François Faucher

Jaqueline Wendland

Marie-Aline Charles

Véronique V, Véronique P, Laetitia Valérie

Monique

Marie-Jo Thomas

Babak Khoshnood

David Courtin

Michel Cot

André Garcia

Jules Alao

Nadine Fievet

Roméo Zoumenou

Achille Massougbodji

Michael Boivin

Michael Mireku

Moudachirou Ibikounle

Asia Maselko

Marie-Josèphe Saurel-Cubizolles

Suzanne Maman

Jacqueline Milet

I knew that she was a professor and researcher who was gifted in mentoring and helping young, aspiring students

Figure 10. Follow-up of pregnant women from second trimester until delivery. Table 1. WHO treatment guidelines for P. falciparum infection in high-transmission areas of SSA.

List of Tables

Table 2. Infant-Toddler HOME inventory sub-scales (ages 0 to 3).

Table 3. Summary of all studies and variables collected during follow-up.

Abstract

Soil-transmitted helminth and malaria infection, two parasitic infections found primarily in low-and middle-income populations, impact up to 50% of pregnant women in Sub-Saharan Africa. Parasitic infection in pregnant women is known to have negative consequences on maternal and infant health, such as increased risk of maternal anemia, pre-term birth, low birth weight, and childhood parasitic infection. However, its impacts on child neurocognitive and behavioral development remain widely unknown. Therefore, this thesis sought to study the impacts of soil-transmitted helminth infection in pregnancy (SHIP) and Plasmodium falciparum malaria in pregnancy (MIP) on child neurocognitive and behavioral development in a mother-child cohort from Benin, as well as investigate additional risk factors for parasitic infection that could impact associations. Our mother-child cohort included pregnant women enrolled in a clinical trial in their second trimester comparing the efficacy of two intermittent preventive treatments in pregnancy against malaria, sulfadoxine-pyrimethamine and mefloquine, in Allada, Benin. Blood and stool samples were collected from women at three antenatal care visits (ANVs); SHIP was diagnosed using the Kato-Katz technique and MIP was diagnosed by thick blood smear tests at ANVs and quantitative polymerase chain reaction (qPCR) methods at delivery. Women received treatment for infection according to existing national guidelines, as well as the standard antenatal care package for Benin at the first ANV.

Surviving offspring were assessed by trained investigators for neurocognitive and motor development at one year using the Mullen Scales of Early Learning and again at six years using the Kaufman Assessment Battery in Children 2 nd Edition (KABC-II), the Bruininks-Oseretsky Test of Motor Proficiency Edition 2, the Test of Variables of Attention, and the Strengths and Difficulties Questionnaire (SDQ). A sub-cohort of mothers and children were included in independent analyses to test the role of child blood lead level and maternal depression on subsequent risk of parasitic infection. Prevalence of SHIP was 13% at the 1st ANV, 9% at the 2nd ANV, and 1% at delivery. According to thick blood smear tests, 122 (25%) women were infected with malaria at least once during pregnancy and 121 (31%) were infected with placental malaria diagnosed by qPCR at delivery. Within the sub-cohort, median blood lead level among children measured at 12 months was 56.50 (4.81-578) μg/L and 39/302 (13%) mothers had depressive symptoms 12-months post-partum according to the Edinburgh Postnatal Depression Scale. According to multiple linear regression analyses, SHIP was not associated with low neurocognitive scores in children at six years. Higher SDQ internalizing scores, indicating increased emotional impairments in children, were associated with helminth infection at the 2nd ANV/delivery [1.07 Confidence Interval (CI) (0.15, 2.00)] and at least once during pregnancy [0.79 CI (0.12,1.46)] in adjusted models. Beta coefficients showed that impaired gross motor scores at one year were associated with MIP at least once 0.05)], placental malaria by qPCR ], and high parasite density at delivery 0.02)]. Malaria and high parasite density at the 2 nd ANV were associated with lower KABC-II Non-Verbal Index scores at six years ]

and ], respectively. Neither blood lead level nor maternal depression were found to impact subsequent risk of parasitic infection in children according to regression analyses, before and after adjustment of relevant factors. This thesis provides evidence of longterm behavioral and neurocognitive consequences of parasitic infection in pregnancy in a mother-child cohort with extensive, prospective biological and neurocognitive assessments in West Africa. A better understanding of the mechanisms behind these associations is necessary in order to add to existing evidence that sufficient and early diagnosis and treatment of these infections is imperative and of international public health concern.

Keywords: soil-transmitted helminth infection; malaria; pregnancy; child neurocognitive development; Sub-Saharan Africa

Résumé court

Les infections par helminthes transmis par le sol et le paludisme, deux infections parasitaires que l'on trouve principalement dans les populations à faible et moyen revenu, femmes étaient infectées par le paludisme au moins une fois pendant la grossesse et 121 (31%) étaient infectées par le paludisme placentaire diagnostiqué par qPCR à l'accouchement. Au sein de la sous-cohorte, la plombémie médiane des enfants mesurée à 12 mois était de 56,50 (4,81-578) μg/L et 39/302 mères (12,9%) présentaient des symptômes dépressifs 12 mois après l'accouchement selon l'échelle de dépression postnatale d'Édimbourg. Selon les analyses de régression linéaire multiple, le SHIP n'était pas associé à de faibles scores neurocognitifs chez les enfants à six ans. Des scores d'intériorisation SDQ plus élevés, indiquant des déficiences émotionnelles accrues chez les enfants, étaient associés à une infection helminthique lors de la 2e CPN/accouchement [1,07 Intervalle de Confiance (IC) (0,15, 2,00)] et au moins une fois pendant la grossesse [0, 79 IC (0,[START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF]1,[START_REF] Mutombo | Diagnosis and drug resistance of human soil-transmitted helminth infections: A public health perspective[END_REF]] dans les modèles ajustés. Les coefficients bêta ont montré que l'altération des scores de motricité globale à un an était associée à une MIP au moins une fois [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF]0,05)], au paludisme placentaire par qPCR [START_REF] Sun | Anemia in Pregnancy: A Pragmatic Approach[END_REF][START_REF] Uneke | Impact of Placental Plasmodium falciparum Malaria on Pregnancy and Perinatal Outcome in Sub-Saharan Africa[END_REF]] et à une densité parasitaire élevée à l'accouchement [START_REF] Ndibazza | Associations Between Maternal Helminth and Malaria Infections in Pregnancy and Clinical Malaria in the Offspring: A Birth Cohort in Entebbe, Uganda[END_REF]0,02)]. Le paludisme et une densité parasitaire élevée lors de la 2e CPN étaient associés à des scores inférieurs à l'indice non verbal KABC-II à six ans [START_REF] Ndibazza | Associations Between Maternal Helminth and Malaria Infections in Pregnancy and Clinical Malaria in the Offspring: A Birth Cohort in Entebbe, Uganda[END_REF][START_REF] Giollabhui | Maternal inflammation during pregnancy and offspring psychiatric symptoms in childhood: Timing and sex matter[END_REF]] et [START_REF]Deworming in children[END_REF][START_REF] Stein | The relationship between post-natal depression and mother-child interaction[END_REF]],

respectivement. Ni la plombémie ni la dépression maternelle ne se sont avérées avoir un impact sur le risque ultérieur d'infection parasitaire chez les enfants selon les analyses de régression, avant et après ajustement des facteurs pertinents. Cette thèse fournit des informations des conséquences comportementales et neurocognitives à long terme de l'infection parasitaire pendant la grossesse dans une cohorte mère-enfant avec des évaluations biologiques et neurocognitives exhaustives et prospectives en Afrique de l'Ouest. Une meilleure compréhension des mécanismes à l'origine de ces associations est nécessaire afin d'ajouter aux preuves existantes qu'un diagnostic et un traitement suffisants et précoces de ces infections sont impératifs et constituent une préoccupation de santé publique internationale.

Mots-clés: infection aux helminthes transmis par le sol ; paludisme ; grossesse ; développement neurocognitif de l'enfant ; Afrique subsaharienne

Résumé extensif

Introduction Deux des infections parasitaires les plus fréquentes en Afrique subsaharienne sont les infections par helminthes transmises par le sol, qui affecte 1,5 milliard d'individus chaque année (1), et le paludisme, l'infection parasitaire la plus courante en Afrique d'où proviennent plus de 94 % des 229 millions de cas diagnostiqués en 2019; les autres 6 % des cas provenant d'Asie et d'Amérique du Sud. (2). Le terme "helminthes transmis par le sol" désigne plusieurs espèces de vers parasites, dont les plus courants sont les ascaris (Ascaris lumbricoides), les trichures (Trichuris trichiura) et les ankylostomes (Ancylostoma duodenale et Necator americanus) [START_REF] Alelign | Soil-Transmitted Helminth Infections and Associated Risk Factors among Schoolchildren in Durbete Town, Northwestern Ethiopia[END_REF].

Les femmes enceintes et les enfants de moins de cinq ans sont particulièrement vulnérables à ces infections parasitaires et à leurs conséquences sur la santé telles que l'anémie maternelle, l'insuffisance pondérale à la naissance, la naissance prématurée, le retard de croissance foetale et la malnutrition [START_REF] Cottrell | Submicroscopic Plasmodium falciparum Infections Are Associated With Maternal Anemia, Premature Births, and Low Birth Weight[END_REF][START_REF] Dreyfuss | Determinants of low birth weight among HIV-infected pregnant women in Tanzania[END_REF][START_REF] Kalilani-Phiri | Timing of Malaria Infection during Pregnancy Has Characteristic Maternal, Infant and Placental Outcomes[END_REF][START_REF] Mahande | Prevalence of parasitic infections and associations with pregnancy complications and outcomes in northern Tanzania: a registry-based crosssectional study[END_REF]. L'Organisation mondiale de la santé (OMS) recommande actuellement le traitement prophylactique de l'infection par les helminthes et du paludisme lors des visites de routine des soins prénatals dans les pays d'Afrique subsaharienne où les taux de morbidité et de mortalité dus aux infections parasitaires sont élevés. Au Bénin, une petite nation francophone d'Afrique de l'Ouest, les femmes enceintes reçoivent une dose unique de mébendazole (500 mg) ou d'albendazole (400 mg) en prophylaxie pour prévenir les infections par helminthes pendant la grossesse et trois doses de traitement préventif intermittent pendant la grossesse par sulfadoxine-pyriméthamine contre le paludisme (2,[START_REF]Deworming in pregnant women[END_REF]. Même en présence de soins prénatals de routine et de prévention de ces infections, pour lesquels l'adhésion peut souvent être faible [START_REF] Badirou | Adherence to Intermittent Preventive Treatment of Malaria in Pregnancy with Sulfadoxine-Pyrimethamine and Associated Factors: A Cross-Sectional Survey in Benin's Public Hospitals[END_REF], la morbidité chez les femmes enceintes et leur enfants survivants continue d'être une grande préoccupation de santé publique internationale (10,[START_REF] Nicinski | Neglected Tropical Disease Program[END_REF].

Les conséquences de ces infections sur le développement neurocognitif du foetus restent largement inconnues. Une étude précédente a révélé que les infections par helminthes pendant la grossesse, en particulier les ankylostomes, altère négativement le développement de la motricité globale de la progéniture un an après l'accouchement dans une cohorte d'enfants béninois [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF]. En ce qui concerne le paludisme pendant la grossesse, une étude en laboratoire a trouvé une corrélation entre la progéniture exposée au paludisme expérimental et les déficits neurologiques [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF]. En outre, les femmes enceintes et les enfants des pays à faibles revenus ou à revenus intermédiaires sont exposés à un large éventail de facteurs de risque d'infections parasitaires. Des études récentes ont montré des résultats inédits liant l'exposition environnementale au plomb (Pb) à une diminution du risque d'infection par le paludisme [START_REF] Moya-Alvarez | Elevated Blood Lead Levels Are Associated with Reduced Risk of Malaria in Beninese Infants[END_REF], et la dépression maternelle à une augmentation du risque d'infection du nourrisson [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF].

Cependant, des recherches supplémentaires sur ces sujets sont nécessaires afin de mieux comprendre les facteurs de risque d'infection parasitaire dans les zones de forte transmission.

Compte tenu de ces lacunes dans la littérature et dans la compréhension épidémiologique des infections parasitaires pendant la grossesse dans les régions du monde où ces infections sont fréquentes, nous avons cherché à étudier ces questions dans le cadre de cette thèse. Cette thèse a donc cherché à étudier l'impact des infections par helminthes pendant la grossesse (SHIP) et du paludisme à Plasmodium falciparum pendant la grossesse (MIP) sur le développement neurocognitif et comportemental de l'enfant dans une cohorte mère-enfant du Bénin suivie du deuxième trimestre de la grossesse jusqu'à six ans après l'accouchement. Les objectifs principaux de cette thèse étaient d'étudier l'impact de deux infections parasitaires pendant la grossesse, l'infection par helminthes et le paludisme, sur le développement neurocognitif et comportemental de l'enfant à court et à long terme dans une cohorte mère-enfant du district d'Allada, au Bénin. Les objectifs secondaires de cette thèse visaient à étudier les facteurs sociaux et environnementaux potentiels liés au risque d'infection parasitaire qui auraient pu avoir un impact sur les associations entre l'infection parasitaire pendant la grossesse et le développement neurocognitif de l'enfant dans notre population.

Objectifs principaux

1. Étudier l'impact des infections par helminthes pendant la grossesse sur le développement neurocognitif et comportemental de l'enfant à l'âge de six ans.

2. Étudier l'impact de l'infection palustre à P. falciparum pendant la grossesse sur le développement neurocognitif de l'enfant à un et six ans. (0,[START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF]1,[START_REF] Mutombo | Diagnosis and drug resistance of human soil-transmitted helminth infections: A public health perspective[END_REF]], respectivement, après ajustement pour les facteurs de risque identifiés dans les graphiques acycliques directs. L'imputation multiple des variables manquantes et des paires mère-enfant perdues de vue a confirmé ces résultats. Une analyse de médiation par des techniques de modélisation par équations structurelles a permis d'évaluer le rôle de facteurs médiateurs potentiels, tels que l'inflammation, dans cette association. Cependant, l'inflammation mesurée par la protéine C-réactive pendant la grossesse ne s'est pas avérée être un facteur médiateur significatif.

Alors que le MIP diagnostiqué au moins une fois pendant la grossesse n'a pas altéré de manière significative les scores ELC chez les enfants, il a altéré de manière significative les scores de développement de la motricité globale à un an après ajustement sur les facteurs de risque [-2,55 (-5,15, 0,05)]. En particulier, le paludisme diagnostiqué par qPCR et une densité parasitaire plus élevée à l'accouchement étaient associés à une altération significative des scores de développement de la motricité globale chez les enfants après ajustement, [-4,95 (-7,65, -2,24)] et [-1,92 (-3,86, 0,02)], respectivement. Le MIP diagnostiqué lors d'une CPN ou d'un accouchement par goutte épaisse n'était pas associé à une altération des scores de développement cognitif ou moteur chez les enfants à un an. Une densité parasitaire élevée à l'accouchement était associée à une altération des scores de l'IPM dans les modèles ajustés [-1,25 (-2,46, -0,05)]. Le paludisme diagnostiqué par microscopie et la densité parasitaire élevée lors de la 2e CPN étaient tous deux associés de manière significative à une altération des scores INV du KABC-II dans les modèles ajustés [START_REF] Ndibazza | Associations Between Maternal Helminth and Malaria Infections in Pregnancy and Clinical Malaria in the Offspring: A Birth Cohort in Entebbe, Uganda[END_REF][START_REF] Giollabhui | Maternal inflammation during pregnancy and offspring psychiatric symptoms in childhood: Timing and sex matter[END_REF]] et [START_REF]Deworming in children[END_REF][START_REF] Stein | The relationship between post-natal depression and mother-child interaction[END_REF]], respectivement. Les infections palustres tardives lors de la deuxième CPN et de l'accouchement ont montré des tendances à la détérioration des scores de l'IPM et de l'INV à six ans, bien que statistiquement non significatives.

