusion, a measurement of anti-T. gondii IgG was performed on available samples corresponding to the end of the study, usually at delivery, or the previous consultation not exceeding one month before delivery (Fig 1). Platelia™ TOXO IgG (Bio-Rad), an indirect ELISA immunoassay for quantitative determination of anti-T. gondii IgG in human serum or plasma, was used according to the manufacturer’s recommendations. The absorbance was read at 450/620nm on Asys UVM 340 spectrophotometer (Biochrom). Results corresponding to IgG titers <6 IU/mL were negative; they were equivocal for IgG titers comprised between 6 and 9 (excluded) IU/mL and positive for IgG titers ≥ 9 IU/mL.

When the test was positive at the end of pregnancy for a woman previously diagnosed as seronegative, Platelia™ TOXO IgG was carried simultaneously on both inclusion and exit study plasma samples of each individual to check the transition from negativity to positivity using the same immunoassay. For the rare women with discrepant results on the sample of the end of the study (i.e., the plasma of the end of the study was tested positive for the first assay and then negative during the second run, both runs being assessed with Platelia™ TOXO IgG), LDBIO Toxo II IgG confirmation (LDBIO Diagnostics) was used according to the manufacturer’s recommendations. This immunoblot qualitative assay is proposed to confirm positive or equivocal results obtained by classical serological tests. Presence of specific anti-T. gondii IgG was confirmed if at least three bands were observable at either 30, 31, 33, 40 or 45 kDa, including the band at 30 kDa in all cases.

Pregnant women who evolved from negative to positive anti-T. gondii IgG between inclusion and exit of the study were analyzed more specifically. Namely, all available plasma samples that had been sequentially collected at each visit (an average of six visits) during pregnancy were tested in a single experiment with Platelia™ TOXO IgG, together with the initial analyzed samples (inclusion and exit) to evaluate the seroconversion period. The determination was refined by detecting anti-T. gondii IgM on the plasma samples corresponding to the period that circumscribed the IgG seroconversion. For this purpose, the qualitative Platelia™ TOXO IgM (Bio-Rad) detection assay was used, according to the manufacturer’s recommendations. The absorbance was read at 450/620nm under the same conditions as Platelia™ TOXO IgG. Results were expressed as a ratio, which may be negative (<0.8), equivocal (from 0.8 to <1) or positive (≥ 1).

The IgG avidity test described by Robert-Gangneux et al. [31] was applied using Platelia™ TOXO IgG (Bio-Rad) for one woman (pregnant woman #1, or PW1) with equivocal IgG results throughout the pregnancy. This method allows for the determination of an avidity index expressed in percentage depending on the age of infection: a high avidity index indicates a formerly acquired immunity (≥ 10 months) and a lower avidity index indicates a possible recently contracted toxoplasmosis infection. Then it is even possible to estimate the period of infection to be less than 3 months, 3 to 5 months and 5 to 10 months.

Detection of cases of Congenital Toxoplasmosis (CT)

The detection of CT in newborns was made for the group of women who had seroconverted. Qualitative Platelia™ TOXO IgM (Bio-Rad) detection assay was used on cord blood plasmas, according to the manufacturer’s recommendations. Also, an immunoblot toxoplasmosis assay (Toxoplasma Western Blot IgG IgM, LDBIO Diagnostics) was performed for the comparison of mother-and-cord immunological IgG profiles. Maternal plasma samples (from circulating blood at delivery) and fetal plasma samples (from cord blood) were used. According to the manufacturer’s recommendations, maternal and cord plasma samples were incubated on two separate and contiguous strips from the same nitrocellulose transfer membrane on which T. gondii antigens separated by electrophoresis were bound. A polyclonal goat anti-human IgG coupled with alkaline phosphatase was used as conjugate. CT is suggested by the presence of a band in the cord but not the maternal plasma at a molecular weight less than 120 kDa. Because of insufficient plasma quantities, a similar immunoblot toxoplasmosis assay using an IgM conjugate was not performed.

Methodological constraints

This sero-epidemiological study was constrained by sample availability, quantity and quality. First, some plasma samples were fully utilized following the initial malaria research program (Fig 1). Then, to avoid damaging the analysis automat for the ToRC assay, due to insufficient volume or too high viscosity, a number of enrollment plasma samples were diluted 1/3 in washing buffer, as recommended by the manufacturer. More precisely, this adaptation concerned 348 out of the 979 processed samples (35.5%). Five of them ended in technical failure. Without applying any dilution corrective factor, these samples yielded 189 seropositive and 154 seronegative results. The interpretation of the seronegative results could have been tricky, due to the 0–9 scale of values corresponding to the negativity threshold of the test (≤ 9 IU/mL), but matching with the serological results at study exit (realized with Platelia™ TOXO IgG on non-diluted samples) allowed for confirmation of seronegativity for 105/154 of them. The remaining 49 plasma samples at exit were not available because having been depleted: it was decided to consider these 49 plasma samples as seronegative, insofar as their inclusion in or exclusion from the analysis had little influence on the seronegativity rate of the entire cohort (n = 462/974, 47.4% vs. n = 413/925, 44.6%).

Statistical analysis

Firstly, we calculated the prevalence of maternal toxoplasmosis based on IgG levels to T. gondii at the start of the study; IgG titers (UI/mL) were transformed into binary variables (seronegative/seropositive) according to the thresholds described above. Toxoplasmosis seroconversion was defined as the transition from seronegativity at inclusion to seropositivity at the end of the follow-up. The timing of seroconversion, as well as maternal and newborn characteristics in case of seroconversion were described. Finally, the prevalence of CT (95% CI), defined as mother-and-cord immunological IgG different profiles, was calculated.

Maternal and newborn characteristics associated with T. gondii serological maternal status at inclusion were determined using chi-square test or t-tests. For these analyses, women who had seroconverted during pregnancy (n = 12) were excluded from these analyses and were considered as a separate group.

The following characteristics were tested for their possible association with serological toxoplasmosis maternal status: 1) socio-epidemiological data: village of residence, season at enrolment, mother’s education level, bednet possession and number of medical visits (ANC and emergency) during follow-up; 2) maternal clinical data: age, gravidity, gestational age at enrolment and at delivery determined by ultrasound [32]; and 3) neonatal data: prematurity (<37 weeks gestation), stillbirth, birth weight and low birth weight (<2,500 grams).

Secondly, we assessed the association between T. gondii serological maternal status at inclusion and maternal malaria infection (at inclusion, during pregnancy and at delivery). Malaria infection during pregnancy was defined as at least one positive thick blood smear (TBS) during ANC and emergency visits. Malaria infection at delivery was considered positive when parasites were detected either in peripheral or placental blood. A logistic univariate analysis was performed to assess this association, and a following multivariate logistic model adjusted on maternal socio-demographic and clinical characteristics was conducted to ensure an association existed. At least, the mean of maternal plasma antibody levels to the Apical Membrane Antigen 1 of P. falciparum (anti-PfAMA1 IgG), both at inclusion and delivery, was log-transformed and compared according to the T. gondii serological maternal status at inclusion. IgG directed to PfAMA1, which is a merozoite antigen from the erythrocytic asexual stages of P. falciparum and also a conserved apicomplexan protein, were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA) as previously described [33]. Associations were then assessed using a linear univariate analysis followed by a multivariate linear regression model to account for potential confounding factors such as maternal socio-demographic and clinical characteristics. The differences of anti-PfAMA1 IgG levels between inclusion and delivery were established using a multivariate mixed model where antibody levels at inclusion and at delivery were paired for each woman.

Data were analyzed with Stata® Software, Version 13 (StatCorp LP, College Station, TX, USA) and the graphics were done using Graph Pad Prism (Version 8.1.2).

Ethics

The STOPPAM project was approved by the ethics committees of the Research Institute for Development (IRD) in France and of the Science and Health Faculty (University of Abomey Calavi) in Benin. Informed written consent with the possibility of withdrawing from the study at any time was obtained from all women before enrollment. The retrospective sero-epidemiological investigation of toxoplasmosis during pregnancy performed on the STOPPAM cohort was approved by the STOPPAM project committee. All the methods were performed in accordance with the institutional guidelines and regulations pertaining to research involving humans.

Results

Toxoplasmosis seroprevalence, seroconversion and CT

The different steps of the serological survey are presented in Fig 1. Among plasma samples collected at the inclusion of 1,037 pregnant women in the STOPPAM study, 979 samples from the first visit were available for the determination of toxoplasmosis seroprevalence. Excluding 5 samples due to technical failure, 512 and 462 women were found serologically positive and negative to toxoplasmosis, respectively. Therefore, the toxoplasmosis IgG seroprevalence was 52.6% (512/974). Among the 462 toxoplasmosis seronegative women at enrolment, 350 samples at delivery (or the preceding period when plasma samples at delivery were depleted) corresponding to 75.7% of them, were available to establish the toxoplasmosis seroconversion rate. Twelve women were found to have contracted a primo-infection by T. gondii between these two measurement points, their serological IgG toxoplasmosis status having changed from negative to positive. Therefore, a toxoplasmosis seroconversion rate of 3.4% (12/350, 95% CI [0.0178; 0.059]) was observed when considering seronegative pregnant women with available plasma samples at delivery. Among the 12 cases identified with seroconversion during pregnancy, 9 mother-and-cord plasma pairs were available for highlighting potential cases of CT. Since 2 CT cases could be observed only based on IgG immunoblot results (qualitative Platelia™ TOXO IgM cord blood results being negative and IgM immunoblotting could not be performed), the congenital toxoplasmosis rate was 0.2%, corresponding to 2/835 live births (95% CI [0.0003; 0.086]), i.e., a ratio of 2.39 per 1,000 live births, among the initial group of 974 women with known toxoplasmosis serological status; knowing that 19 births were twins, 163 deliveries remained unrecorded, for 816 women (i.e., 835–19) among the whole study group of 979 women. These 163 situations were distributed into 80 women lost to follow-up or having stopped the follow-up (49.1%), 35 stillbirths (21.5%), 32 miscarriages (19.6%) and 16 women whose pregnancy has been invalidated (9.8%).

Women’s and newborns’ characteristics according to their toxoplasmosis serological profile

Environmental and clinical characteristics recorded during the STOPPAM study for the mothers and their newborns were used for the present analysis. It appears that T. gondii seropositive women were older (27.6 ± 6.2 years vs. 25.1 ± 6.0 years; P<0.001) and had higher gravidity (3.7 ± 2.2 vs. 3.1 ± 1.8; P = 0.003) than those who remained seronegative all along their pregnancy (Table 1). Gestational age at delivery did not differ between the two groups. Regarding living conditions, no difference was observed between groups of women regarding either their rural or semi-rural location or their bednet possession. Also, no difference regarding presence of anemia was evidenced between groups. Finally, T. gondii seropositive women had completed a lower education level than T. gondii seronegative ones (P = 0.006).

Table 1

Clinical and environmental characteristics of pregnant women according to their T. gondii serological status.