Concernant les objectifs secondaires de cette thèse, la plombémie médiane parmi les 204 enfants de nos analyses était de 56,50 (±56,83) Il a été démontré que l'infection maternelle pendant la grossesse active la réponse immunitaire inflammatoire de la mère, qui peut à son tour entraîner un risque accru de problèmes et de troubles du comportement chez la progéniture [START_REF] Meyer | In-vivo rodent models for the experimental investigation of prenatal immune activation effects in neurodevelopmental brain disorders[END_REF][START_REF] Shi | Maternal influenza infection is likely to alter fetal brain development indirectly: the virus is not detected in the fetus[END_REF]. Une étude en laboratoire a apporté la preuve d'un lien de causalité entre une infection expérimentale par le paludisme pendant la grossesse et une altération du développement neurocognitif du foetus [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF][START_REF] Weckman | The Impact of Infection in Pregnancy on Placental Vascular Development and Adverse Birth Outcomes[END_REF]. Une étude précédente a spécifiquement révélé que des marqueurs élevés d'inflammation chez les femmes enceintes étaient associés à davantage de symptômes d'internalisation et d'externalisation chez leur progéniture plus tard dans l'enfance [START_REF] Giollabhui | Maternal inflammation during pregnancy and offspring psychiatric symptoms in childhood: Timing and sex matter[END_REF]. Les mécanismes entre le SHIP et le développement comportemental ont été explorés dans les analyses ; cependant, ces mécanismes n'ont pas été explorés dans les modèles de paludisme.

Les recherches sur le moment des infections maternelles pendant la grossesse et leur impact sur la santé du foetus restent équivoques [START_REF] Kalilani-Phiri | Timing of Malaria Infection during Pregnancy Has Characteristic Maternal, Infant and Placental Outcomes[END_REF][START_REF] Mahande | Prevalence of parasitic infections and associations with pregnancy complications and outcomes in northern Tanzania: a registry-based crosssectional study[END_REF]. Nos analyses montrent une tendance entre une infection palustre tardive et des scores neurocognitifs plus faibles chez les enfants à un et six ans.

Cette thèse apporte la preuve que l'exposition au plomb, un métal lourd neurotoxique, est toujours un problème de santé publique dans les milieux à faibles ressources comme le Bénin, même si l'exposition au plomb via la peinture et l'essence a été éradiquée dans les pays à hauts revenus pour la plupart [START_REF] Akkus | Exploring Childhood Lead Exposure through GIS: A Review of the Recent Literature[END_REF]. Il est essentiel d'assurer une surveillance régulière de l'exposition au plomb dans cette population car les munitions utilisées pour chasser la viande de brousse ou encore la présence de peinture dans les maisons étaient associées à une augmentation de la plombémie des enfants [START_REF] Ahmadi | Follow-Up of Elevated Blood Lead Levels and Sources in a Cohort of Children in Benin[END_REF][START_REF] Lysaniuk | Types of homes and ways of life: a territorial analysis of the environmental determinants that factor into the proliferation of malaria vectors in the rural region of Allada in Benin[END_REF]

Introduction

Justification of thesis

Morbidity and mortality from parasitic infections in Sub-Saharan African (SSA) countries remain unacceptably high despite improvements in public health surveillance and treatment [START_REF] Murray | Disabilityadjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010[END_REF]. Two of the most prevalent parasitic infections are soil-transmitted helminth infections, which impact 1.5 billion individuals every year (1), and malaria infection, the parasitic infection most commonly found in SSA where more than 94% of the 229 million cases diagnosed in 2019 originated from, with the other 6% of infections coming from Asia and South America (2). Pregnant women and children under five years of age are particularly vulnerable to these parasitic infections and their dire health consequences such as anemia, low birth weight, pre-term birth, fetal growth restriction, and malnutrition [START_REF] Cottrell | Submicroscopic Plasmodium falciparum Infections Are Associated With Maternal Anemia, Premature Births, and Low Birth Weight[END_REF][START_REF] Dreyfuss | Determinants of low birth weight among HIV-infected pregnant women in Tanzania[END_REF][START_REF] Kalilani-Phiri | Timing of Malaria Infection during Pregnancy Has Characteristic Maternal, Infant and Placental Outcomes[END_REF][START_REF] Mahande | Prevalence of parasitic infections and associations with pregnancy complications and outcomes in northern Tanzania: a registry-based crosssectional study[END_REF]. The World Health Organization (WHO) currently recommends prophylaxis treatment of soil-transmitted helminth and malaria infection during routine antenatal care visits in SSA countries with high morbidity and mortality rates from parasitic infection. In Benin, a small French-speaking West African nation, pregnant women receive single-dose mebendazole (500 mg) or albendazole (400 mg) prophylaxis to prevent soil-transmitted helminth infection in pregnancy (SHIP) and three doses of intermittent preventive treatment in pregnancy with sulfadoxinepyrimethamine (IPTp-SP) for malaria in pregnancy (MIP) (2,[START_REF]Deworming in pregnant women[END_REF]. Even in the presence of routine antenatal care and prevention of these infections, for which adherence can often be low [START_REF] Badirou | Adherence to Intermittent Preventive Treatment of Malaria in Pregnancy with Sulfadoxine-Pyrimethamine and Associated Factors: A Cross-Sectional Survey in Benin's Public Hospitals[END_REF], morbidity in pregnant women and their surviving offspring continues to be of great international public health concern (10,[START_REF] Nicinski | Neglected Tropical Disease Program[END_REF].

While the consequences of parasitic infection during pregnancy on maternal and birth outcomes have been widely studied, the consequences of these infections on fetal neurocognitive development remain largely unknown. One previous study found SHIP, particularly from hookworm species, to impair gross motor development in offspring one-year post-partum in a cohort of Beninese children [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF]. In terms of MIP, one laboratory study found a correlation between experimental malaria-exposed offspring and neurological deficits [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF]. Previous literature has hypothesized that maternal infection during pregnancy may lead to placental inflammatory immune responses that can alter brain development of the fetus and have long-term neurodevelopment consequences [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF][START_REF] Khan | Long-term effects of maternal immune activation on depression-like behavior in the mouse[END_REF]; however, this pathway as it relates to maternal infection from SHIP and MIP have yet to be investigated.

Additionally, pregnant women and children in low-and middle-income countries are exposed to a wide array of factors increasing their risk of parasitic infections, such as tropical climates, iron deficiency, inadequate hygiene and sanitation, and conditions of poverty.

Recent studies have shown novel results linking environmental exposure to lead (Pb) to a decreased risk of malaria infection [START_REF] Moya-Alvarez | Elevated Blood Lead Levels Are Associated with Reduced Risk of Malaria in Beninese Infants[END_REF], and maternal depression to an increased risk of infant infection [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF]. However, more research is needed in order to better understand the risk factors of parasitic infection in high-transmission areas. Given these gaps in the literature and in the epidemiological understanding of parasitic infection in pregnancy in areas of the world with a high burden of disease, we sought to address these questions within this thesis.

Objectives and hypotheses

The main objectives of this thesis were to study the impact of two parasitic infections during pregnancy, soil-transmitted helminth infection and malaria, on child neurocognitive and behavioral development in a mother-child cohort in the district of Allada, Benin.

Secondary objectives of this thesis sought to investigate factors potentially related to risk of parasitic infection that could have an impact on associations between parasitic infection in pregnancy and child neurocognitive development in our population, but that have been widely b. To investigate the association between child blood lead levels and risk of malaria infection.

We hypothesized that parasitic infection, both soil-transmitted helminth infection and malaria infection, during pregnancy would negatively impact child neurocognitive development. Based on results by [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF] that demonstrated an inverse association between SHIP and child gross motor scores as measured by the Mullen Scales of Early Learning (MSEL) at one year of age in this same cohort [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF], we hypothesized that SHIP would continue to impair long-term neurocognitive and behavioral development measured by the Kaufman Assessment Battery in Children 2 nd Edition (KABC-II) and the Strengths and Difficulties Questionnaire (SDQ), respectively, in this cohort at six years of age. Based on previous hypotheses of the role of maternal inflammation markers as the mechanism for this relationship, we hypothesized that maternal inflammation was a mediating factor between SHIP and impaired child development. There were no previous studies conducted on the impact of MIP on child neurocognitive development; however, one laboratory study on rodent-models found that experimental malaria-exposed offspring had persistent neurological deficits compared to the control group in their analyses [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF]. Due to this evidence, and the previously mentioned hypothesis that maternal infection may induce an inflammatory response that hinders fetal brain development, we hypothesized that MIP would be associated with impaired neurocognitive development measured by the MSEL and the KABC-II in children in our cohort at both one and six years of age, respectively.

The role of social and environmental factors on risk of parasitic infection was studied in a sub-cohort of 400 mother-child pairs for secondary objectives in order to identify whether these factors should be considered in our main analyses and in order to add to the limited existing epidemiological research on the link between parasitic infection and child development. We hypothesized that maternal depression and poorer quality of parent-child interactions as measured by the Edinburgh Postnatal Depression Scale (EPDS) and the Home Observation Measurement of the Environment (HOME), respectively, would be associated with higher risk of parasitic infection [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF]. One previous study had identified an inverse association between child blood lead level and risk of malaria infection in this cohort of Beninese children at 12 months of age ( 14), therefore we hypothesized that we would continue to see this association between high blood lead level and lower risk of malaria infection in a prospective follow-up of the same children from 12-24 months of age in this cohort.

Thesis Overview

This thesis is divided into seven chapters: Chapter 6 presents the fourth article of this thesis, published in Plos One, which studied the association between blood lead level and subsequent risk of malaria infection from P.falciparum in a prospective sub-cohort of children from our population.

Chapter
Finally, Chapter 7 discusses the main findings for each objective of the thesis, the overall strengths and limitations of findings, and their implications across public health domains. Following this chapter is a conclusion that offers recommendations for policy and future research.

Chapter 1. Background: Parasitic infection, pregnancy, and child development

This first chapter provides background information necessary to understand the justification of the objectives of this thesis. The primary objectives sought to study the impact of two parasitic infectious diseases during pregnancy, soil-transmitted helminth infection and malaria, on child neurocognitive development. Secondary objectives explored hypothesized risk factors for parasitic infection that remain largely unstudied in order to assess their importance for models used in the primary objectives. This chapter explains the epidemiology of these two parasitic infections in pregnancy, as well as their known risk factors and consequences on maternal and infant health. Finally, this chapter discusses the factors known to influence child neurocognitive development, and the particular difficulties faced by children in low-and middle-income countries. The purpose of this chapter is to explain the existing literature related to parasitic infection in pregnancy and child neurocognitive development in order to highlight the gaps in the existing literature that this thesis addresses.

Soil-transmitted helminth infection

Soil-transmitted helminth (STH) infections are infections caused by several species of parasitic worms that affect almost a quarter of the world's population. Each year, more than 1.5 billion people, primarily from low-and middle-income countries, are infected by STH species, contributing to over 5 million disability-adjusted life years (DALYs) [START_REF] Pullan | Global numbers of infection and disease burden of soil transmitted helminth infections in 2010[END_REF]. Symptoms of these infections include gastrointestinal illness such as diarrhea and abdominal pain, as well as overall weakness and malaise. Morbidity from STH infections is directly related to the species of worm and the number of worms, or the parasite density, found in infected individuals. Those with a lighter parasitic density may not show symptoms; however, those with higher density infections may show severe symptoms which can lead to long-term health and developmental consequences [START_REF] Organization | Soil-transmitted helminthiases : eliminating as public health problem soil-transmitted helminthiases in children : progress report 2001-2010 and strategic plan 2011-2020[END_REF]. As their name suggests, these helminth species transmit infected eggs to individuals through contaminated soil in areas of the world with poor hygiene, sanitation, and access to water. The majority of those infected by STH each year are from Asian and African countries of low-and middle-income. Populations living in these areas of the world can exhibit polyparasitism, meaning individuals may be infected with more than one species of helminth at a time [START_REF] Dawaki | The burden and epidemiology of polyparasitism among rural communities in Kano State, Nigeria[END_REF]. Soil-transmitted helminth infections are among 17 diseases on the list of Neglected Tropical Diseases, established by the WHO as diseases that impact tropical and sub-tropical low-income countries and have not yet been eradicated due to low funding and low attention from public health stakeholders [START_REF]Neglected tropical diseases[END_REF].

Life cycle and transmission

The term 'soil-transmitted helminths' refers to several species of parasitic worms, the most common of which are roundworms, Ascaris lumbricoides, whipworms, Trichuris trichiura, and hookworms, Ancylostoma duodenale and Necator americanus [START_REF] Alelign | Soil-Transmitted Helminth Infections and Associated Risk Factors among Schoolchildren in Durbete Town, Northwestern Ethiopia[END_REF].

A.lumbricoides infects an estimated 800 million individuals per year, primarily children and adolescents [START_REF] Vos | Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[END_REF]. This species infects individuals through fecal-oral transmission of eggs, as seen in Figure 1 [START_REF] Prevention | CDC -Ascariasis -Biology[END_REF]. Adult worms live in the small intestines of infected individuals, and female worms are able to produce hundreds of thousands of eggs per day which are then passed through feces during defecation. In areas of the world with poor sanitation and access to running water, defecation often occurs outside and in unprotected areas where feces can contaminate soil, which creates the perfect life-cycle environment for fertilized helminth eggs.

Third-stage larvae develop to infectious levels in fertile eggs within a few weeks. Larvaeinfected eggs in the soil are then swallowed, primarily by children playing in the soil, who ingest the soil or fruits and vegetables improperly washed beforehand that still contain traces of soil [START_REF] Mireku | Consequences of prenatal geophagy for maternal prenatal health, risk of childhood geophagy and child psychomotor development[END_REF]. Once these eggs are swallowed, the larvae hatch and travel from the intestinal mucosa into the lungs where larvae further mature and finally redescend back down into the small intestine to complete the life cycle and reach their adult form. Adult worms can live in the intestines of infected individuals for up to two years if left untreated. T.trichiura, named whipworm for its shape, infects close to 480 million people per year; again children are found to be the most heavily burdened [START_REF] Vos | Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[END_REF]. Similar to Ascaris, these whipworm species infect hosts through fecal-oral transmission. However, unlike Ascaris, T.trichiura larvae do not migrate to the lungs to reach their adult form, but rather attach to intestinal villi and mature into their adult forms within the ascending colon of the intestines [START_REF] Viswanath | Trichuris Trichiura[END_REF].

Hookworm species A.duodenale and N.americanus infect an estimated 470 million people per year, and are most prevalent in adult populations rather than in children, although children may still become infected by these species [START_REF] Vos | Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013[END_REF]. Hookworms have free-living larvae found in soil that penetrate human skin, usually bare feet, during transmission (Figure 2) (44).

Adult worms living in the intestines of infected individuals pass their eggs through feces defecated in nearby soil. Larvae then hatch within a few days and are free-living within the contaminated soil and mature until they reach their infective larval stage. Once infective, these larvae can live in contaminated soil for several weeks until coming into contact with bare skin, normally bare feet of individuals walking in contaminated soil, where they penetrate the skin and travel through blood vessels to the heart and lungs. Once in the lungs, the larvae penetrate the pulmonary alveoli and ascend the bronchial tree to the pharynx where they are swallowed. Finally, the larvae reach the small intestines where they mature into their adult worm stage and attach themselves onto the intestinal wall and can cause significant blood loss in hosts [START_REF] Wei | Hookworm Infection: A Neglected Cause of Overt Obscure Gastrointestinal Bleeding[END_REF].

Figure 2. Life-cycle and transmission of hookworm species.

Diagnosis, prevention, and treatment

A variety of diagnostic methods are available to detect STH infections, ranging from microscopic methods to antigen/antibody assays to more advanced polymerase chain reaction (PCR) methods [START_REF] Mutombo | Diagnosis and drug resistance of human soil-transmitted helminth infections: A public health perspective[END_REF]. The most widely-used method to date is the Kato-Katz technique, developed by Dr. Kan Kato and Naftale Katz in the mid 20 th century, which is considered the gold standard for diagnosis of STH infection of multiple species by the WHO [START_REF] Katz | A simple device for quantitative stool thick-smear technique in Schistomiasis Mansoni[END_REF]. This microscopy technique involves the examination of stool samples for the presence of helminth eggs under a light microscope; information on the number of eggs and the type of eggs are recorded in order to diagnose STH infection. The Kato-Katz technique is widely accepted for use in low-income countries with a high-burden of disease from STH due to its low-cost, ability to diagnose multiple species of STH, relatively simple application given the limited laboratory resources in low-income countries, easily learned techniques by laboratory technicians, and high specificity for detection of infection. However, there are some limitations to this diagnostic tool, such as that it is less reliable in detecting certain species of helminths (Strongyloidiasis) and in detecting low egg densities, therefore potentially underestimating the true burden of disease from STH infection in endemic populations [START_REF] Keller | Performance of the Kato-Katz method and real time polymerase chain reaction for the diagnosis of soiltransmitted helminthiasis in the framework of a randomised controlled trial: treatment efficacy and day-to-day variation[END_REF].

Several preventive measures are available for endemic populations to reduce the burden of disease from STH infection, such as Water, Sanitation, and Hygiene (WASH) campaigns carried out through UNICEF [START_REF]Water, Sanitation and Hygiene (WASH)[END_REF]. WASH aims to provide clean water and sanitation to low-income areas to improve overall health and wellbeing of under-served populations. These campaigns, which include a focus on hand-washing practices, have been associated with a reduced odds of STH transmission by at least 33% according to a metaanalysis [START_REF] Strunz | Water, Sanitation, Hygiene, and Soil-Transmitted Helminth Infection: A Systematic Review and Meta-Analysis[END_REF]. Additionally, wearing adequate footwear while outside has been shown to prevent hookworm infection in particular, due to their mode of transmission via cutaneous penetration [START_REF] Strunz | Water, Sanitation, Hygiene, and Soil-Transmitted Helminth Infection: A Systematic Review and Meta-Analysis[END_REF]. While these preventive measures are long-term and require substantial political and societal involvement, systematic anti-helminthic prophylaxis is also used to prevent STH infection in the shorter term among high-risk groups of morbidity and mortality such as children under the age of five, women of reproductive age, and pregnant women.

For children ages 12 months to 12 years and for non-pregnant women of reproductive age, the WHO recommends a single-dose of albendazole (400 mg) or mebendazole (500 mg), two inexpensive and easy-to-administer treatments, given once a year when the baseline prevalence of STH infections in the community is over 20%, and twice a year when the prevalence in the community is over 50% [START_REF]Deworming in children[END_REF][START_REF]WHO | Deworming in non-pregnant adolescent girls and women of reproductive age[END_REF]. For pregnant women, the WHO recommends a single-dose of albendazole (400 mg) or mebendazole (500 mg) given to women at their first antenatal care visit after the first trimester of pregnancy in areas where the baseline prevalence of hookworm infection is 20% or more and where the prevalence of anemia in pregnant women is 40% or more [START_REF]Deworming in pregnant women[END_REF], given that hookworm infection in particular can be a major cause of anemia. The prevention and treatment of STH infection in pregnancy is of particular public health importance due to the elevated risks of morbidity and mortality in pregnant women and their offspring.

Soil-transmitted helminth infection in pregnancy

Up to 30% of women in SSA are estimated to be diagnosed with SHIP at least once during routine antenatal care follow-up [START_REF] Ziegelbauer | Effect of Sanitation on Soil-Transmitted Helminth Infection: Systematic Review and Meta-Analysis[END_REF]; however, this prevalence ranges between countries, with studies reporting 11% of women diagnosed in Benin and 32% in Ethiopia [START_REF] Derso | Prevalence of intestinal parasitic infections and associated risk factors among pregnant women attending antenatal care center at Felege Hiwot Referral Hospital, northwest Ethiopia[END_REF][START_REF] Ouédraogo | Maternal anemia at first antenatal visit: prevalence and risk factors in a malariaendemic area in Benin[END_REF] for example. Preventive prophylaxis efforts target pregnant women in particular since they and their fetus are most at risk of the various consequences of infection during the prenatal period.

Demographic and clinical characteristics have been shown to put certain pregnant women at higher risk of infection than others. A multitude of literature has shown that primigravida women, women who are pregnant for the first time, have an increased susceptibility to SHIP compared to multigravida women [START_REF] Adegnika | Increased prevalence of intestinal helminth infection during pregnancy in a Sub-Saharan African community[END_REF][START_REF] Eijk Am Van, Lindblade | Geohelminth Infections among Pregnant Women in Rural Western Kenya; a Cross-Sectional Study[END_REF][START_REF] Ojurongbe | Prevalence and associated factors of Plasmodium falciparum and soil transmitted helminth infections among pregnant women in Osun state, Nigeria[END_REF][START_REF] Woodburn | Risk factors for helminth, malaria, and HIV infection in pregnancy in Entebbe, Uganda[END_REF]. Maternal age, education, and socio-economic status also influence infection, with lower maternal age being associated with increased infection risk and higher maternal education and socio-economic status associated with lower infection risk [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF][START_REF] Woodburn | Risk factors for helminth, malaria, and HIV infection in pregnancy in Entebbe, Uganda[END_REF][START_REF] Mpairwe | Pregnancy and helminth infections[END_REF][START_REF] Wekesa | Intestinal Helminth Infections in Pregnant Women Attending Antenatal Clinic at Kitale District Hospital, Kenya [Internet[END_REF].

Consequences on maternal health

SHIP can have several negative consequences on maternal health, including increased risk of maternal anemia, co-infection with other parasites, inflammation, malnutrition, miscarriage, and mortality [START_REF] Mahande | Prevalence of parasitic infections and associations with pregnancy complications and outcomes in northern Tanzania: a registry-based crosssectional study[END_REF][START_REF] Anderson | Old friends and friendly fire: Pregnancy, hookworm infection, and anemia among tropical horticulturalists[END_REF][START_REF] Kurtis | Maternal Schistosomiasis japonica is associated with maternal, placental, and fetal inflammation[END_REF][START_REF] Nurdia | Impact of intestinal helminth infection on anemia and iron status during pregnancy: a community based study in Indonesia[END_REF]. Anemia is a deficiency in functional red blood cells and their hemoglobin concentration, which causes poor tissue oxygenation and can have serious health implications, particularly in pregnant women who are in greater need of blood supplies in order to adequately nourish mother and developing fetus. While multifactorial, one major cause of anemia is hookworm infection, due to chronic blood loss from the attachment of worms onto the intestines [START_REF] Anderson | Old friends and friendly fire: Pregnancy, hookworm infection, and anemia among tropical horticulturalists[END_REF]. In fact, while the estimated DALYs from STH infection are around 5 million [START_REF] Pullan | Global numbers of infection and disease burden of soil transmitted helminth infections in 2010[END_REF], if taking into account anemia due to hookworm infection, those estimates increase 7-fold to more than 39 million DALYs (50) due to STH infection. Anemia in turn can contribute to low birth weight, pre-term birth, and other adverse pregnancy outcomes for mother and fetus [START_REF] Sun | Anemia in Pregnancy: A Pragmatic Approach[END_REF].

Infections with helminth species and with malaria are two of the most prevalent parasitic infections in African nations and often overlap in their geographical distribution [START_REF] Brooker | The codistribution of Plasmodium falciparum and hookworm among African schoolchildren[END_REF][START_REF] Brooker | Epidemiology of plasmodium-helminth co-infection in Africa: populations at risk, potential impact on anemia, and prospects for combining control[END_REF]. Due to this, individuals are often co-infected with helminth and malaria species, particularly P.

falciparum. However, previous research has found conflicting evidence on whether or not infection with one parasite increases, decreases, or has no change on the risk of infection with a subsequent parasite [START_REF] Eijk Am Van, Lindblade | Geohelminth Infections among Pregnant Women in Rural Western Kenya; a Cross-Sectional Study[END_REF][START_REF] Adegnika | Epidemiology of malaria and helminth interaction: a review from 2001 to 2011[END_REF][START_REF] Degarege | Malaria and related outcomes in patients with intestinal helminths: a cross-sectional study[END_REF][START_REF] Shapiro | Epidemiology of helminth infections and their relationship to clinical malaria in southwest Uganda[END_REF]. A meta-analysis in 2013 pooled results from these existing studies and concluded that pregnant women infected with helminths had an increased risk of malaria infection compared to uninfected pregnant women [START_REF] Naing | Malaria and soil-transmitted intestinal helminth co-infection and its effect on anemia: a metaanalysis[END_REF]. Researchers hypothesized that SHIP, which tends to be a more chronic infection than malaria, may compromise the maternal immune system and its ability to react to and fight off subsequent infections. Of course, due to the differing routes of transmission of different helminth species, their effect on risk of malaria infection varies between species, which could be the reason for conflicting results on the impact of helminth infection on risk of malaria infection [START_REF] Adegnika | Epidemiology of malaria and helminth interaction: a review from 2001 to 2011[END_REF].

Exposure to helminths induces an inflammatory immune response in infected individuals, which can subsequently impact fetal development [START_REF] Mpairwe | Pregnancy and helminth infections[END_REF]. Hookworm infection has been shown to increase systemic inflammation, more specifically increase erythrocyte sedimentation rate, a general marker of inflammation [START_REF] Anderson | Old friends and friendly fire: Pregnancy, hookworm infection, and anemia among tropical horticulturalists[END_REF]. Inflammation is thought to be the mechanism through which SHIP and other maternal infections can lead to adverse birth outcomes and impaired fetal brain development, which will be discussed later in this thesis.

Finally, SHIP can lead to malnutrition in chronically infected individuals in endemic regions of the world [START_REF] Stephenson | Malnutrition and parasitic helminth infections[END_REF]. Anemia and iron deficiency due to helminth infection in pregnancy greatly contribute to this burden of disease from malnutrition [START_REF] Arinola | Serum Micronutrients in Helminth-infected Pregnant Women and Children: Suggestions for Differential Supplementation During Anti-helminthic Treatment[END_REF], which is why iron and folate acid supplements are vital during prenatal care in high-risk populations. Maternal malnutrition can subsequently impact fetal growth and development, as well as increase risk of miscarriage and mortality [START_REF] Stephenson | Malnutrition and parasitic helminth infections[END_REF][START_REF] Bleker | Malnutrition and depression in pregnancy and associations with child behaviour and cognitive function: a review of recent evidence on unique and joint effects 1[END_REF].

Consequences on birth outcomes

Several studies have shown a detrimental impact of SHIP on birth outcomes such as preterm birth, low birth weight, and fetal growth restriction due to the increased risk of maternal anemia from infection [START_REF] Mpairwe | Pregnancy and helminth infections[END_REF][START_REF] Passerini | Increased Birth Weight Associated with Regular Pre-Pregnancy Deworming and Weekly Iron-Folic Acid Supplementation for Vietnamese Women[END_REF][START_REF] Friedman | Schistosomiasis and pregnancy[END_REF]. However, the mechanism behind this link remains unclear and several studies have in fact shown that the impact of helminth-malaria co-infection during pregnancy is primarily to blame for these negative birth outcomes [START_REF] Boel | Complex Interactions between Soil-Transmitted Helminths and Malaria in Pregnant Women on the Thai-Burmese Border[END_REF]. Many clinical trials have sought to investigate whether helminth prophylaxis in pregnancy can improve these birth outcomes, with some showing promising results [START_REF] Christian | Antenatal anthelmintic treatment, birthweight, and infant survival in rural Nepal[END_REF][START_REF] De Silva | Effect of mebendazole therapy during pregnancy on birth outcome[END_REF][START_REF] Torlesse | Albendazole therapy and reduced decline in haemoglobin concentration during pregnancy (Sierra Leone)[END_REF]. However, other clinical trials have found conflicting results that show no impact of helminth treatment during pregnancy on improving adverse birth outcomes [START_REF] Larocque | A doubleblind randomized controlled trial of antenatal mebendazole to reduce low birthweight in a hookworm-endemic area of Peru[END_REF][START_REF] Ndibazza | Effects of deworming during pregnancy on maternal and perinatal outcomes in Entebbe, Uganda: a randomized controlled trial[END_REF][START_REF] Ndyomugyenyi | Efficacy of ivermectin and albendazole alone and in combination for treatment of soil-transmitted helminths in pregnancy and adverse events: a randomized open label controlled intervention trial in Masindi district, western Uganda[END_REF].

Consequences on child health and development

Maternal infection with helminths can impact fetal and infant immunity to helminth and other parasitic infections. As previously stated, maternal infection with helminths elicits an immuno-regulatory response, which can impact the attached fetus in the placenta [START_REF] Petersen | Protozoan and helminth infections in pregnancy. Short-term and long-term implications of transmission of infection from mother to foetus[END_REF]. Fetal exposure to maternal antibodies produced in response to infection could in fact increase susception to helminth and other parasitic infection, compared to unexposed fetuses, according to some literature [START_REF] Petersen | Protozoan and helminth infections in pregnancy. Short-term and long-term implications of transmission of infection from mother to foetus[END_REF][START_REF] Maizels | Helminth parasites--masters of regulation[END_REF]. This could be due to the fact that helminth infection has been shown

to induce T-cell hyporesponsiveness, a down-regulation of the immune response, making for ideal physiological conditions for parasite survival within human hosts [START_REF] Ndibazza | Associations Between Maternal Helminth and Malaria Infections in Pregnancy and Clinical Malaria in the Offspring: A Birth Cohort in Entebbe, Uganda[END_REF]. However, other research has demonstrated that fetuses acquire immunity from infected mothers which prevents them from experiencing severe forms of disease from helminth infection otherwise seen in infants without this acquired immunity [START_REF] Da Paz | Infection by Schistosoma mansoni during pregnancy: Effects on offspring immunity[END_REF][START_REF] Elliott | The impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in Uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood [ISRCTN32849447[END_REF]. Subsequently, a large body of evidence is available on the detrimental impact of STH infection in childhood on child cognition and development [START_REF] Dickson | Effects of treatment for intestinal helminth infection on growth and cognitive performance in children: systematic review of randomised trials[END_REF][START_REF] Ezeamama Ae | HELMINTH INFECTION AND COGNITIVE IMPAIRMENT AMONG FILIPINO CHILDREN[END_REF]. However, evidence on the ability of routine helminth treatment in children to negate or reverse these consequences remains insufficient [START_REF] Dickson | Effects of treatment for intestinal helminth infection on growth and cognitive performance in children: systematic review of randomised trials[END_REF][START_REF] Nampijja | Effects of Maternal Worm Infections and Anthelminthic Treatment during Pregnancy on Infant Motor and Neurocognitive Functioning[END_REF].

Two previous studies, to our knowledge, have investigated the impacts of SHIP on child neurocognitive development. The first, within a Ugandan mother-child cohort in the early 2000s, found that maternal infection from Mansonella perstans and Strongyloides stercoralis worm species were associated with impaired early executive function and language in 15-month old infants [START_REF] Nampijja | Effects of Maternal Worm Infections and Anthelminthic Treatment during Pregnancy on Infant Motor and Neurocognitive Functioning[END_REF]. The second study took place in the same mother-child cohort analyzed within this thesis from Allada, Benin. This study found that SHIP diagnosed at the first antenatal care visit and at any point during pregnancy was associated with impaired gross motor scores and cognitive ability in children as measured by the MSEL at 12 months of age. Authors of these studies hypothesized that maternal inflammation and/or maternal anemia could be the mechanisms behind these associations, as both have been shown to impact fetal brain development [START_REF] Smith | Maternal Immune Activation Alters Fetal Brain Development through Interleukin-6[END_REF][START_REF] Chang | Effect of iron deficiency anemia in pregnancy on child mental development in rural China[END_REF]. In addition to these studies, there is a growing body of literature showing negative associations between other types of maternal infection, such as human immunodeficiency virus (HIV) or influenza, and impaired cognitive and behavioral development in offspring [START_REF] Doaré | Neurodevelopment in children born to HIVinfected mothers by infection and treatment status[END_REF][START_REF] Murphy | Maternal infection and stress during pregnancy and depressive symptoms in adolescent offspring[END_REF]. This research also suggests that the materno-fetal immunoinflammatory response to infection disrupts placental vasculogenesis and angiogenesis necessary for fetal development [START_REF] Weckman | The Impact of Infection in Pregnancy on Placental Vascular Development and Adverse Birth Outcomes[END_REF].

In addition to the direct impact of SHIP on maternal and fetal outcomes, the use of preventive and treatment drugs for infection during pregnancy could also have consequences for the fetus; however, this subject remains highly debated amongst public health actors and research on the subject remains equivocal. Some studies have shown that helminth prophylaxis has no impact (either positive or negative) on adverse birth outcomes [START_REF] Nampijja | Effects of Maternal Worm Infections and Anthelminthic Treatment during Pregnancy on Infant Motor and Neurocognitive Functioning[END_REF][START_REF] Lau | Treatment of soil-transmitted helminth infections in pregnancy: a systematic review and metaanalysis of maternal outcomes[END_REF]. While observational studies have shown benefits of anti-helminthic treatment on maternal and birth outcomes, no clinical trials have found such benefits [START_REF] Imhoff-Kunsch | Antihelminthics in pregnancy and maternal, newborn and child health[END_REF].

Gaps in the research

Based on the existing literature of the consequences of SHIP, we found that evidence was needed to support the limited studies on its impacts on child neurocognitive development, especially in school-aged children. We also sought to confirm the negative associations between SHIP and child cognitive development seen in children at 12 months from this birth cohort in Benin prospectively at six years of age. Additionally, the hypothesized role of the maternal inflammatory response, particularly due to helminth infection, needed to be further investigated.