	Serological toxoplasmosis status at inclusion
Maternal characteristics	T. gondii seropositive group (n = 512)		T. gondii seronegative group (n = 450)		P a	T. gondii seroconverting group (n = 12)
	n	Mean (± SD) or %	n	Mean (± SD) or %		n	Mean (± SD) or %
Age (years)	505 b	27.6 (± 6.2)	443 c	25.1 (± 6.0)	<0.001 *	12	28.6 (± 5.5)
Gravidity (n)	512	3.7 (± 2.2)	450	3.1 (± 1.8)	0.003	12	3.8 (± 1.9)
Primigest	77	15.0%	101	22.4%	<0.001 *	1	8.3%
Secondigest	110	21.5%	107	23.8%		2	16.7%
Multigest	325	63.5%	242	53.8%		9	75.0%
Gestational age at enrolment (weeks)d	512	16.1 (± 4.7)	450	16.8 (± 4.9)	0.03 *	12	15.9 (± 5.9)
Gestational age at delivery (weeks)	440 b	39.2 (± 2.9)	395 c	39.4 (± 2.4)	0.31*	11 e	39.9 (± 1.6)
Living site
Rural sites (Akodeha and Ouedeme Pedah)	289	56.5%	261	58.0%	0.63	8	66.7%
Semi-rural site (Comé)	223	43.5%	189	42.0%		4	33.3%
Maternal education
None	317	62.0%	228	50.7%	0.006	8	66.7%
Partial primary	98	19.1%	107	23.8%		4	33.3%
Complete primary	34	6.6%	42	9.3%		-	-
Beyond primary	63	12.3%	73	16.2%		-	-
Number of visits (ANC + urgency)	512	5.31 (± 2.2)	450	5.12 (± 2.2)	0.19*	12	5.2 (± 1.8)
Season at enrolment
Dry season	212	41.4%	206	45.8%	0.17	2	16.7%
Not dry season	300	58.6%	244	54.2%		10	83.3%
Bednet possession
Yes	166	32.4%	139	31.0%	0.61	4	33.3%
No	346	67.6%	311	69.0%		8	66.7%
IPTp-SP dose
No dose	36	7.0%	26	5.8%	0.61	0	0%
One dose	38	7.4%	39	8.7%		0	0%
Two doses	435	85.0%	384	85.3%		12	100%
Three doses	3	0.6%	1	0.2%		0	0%
Anemia
At inclusion	311/509b	61.1%	271/446c	60.8%	0.91	6	50.0%
At delivery	144/328b	43.9%	138/295c	46.8%	0.47	3/7e	42.8%

a: P value of the t-test

* or chi-squared test

P < 0.05 in bold.

b: available values out of 512 women.

c: available values out of 450 women.

d: gestational age determined by ultrasound.

e: available values out of 12 women.

On their own, newborns did not differ in terms of prematurity, stillbirth, mean birth weight or low birth weight prevalence according to the toxoplasmosis serological status of their mothers during pregnancy (Table 2).

Table 2

Clinical characteristics of newborns according to the T. gondii serological status of their mothers during pregnancy.

Newborn clinical characteristics	T. gondii seropositive mothers (n = 440a/512)		T. gondii seronegative mothers (n = 395a/450)		P b	T. gondii seroconverting mothers (n = 11a/12)c
	n	Mean (± SD) or %	n	Mean (± SD) or %		n	Mean (± SD) or %
Prematurity (< 37 weeks)	44	10.0%	41	10.4%	0.86	1	9.1%
Stillbirth	21	4.8%	14 d	3.5%	0.38	0	0%
Birth weight (g)	435 e	2953 (± 536)	385 f	2961 (± 503)	0.88	11	3040 (± 631)
Low birth weight (< 2500g) g	62	14.2%	47	12.2%	0.39	2	18.2%

a: available newborn values.

b: P value of the univariate logistic regression.

c: the values mentioned here are indicative.

d: 1 missing value.

e: 5 missing values.

f: 10 missing values.

g: calculated on the number of infants with documented birth weight.

Seroconversion maternal group and infants

Table 1 also presents the clinical data of women who contracted toxoplasmosis during pregnancy (n = 12). Their mean age at inclusion was 28.6 ± 5.5 years, their mean number of previous pregnancies was 3.8 ± 1.9; they were enrolled at 15.9 ± 5.9 gestation weeks and gave birth at 39.9 ± 1.6 gestation weeks. For 7 out of these 12 women, plasma samples at inclusion were diluted 1/3 for the ToRC assay, and their negativity confirmed later using the Platelia™ TOXO IgG assay on non-diluted plasma samples. Among infants born to this seroconversion maternal group, one case of prematurity and two cases of low birth weight were recorded (Table 2).

Ultrasonography performed during the follow-up did not reveal any abnormality in fetuses suspected of CT.

Since an average of six blood samples were collected for this group during pregnancy, this allowed over time anti-T. gondii IgG measurements for an individual estimate of the seroconversion period, as illustrated in Fig 2. Anti-T. gondii IgM levels were measured on samples circumscribing the seroconversion time-point, identifiable by the change of anti-T. gondii IgG results from negative to positive, in order to strengthen the diagnostic argument. Results were negative in all cases except one for which IgM levels changed from borderline to positive (PW5). Three women may have seroconverted towards the end of the first trimester of pregnancy (PW1, PW2 and PW3). For one of them (PW1), anti-T. gondii IgG doubtful results needed an assessment using an IgG avidity test. Low and crescent results from the first to the third measure led to a broad estimate for the seroconversion period covering the end of the first trimester and the whole second trimester (Fig 2). Two women were infected during the 6th month (PW4 and PW5), and their newborns had low birth weight, including one (PW5) who was born prematurely with no apparent link with toxoplasmosis. Seven women (from PW6 to PW12) seroconverted during the third trimester of pregnancy, including two (PW9 and PW10) during the end of the eighth month and two (PW11 and PW12) during the ninth month. The two congenital toxoplasmosis cases that were identified were the result of a maternal infection occurring between the third and fourth months of pregnancy for one case (PW2) and at the end of the pregnancy for another (PW9).

Fig 2

Schematic representation of the timing of toxoplasmosis seroconversion in the subgroup of 12 pregnant women.

The representation of the gestational period begins from the second month of pregnancy. Each pregnant woman (PW) corresponds to a line. For each line, the antenatal care visits (ANC) are represented either by one or two serological results or by an “x” when the sample was not available; emergency visits (visit excluding ANC) are represented by an asterisk (*). Each follow-up starts by an inclusion and finishes by delivery excepted for the PW6 woman who was lost to follow-up after her emergency visit. On each line, qualitative anti-T. gondii IgG and IgM results are mentioned as negative (N), positive (P) or doubtful (D); anti-T. gondii IgG is mentioned in all cases and is followed (/) by anti-T. gondii IgM result when it has been achieved, i.e., IgG or IgG/IgM. Colored areas correspond to confirmed (grey) or estimated (hatched grey) seroconversion period. Regarding possible CT cases, mother-and-cord immunological IgG profiles were identical except for PW mentioned in bold where distinct profiles were in favor of CT. Mother-and-cord immunological IgG profiles were not performed for underlined PWs because of unavailability of either mother or cord blood sample. a: maternal seroconversion associated with a strong CT suspicion. b: maternal seroconversion associated with newborn low birth weight. c: maternal seroconversion associated with premature birth. d: case of maternal seroconversion lost to follow-up before delivery.