Malaria

Malaria is an infectious disease caused by parasites that are transmitted to humans through the bite of infected female Anopheles mosquito vectors [START_REF] Bloland | Population NRC (US) C on, Program on Forced Migration and Health at the Mailman School of Public Health CU. Epidemiology of Malaria [Internet[END_REF]. Malaria occurs in tropical and sub-tropical areas of the world, where wet and humid meteorological conditions provide a favorable environment for mosquito populations (2). In non-immune individuals, malaria symptoms include fever, chills, headache, and diarrhea, among other non-specific symptoms.

These symptoms can occur anywhere from 9-30 days after being bitten by an infected mosquito.

If not treated quickly with antimalarial drugs, infection can progress to severe illness, including cerebral malaria, and death. The progression of malaria in infected individuals depends highly on the immune status of the individual, the type of parasite they are infected with, and the use of antimalarial drugs after infection. In areas of the world where transmission of malaria is common, individuals acquire immunity after repeated infections of malaria and eventually exhibit asymptomatic infections [START_REF] Bloland | Population NRC (US) C on, Program on Forced Migration and Health at the Mailman School of Public Health CU. Epidemiology of Malaria [Internet[END_REF].

According to the World Malaria Report in 2020, there were over 229 million cases of malaria reported in 2019, with 409,000 reported deaths due to malaria infection [START_REF]World malaria report[END_REF]. An estimated 94% of these malaria infections and deaths occurred in SSA, with the remaining infections occurring primarily in South America and Asia, which is why these regions are known as the 'malaria belt' of the world (Figure 3) (100). The most at-risk populations in hightransmission areas are children under five years of age and pregnant women, as well as patients with HIV/AIDS (2). In 2019, over 67% of deaths due to malaria occurred in children under the age of five [START_REF]World malaria report[END_REF].

Life cycle and transmission

As previously mentioned, malaria is transmitted through the bite of infected mosquito vectors from the Anopheles genus. There are over 400 different species of Anopheles mosquitoes, with 30 of these species capable of transmitting the malaria parasite to humans.

One of five parasites from the Plasmodium genus can cause human malaria infection: P. falciparum, P. vivax, P. ovale, P. knowlesi and P. malariae. The geographical distribution of these mosquito and parasite species varies; however, P. falciparum parasites are found in almost all areas of the world where malaria transmission occurs and account for over 90% of malaria infections in SSA. This species of parasite is also most responsible for severe malaria infection and death [START_REF] Bloland | Population NRC (US) C on, Program on Forced Migration and Health at the Mailman School of Public Health CU. Epidemiology of Malaria [Internet[END_REF].

The malaria parasite life cycle involves two hosts, the mosquito vector where the sexual phase of the life cycle occurs and the human host where the asexual phase occurs (Figure 4) (101). When a female Anopheles mosquito takes a blood meal from a human infected with malaria, they ingest the male and female gametocytes of the parasite along with their meal, which then make their way into the gut of the mosquito. From there, the male and female gametes of the parasite form zygotes, which continue through the sexual life cycle to eventually develop into oocysts which grow and divide to form sporozoites. This growth and division, known as the sporogonic phase, continues for 8-15 days until the oocysts burst and release these infectious sporozoites into the body of the mosquito, which then travel to the mosquito salivary glands, ready to be injected into their hosts. This stage of the life cycle occurring within the mosquito vector does not lead to clinical infection in mosquitoes, in fact the malaria parasite and mosquito have a symbiotic, or mutually beneficial, relationship. When an infected mosquito draws a blood meal from a human host, the sporozoites that are present within the salivary glands are injected into the host (102).

Once within the human host, there are two parts of the malaria parasite life cycle that occur, one in the human liver and one in the red blood cells (RBCs). The injected sporozoites travel to and infect the liver cells of the human, usually within a few hours of biting, and mature into schizonts which contain tens of thousands of merozoites, the stage that invades the RBCs.

This phase of the life cycle, known as the pre-erythrocytic phase, lasts 5-16 days and an infected individual does not experience clinical symptoms of infection at this point. (Side note: parasites from P. vivax and P. ovale species can remain dormant in the liver for months or years and cause repeated malaria attacks in infected individuals long after treatment of initial infection).

When the schizonts in the liver are fully matured, they burst and release the merozoites that travel to and invade RBCs; a process that happens too quickly for the host immune system to react and defend itself from infection (103). Once within the RBCs, merozoites ingest hemoglobin to grow and multiply within the cell as they pass through the ring, trophozoite, and schizont stages of the life cycle. This part of the life cycle, known as the blood stage or the erythrocytic phase, is when infected hosts exhibit the clinical symptoms of malaria infection.

At the end of this cycle, RBCs burst and release new merozoites to infect other RBCs within the human host, allowing parasite density to increase in the absence of treatment. A few of the parasites from the RBCs develop into the sexual male and female gametocytes which are then ingested by Anopheles mosquitoes during blood meals (Meibalan & Marti, 2017).

Another important factor of malaria transmission is mosquito vector behavior and environment. For example, the most prevalent malaria vector species in SSA, A. gambiae, breeds in small pools of stagnant water, meaning that during and just after rainy seasons are the periods of the year when transmission of malaria is highest, and therefore vector control strategies could be the most beneficial in order to prevent infection. Mosquitoes also tend to feed at night, when human hosts are sleeping, meaning that certain preventive measures can be taken to reduce the risk of being bitten by an infected mosquito vector at night. These methods of prevention and treatment will be discussed in detail later. Most often, individuals living in high-transmission areas are cognizant of malaria symptoms and know to get treatment when exhibiting symptoms, if treatment is easily accessible. Severe malaria refers to infections with the previously mentioned symptoms, as well as more serious complications such as organ system failure, cerebral malaria, severe anemia, respiratory distress, or hypoglycemia. Severe malaria can result from untreated uncomplicated malaria and is often the result of infection from P. falciparum species. This type of malaria infection can be fatal if not treated urgently, and can have long-lasting health consequences for survivors. As briefly mentioned earlier in this section, individuals infected with malaria can develop an immunity after repeated infections. This is typical in high-transmission areas of the world, like SSA, and is the reason why children under five years of age are the most at risk for serious disease and death, since their immune systems are less developed and less capable of fending off malaria infection. Once an individual has survived multiple malaria infections, they develop an acquired immunity to clinical infection and in most cases exhibit asymptomatic malaria infections, which is most common in adults living in malaria-endemic areas.

Diagnosis, prevention, and treatment

The prompt diagnosis of malaria is crucial in providing adequate care to infected individuals and reducing the risk of severe infection or death, since malaria is a curable disease if diagnosed and treated effectively. There are several methods for malaria diagnosis, including clinical, laboratory, and molecular methods, and the use of one method over another largely depends on the availability of laboratory equipment and trained technicians, the cost, and the context in which malaria is being diagnosed (i.e., an endemic area vs. non-endemic area) (104).

The clinical manifestations of malaria can be used to diagnose disease in settings with sparce laboratory equipment and technicians; however, due to the non-specific symptoms of malaria illness that may overlap with other febrile illnesses common in high-transmission areas, this clinical diagnosis sometimes leads to over-diagnosis of malaria and over prescription of antimalarial medications (105). The gold standard for malaria diagnosis is the microscopic examination of thick and thin blood smears to identify the presence of malaria parasites in a laboratory (106). This method of diagnosis is relatively simple to carry out and provides results within a few hours of blood collection from an individual. The examination of thick and thin blood smears allows technicians to identify the presence of malaria parasites, including the species of parasite and the parasitemia (the quantity of parasites in the blood). In settings where laboratory equipment is not as readily available, and when diagnostic results are needed quickly in order to provide treatment, Rapid Diagnostic Tests (RDTs) can be used. These tests detect specific malaria antigens created in response to malaria infection using a blood sample; results can be obtained within 15 minutes of administering this test. While this method of diagnosis is beneficial in low-income, rural settings, it does not permit medical professionals to identify the parasite species or parasitemia in infected individuals, it simply identifies whether or not an individual is infected. Additionally, those with low parasitemia may have false negative RDT results and therefore go untreated for malaria infection. The Centers for Disease Control (CDC)

recommends that all RDT diagnostic results, whether negative or positive, be followed up with microscopy techniques when available (107). Finally, the most sensitive and specific diagnostic method for malaria is the use of PCR-based techniques. These techniques require more sophisticated laboratory resources, which are not available in many low-income settings, and involve higher costs and more rigorous training for laboratory technicians to accurately carry out procedures. However, PCR methods are able to identify extremely low parasitemia, as few as 1-5 parasites/µl of blood (≤ 0.0001% of infected red blood cells) compared with around 50-100 parasites/µl of blood by microscopy or RDT (104).

The most effective ways to prevent malaria infection are vector control strategies, such as insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS) with insecticides. Bed nets are the most commonly used vector control method because of this ease of use and cost effectiveness. The most-used class of insecticides on bed nets are pyrethroids, and ITNs must be retreated with this insecticide every 6-12 months in order to remain effective at preventing mosquito bites and killing mosquitoes that enter the home (108). Studies have shown that using

ITNs reduces malaria incidence in children and pregnant women, the two most at risk groups of malaria and its consequences (109). The WHO now recommends the universal access and use of long-lasting insecticide-treated bed nets (LLITNs) which can last up to 3 years and have been shown to be more effective than simple ITNs (110). IRS control strategies involve the spraying of indoor walls and ceilings of homes with insecticides to kill mosquitoes that try to enter the home; IRS must be carried out every 4-6 months in homes in order to remain effective.

Non-pyrethroid insecticides can be used in IRS campaigns, often in addition to the use of LLITNs in order to provide added protection against mosquito vectors (111). Unfortunately, evidence is now emerging of insecticide resistance within certain mosquito species, meaning that new vector control efforts must be prioritized along with existing strategies (112).

Additionally, due to the toxic nature of pesticides in general, concerns about the safety and potential long-term consequences of insecticide-use for malaria vector control have become more frequent (113,114).

In addition to vector control, preventive antimalarial drugs can be administered to particularly at-risk groups, such as pregnant women and children under the age of five. There are several different types of antimalarial drugs available, and the type of drug used in a given population depends on the prevalent parasites in the area, as well as on the antimalarial drug resistance in that country. In Benin, the WHO recommendations call for intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) to be given 3 times during the first year of life at approximately 10 weeks, 14 weeks, and 9 months of age, corresponding to the routine vaccination schedule of the Expanded Program on Immunization (EPI) (115). For pregnant women, the WHO recommends at least 3 doses of IPTp-SP during pregnancy, beginning in the 2 nd trimester, with doses being given at least one month apart. As mentioned before, drug resistance of certain antimalarials is becoming more common, and researchers and doctors around the world are increasingly investigating new drugs or combinations that could help offset this drug resistance in high-transmission areas [START_REF] González | Intermittent Preventive Treatment of Malaria in Pregnancy with Mefloquine in HIV-Negative Women: A Multicentre Randomized Controlled Trial[END_REF]. WHO treatment guidelines for malaria infection from P. falciparum in SSA are displayed in Table 1 (116).

Malaria in pregnancy

An estimated 125 million pregnancies occur in malaria-endemic regions of the world each year, and research demonstrates that one in four women in these regions are infected with malaria over the course of pregnancy (118,119). In SSA, pregnant women are at an increased risk of malaria infection from P. falciparum and P. vivax species due to immunological changes during pregnancy and because a unique phenomenon occurs during pregnancy where P.

falciparum erythrocytes sequester in the placenta in infected women (120). This type of placental malaria infection allows parasites to evade the host immune response and is typically characteristic of more chronic maternal malaria infection that can be particularly harmful for mothers and the developing fetus (118).

The risk of MIP differs between low-and high-transmission regions, with pregnant women in low-transmission areas more susceptible to severe malaria infection and death due to their insufficient immunity to malaria infection. Additionally, women of all gravidities in lowtransmission areas are at equal risk of infection, while primigravidae women are the most at risk of malaria infection in high-transmission areas (118,120) since immunity is acquired with increasing numbers of pregnancies in these areas. While MIP is more common in hightransmission areas, women do not necessarily exhibit pathological symptoms of disease, particularly in the case of placental malaria infection (120). Younger maternal age has been

shown to be independently associated with increased risk of malaria infection in pregnancy in high-transmission areas (121) and HIV infection also increases susceptibility to malaria infection in pregnant women, as it decreases variant-specific immunity and impairs cytokine response to malaria (120,122). Studies have shown that pregnancy trimester may also impact risk of infection, with women in the second trimester most at risk of infection (118). However, there is little available research on malaria during the first trimester, especially since women in SSA often do not attend antenatal care clinics before the second trimester (123).

Diagnosis of MIP is commonly done using microscopy and PCR methods, where laboratory settings permit such sophisticated techniques. As previously mentioned, PCR tests are able to detect low parasitemia thresholds, lower than those detected by microscopy or RDTs, and in pregnant women from high-transmission areas often reflect chronic, asymptomatic, submicroscopic infections. Placental histology is also used to detect malaria in pregnant women, and involves the examination of placental tissue collected at delivery in order to diagnose past or current malaria infections through identification of malaria pigment leukocyte infiltrates (124).

Consequences on maternal health

MIP, both peripheral and placental, contributes to an estimated 200,000 infant deaths and 10,000 maternal deaths each year (120,125); however, mortality rates differ significantly between low-and high-transmission areas (118). In low-transmission areas, pregnant women are at increased risk of severe malaria infection, often leading to cerebral malaria and/or death (126). Overall, pregnant women are three times more likely to develop severe malaria than non-pregnant women, and therefore more at risk of severe anemia, hypoglycemia, respiratory distress, renal failure, and cerebral malaria (127). The consequences of MIP, including maternal and infant outcomes, vary depending on the time of infection and the immune status of the pregnant woman, as illustrated in Figure 5 (118).

Consequences on birth outcomes

MIP has been associated with increased risk of stillbirth, pre-term birth, fetal growth restriction, and low birth weight (136,137). In malaria-endemic regions, MIP contributes to up to 20% of stillbirths and risk of stillbirth is even higher still in areas of low endemicity (138,139). An estimated 20% of infants born with low birth weight, defined as weight at birth less than 2500g, can be attributable to placental malaria in SSA (140). MIP increases the risk of low birth weight due to a multitude of factors including maternal anemia, intrauterine growth restriction (IUGR), and pre-term birth (127). MIP is thought to impair oxygen and nutrient delivery to the fetus, resulting in IUGR (141). Malaria also substantially contributes to pre-term birth in endemic areas, up to 36% of pre-term births being attributable to infection, presumably due to the hosts immune response to infection which triggers early labor (139). Additionally, women in malaria endemic-regions are more likely to be malnourished, and the interaction of these two factors can increase risk of low birth weight (142).

MIP, particularly placental malaria, has been shown to impede placental vascular development, a process essential for a healthy pregnancy and proper development of the fetus (143). In cases of placental malaria, parasitized erythrocytes sequester in the placental intervillous space and induce a systemic inflammatory immune response in the mother (144).

This increase in inflammation as a result of infection has been shown to dysregulate vasculogenesis and angiogenesis pathways between mother and fetus necessary for proper fetal development (145), therefore increasing risk of low birth weight, pre-term birth, and stillbirth.

In addition, the impairment of these processes in the placenta could impact overall fetal growth and development and have long-term consequences on infant and child growth and development [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF].

Consequences on child health and development

As previously mentioned, MIP is estimated to contribute to over 200,000 infant deaths, and can also continue to impact the lives of surviving offspring after birth in several ways (120).

Pregnant women infected with malaria have infants at greater risk of congenital malaria, when P. falciparum parasites infect the cord or peripheral blood of newborns within the first seven days after birth (146). Newborns, who have underdeveloped immune systems, can develop severe symptoms from congenital malaria and have an increased risk of death (147). Placental malaria has also been shown to increase the risk of malaria disease in infants in the first few months of life through interference of the transfer of maternal antibodies to the developing fetus. Infants exposed to malaria in utero have been shown to acquire a tolerance to malaria blood-stage antigens that continues into childhood, and therefore increases their susceptibility to malaria infection (148). However, recent literature suggests that fetal exposure to malaria may in fact prime the malaria-specific T-cell immune response in infants, therefore providing a type of acquired immunity to malaria in infants in the first few months of life (149).

This increased risk of malaria infection in early childhood is of significant public health importance due to the fact that children under five years of age are the most at risk of mortality from malaria, with the WHO estimating almost 300,000 infant deaths per year due to childhood malaria infection ( 115). Research has also shown that children are at a greater risk for severe malaria, which can present as cerebral malaria, and can have long-term implications for neurocognitive development in children ( 150). Novel research has also shown an association between child malaria infection and lead poisoning, lead being a neurotoxic heavy metal known to impair child growth and development ( 14). This research suggests that children living in areas burdened by both malaria and lead exposure may face significant challenges in terms of sufficient neurocognitive development. MIP has also been found to increase risk of anemia in children after birth, with placental and peripheral malaria being found as the main contributors to hemoglobin concentrations in infants in the first year of life (151).

As discussed earlier, maternal infection with malaria activates the inflammatory immune response which can subsequently impair placental vasculogenesis vital for proper fetal growth and development (145). Placental malaria also involves the complementary immune response, which is known to play a role in fetal neurodevelopment (152). Recent research has investigated this mechanism and found that experimental malaria-exposed offspring had persistent neurocognitive deficits in memory and affective-like behavior compared to the unexposed controls [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF], providing evidence that placental malaria disrupts this complement immune response, particularly the C5a receptor, and has long-term implications for fetal neurocognitive development.

Gaps in research

While MIP is associated with several negative consequences in terms of maternal and fetal outcomes, its impacts on fetal neurocognitive development remain widely unknown.

Recent research provides laboratory evidence of a negative association between placental experimental malaria and fetal neurocognitive development; however, studies of this association in large mother-child cohorts have yet to be carried out. This thesis provides the first known study to investigate the relationship between MIP and child development in a large mother-child cohort from a malaria-endemic area in SSA.

Child neurocognitive development

Neurocognitive development refers to the processes in the brain involved in the development of neurological pathways necessary for cognitive, behavioral, and emotional functioning in individuals (153). These processes begin during the prenatal phase of life and continue for the first several years after birth, with the first 8 years of life considered to be the most critical period for this social, emotional, cognitive, physical, and behavioral development in children (154). Early models of child development by well-known developmental psychologists, such as Jean Piaget and his counterparts, consisted of age-specific stages governed primarily by maturation during childhood and biological processes (155). However, cognitive development begins at the fetal stage (156), as seen in Figure 6, and is influenced by a myriad of factors.

Within the first 10 weeks of conception, stem cells develop into neurons and glia, the primary cells that compose the brain. As these cells multiply, they then migrate to the part of the fetus where the brain will develop, the proper positioning of these cells being a crucial step in fetal brain development. As neurons continue to develop in the second and third trimesters, they develop branches, an axon, and undergo myelination, processes that allow for neurons to receive and transmit information throughout the body. Neurotransmitters, the chemicals responsible for communication between neurons, establish synapses between neurons to facilitate their communication. By the third trimester, neurons undergo "pruning", otherwise known as apoptosis (cell death), when the brain signals that certain neurons make inappropriate or unneeded connections (154). Duke University Wordpress (2020).

The foundations of child brain development continue throughout early and middle childhood, when up to 80% of the brain's grey matter is created through an interplay of genes, environment, and life experiences (156) (Figure 7). Several factors including learning environment, social climate, family dynamics, educational opportunities, sensory stimulation, adverse life events, and socio-economic and social conditions can all play a role during this phase of development and have long-lasting implications for individuals. Further information on these influential factors for child development is detailed later in this chapter. Several tools are available to assess and measure sensory development, language ability, and cognitive and motor functioning in children of all ages. These assessments can be carried out by parents, educators, or health professionals in young children to assess various aspects of development within one tool, with infants receiving one or more developmental scores for each domain (157). For example, there is the Ages and Stages Questionnaire (ASQ), a parental screening tool for developmental delays in infants 1-66 months of age.

Another well-known tool is the Bayley Scales of Infant and Toddler Development, a tool used to assess cognitive, motor, language, interaction, social-emotional, and adaptive behavior development in infants less than 1-42 months of age. Finally, the MSEL assesses child gross motor functioning and four cognitive scales to deliver a final, early learning composite (ELC) score. Once children are old enough, developmental assessment tools involve tasks and activities that children can complete under the supervision of test administrators to measure cognitive, motor, and behavioral processes. Among the most well-known are the Kaufman Assessment Battery for Children (KABC) and the Early Screening Inventory-Revised (ESI-R) tests that assess overall cognitive ability and processing in middle childhood (158).

Risk factors of impaired neurocognitive development

The Developmental Origins of Health and Disease (DOHaD) conceptual theory

proposes that an array of factors, exposures, and experiences influence health throughout the life course, especially in adult life (159). There are several factors, both prenatal and postnatal, that begin to influence and negatively impact infant and child health and development, which can have long-term implications. Among these are socio-economic conditions, genetic predispositions, adverse birth outcomes, the home-learning environment, parental psychological well-being, environmental exposures, infectious disease, and nutritional deficiencies.

Socio-economic conditions

It is estimated that one third of children under five years of age in low-and middleincome countries are at risk of developmental impairments due to conditions of poverty and stunting from malnutrition (160). Populations in developing nations are more often exposed to political unrest, conflict, extreme poverty, migration, reduced access to education, and other stressful events that can all have an impact on growing infants and children, as well as on the population as a whole. Low socio-economic status is often linked with lower parent education levels and support, both of which have been shown to impact child development. Studies have shown that lower levels of parent education level, particularly that of the mother, are associated with impaired child development in developing countries (161,162).

The difficulties faced in developing nations are also a major contributor to decreased school attendance rates, according to available studies. In 2015, the Global Monitoring Report indicated that 58 million primary school-aged children were not enrolled in school in lowincome countries (163). National statistics on school enrollment rates and child cognitive development in developing countries is extremely lacking, thus contributing to the issue of poor development for which there are no clear statistics or testing norms outside of available Western ones (160).

Genetics

As molecular biology continues to advance, scientists are identifying more and more genes associated with behavioral and cognitive disfunctions and disorders (164). The inheritance of these genes from parents to children can have a substantial effect on cognitive development in infants before they are even born. Some well-documented, but rare, genetic developmental disorders include Prader-Willi Syndrome (PWS) and Williams syndrome (165,166). Novel research has also begun to show certain genetic markers increase risk of autism and attention deficient and hyperactivity disorder (ADHD) (167).

Adverse birth outcomes

Infants born pre-term (before 37 weeks' gestation) and/or with a low birth weight (under Similarly, infants born with a low or very low birth weight have growth and developmental deficits, sometimes as a result of intrauterine growth restriction during pregnancy (177,178).

Learning environment and parental well-being

In addition to genetic and obstetrical factors, the environment in which infants and children live and interact with others during the very early years of life are crucial for their development. Multiple studies have shown that stimulating home environments, in which there are a variety of toys and objects for infants to use to help with gross and fine motor skills and an active participation from family members to stimulate language and emotional skills, significantly increase the developmental capacities of growing infants, even in settings of poverty (179-181). One study even found that home environment contributed more to child intellectual performance than did socio-economic status or maternal education level (182).

However, these factors more than likely act synergistically to impact child cognitive and behavioral development [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF].

Maternal depression, also referred to as post-partum depression, that normally occurs within the first year after giving birth, has also been shown to negatively impact the cognitive and behavioral development of infants (183,184). Research has shown that mothers suffering from depression interact with their children differently than non-depressed mothers, and they provide less stimulation and attention to children that is needed for their neurocognitive and psychosocial development (185). The link between maternal depression and impaired child neurocognitive development is even thought to be mediated by the quality of the home environment and parent stimulation (186). Recent evidence has also emerged that maternal depression can even impact the physical health of developing children, increasing their risk of diarrheal disease and febrile illness [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF]187).

Environmental exposures

Environmental exposure from chemicals, pollutants, and heavy metals during pregnancy or childhood has been shown to negatively affect crucial developmental processes.

The most studied contaminants in prenatal and postnatal exposure studies are persistent organic pollutants (organochlorinated pesticides, polychlorinated biphenyls), non-persistent pollutants (phthalates, bisphenol A), and toxic heavy metals (cadmium, lead and mercury) (188). Pre-natal exposure to these chemicals can occur through trans-placental passage, particularly in heavily polluted urban areas, or postnatally through air or dust inhalation or through hand-to-mouth ingestion (189,190). Studies have shown that even low levels of exposure to certain pollutants, such as organophosphate pesticides, during fetal developmental phases can impair cognitive, social, and behavioral development (191,192).

Lead (Pb) is a neurotoxic heavy metal well-known for its irreversible and highly detrimental effects on child health and development (193)(194)(195). Prenatal exposure to lead has been particularly linked to disruptions in child cognitive and neurobehavioral development (196). Pollutant co-exposures can also culminate in the body, primarily in individuals living in low-income and low-resource settings, thus further putting these populations at risk of severe neurodevelopmental disorders (197).

Infectious disease in childhood

While exposure to infectious disease during pregnancy and its association with child development have been understudied, there is evidence linking childhood exposure to infections with impaired neurocognitive and behavioral development. STH infection in schoolage children has been found to impair academic performance in numerous studies, with these studies also demonstrating the cognitive and educational benefits of school-focused deworming programs in burdened populations (198,199). Cerebral malaria, as mentioned earlier in this thesis, can also be most common amongst children under the age of five and can have long-term neurocognitive consequences for survivors. Several cohort studies have shown persistent, significant cognitive impairments in children infected with severe forms of P.

falciparum malaria, with one study referring to these impairments as the "hidden burden" of malaria in endemic countries (200)(201)(202). Finally, HIV-infected infants and children have been shown to have impaired growth and development compared to HIV-uninfected counterparts, even in the presence of antiretroviral therapies (203,204). Prenatal exposure to HIV and materno-fetal transmission of HIV has also been linked to cognitive and developmental delays in surviving infants (205).

Nutritional deficiencies

Pre-and post-natal nutrition is one of the major contributing factors for efficient child development. Maternal health and nutrition are vital for adequate fetal development and growth, with many studies demonstrating that proper maternal levels of folic acid, iron, vitamin D, zinc, and long-chain polyunsaturated fatty acids are particularly important in optimizing pregnancy and birth outcomes (206)(207)(208). The overall dietary nutrition of mothers has also been greatly studied, since inadequate or excessive gestational weight gain can be suboptimal for offspring development (209,210). Child nutrition has also been a widely studied public health issue and is known to be associated with cognition, with malnourished children more at risk of growth stunting and developmental impairments (208,211,212).

Nutritional deficiencies in iron are the most significant contributors to maternal and child anemia, which have been subsequently linked to impaired fetal and child development.

Pregnant women have higher iron requirements than non-pregnant women, in order to provide adequate nutrition and blood flow to the fetus in the placenta. Therefore, iron deficiency and iron deficiency-anemia (IDA) are of great public health concern in this population, especially in low-income countries that may not have access to food sources with adequate iron stores (213). Studies have suggested that iron plays an integral role in fetal development, therefore establishing a connection between prenatal iron deficiency and IDA and impaired fetal growth and development (214). Prenatal anemia increases the risk of childhood anemia, which in turn can also have negative effects on child development if left untreated (215). Observational and experimental studies have shown that iron deficient children have long-term cognitive, social, and emotional deficits compared to iron-replete children (216,217).

Gaps in research

The influence of infectious disease during pregnancy has not been widely studied and is of interest to public health factors particularly in heavily burdened areas of the world. Figure 8 shows a Direct Acyclic Graph (DAG) demonstrating the complex documented pathways between parasitic infection in pregnancy and impaired child neurocognitive development, as well as the pathways that were previously unexplored in the literature and that were addressed within the objectives of this thesis to help better our epidemiological understanding of these relationships and the mechanisms behind them.

Chapter 2. Materials and Methods

The population of mothers and their children involved in this thesis are part of a larger, multi-study cohort from the district of Allada, Benin that was followed prospectively from the second trimester of pregnancy until offspring were six years of age. This chapter details the study sites, population, objectives, and procedures of the five studies from which data on participants were used for the purposes of this thesis. Biological, neurocognitive, sociodemographic, and anthropometric assessments of participating women and children are The scientific context, objectives, population, and assessments of each of the previously mentioned studies are further detailed in the remainder of this chapter.

The MiPPAD clinical trial

Context & Implementation

In 2012, the WHO guidelines for malaria prevention in HIV-negative pregnant women living in SSA countries with stable transmission of P. falciparum recommended at least two doses of IPTp-SP, in conjunction with the use of LLITNs, starting from the second trimester and at least one month apart . While IPTp-SP proved to be cost-effective and reduced maternal morbidity, parasite resistance to the drug had been documented and could rapidly gain in momentum if no alternative strategies were identified (218). Based on previous evidence, mefloquine (MQ) proved to be a valid substitute for SP in this population due to its long halflife, ability to be administered in a single dose, and acceptable reproductive toxicity profile As part of standard antenatal care guidelines in Benin, women received LLITNs (PermaNet, Vestergaard Fransen) at recruitment. Women were assessed for hemoglobin, HIV, and the syphilis rapid plasma regain test at recruitment per local standard procedures. Women also received ferrous sulphate-folic acid supplements at each ANV for anemia prevention.

Allada district

Direct consumption of supplements was not monitored by study personnel. At the first ANV, women received 600mg of mebendazole (100mg to take twice daily for three days) for prevention of helminth infections and received treatment again at the 2 nd ANV if diagnosed with helminth infection. At each ANV, women received their respective IPTp treatments according to their designated intervention arm. Women in the standard IPTp with SP group were treated with three tablets of the fixed combination therapy containing 500mg of sulfadoxine and 25mg of pyrimethamine (Malastop, Sterop).

Participants were encouraged to seek medical care at the ANV clinics whenever needed and a passive surveillance system was put into place to monitor symptoms of illness, malaria episodes, or other adverse events which were documented within the clinical trial. Clinical malaria episodes were treated with oral quinine in the first trimester or with artemetherlumefantrine in subsequent trimesters for uncomplicated malaria; severe malaria was treated with parental quinine [START_REF] Ouédraogo | Placental impression smears is a good indicator of placental malaria in sub-Saharan Africa[END_REF]. At delivery, if women were diagnosed as anemic, they were treated with oral ferrous sulphate 200 mg/8 hours for three months or by blood transfusion in severe cases (124).

Socio-demographic and anthropometric information during pregnancy

Participating pregnant women were seen at two ANVs in one of the three maternity clinic locations at least one month apart, as well as at delivery, for a total of three scheduled ANVs. At the first ANV, sociodemographic information was collected via a questionnaire administered to women. Information collected included maternal age, gravidity, marital status, occupation and education level of mother and father, area of residence, and information related to the use of LLITNs the previous night.

Maternal weight, height, and gestational age were measured at each ANV by trained health professionals. Weight was measured using an electronic scale to the nearest 0.1kg (SeaCorp, Hanover, Maryland) and height was measured using a body meter device to the nearest 0.1cm (Seca 206 Bodymeter, SeaCorp). Gestational age was determined by measuring fundal height using manual palpation at each ANV. Maternal pre-pregnancy Body Mass Index At delivery, in addition to the previously mentioned information collected for women, newborn length was measured to the nearest 1mm using a locally manufactured wooden measuring scale validated by the WHO ( 226) and newborn weight was assessed to the nearest 10g using an electronic baby scale (Seca type 354). Newborn sex was noted, as well as gestational age at birth according to the Ballard's score (227).

Malaria infection during pregnancy

At each ANV, 4mL of venous blood was collected from pregnant women and divided into two tubes for examination and storage; 2mL was introduced into an iron-free dry tube, and the other 2mL was stored in a tube with potassium Ethylenediaminetetraacetic acid (EDTA).

Malaria infection by P. falciparum was determined at each ANV, including delivery, by thick and thin blood smear tests using the Lambaréné technique. From collected samples, 10l of blood was smeared on a 1.8cm x 1cm rectangular slide and stained with Giemsa. Stained slides were read with an oil immersion lens at 1000X magnification. The number of parasites per l of blood was obtained by applying a multiplication factor to the average parasitemia/field.

In addition to microscopy techniques for malaria diagnosis, 50l of maternal peripheral and placental blood was spotted onto filter papers for parasitological assessment at delivery.

Filter papers were tested for P. falciparum presence and density by a real-time quantitative polymerase chain-reaction (qPCR) assay targeting 18S ribosomal deoxyribonucleic acid (rDNA) [START_REF] Mayor | Changing Trends in P. falciparum Burden, Immunity, and Disease in Pregnancy[END_REF]. Parasitemia was quantified by extrapolation of cycle thresholds, the value for which DNA is amplified, from a standard curve of P. falciparum ring-infected erythrocytes.

Samples where no cycle thresholds were detected were considered negative, and a density of 2 parasites/μL was assigned if amplification was lower than the standard curve of 5 parasites/μL (124).

Hematological assessments

From blood samples taken at each ANV, 10l was used to determine the concentration of Hemoglobin (Hb) using the Hemo-control photometer device (EKF Diagnostics, Barleben/Magdeburg, Germany). The device was calibrated daily for quality control and one in ten Hb concentrations was crosschecked by a Hematology analyzer (Erma Laboratory, Japan) at the Allada Central Hospital laboratory. Hb concentration was recorded in g/L and women with a concentration less than 110 g/L were considered anemic.