Association between T. gondii serological status and P. falciparum infection during pregnancy

Fig 3 presents the percentages of malaria-infected women at three time points (inclusion, follow-up and delivery). Presence of a Plasmodium infection at enrollment did not differ between T. gondii seronegative (ninclusion = 84/450, 18.7%) and seropositive (ninclusion = 78/512, 15.2%, P = 0.16) pregnant women. Nevertheless, during follow-up and delivery, T. gondii seronegative women had more malaria infections than T. gondii seropositive ones (nfollow-up = 186/450, 41.3% vs. nfollow-up = 179/512, 35.0%, P = 0.04 and ndelivery = 48/450, 10.7% vs. ndelivery = 32/512, 6.3%, P = 0.01). However, although this association did not reach significance in a multivariate analysis during the follow-up, this observation was supported by the negative association between the toxoplasma serological status and presence of a malaria infection at delivery (OR = 0.531 and P = 0.012; Table 3). This result shows that women seropositive for T. gondii develop less malaria infections at delivery than women seronegative for T. gondii. This analysis has been conducted in a multivariate model meaning that the negative association between T. gondii serology and malaria infections at delivery is independent from the effect of adjustment variables on malaria infections at delivery. This was namely the case for IPTp which did not impact on the presence of a malaria infection at delivery, whatever the number of IPTp doses administered during pregnancy (S1 Table).

Fig 3

Maternal malaria events in relation to the T. gondii serological status.

Maternal malaria infection determined by TBS during the STOPPAM study was used to compare pregnant women according to their toxoplasmosis serological status. T. gondii seronegative women are in the grey bar (n = 450) and T. gondii seropositive women in the black bar (n = 512).

Table 3

Multivariate analysis exploring the association between malaria infection and T. gondii maternal serological status.

		Malaria infectiona during the follow-up (n = 948)			Malaria infectiona at delivery (n = 948)
Independent variables	Categories	Odds Ratiob	[95% CI]	P valuec	Odds Ratiob	[95% CI]	P valuec
T. gondii positive serological statusd		0.835	[0.626; 1.116]	0.223	0.531	[0.324; 0.87]	0.012
Adjustment variables:
Maternal agee	[15–22]
	[23–29]	0.717	[0.488; 1.053]	0.090	0.768	[0.387; 1.526]	0.451
	[30–35]	0.434	[0.285; 0.661]	<0.001	1.198	[0.592; 2.421]	0.616
Primigest vs. multigest women		1,480	[0.97; 2.257]	0.069	1.839	[0.932; 3.629]	0.079
Living site	Akodeha
	Ouedeme Pedah	1.756	[1.195; 2.582]	0.004	0.788	[0.429; 1.447]	0.443
	Comé	0.430	[0.308; 0.6]	<0.001	0.404	[0.222; 0.733]	0.003
Maternal educationf	None
	Partial primary	0.997	[0.688; 1.444]	0.987	1.373	[0.752; 2.507]	0.302
	Complete primary	1,285	[0.741; 2.227]	0.371	1.744	[0.735; 4.138]	0.207
	Beyond primary	0.651	[0.404; 1.047]	0.077	0.838	[0.363; 1.932]	0.678
Number of visits (ANC + emergency)g	[0–4
	[5,6]	2.075	[1.283; 3.355]	0.003	4.679	[1.498; 14.62]	0.008
	[7–12]	3.023	[1.86; 4.912]	<0.001	6.127	[1.958; 19.172]	0.002
Dry season		0.859	[0.643; 1.148]	0.305	1.120	[0.691; 1.816]	0.646
Bednet possession		1.119	[0.813; 1.539]	0.491	0.943	[0.547; 1.624]	0.831
Number of IPTp-SP dosesh	[0,1]
	[2,3]	0.642	[0.379; 1.088]	0.100	0.984	[0.316; 3.066]	0.977

a: malaria infection was defined by at least one positive TBS during pregnancy (follow-up or delivery); 365 women had at least one malaria infection during the follow-up.

b: an Odds Ratio <1 shows a negative association between the variable and malaria infection whereas an Odds Ratio >1 shows a positive association.

c: significant P value <0.05 is in bold.

d: Toxoplasmosis serological status defined at inclusion (n = 948).

e: age has been divided into 3 periods.

f: maternal education was sequenced into 4 categories.

g: number of visits has been divided into 3 categories.

h: number of IPTp-SP doses has been classified into 2 categories.

The logistic analysis was adjusted for maternal age, gravidae, living site, maternal education, number of visits, dry season, bednet possession and number of IPTp-SP doses.

Lastly, T. gondii seropositive women tended to have lower anti-PfAMA1 IgG levels both at inclusion (P = 0.05) and at delivery (P = 0.002) compared to seronegative ones (Fig 4). In order to ensure the robustness of this association, a multivariate analysis adjusted for possible confounders was performed for anti-PfAMA1 IgG levels at inclusion and at delivery (Table 4). As expected, malaria infections during the follow-up were associated with increased levels of anti-PfAMA1 IgG in mothers at delivery. Also, and independently of malaria infection, anti-PfAMA1 IgG levels were lower at delivery for T. gondii seropositive women compared to seronegative ones (P = 0.008; Table 4). A second analysis was performed to clarify when the levels of anti-PfAMA1 IgG differ between T. gondii seropositive and seronegative pregnant women. Again, as expected, between inclusion and delivery, the levels of anti-PfAMA1 IgG increased if women were infected by P. falciparum during the follow-up. Independently of malaria infections, the levels of anti-PfAMA1 IgG decreased more sharply in T. gondii seropositive women than in seronegative women (S2 Table).

Fig 4

Maternal IgG levels to PfAMA1 in relation to T. gondii serological status.

Anti-PfAMA1 IgG levels measured in STOPPAM study were compared according to maternal toxoplasmosis serological status. Only women with complete results (anti-PfAMA1 and anti-T. gondii IgG at inclusion or delivery) were taken into account for the calculation. The bars represent the mean of anti-PfAMA1 IgG levels, and the whiskers represent the standard deviations. a: 13 missing values at delivery.

Table 4

Multivariate analysis exploring the association between anti-PfAMA1 IgG levels at inclusion and delivery and T. gondii maternal serological status.

		anti-PfAMA1 IgG at inclusion (n = 650) a			anti-PfAMA1 IgG at delivery (n = 627) a
Independent variables	Categories	Coefb	[95% CI]	P valuec	Coefb	[95% CI]	P valuec
T. gondii positive serological status		-0.141	[-0.372; 0.09]	0.230	-0.295	[-0.514; -0.075]	0.008
Malaria infectiond during follow-up					0.337	[0.107; 0.566]	0.004
Adjustment variables:
Maternal agee	[15–22]
	[23–29]	-0.081	[-0.395; 0.232]	0.610	-0.023	[-0.318; 0.273]	0.881
	[30–35]	-0.499	[-0.827; -0.17]	0.003	-0.230	[-0.543; 0.084]	0.151
Primigest vs. multigest women		-0.092	[-0.446; 0.262]	0.609	-0.030	[-0.354; 0.295]	0.858
Living site	Akodeha
	Ouedeme Pedah	0.646	[0.346; 0.947]	<0.001	-0.371	[-0.659; -0.083]	0.012
	Comé	-0.479	[-0.742; -0.217]	<0.001	-1.156	[-1.412; -0.9]	<0.001
Maternal educationf	None
	Partial primary	-0.258	[-0.56; 0.043]	0.093	-0.222	[-0.509; 0.064]	0.128
	Complete primary		[-0.326; 0.584]	0.577	0.168	[-0.258; 0.595]	0.438
	Beyond primary	0.129–0.300	[-0.667; 0.067]	0.109	-0.149	[-0.489; 0.191]	0.389
Number of visits (ANC + emergency)g	[0–4]
	[5,6]				-0.096	[-0.518; 0.326]	0.656
	[7–12]				-0.104	[-0.532; 0.325]	0.635
Dry season		0.117	[-0.112; 0.347]	0.316	0.019	[-0.199; 0.238]	0.863
Bednet possession		0.025	[-0.228; 0.279]	0.844	-0.028	[-0.268; 0.212]	0.821
Number of IPTp-SP dosesh	[0,1]
	[2,3]				0.008	[-0.509; 0.524]	0.977

a: Data on age and/or toxoplasmosis status and/or PfAMA1 dosage were missing for 387 women at inclusion and for 410 women at delivery.

b: a coefficient <0 shows a negative association between the variable and malaria infection whereas a coefficient >0 shows a positive association.

c: significant P value <0.05 is in bold.

d: malaria infection was defined by at least one positive TBS during pregnancy (follow-up or delivery).

e: age has been divided into 3 periods.

f: maternal education was sequenced into 4 categories.

g: number of visits has been divided into 3 categories.

h: number of IPTp-SP doses has been classified into 2 categories.

The linear analysis was adjusted for quantitative TBS, maternal age, gravidae, living site, maternal education, number of visits, dry season, bednet possession and number of IPTp-SP doses.

Discussion

To provide additional data on toxoplasmosis during pregnancy in Benin, we carried out a retrospective sero-epidemiological study on a longitudinal cohort of pregnant women. Performed between 2008 and 2010 on 974 women living in a rural area of southern Benin, this study showed a toxoplasmosis seroprevalence rate of 52.6% with a seroconversion rate of 3.4% among seronegative women, also interpretable as 1.4% among all women screened (value used subsequently in order to have the same comparison basis as that used in bibliographic references cited below), and a congenital toxoplasmosis rate of 0.2% among live births.

Although the follow-up of the cohort started early in pregnancy, with a mean gestational age of 17 weeks at inclusion [29], seroconversion and congenital toxoplasmosis rates were potentially underestimated for three main reasons. Firstly, the follow-up design began at the first ANC around the fourth month of pregnancy, so it cannot be excluded that seroconversion cases having occurred during the periconceptional period or at the beginning of pregnancy went unnoticed, especially since we did not measure the anti-T. gondii IgM. However, the risk of not having detected these cases is low since during this period, toxoplasmosis infections are rarely involved in maternal-fetal transmission and, if transmitted, frequently result in spontaneous miscarriage or malformations that would have been detected during ultrasound monitoring performed throughout the study [34]. Secondly, the depletion in some samples at delivery, as well as the lack of some samples due to miscarriages or stillbirths, contributed surely to an underestimation of the number of T. gondii seronegative women having seroconverted. Lastly, the non-monitoring of serological and ophthalmological follow-up of infants during their first year of life may have hampered the detection of tardive CT.

Despite these potential biases, the 1.4% seroconversion rate observed in the study aligns with the 1.6% value found overall in sub-Saharan Africa [14]. On a smaller scale in Benin, this rate of 1.4% in 2008–2010 is significantly higher than the values resulting from successive studies carried out later on smaller numbers of women, namely in 2011 (30% seroprevalence and 0.4% seroconversion rates among 283 women screened in the northern rural area) [25], in 2012 (48% seroprevalence and 0.7% seroconversion rates among 266 women screened in the southern urban area) [23] and in 2016 (36% seroprevalence and 0.5% seroconversion rates among 399 women screened in the southern rural area) [26]. Some factors can explain these differences: 1) two of the cited studies focused on toxoplasmosis, and therefore seronegative women were made aware of the risk of infection through the adoption of preventive measures [23, 26]; and 2) spatial and temporal heterogeneity in the distribution of the parasite may have occurred, as described in the introduction for Benin, and in agreement with observations reported in the annual reports of the French National Reference Center for Toxoplasmosis [35]. In view of these factors, and knowing that different serological techniques were used, the higher seroconversion rate found in our study can also be explained by its retrospective aspect: the original women’s follow-up focused on malaria without any attention being paid to toxoplasmosis, so this infection followed its natural course without any preventive measures. The absence (except in one case) of specific anti-T. gondii IgM has not been explained. It may be due to their lability, i.e., transient or short time presence, during the specific IgG positivation period [36]. However, an absence of specific IgM to toxoplasmosis is not uncommon, with no precise explanation provided [37].