Iron stores in pregnant women were determined by serum ferritin concentration after samples were centrifuged for serum analysis. A rapid test (CRP Latex; Cypres Diagnostics Inc., Campbellville, Ontario, Canada) measured concentrations of C-reactive protein (CRP) in blood samples. Women were considered to have markers of inflammation if their CRP concentration was less than 5mg/L. Iron deficiency (ID) was defined as a serum ferritin concentration less than 12μg/L in the absence of inflammation or a serum ferritin concentration less than 70μg/L in the presence of inflammation measured by CRP.

Funding & Ethics Statement

The

The APEC nested-cohort study

Context & Implementation

Pregnant women in developing countries are at increased risk of anemia during pregnancy due to a multitude of factors such as parasitic infections and nutritional deficiencies.

A previous study found 60% of pregnant women in a Beninese cohort to be anemic (228). Even with prevention and treatment measures in place in high-risk populations in SSA endemic to malaria parasitic infection, the effectiveness of these interventions is still widely undocumented (229). Therefore, a nested prospective cohort study, APEC, was launched as an ancillary study to the MiPPAD clinical trial with the primary objective to investigate the etiological factors of anemia in pregnancy and its consequences on maternal and child health in a malaria endemic population.

The APEC study ran parallel to MiPPAD and enrolled the first 1,005 pregnant women who agreed to participate in the study at the first ANV within the clinical trial from January 2010 until May 2011. Follow-up of pregnant women from recruitment until delivery in the MiPPAD and APEC cohorts is depicted in Figure 10. In addition to the previously mentioned biological, socio-demographic, and anthropometric data collected from pregnant women in the MiPPAD trial, participants in APEC were also assessed for additional parasitic infection caused by soil-transmitted helminths and for vitamin B12 and folic acid concentrations to identify relevant nutritional deficiencies. A sub-cohort of 400 children were followed from birth to 12 months of age in the APEC study the impact of prenatal anemia in mothers on child malarial and hematological statuses. Participants were chosen based on maternal anemia status; 200 infants were born to anemic mothers and 200 were born to non-anemic mothers.

Figure 10. Follow-up of pregnant women from second trimester until delivery.

Soil-transmitted helminth infection during pregnancy

At each ANV, pregnant women received a container to collect stool samples from the morning following the visit. Stool samples were also collected either 15 days prior to the expected delivery date of women or within a week after delivery. Containers were retrieved from pregnant women within six hours of defecation in order to test for the presence of soiltransmitted helminth eggs.

Thick smears of stool samples were prepared and assessed using the Kato-Katz technique to obtain a quantitative estimate of the intensity of helminth infection according to WHO standards [START_REF] Organization | Bench aids for the diagnosis of intestinal parasites[END_REF]. Soil-transmitted helminth eggs of A.lumbricoides, N.americanus, A.duodenale, and T.trichiura species were identified. Quality control was performed by two laboratory technicians who systematically and independently examined each of the two prepared slides of thick smears under a microscope; the mean egg count of the two results was calculated. The number of eggs in each individual sample was calculated, recorded, and then the number of eggs per species was multiplied by a factor of 24 in order to obtain the standardized fecal egg count (FEC), expressed in eggs per gram (epg). An individual was considered to be infected with soil-transmitted helminths if the FEC was at least 24 epg for any species.

Nutritional assessments

From the venous blood collected from pregnant women at ANVs and centrifuged, 500μL of serum ferritin and vitamin B12 concentrations were measured using a microparticle enzyme immunoassay (MEIA) method. Folic acid concentrations were determined using fluorescence polarization immunoassay (FPIA) technique with an AxSym Immuno-Assay Analyzer (Abbott Diagnostics, Frankfurt, Germany) (229). Women were considered to have a folic acid deficiency if serum ferritin concentrations were less than 6ng/mL and were considered to be vitamin B12 deficient with vitamin concentrations less than 150 pg/mL.

Infant malaria assessments

The sub-cohort of 400 infants were followed from birth to 12 months and were systematically assessed for malaria status at birth, 3, 6, 9, and 12 months. Infants also had access to free health care services for passive detection of malaria during the follow-up period. At each systematic visit, 8 mL of venous blood was taken from infants and the Lambaréné technique was used to diagnose malaria on thick blood smears, described in detail within the MiPPAD clinical trial protocol. If diagnosed with malaria, infants were treated with artemetherlumefantrine according to existing Beninese guidelines at the time.

Funding & Ethics Statement

The APEC study was supported by the Malaria in Pregnancy Consortium, which is

The TOVI study

Context & Implementation

Prenatal anemia is known to have severe consequences on maternal and birth outcomes, such as low birth weight, pre-term birth, and perinatal mortality (230); U-shaped fluctuations in maternal hemoglobin during pregnancy have also been associated with adverse birth outcomes (231). However, little is known about the impact of prenatal Hb concentrations on infant neurocognitive development (232). Given this research gap and the high prevalence of prenatal anemia in the population of pregnant women enrolled in the MiPPAD clinical trial, researchers sought to follow-up with singletons born to these women at 1 year of age and investigate the impacts of prenatal anemia on infant neurocognitive development scores through the TOVI study. This was a cross-sectional study conducted between June 2010 and June 2013 in the district of Allada, Benin.

All 863 surviving one-year-old singletons born to pregnant from the MiPPAD trial were eligible to participate in the TOVI study. Infant neurocognitive and motor development was measured by a trained research nurse at a scheduled 1-year post-partum visit at a local maternity clinic using the American Guidance Service edition of the MSEL (20). Within three days after cognitive assessments, a research nurse different from the one who administered the MSEL visited family homes to collect information related to socio-demographic factors, maternal depression, home environment, and environmental exposures that could impact infant development.

Mullen Scales of Early Learning (MSEL)

The MSEL is an individually administered, norm-referenced measurement tool used to assess cognitive and motor function in children from birth to 68 months of age. The tool consists of five scales to determine functioning: the gross motor scale, fine motor scale, receptive language scale, expressive language scale, and the visual perception scale. The gross motor scale is designed specifically for use in children age 1-36 months in order to assess basic mobility and balance important for young infants. Therefore, children in our population were assessed on all five scales of the MSEL. Raw scores of each scale were computed and then converted into normative t scores; t scores for the fine motor, receptive language, expressive language, and visual perception scales were converted into the Early Learning Composite (ELC) score, indicative of overall cognitive functioning in children. Only trained professionals are permitted to administer the tasks of the MSEL.

The MSEL, previously only available in English and never before used in Benin, was translated and adapted for use in this population prior to the onset of the study in four steps: linguistic translation, training, pilot testing, and review [START_REF] Koura | Usefulness of child development assessments for low-resource settings in francophone Africa[END_REF]. Quality control of assessment administration was completed during the study by the field coordinator and research nurses. Inter-rater reliability was assessed among the three research nurses who administered the MSEL; the gold-standard assessor, the nurse who administered the most assessments, was identified and ELC scores were compared among all three nurses. The correlation score for inter-rater reliability between the gold standard interviewer and other assessors ranged from 94 to 100% with a mean correlation score of 98% [START_REF] Koura | Usefulness of child development assessments for low-resource settings in francophone Africa[END_REF].

Socio-demographic information

During the home visit after the MSEL administration, a questionnaire was given to mothers or guardians of children to assess family possession scores. These scores were computed using a checklist of material possessions that families could own and indicated family wealth: radio, television, bicycle, motorbike, car, at least two cows, and electricity. Maternal education level was also noted during this home visit.

Edinburgh Postnatal Depression Scale (EPDS)

Maternal post-partum depression symptoms were measured using the EPDS, a 10-item

Likert-style questionnaire used to measure symptoms of depression by inquiring about feelings of sadness or hopelessness, changes in appetite or activity, and ideations of self-harm within the previous seven days (233). Each item of the EPDS is scored between 0-3, therefore total scores range between 0-30, with higher scores indicating the presence of depressive symptoms.

The validated French version of the EPDS was translated into a written version in Fon and administered by trained research nurses. The EPDS was evaluated for adaptability and validity in this population during the previously mentioned pilot study, given that there was no previously identified gold standard clinical diagnostic test for maternal depression in Benin.

Home Observation for Measurement of the Environment (HOME)

The HOME inventory, developed by Bettye Caldwell and Robert Bradley, is a systematic assessment of the quantity and quality of stimulation, support, and structure available to a child in their home environment (234). The inventory for Infant-Toddler assessment is a combination of interview questions and observations of mother-child interactions in the home environment and consists of six subscales, detailed in Table 2 (235). The HOME was adapted for use in this population through formal training of research staff, a pilot study involving ten home visits, adaptation of certain items according to the findings of the pilot study, and a final review with research staff [START_REF] Koura | Usefulness of child development assessments for low-resource settings in francophone Africa[END_REF]. Six items of the HOME inventory were adapted for use in this population: the 'Learning Materials' scale was reduced to four questions due to the fact that only one family in the pilot study owned a toy, a question related to the number of books owned by the child was changed from 'At least two books are present or can be seen' to 'The child owns one or more books', an item related to the amount of time the child spends outside was removed since children in this population send most of their days outside. Assessors were trained to administer the adapted HOME inventory in Fon by trained research staff.

Infant blood lead levels

Nurses collected 8mL of venous blood from each child, with 4mL stored in a tube containing dipotassium EDTA and 4mL put into an iron-free dry tube. An aliquot of EDTA blood was diluted 20-fold in ammonia 0.5% v/v and 0.1% v/v surfactant Triton-X and analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer Sciex Elan DRC II ICP-MS instrument) at the Centre de Toxicologie, Institut National de Santé Publique du Québec (Québec, Canada). The limit of detection for blood lead analysis was 0.2 μg/L (236).

Funding & Ethics Statement

The TOVI study was funded by the Eunice Kennedy Shriver National Institute of Child

Health & Human Development (no. R21-HD060524). This study was approved by the institutional review boards of the University of Abomey-Calavi in Benin and New York University in the United States (IRB#09-1253). At recruitment, researchers obtained informed consent from all women and guardians who participated.

The TOLIMMUNPAL study

Context & Implementation

In malaria-endemic areas of the world, children and adults are regularly infected with malaria parasites throughout their lives, leading to an eventual acquired immunity over time that protects many adults from overt illness caused by malaria (237). Antibodies produced in response to P. falciparum parasites play an essential role in this acquired immunity (238); however, the specific antigens that illicit these antibody responses are not well known.

Understanding these antigens is vital for effective vaccine development against P. falciparum infection during the blood stages, particularly in areas like Benin. Therefore, the TOLIMMUNPAL study sought to study antibody responses to candidate vaccine antigens and malaria infection in children from birth to 24 months of age in Benin.

The study recruited the same 400 children, 200 anemic and 200 non-anemic, followed after birth in the APEC study between 2010 and 2013. TOLIMMUNPAL systematically collected information related to P. falciparum infection in childhood, as well as hematological and environmental factors relevant to risk of malaria infection in the study area. For the purposes of this thesis, only the data collected on malaria infection and related risk factors in children between 12-24 months was used.

Malaria infections in early childhood

Malaria infection was diagnosed during the 12-month follow-up (from 12 to 24 months of age) using both positive thick blood smear tests and RDTs. Participating children were visited by a nurse during a monthly systematic visit, where children were given both a thick blood smear and an RDT, whether they presented with symptoms or not. Children also had a systematic visit at a healthcare center in Benin at 18 and 24 months of age, where venous blood samples were drawn in addition to the administration of a thick blood smear and RDT. A nurse visited the homes of participating children every 15 days, during which a thick blood smear test was administered, regardless of whether children presented with symptoms or not, and an RDT if children had fever or history of fever in the previous 24 hours (temperature > 37.5°C).

Children had access to free emergency care and were able to present at the hospital anytime symptoms occurred, and a thick blood smear test and an RDT were administered to diagnose malaria. Malaria episodes were identified as symptomatic if children presented with fever at the time of diagnosis or within three days of diagnosis, and episodes were identified as asymptomatic in the absence of fever at time of diagnosis or in the three days following diagnosis. P.falciparum density was determined if children tested positive for malaria through thick blood smear or RDT.

Hematological Assessments

At 12 months, blood samples were collected from children to assess iron deficiency.

Iron deficiency was defined as low ferritin concentration (<12 μg/L) corrected for CRP (239), and/or iron deficient tissue in the body indicated by soluble transferrin receptors (sTfR) in the blood (>8.3 mg/L) (240).

Environmental Assessments

To account for the potential difference in risk of malaria outcome among children, researchers collected information on mosquito populations in 180 out of the 400 participating homes of children based on geographical equi-distribution criteria [START_REF] Lysaniuk | Types of homes and ways of life: a territorial analysis of the environmental determinants that factor into the proliferation of malaria vectors in the rural region of Allada in Benin[END_REF]. CDC mosquito-light traps were installed in selected homes at intervals of two consecutive nights per month between April 2011 and February 2013, where mosquitoes were trapped and then analyzed by researchers for mosquito species and density. The average number of mosquitoes found per home per night was calculated for all children using multiple imputation.

At each visit, mothers were asked if their children slept under an LLITN the previous night. Based on the number of times children were said to have slept under a bed net during the follow-up, they were classified as having using LLITNS rarely, occasionally, frequently, or always.

Funding & Ethics Statement

The TOLIMMUNPAL study was funded by the Agence Nationale de le Recherche

The EXPLORE study

Context & Implementation

Lead (Pb), a neurotoxic heavy metal, can be found in gasoline, paint, out-of-date water pipes, ammunition, and contaminated soil, particularly in developing countries where these

sources have yet to be eradicated (241). Child exposure to lead through inhalation or ingestion is known to have permanent and serious consequences on neurodevelopment, child growth, and risk of infection [START_REF] Moya-Alvarez | Elevated Blood Lead Levels Are Associated with Reduced Risk of Malaria in Beninese Infants[END_REF]242). The CDC recommends intervention when blood lead levels are 50 μg/L or more; however, even blood lead levels under this threshold can be detrimental to child health and development (193). However, the sources of lead and its burden in SSA countries has not been as widely studied as in Western countries. During the TOVI study, 58% of children had elevated blood lead levels (>50μg/L) at one year, indicating high levels of exposure in this population.

Therefore, researchers conducted the EXPLORE study to follow-up with these children prospectively at six years of age to assess the impact of heavy metal exposure (from Pb and manganese (Mn)) on child neurocognitive and behavioral development, as well as to study the sources of these heavy metals in this population. All children who participated in the TOVI study at one year of age were invited to participate in EXPLORE. The cross-sectional study took place in Allada, Benin from May 2016 until June 2018. Table 3 provides a summary of all studies that pregnant women and children participated in from the second trimester of pregnancy until six years of age, as well as the relevant collected and used within this thesis.

P. falciparum infection

Neurocognitive development

Sociodemographic information

Adapted from S. [START_REF] Ahmadi | Follow-Up of Elevated Blood Lead Levels and Sources in a Cohort of Children in Benin[END_REF].

Before recruitment in EXPLORE, study personnel were trained by the Principal The day before assessments, participating families were visited by study personnel where protocol was explained to them and they were given containers to collect child stool samples. The following day, mothers and their children visited one of the three participating health centers in Allada (Allada, Attogon, Sékou) where all assessments were carried out. Upon arrival, if the child was visibly ill or had symptoms, their temperature was taken. If children had a fever, a blood sample was taken to test for parasitic infection and anemia, and appropriate treatment according to national guidelines was provided. Mothers and children were then asked to return to the health center in a few days when the child was feeling better in order to complete the neurocognitive assessments.

Biological Assessments

The day of neurocognitive assessments at the health clinic, families arrived and returned the containers given to them the previous day with a child stool sample to be analyzed for helminth infection. The Kato-Katz technique was used to detect the presence of soil-transmitted helminth eggs, as described in detail within the APEC protocol. Eggs belonging to A.lumbricoides, N.americanus, A.duodenale, and Taenia species were identified. Samples of 8mL of venous blood were collected from children; 4mL was stored in a tube containing dipotassium EDTA and 4mL put into an iron-free dry tube. Iron deficiency status was determined, with children considered as iron deficient with serum ferritin concentrations <15 μg/L corrected for CRP. P. falciparum malaria infection was diagnosed using a thick blood smear test and parasite density was calculated according to the average parasitemia/field. Blood lead level of children was also assessed using inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer Sciex Elan DRC II ICP-MS instrument) on an aliquot of EDTA blood diluted 20-fold in ammonia 0.5% v/v and 0.1% v/v surfactant Triton-X at the laboratory of the Ecole des Hautes Etudes en Santé Publique (EHESP) (Rennes, France). The limit of detection for blood lead analysis was 0.2 μg/L (236).