In terms of public health, it is] ing to consider the situation with respect to other countries that manage toxoplasmosis in pregnancy differently. During the same 2008–2010 period as considered here, the United States, where no routine toxoplasmosis gestational screening was conducted, reported a low toxoplasmosis seroprevalence rate of 9% [38] associated with high CT rates ranging from 0.01% to 0.1% depending on the states [39, 40]. In comparison, France reported a higher 37% seroprevalence rate associated with a lower 0.02% CT rate, as an outcome of its toxoplasmosis gestational screening [41]. In Benin, where the screening is limited to hospitals or large private clinics and is costly [23, 26], one would expect the high prevalence rate of 52.6% to be associated with a much higher rate of CT than in the United States where the circulation of the parasite is low. But the fairly close value of 0.2% is compelling and suggests a possible reducing effect on in utero T. gondii transmission, which could be attributable to the IPTp-SP administered during pregnancy, a treatment that is inexistent in non-tropical countries such as in the United States.

Investigating the impact of IPTp-SP on T. gondii infection is not new [37]. Despite ethical impossibilities precluding experiments on it, we can hypothesize that IPTp-SP administered against malaria could reduce the impact of pregnancy-associated toxoplasmosis on fetuses. The doses would not prevent infection but would decrease the serious parasitic effects. Currently, IPTp-SP is administered as often as possible from the second trimester of pregnancy in malaria endemic areas [27], and WHO currently recommends at least three SP doses during pregnancy. When the STOPPAM project was implemented in 2008–2010, the current national guidelines were followed and, under the supervision of midwives, 86% of the women received two SP doses (each dose corresponding to 1,500mg sulfadoxine and 75mg pyrimethamine) spaced at least one month apart from the second trimester of pregnancy [42-44]. In parallel, fetal ultrasonography was performed three times at gestational weeks 26, 30 and 36 [32] and did not reveal any abnormality of the cephalic structure among the fetuses from the seroconversion maternal group. Similarly, no birth defects were observed. Despite this low number of observations, it is tempting to hypothesize that the IPTp-SP may have contributed to limiting the severe adverse consequences due to toxoplasmosis, as in the case of PW2. To illustrate this hypothesis, even if the aim of the study was different, a multicenter randomized trial was conducted to compare the efficacy and tolerance of pyrimethamine (50mg/day) associated with sulfadiazine (3x1g/day) and folinic acid versus spiramycin (3x1g/day), in order to reduce T. gondii placental transmission [45]. Although few individuals were in the compared groups due to the interruption of the trial, this study found a trend toward lower T. gondii placental transmission in the SP group, in which no fetal cerebral toxoplasmosis lesions were observed. Thus, although the sulfamide-pyrimethamine regimens were very different between this latter study and ours (since the parasite targets were different), it led to a reduced severity of the fetal sequelae, which is in agreement with the absence of ultrasonographic signs in our PW2 case despite a maternal infection having occurred at around 4 months of gestation.

Strikingly, this study observed a negative relationship between T. gondii seropositivity and both the number of malaria infections during pregnancy and the presence of placental malaria at delivery. Paradoxically, this finding was accompanied by lower anti-PfAMA1 IgG levels in T. gondii seropositive women, compared to others, especially at delivery. Would T. gondii presence provide protection to the host against malaria? Several elements may be proposed to explain this:

Seropositivity to T. gondii reflects not only a past infection with this parasite but also the maintenance of a control immunity against the encysted forms of the parasite, mainly in the brain and muscle tissues [46]. On the other hand, the AMA1 protein is a conserved element of Apicomplexa parasites [47-49] (with about thirty percent homology between T. gondii and Plasmodium) for which it would play an essential role in the parasite positioning on the host cell’s membrane for favoring cell invasion [47]. Thus, toxoplasmosis infection could generate anti-AMA1 IgG potentially cross-reacting with PfAMA1 antigen sites. Consequently, this could either down-regulate the production of specific anti-PfAMA1 IgG necessary to fight against malaria or slow down/prevent the formation of the tight attachment zone between Plasmodium and the host cell, created by a parasitic protein complex [47]. Indeed, the production of anti-AMA1 Ig would block the interaction between RON (rhoptry neck protein) and AMA1 localized in the moving junction necessary for parasite invasion [50, 51]. To further investigate this hypothesis, it would be necessary to measure anti-TgAMA1 IgG levels and to introduce these new data in the global analysis.

Another explanation could be that the persistent installation of T. gondii in its host involves a dynamic regulation of combined cellular and molecular immunoregulatory networks [52-55], which can impact the newly infecting Plasmodium parasite using similar pathways. This could result in less production of anti-PfAMA1 IgG to effectively fight plasmodial parasites. This hypothesis seems paradoxical due to the clearly demonstrated association between anti-PfAMA1 IgG levels and the clinical and parasitological protection against malaria [56]. Nevertheless, it may be consistent in that these IgG levels, measured at delivery, reflected a lower need for T. gondii seropositive women to react against P. falciparum infection during their pregnancy.

Finally, and without excluding the previous explanations, another suggestion is based on experimental observations where mice primo-infected with P. berghei, treated with antimalarial drugs and then infected with T. gondii, developed immune benefits such as faster production of anti-T. gondii IgG in comparison to mice infected with T. gondii alone [53].

Altogether, these possible explanations may support the observation in this study of better immune control towards malaria for pregnant women chronically infected by T. gondii.

Except for three previous studies—two of which used PCR methods on Ghanaian pregnant women only at delivery [28] or at the recruitment on the third trimester of pregnancy [37] and the third study investigating hematological parameters in young children in Cameroon [57]—to our knowledge, this study is the first to simultaneously investigate T. gondii and P. falciparum in a longitudinal human cohort. This retrospective sero-epidemiological study on toxoplasmosis in Benin was primarily dedicated to drawing the medical staff’s attention to this pathology, which is underestimated during pregnancy in African countries where other infectious pressures such as that exerted by malaria is extremely concerning. For the first time in Benin, it offered the possibility of investigating a cohort of nearly 1,000 pregnant women. Although this observation goes back 10 years, the high CT rate concluded in the findings reinforces the importance of implementing preventive measures as much as possible against toxoplasmosis to pregnant women in the absence of knowledge of their serological status and when no screening program exists. Indeed, this study also put forward that women with a low education level were more likely to be seropositive to T. gondii. This aspect, also highlighted in a study in the United States [58], is undoubtedly the cause and the consequence of less access to and less awareness of prevention messages among these women.