Anthropometric Assessments

Height and weight of six-year-old children was collected; height was measured by a locally manufactured length board and weight was determined using the TANITA analogue scale. Weight-for-age z-scores for children were calculated according to the WHO z-score classification system (243).

Developmental Assessments Kaufman Assessment Battery for Children 2 nd edition (KABC-II)

The KABC-II, first published in 2004, is a psychological diagnostic tool used to assess cognitive ability in children 3-12 years of age. The second edition of the tool measures simultaneous and sequential processing, as in the first edition, as well as planning and learning abilities in children. There are four subscales of the KABC-II, the Simultaneous Processing scale, the Sequential Processing scale, Learning ability, and Planning ability. For the purposes of assessments in this population, the Planning ability scale was not used and certain items from each scale that were not culturally appropriate were not included. The KABC-II is a Western measure of cognition that has been widely used in SSA countries, particularly in the context of malaria-endemic populations (200,244). The construct validity of the French version of the KABC-II (by Les Editions du Centre de Psychologie Appliquée (ECPA); French translation published and distributed exclusively by ECPA with the permission of AGS Publishing, Pearson Products Inc.) was verified in this population in Benin during a pilot study (245), and instructions were orally translated from French to Fon by trained study personnel. The standardized Mental Processing Index (MPI), a measure of global cognitive ability, and the Non-Verbal Index (NVI), a non-verbal assessment of child processing and cognitive abilities, both generated by KABC-II scores, were used for the purposes of this thesis and its analyses.

A quality control of the KABC-II was completed at the Global Health Assessment (GHA) center in Uganda. Videos of investigators administering the KABC-II were sent to the GHA center each month for six months and each investigator received individualized feedback regarding their assessment of children.

Bruininks-Oseretsky Test of Motor Proficiency Edition 2 (BOT-2)

The BOT-2 is a comprehensive, standardized, norm-referenced diagnostic tool used to assess gross and fine motor skills in school-aged children. The short form of the BOT-2 was used for motor skill assessment in this population, and included asking children to perform activities such as folding a piece of paper, jumping in place, and balancing on one leg. Final composite scores for fine and gross motor skills were standardized by age and sex and used for the purposes of this thesis. The BOT-2 was not validated for use in this population prior to the study onset; however, the BOT-2 short form has been used in other similar SSA populations to assess motor development in children (246).

Test of Variables of Attention (TOVA)

The TOVA, a non-language-based performance test, was administered to identify ADHD in children. Children were seated in front of a computer and completed the test by reacting to various stimuli that appeared on the screen. Children were instructed to press a certain button when a specified target would appear on the screen. Omission errors are the number of times the child does not click the button when the target appears on the screen and commission errors are the number of times the child clicks the button too early (before the target appears on the screen). Response time is the amount of time it takes the child to press the button once the target appears on the screen. Variability is the consistency in the child's response times. The final score was calculated based on response times, variability of response times, and rate of deterioration of responses to the computerized stimuli.

Strengths and Difficulties Questionnaire (SDQ)

Mothers and guardians received a questionnaire to fill out which included the SDQ, a brief 25-question behavioral screening tool to assess behavioral development in children 3-16 years of age, developed by child psychiatrist Robert N. Goodman (247). The questionnaire screens for issues related to five subscales: emotional problems, conduct problems, hyperactivity, peer problems, and pro-social behaviors; with the four former subscales used to generate an overall difficulties score. The SDQ further differentiates between emotional and behavioral conduct problems in children through internalizing and externalizing scores generated by its subscales. Scores from the conduct problems and hyperactivity subscales are combined to create the externalizing (behavioral issues) score, while emotional and peer problems are combined to form the internalizing (emotional issues) score. Higher scores indicate more behavioral problems in children.

EPDS

Within the maternal questionnaire, mothers completed the EPDS to measure symptoms of depression. Each item of the EPDS is scored between 0-3, therefore total scores range between 0-30, with higher scores indicating the presence of depressive symptoms. The validated French version of the EPDS was translated and the written version in Fon was administered by trained research nurses. If a woman indicated within the EPDS that they had had thoughts of self-harm, she was seen by a psychologist on the research team for further follow-up and aide.

Socio-demographic information

Similar to protocol used at the one-year follow-up, maternal questionnaires assessed family possession scores using a checklist of material possessions owned by the family to indicate wealth: a radio, television, bicycle, motorbike, car, at least two cows, and electricity.

Women were also asked about their education level.

Funding & Ethics Statement

Funding for EXPLORE was provided by the Fondation de France (2015 00060746).

Ethical approval was obtained by the Committee of Ethical Research of the Applied Biomedical Sciences Institute (CER-ISBA) in Benin. Written informed consent was obtained by mothers and guardians of children in the presence of a witness, with thumbprints provided for consent if women could not read and/or write.

Statistical Software

Statistical analyses were completed using STATA 13.1 (StataCorp. 2013

Chapter 3. The effect of soil-transmitted helminth infection in pregnancy on child neurocognitive development

This chapter presents the published article studying the impact of soil-transmitted helminth infections in pregnancy on child neurocognitive and behavioral development at six years of age. The analyses within this paper are a continuation of a previous article published by [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF] that showed impaired gross motor function in one-year-old children born to mothers who were infected by hookworms during pregnancy in our population. Our results show that SHIP no longer impairs neurocognitive development in children long-term as measured by the KABC-II, BOT-2, and TOVA assessments at six years of age. However, further analyses show that SHIP was associate with impaired behavioral development in sixyear-old children as measured by the SDQ, particularly from the internalizing score indicating emotional development in children. Mediation analyses explored the hypothesized mechanism of this association via activation of the maternal inflammatory immune response; however, the limited inflammation markers that are non-specific to helminth infection available for our analyses did not confirm this hypothesis. This is the first publication of its kind to address the long-term impacts of SHIP on child development, and to explore the mechanism of maternal inflammation. Additionally, this paper serves as evidence that SHIP and helminth infection in childhood carry a large burden of disease in this population, even in the context of a clinical trial and prospective follow-up of children, thus reinforcing the need for sufficient preventive and treatment options in heavily burdened areas of the world. This article was published in Plos Neglected Tropical Diseases as an original research article.

Chapter 4. The effect of malaria in pregnancy on child neurocognitive development

This chapter presents the second article of this thesis studying the impact of Plasmodium falciparum malaria infection in pregnancy on child neurocognitive development at one and six years of age. While the consequences of MIP on birth outcomes, such as pre-term birth, low birth weight, and fetal growth restriction, are well known, little is known about the long-term consequences on development of surviving offspring. Other types of maternal infection in pregnancy, including SHIP, influenza, and sexually-transmitted infections have been linked to increased risk of motor and behavioral impairments [START_REF] Mireku | Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children[END_REF]248,249); however, only one previous study investigated experimental malaria infection in rodents and its risks for developmental impairment in offspring [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF]. This paper is the first to investigate this relationship in a large mother-child cohort in SSA and provides evidence that MIP, particularly later in pregnancy, can have detrimental impacts on child motor and neurocognitive development at both one and six years of age. Our article also shows that infections diagnosed by qPCR methods, often only available in areas with access to advanced laboratory techniques, are especially harmful for long-term child development. These infections reflect chronic, asymptomatic infections that often go untreated, this article therefore serves as evidence that diagnosis and treatment of these infections must be reinforced in low-income areas of the world like Benin in order to avoid their long-term consequences on child development. This article has been accepted for publication in Clinical Infectious Diseases.

Chapter 5. Maternal depression and parasitic infection

This chapter includes the third article of this thesis investigating the potential confounding role of maternal depression and parent-child interactions on risk of soiltransmitted helminth and malaria infection. Maternal depression has been shown to impact child development, with maternal psychological wellbeing and quality of care given to children two important determinants of healthy child development (186,250). However, the role of maternal depression and parent-child interactions on subsequent infection has been less studied. We conducted analyses within a sub-cohort of mothers and children from our birth cohort in order to test the hypothesis that women with more depressive symptoms and lower quality parentchild interactions would be associated with higher risk of parasitic infection in children. This hypothesis was based off of previous work by [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF] which demonstrated an association between post-partum depression in mothers and increased risk of diarrheal disease in their infants [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF]. Our analyses revealed that while maternal depression, as measured by the EPDS, was associated with lower HOME scores, indicating lower quality of parent-child interactions and home learning environment of children, neither of these factors were found to influence subsequent parasitic infection in children. These analyses add to our understanding of the mechanisms behind the relationship between parasitic infection during pregnancy and child development, since parasitic infection during pregnancy has been found to increase the risk of infection in surviving offspring according to some studies [START_REF] Ndibazza | Associations Between Maternal Helminth and Malaria Infections in Pregnancy and Clinical Malaria in the Offspring: A Birth Cohort in Entebbe, Uganda[END_REF]251). Understanding how maternal depression and parent-child interactions may influence risk of childhood infection, which subsequently is known to impair neurocognitive and motor development (199,252,253), is important for better interpreting the results of the primary objectives of this thesis. This quantitative work was done in tandem with a qualitative study exploring the experiences, barriers, and perceptions of risk of pregnant women in this cohort regarding prevention and treatment of parasitic infection during their pregnancies. This qualitative study, involving semidirect interviews of participating women, also explored the role of maternal depression in the prevention and treatment of parasitic infection in children in this population. These interviews are set to take place in June 2021, with a qualitative analysis of interviews taking place shortly thereafter. The quantitative analyses for this subject are currently under review for publication.

Chapter 6. Blood lead level and risk of malaria infection

In this chapter, I present the fourth article of this thesis investigating the potential confounding role of blood lead levels from environmental exposure on risk of malaria infection. Moya-Alvarez et al. (2016) demonstrated an inverse relationship between elevated blood lead levels (> 50 μg/L) and malaria risk in a sub-cohort of 203 12-month-old Beninese children in our population. We therefore sought to investigate whether or not this relationship would continue if the prospective follow-up of these same children from 12-24 months of age was taken into account, thus offering stronger evidence of this association. However, our analyses revealed that elevated blood lead levels in children at 12 months of age did not impact subsequent risk of malaria infection in a prospective follow-up from 12-24 months. While these analyses were completed within a sub-cohort of children for whom information on lead exposure and malaria infection was available, this study aided our overall understanding of risk factors for parasitic infections and provided evidence that the previously hypothesized inverse effect of lead exposure on malaria risk should not be taken into account in models investigating the relationship between parasitic infection in pregnancy and child development, even given the strong evidence linking lead exposure to impaired child development (254). This paper was published in Plos One as an original research article.

Previous work by

Chapter 7. Discussion

This chapter details and interprets the main findings of each objective presented in preceding chapters, as well as discusses the major strengths and limitations of the work done for this thesis as a whole, in an effort to not repeat elements of the discussions found in each individual article. This chapter also presents the implications of these findings across public health domains and suggests further research that is needed on this and related subjects.

Main findings

Multiple linear regression analyses analyzed the impact of SHIP and MIP on child neurocognitive and behavioral development. Our analyses revealed that SHIP diagnosed at each ANV and at least once during pregnancy, as well as hookworm density during pregnancy were not found to be associated with neurocognitive development, motor function, or attention/hyperactivity disorder in children at six years of age. However, SHIP was associated with impaired emotional development. Helminth infection at the 2 nd ANV/delivery and at any point in pregnancy were associated with higher internalizing scores, indicating more emotional problems. Mediation analyses revealed that inflammation measured by CRP in pregnancy was not found to be a significant mediating factor of this association, as hypothesized. Regarding MIP, we found that infection at least once during pregnancy diagnosed by microscopy, placental malaria diagnosed by qPCR, and high parasite density at delivery were associated with impaired gross motor development scores measured by the MSEL in children at one year. MIP diagnosed by microscopy and high parasite density at the 2 nd ANV negatively impacted NVI scores of the KABC-II, and analyses show a trend that infections occurring later in pregnancy more negatively impact KABC-II scores than earlier infections.

Parasitic infection during pregnancy is known to increase the risk of infection in childhood [START_REF] Petersen | Protozoan and helminth infections in pregnancy. Short-term and long-term implications of transmission of infection from mother to foetus[END_REF]148), which in turn can impair their neurocognitive development according to observational studies (198,200). Therefore, this thesis investigated other hypothesized factors that could influence parasitic infection in childhood, that are well-established risk factors of impaired neurocognitive development (184,195), and contribute to the main findings of this thesis.

We conducted a quantitative analysis of the impact of maternal post-partum depression measured by the EPDS on parasitic infection in childhood. This quantitative analysis is being done in tandem with a qualitative analysis, which has unfortunately been re-scheduled several times over the course of this thesis due to ongoing political unrest in Benin in 2019 and the coronavirus pandemic in 2020. A summary of the qualitative study beginning in Allada, Benin in June 2021 is included in Appendix 1. Briefly, the aim of this mixed-methods study is to investigate the experiences, barriers, and challenges pregnant women have in taking preventive treatments for anemia, malaria, and soil-transmitted helminth infection during antenatal care follow-up, including an in-depth analysis of the impact of maternal depression on pre-and postnatal preventive care. The quantitative analysis of this research question looked more closely at the link between maternal depression, parent-child interactions, and parasitic infection. One previous study had found an association between maternal depression and increased risk of diarrheal disease in children [START_REF] Rahman | Maternal depression increases infant risk of diarrhoeal illness: --a cohort study[END_REF], hypothesizing that depressed women have more difficulty caring for themselves and their children, including noticing when illness occurs and providing timely treatment, thus leading to greater risk of infection. Our regression analyses did not find maternal depression one-year post-partum to impact risk of parasitic infection in children.

However, our analyses did confirm that maternal depression was associated with lower quality and quantity of parent-child interactions as measured by the HOME, as seen in previous research [START_REF] Stein | The relationship between post-natal depression and mother-child interaction[END_REF], with many hypothesizing that this association is a mediator for poor neurodevelopmental outcomes in children.

Recently, two cross-sectional cohort studies revealed an inverse relationship between blood lead level and risk of malaria infection in African cohorts. These studies, conducted in children, hypothesized that absorption of lead into red blood cells could inhibit the life cycle of the P. falciparum parasite, which also occurs in red blood cells, and therefore "protect" against malaria infection [START_REF] Moya-Alvarez | Elevated Blood Lead Levels Are Associated with Reduced Risk of Malaria in Beninese Infants[END_REF]255). In response to this previous research, as well as a study showing high blood lead levels (50 μg/L or more according to the CDC) in our population of mothers and children in Benin (20), we studied whether or not we would see this same inverse association between blood lead level and malaria infection in our population prospectively over the course of a 12-month follow-up of a sub-cohort of children for whom blood lead level and repeated malaria incidence was available. Median blood lead level among the 204 children in our analyses was 56.50 (±56.83) μg/L, above the CDC intervention threshold. Our analyses found that blood lead level measured at one year of age in this population of children was not associated with increased risk of malaria episodes between 12-24 months of age, even after adjusting for other relevant risk factors. The previous significant association found by Moya-Alvarez et al. (2016) could be due to the fact that at 12 months of age, children in malariaendemic countries begin to lose P.falciparum immunity acquired from their mothers and they experience their first episodes of malaria and have higher parasite densities in their red blood cells. These results suggested to us that in the context of routine infection screening and treatment, as recommended by the WHO, environmental exposure from lead does not seem to impact long-term risk of malaria infection in children, and therefore this exposure was not taken into account as a potential risk factor for infection in pregnant women in our primary analyses either.

Strengths & Limitations of thesis

Each article of this thesis details the strengths and limitations for each objective and the analyses used within them. Overall, the studies conducted within this thesis are the first of their kind that explore the long-term neurocognitive consequences of parasitic infection in pregnancy, thus adding significantly to existing epidemiological research. Perhaps the greatest strength of this work is the extensive follow-up of participants in the Allada mother-child cohort, a rarity in SSA populations. The quality and validity of biological, neurocognitive, and anthropometric assessments used during follow-up contribute to this strength.

In addition to the limitations of the analyses for each objective, there are some overarching limitations to what we were able to accomplish within the confines of this thesis.

One such limitation is the fact that we did not include maternal or infant iron deficiency/anemia in many analyses due to its complex and multifactorial nature. This thesis sought to investigate the impact of parasitic infection in pregnancy independently from maternal and infant anemia, and further studies are currently underway to provide more insight into the risk factors of anemia and how it impacts child neurocognitive development in this population. Secondly, the objective studying the association between SHIP and neurocognitive and behavioral development was done in the presence of regular helminth prophylaxis and treatment during pregnancy and childhood, therefore our study was unable to investigate whether or not prophylaxis could attenuate or even reverse the negative effects of SHIP or child helminth infection, as seen in some previous studies. The impact of anti-helminthic prophylaxis treatments has been widely studied; however, results remain conflicting on how they may affect neurocognitive development in children.