This study also concluded that a preexisting infection by T. gondii plays a potential protective role against malaria. It follows that it is essential to understand the interactions between pathogens infecting a single human host, in order to properly measure their effects, which are neither strictly protective nor strictly deleterious but imbalanced. In this sense, further investigations on the cross-reactivity between IgG specific for T. gondii and P. falciparum would be necessary in groups representative of the general population in malaria endemic areas, in order to remove any bias linked to the IPTp delivered to pregnant women.

Multivariate analysis of effect of toxoplasmosis serological status and IPTp on malaria infection at delivery.

(DOCX)

Click here for additional data file.

Multivariate analysis of differences in anti-PfAMA1 IgG levels between inclusion and delivery.

(DOCX)

Click here for additional data file.

22 Jun 2021

PONE-D-21-15174

Retrospective sero-epidemiological study of toxoplasmosis during pregnancy in Benin and potential relationships with malaria

PLOS ONE

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Reviewer #1: This is an interesting paper providing insights into the prevalence of toxoplasmosis in pregnant women and the incidence of congenital toxoplasmosis in Benin. The study is well-conducted and the paper reads very well. However, I have some important comments and some questions remain to be addressed.

First, the title is not self-explanatory and does not fully reflect the content of the paper. Maybe try: “Retrospective study of prevalence of toxoplasmosis in pregnant women in Benin and its relation with malaria diagnosis at delivery” ?

Results:

Line 161: the occurrence of a “positive (result) for the first assay and negative for the second”, is not straightforward, as it is mentioned (line 151) that only seronegative women were re-tested ate delivery. Please explain how this sequence of results could happen.

Line 206: what do you mean by “depleted”? Were these 49 samples negative when tested diluted to 1/3?

Line 267: I don’t share this viewpoint. The rate of seroconversion should be calculated on the seronegative only. Seropositive at first serology cannot “seroconvert”.

Line 271: it would be interesting here to convert this ratio into x/1000 live births, which is a widely rate used to compare incidence of diseases.

Line 278: it is not adapted to cite the gestational age here, especially as seropositive women were enrolled at an earlier stage (and the means are very close, 16.1 and 16.8 !). If seropositive had been enrolled at a later stage, it could have been an issue, as they might have acquired infection in early pregnancy (which would have been unnoticed as IgM were not tested).

Line 293: were the prevalence of prematurity (1/12) and low birth weight (2/12) similar to that observed in the whole group of women?

Line 302: it is very surprising that IgM were detected in only 1/12 patients. As it is the hallmark of Toxoplasma acute infection, it casts doubt on the results of IgG. It cannot be excluded that the technique used for the first sample (Bioplex) lacks sensitivity. Were these samples re-analyzed using the WB Toxo IgGII to confirm that they were indeed negative at the beginning of pregnancy? This could also explain why ultrasound surveillance did not detect any abnormalities. By contrast to the authors’ hypothesis (lines 367-70), it is unlikely that long-term storage of sera led to an alteration of antibody, as banks of sera are widely used to evaluate new serological assays with no such issue. Besides, infection with no IgM is a rare event (see Fricker-Hidalgo, JCM 2013). Or maybe the authors wanted to cite this paper but linked another one (ref #37)? PW is probably not a seroconversion, as there is no move in antibody dosages, and should be probably excluded.

The authors report one infected neonate, following a maternal infection at around 4 months of pregnancy, it is all the more strange that no ultrasonographic signs were detected, as congenital infection at that stage is symptomatic. This should be discussed.

Table 4: were PfAMA1 antibody dosages at inclusion and at delivery paired (same patient)? If not it seems difficult to interpret the decrease.

How was congenital infection diagnosed? Was IgM assessed in the cord blood? Only 2 infants had WB profiles different from their mother, so it is important to know how diagnosis was made or excluded.

Fig. 3&4: use preferably the same color legend. In Fig.4, bars should be grouped in each category.

Discussion:

Line 383: “Investigating the impact of IPTp-SP on T. gondii infection”: this sentence should appear in the objectives, at the beginning of the manuscript.

Lines 397-401: should be deleted, as this trial cannot support, nor be compared to two intakes of sulfadoxine-pyrimethamine. Indeed, it was a clinical trial comparing sulfadiazine-pyrimethamine versus spiramycine, thus did not address the issue of treating or nor treating. Furthermore, threatment was given daily.

Line 432: “new antiparasitic compounds with anti-T. gondii and anti-P. falciparum cross-species efficacy could soon be defined”: such what? Antiparasitic drugs already target the same pathways. Maybe this sentence could be rephrased, if the authors rather think of immune cross-reactivity (vaccine development or immunomodulation therapy?).

How do the authors explain that Toxoplasma seropositivity had a preventive effect on malaria infection at delivery, but no overall effect during follow-up (table 3)?

Minor points:

Line 62 : prefer “inversely correlated to the age of pregnancy at maternal infection”

Line 72: sulfadoxine is no more available for the treatment of toxoplasmosis.

Reviewer #2: General comments

The manuscript presented Toxoplasma seroepidemiology and malaria infection in a large cohort of pregnant women in a region of Benin. The study is interesting and well-written. It showed the epidemiology of congenital toxoplasmosis in a region where is applied an intermittent preventive treatment against malaria during pregnancy using a sulfadoxine-pyrimethamine (SP) combination (IPTp-SP). However, based on the results of this observational study in a specific population, the authors tried to find a relation between toxoplasmosis and malaria.

Specific comments

Title

The data presented in the study did not show any relation between toxoplasmosis and malaria during pregnancy. In consequence, could the authors modify the title by suppressing « potential relationship »?

Abstract

Lines 37-39: Could the authors specify IgG are anti- P. falciparum Apical Membrane Antigen 1?

Mat & Meth

Lines 163-164: Could the authors reconsider this sentence?

According to the reviewer, this assay has been developed and been used to detect low titers of specific anti-Toxoplasma IgG. It is not a screening test, as some assays using a limited number of parasite antigens.

Results

Toxoplasmosis seroprevalence, seroconversion and CT

Could the authors give here or in Seroconversion maternal group and infants the % of CT among infants whose mothers had an infection during pregnancy?

Association between T. gondii serological status and P. falciparum infection during pregnancy

Lines 320-323: The OR is low 0.531 (< 2 fold fewer risks for Toxoplasma seropositive pregnant women to have malaria at delivery). Could the authors specify this in the section Discussion?

Table 3: Could the authors shortly present in the text the results of malaria riks according to the number of visits?

Discussion

Lines 350-351: The authors should add that the lack of opthalmological examination in infants in their study could lead to misdiagnosing CT cases.

Lines 410-412: Could the authors give the % of homology between the proteins of T. gondii and P. falciparum.

The authors should consider that the tendency to observe a lower malaria infection at delivery occurred in a population of women treated with sulfadoxine-pyrimethamine (SP) combination. To support a relation between toxoplasmosis and malaria such a study should be done in non-treated pregnant women and, or the general population. Indeed, we could not exclude that such treatment affected the risk of malaria between Toxoplasma negative and seropositive pregnant women. To support this last hypothesis, the rate of malaria was not different between Toxoplasma negative and seropositive women at the beginning of pregnancy (Figure 3).

Finally, the data are old (2008-2010, more than ten years).

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

30 Jul 2021

Please find the responses to the Editor and to the Reviewers attached to the cover letter.

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

31 Aug 2021

PONE-D-21-15174R1

Retrospective study of toxoplasmosis prevalence in pregnant women in Benin and its relation​ with malaria

PLOS ONE

Dear Magalie,

You improved your revision to the satisfaction of reviewer #2, but reviewer #1 still raised a number of minor points that appear easily addressed, so to give you the opportunity to address the points of reviewer #2 I have marked you ms dpown for "minor revision". If you make it absolutely clear in your rebuttal how you dealt with each point I should be able to make a rapid editorial decision without sending your further revised ms back out for review.,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 15 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Gordon Langsley

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed most comments, but some clarifications are still needed.

About M&M of serology:

- line 160: do you mean that when the Platelia IgG assay was positive at the end of pregnancy for a woman previously diagnosed as seronegative, both sera (inclusion and end) were retested with Platelia IgG? This is still not clear.

- line 163: for the “rare samples with confirmed discrepant results”, do you mean “negative with Bioplex and positive with Platelia at inclusion” ? or “samples at end of study that tested positive, then negative, with Platelia during the first and second run, respectively ” ?

- line 174: “qualitative” should be removed, as the Platelia assay is a quantitative assay.

Results:

- line 212: were any of these 49 women with a negative result obtained on diluted samples at inclusion, diagnosed seropositive at delivery? It should be mentioned, so that there is doubt regarding the 12 announced cases of seroconversion.

- line 269: precise n/N seronegative samples available at delivery

- line 274: precise “available plasma samples at delivery”

- line 275: if there were 12 mother-cord plasma samples, why were only 9 “available for highlighting potential cases of CT” ? It could be rephrased as “from the 12 cases identified with seroconversion during pregnancy, mother and cord blood paired samples were available in only 9”. For better understanding, please mention the results of Platelia Toxo IgM (0/9 positive samples ?) and of WB IgG/IgM, as described in M&M.

- line 277: regarding the rate of CT: 2/820 live births: does it mean that there has been 159 miscarriages? This should be discussed, as some fetal losses might have been due to unrecognized toxoplasmosis.

-the table presented in the response to reviewer 2 should be shown, at least as supplemental material.

Discussion:

-line 367: the rate of seroconversion is 3.4%, not 1.4%, as calculated in the Results section.

- line 383: I would like to insist that seroconversion with no IgM is a rare event, thus it not “not uncommon”, as written, and the sentence must be amended. The study by Fricker-Hidalgo reported 15 cases from 12 centers over 10 years, i.e. 15/4500 (0.3%) cases of seroconversion, which is far different from your results.

- lines 416-19: need to be rephrased. Maybe try “Thus, although the sulfamide-pyrimethamine regimens were very different between this latter study and ours, it led to a reduced severity of the fetal sequelae, which is in agreement with the absence of ultrasonographic signs in our PW2 case despite a maternal infection having occurred at around 4 months of gestation.”

Reviewer #2: The authors answered all the comments of the reviewer.

Minor comment

Lines 364-365: Ophthalmological follow-up (as serological follow-up) during the first year of life is sufficient to diagnose CT.

Long-term ophthalmological follow-up is useful to diagnose ocular toxoplasmosis in newborns previously diagnosed with CT.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Sep 2021

The requested information is provided in the file including both the cover letter and the responses to reviewers.

Submitted filename: Dambrun et al_Response to Reviewers.pdf

Click here for additional data file.

18 Oct 2021

PONE-D-21-15174R2Retrospective study of toxoplasmosis prevalence in pregnant women in Benin and its relation​ with malariaPLOS ONE

Dear Dr. DAMBRUN,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Although referee 1 found your revised manuscript much improved a number of minor clarifications have been requested, so in your rebuttal please make absolutely clear how you addressed the points raised, as this will help me make a rapid editorial decision without sending your manuscript back out for review.

Please submit your revised manuscript by Dec 02 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Gordon Langsley

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Dec 2021

We are responding to the request addressed to us to send the latest version of our revised manuscript without changes (e-mail from the Editorial Manager on November, 24th). This last version corresponds to the R2 version sent on September, 17th. The only modification that the Editorial Manager will have to bring concerns the "Data Availability statement". To comply with the online submission procedure, we have entitled “Dambrun et al_Text” the clean version and “Dambrun et al_Text final” the version “with track changes”. These two texts are identical. We hope to respond as clearly as possible to the request of the Editorial Manager.

16 Dec 2021

Retrospective study of toxoplasmosis prevalence in pregnant women in Benin and its relation​ with malaria

PONE-D-21-15174R3

Dear Dr. MAGALIE DAMBRUN,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gordon Langsley

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

30 Dec 2021

PONE-D-21-15174R3

Retrospective study of toxoplasmosis prevalence in pregnant women in Benin and its relation with malaria

Dear Dr. DAMBRUN:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Gordon Langsley

Academic Editor

PLOS ONE