Epidemiological implications

The findings of this thesis have implications in epidemiology, society, environmental science, and health policy, providing an interdisciplinary perspective on parasitic infection and its consequences in vulnerable populations. By viewing public health issues through this interdisciplinary lens, we as researchers, health professionals, and public health stakeholders can be more prepared to tackle these issues on an international, cooperative scale.

One hypothesized mechanism to explain the adverse neurocognitive and behavioral outcomes due to maternal infection is the effect of inflammation during pregnancy on fetal neurodevelopmental abnormalities and risk of psychiatric disorders. Maternal infection in pregnancy has been shown to activate the mothers' inflammatory immune response, which in turn can lead to increased risk of behavioral problems and disorders in offspring, including autism spectrum disorder and depression [START_REF] Meyer | In-vivo rodent models for the experimental investigation of prenatal immune activation effects in neurodevelopmental brain disorders[END_REF][START_REF] Shi | Maternal influenza infection is likely to alter fetal brain development indirectly: the virus is not detected in the fetus[END_REF]. One previous study specifically found that elevated markers of inflammation in pregnant women were associated with more internalizing and externalizing behavioral symptoms in their offspring later in childhood [START_REF] Giollabhui | Maternal inflammation during pregnancy and offspring psychiatric symptoms in childhood: Timing and sex matter[END_REF]. However, these studies either overlooked inflammation due to parasitic infection entirely or used a composite value for inflammation due to differing types of maternal infection during pregnancy, without independently studying specific parasitic infections. One existing laboratory-based study provides evidence of a causal link between experimental malaria infection in pregnancy and impaired neurocognitive development in the fetus through activation of the inflammatory complement system [START_REF] Mcdonald | Experimental Malaria in Pregnancy Induces Neurocognitive Injury in Uninfected Offspring via a C5a-C5a Receptor Dependent Pathway[END_REF][START_REF] Weckman | The Impact of Infection in Pregnancy on Placental Vascular Development and Adverse Birth Outcomes[END_REF]. Results from this thesis suggest that the general inflammation marker of CRP does not mediate the relationship between SHIP and impaired behavioral development in children. Other cytokines and markers specific to STH and malaria infection should be used when investigating the mediating role of inflammation in these associations, since maternal inflammation measured by CRP in our population could have been the result of maternal infection or co-morbidities other than SHIP.

Societal implications

Impaired neurocognitive development of younger generations can have population-level consequences, particularly in SSA countries with less developed economic and administrative systems. Additionally, populations living in these countries are often exposed to several additional factors that influence child development over the course of life, including anemia, malnutrition, conflict, poverty, and low social support systems. The findings of this thesis show that intervention to improve neurocognitive outcomes in surviving offspring can begin in utero, potentially mitigating the long-term impacts of prenatal parasitic infection.

In 2015, the United Nations General Assembly set the 2030 Sustainable Development Goals (SDGs), a set of 17 global goals designed to achieve a better and more sustainable future (256). As part of these goals, improving maternal and child morbidity from infectious diseases and addressing factors that could inhibit child and economic development remain high priorities. Addressing the social inequalities between nations and the specific challenges faced by developing children in SSA countries is a pivotal step in achieving these goals. While the SDGs do not mention specific infectious diseases that need public attention, the WHO has deemed soil-transmitted helminth infections to be a part of their list of Neglected Tropical Diseases (NTDs), a list of diseases that disproportionally effect poor populations, receive little to no public funding for research and intervention, receive little media attention, and impact large proportions of these under-served populations. Therefore, innovative research, such as the work done within this thesis, on NTDs is important to shed light on the extended burden they bring to millions of individuals around the world.

Data collection and reporting on developmental disabilities and impairments in African populations is greatly needed in order to better understand their health burden (257). To date, there are no standardized, comprehensive developmental diagnostic tools for Beninese and other SSA children. The existing tools used in these populations come from Western societies and norms, and are often adapted to African populations and have their construct validity verified prior to their use [START_REF] Koura | Usefulness of child development assessments for low-resource settings in francophone Africa[END_REF]. This gap in knowledge related to developmental norms, definitions, and educational and clinical treatment protocols in SSA countries further hinders these populations and their overall development as societies. Prospective cohorts such as the one included in this thesis are vital for further research efforts to provide more comprehensive data on neurodevelopment in African populations and their implications for society and public health. Further research on this subject is greatly needed in African cohorts, and in fact one study 11-years post-partum is underway in the Beninese population included in this thesis to better interpret results from neurocognitive assessments and to use these results to understand the clinical representations of impairments in this population.

Environmental implications

This thesis provides evidence that exposure to lead, a neurotoxic heavy metal, is still a public health concern in low-resources settings like Benin, even though exposure to lead via paint and gasoline has been eradicated in high-income countries for the most part [START_REF] Akkus | Exploring Childhood Lead Exposure through GIS: A Review of the Recent Literature[END_REF]. It is critical to provide regular surveillance for lead exposure in this population since ammunition used to hunt bushmeat, dust participles inside homes, and contaminated water pipes have all been found to contain significant amounts of lead in previous studies [START_REF] Ahmadi | Follow-Up of Elevated Blood Lead Levels and Sources in a Cohort of Children in Benin[END_REF]241). There is also a public health interest in studying how these persistent environmental exposures impact populations already vulnerable to several other risk factors of disease and impaired cognitive functioning.

Policy implications

The close follow-up of women during antenatal care in the clinical trial allowed researchers to provide WHO and study protocol recommended prevention and treatment medications for maternal anemia, soil-transmitted helminth infection, and malaria infection.

Even in the presence of this close antenatal follow-up, prevalence of parasitic infection was still in congruence with expected estimates (1). At the time of the clinical trial, only two doses of IPTp-SP were recommended to prevent malaria in high-transmission areas of SSA, like Benin.

We saw in our follow-up that there was a higher prevalence of soil-transmitted helminth infection and malaria infection in pregnant women at delivery compared to the 2 nd ANV, suggesting inadequate preventive treatment for these women who are chronically exposed to the parasites causing these infections. Research on the timing of maternal infections during pregnancy and its impact on fetal health remains equivocal [START_REF] Kalilani-Phiri | Timing of Malaria Infection during Pregnancy Has Characteristic Maternal, Infant and Placental Outcomes[END_REF][START_REF] Mahande | Prevalence of parasitic infections and associations with pregnancy complications and outcomes in northern Tanzania: a registry-based crosssectional study[END_REF]. One prospective mother-child cohort study found that in the presence of IPTp-SP and active malaria screening and treatment throughout pregnancy, maternal infection was not associated with adverse birth outcomes [START_REF] Kalilani-Phiri | Timing of Malaria Infection during Pregnancy Has Characteristic Maternal, Infant and Placental Outcomes[END_REF].

Our analyses show a trend between late-term parasitic infection, particularly malaria infection, and lower neurocognitive and behavioral scores in children at one and six years. In 2013, after the clinical trial, the WHO published a policy brief recommending at least three doses of IPTp-SP during prenatal care in malaria-endemic countries. Studies on the impacts of prenatal malarial infection on child developmental outcomes are needed in order to know the impact this protocol may have on associations found within this thesis and to add to the limited research on the timing of malarial infection in pregnancy on adverse birth outcomes in the presence of more regular screening and treatment.

Our analyses provide evidence that submicroscopic, asymptomatic malarial infections pose a high risk to pregnant women in malaria-endemic countries like Benin, and sufficient IPTp and close follow-up during antenatal care is vital to address its risks on fetal development.

The diagnosis of placental malaria using qPCR techniques, which are more time-and resourceintensive to analyze in low-income settings like Benin, revealed that women with these infections at time of delivery were more likely to have offspring with impaired neurocognition. These submicroscopic, asymptomatic infections are left untreated, due to often undetectable parasitic density of these infections through gold standard diagnostic methods and the inability to diagnose infection based on visible symptoms, and therefore could reflect more chronic infections of women. Diagnostic methods that are quick, reliable, affordable, and able to detect low parasite density thresholds (which is of special importance in endemic populations where acquired immunity is active) and close antenatal care follow-up, are imperative in order to provide adequate and timely treatment of infections and curb potential long-term consequences.

The qualitative study that will explore mothers' experiences, barriers, and attitudes toward infection prevention and treatment will also provide insight into the real-life translation of and adherence to pre-and post-natal prophylaxis treatments and the barriers that women and children face that could be addressed by policy adaptations. Previous qualitative studies interested in women's experience with IPTp have recommended increased community healthcare professional training to address questions and concerns of women during antenatal care visits (258), alternative sources of information regarding IPTp and its risks (259), and better availability and access to free-of-charge antenatal services, particularly in rural areas (260).

Conclusion and Perspectives

It is imperative to mention that advances in clinical practices and international public health policy implementation in recent decades has allowed for vast improvements in terms of morbidity and mortality from parasitic infection in SSA countries. However, parasitic infection in endemic areas of the world, primarily low-and middle-income countries, still carries a large burden of disease in vulnerable groups and has long-term consequences for survivors.

Understanding these long-term consequences using an interdisciplinary approach in public health allows a multitude of innovative initiatives and interventions to emerge. Public health issues today call for global and synergistic approaches to tackle them [START_REF] Kivits | Interdisciplinary research in public health: the 'why' and the 'how[END_REF], therefore participation from stakeholders in various disciplines allows for adequate health intervention analysis and action. While maternal morbidity from parasitic infection has improved over the years, there is still much intervention to be done in order to improve health outcomes and quality of life for both mother and fetus throughout the life course.

This thesis provides novel evidence of these long-term consequences of parasitic infection in pregnancy on child development. Our studies showed that even in the presence of prevention and treatment strategies for parasitic infection during antenatal care, SHIP and MIP are associated with impaired child behavioral and neurocognitive development at one and six years of age. Our analyses revealed that later term infections were more deleterious, even in statistically non-significant results. The numerous sensitivity analyses, including multiple imputation of missing values, mediation analyses, and regression analyses including various additional potential confounding factors, confirm the primary results found for each objective and show the robustness of our results.

Future studies in large, prospective SSA cohorts are needed in order to confirm the results seen in this thesis and should explore the mediating role of anemia and maternal inflammation more in depth with proper markers. Further studies should also continue to explore the mediating relationship of parasitic infection during childhood on the relationship between infection during pregnancy and child development. The qualitative study being carried out in tandem with this thesis will complement the quantitative results on the impacts of maternal depression on child parasitic infection and provide valuable data to better understand the experiences and barriers to adequate preventive treatment for parasitic infection during pregnancy. Morbidity and mortality from malaria has decreased over time with improved prevention, surveillance, and treatment efforts; however, the detection and treatment of asymptomatic infections remains of public health concern.

As previously mentioned, the clinical manifestation and diagnosis of neurocognitive impairments in African populations are not well-documented, and further studies should seek to provide country-specific norms and definitions for developmental diseases and disabilities.

Prevention of parasitic infection during pregnancy using IPTp and helminth prophylaxis, surveillance and active detection of cases through close antenatal follow-up, and treatment of malaria and helminth infection cases in pregnancy are needed in order to reduce the burden of these infections in vulnerable populations. Another future area of interest for researchers is the impact of insecticides, in the form of long-lasting insecticide-treated bed nets, spirals, and insecticide sprays, on long-term child neurocognitive development, as these are effective methods of vector control but their long-term impacts on exposed individuals remain widely unknown.

ForFigure 1 .

 1 Figure 1. Life cycle and fecal-oral transmission of A.lumbricoides species.

Figure 2 .

 2 Figure 2. Life-cycle and transmission of hookworm species.

Figure 3 .

 3 Figure 3. World map showing malaria transmission around the world.

Figure 4 .

 4 Figure 4. Life cycle and transmission of Plasmodium parasites in mosquito vector and human host.

Figure 5 .

 5 Figure 5. Effects of malaria in pregnancy through the life course.

Figure 6 .

 6 Figure 6. Fetal brain development over the course of pregnancy.

Figure 7 .

 7 Figure 7. Cognitive development in early and middle childhood.

Figure 8 .

 8 Figure 8. DAG showing pathways between parasitic infection during pregnancy and child neurocognitive development.

Figure 9 .

 9 Figure 9. Study site of Allada, Benin.

  touchent jusqu'à 50 % des femmes enceintes en Afrique subsaharienne. Les infections parasitaires chez la femme enceinte ont des conséquences négatives sur la santé de la mère et du nourrisson, comme un risque accru d'anémie maternelle, de naissance prématurée, de faible poids de naissance et d'infection parasitaire infantile. Cependant, leur impact sur le développement neurocognitif et comportemental de l'enfant reste largement inconnu. Par conséquent, cette thèse visait à étudier l'impact de l'infection par helminthes transmis par le sol pendant la grossesse (SHIP) et du paludisme à Plasmodium falciparum pendant la grossesse (MIP) sur le développement neurocognitif et comportemental de l'enfant dans une cohorte mère-enfant du Bénin, ainsi qu'à rechercher des facteurs de risque supplémentaires d'infection parasitaire qui pourraient avoir un impact sur ces associations. Notre cohorte mère-enfant comprenait 493 femmes enceintes inscrites à un essai clinique au deuxième trimestre comparant l'efficacité de deux traitements préventifs intermittents contre le paludisme pendant la grossesse, la sulfadoxine-pyriméthamine et la méfloquine, à Allada, Bénin. Des échantillons de sang et de selles ont été prélevés chez les femmes lors de trois consultations prénatals (CPNs) ; le SHIP a été diagnostiqué en utilisant la technique de Kato-Katz et le MIP a été diagnostiqué par des tests gouttes épaisses lors des CPNs et par des méthodes de réaction en chaîne par polymérase quantitative (qPCR) lors de l'accouchement. Les femmes ont reçu des traitements de prévention contre les infections parasitaires conformément aux directives nationales existantes, ainsi qu'un paquet de soins prénatals standard pour le Bénin au premier CPN. Les enfants survivants ont été évalués par des enquêteurs qualifiés sur le plan du développement neurocognitif et moteur à un an avec le Mullen Scales of Early Learning, puis à six ans avec le Kaufman Assessment Battery in Children 2 nd Edition (KABC-II), le Bruininks-Oseretsky Test of Motor Proficiency Edition 2 (BOT-2), le Test of Variables of Attention (TOVA), et le Strengths and Difficulties Questionnaire (SDQ). Une sous-cohorte de mères et d'enfants a été incluse dans des analyses indépendantes pour tester le rôle de la plombémie et de la dépression maternelle dans le risque ultérieur d'infection parasitaire. La prévalence du SHIP était de 13% au 1er CPN, 9% au 2ème CPN, et 1% à l'accouchement. D'après les gouttes épaisses, 122 (25%)

1 .

 1 unstudied in the literature. Below are the main objectives (1 & 2), along with their corresponding secondary objectives (a & b), each of which resulted in a publication within the confines of this thesis and served to explain the overall objective of this thesis: to study the impacts of parasitic infection in pregnancy on child neurocognitive development in a mother-child cohort in Benin. The secondary objectives investigated the potential confounding role of blood lead, maternal depression, and parent-child interactions in the relationship between helminth infections and malaria and child development. Main objectives To study the impact of soil-transmitted helminth infections in pregnancy on child neurocognitive and behavioral development at six years of age 2. To study the impact of Plasmodium falciparum malaria infection in pregnancy on child neurocognitive development at one and six years of age Secondary objectives a. To investigate the association between maternal depression and parentchild interactions and risk of helminth infection and malaria.

1 Chapter 3

 13 presents background information regarding risk factors and consequences of parasitic infection during pregnancy from soil-transmitted helminths and from malaria, as well as relevant information on child development assessments and risk factors of impaired development. This chapter provides adequate background knowledge and vocabulary necessary to understand the remainder of the thesis and its attached publications. Chapter 2 details the materials and methods used within this thesis, describing the study design, population, and data collection of each ancillary study that pregnant women and their children participated in from the second trimester of pregnancy until six years postpartum. The assessment of parasitic infection during pregnancy, child neurocognitive and behavioral development at one and six years, and relevant risk factors such as sociodemographic information, maternal depression, and blood lead level is detailed within this chapter. presents the first article investigating the first objective of this thesis, the impact of soil-transmitted helminth infection during pregnancy on child development at six years of age, published in Plos Neglected Tropical Diseases. Chapter 4 includes the second article of this thesis, corresponding to the second objective, assessing the impact of P.falciparum malaria during pregnancy on child development at one and six years of age, submitted for publication. Chapter 5 presents the third article, showing the results of the secondary objective investigating the potential role of maternal depression and parent-child interactions on risk of parasitic infection in a sub-cohort of mothers and their children from our population. The resulting article was submitted for publication.

Figure 1 .

 1 Figure 1. Life cycle and fecal-oral transmission of A.lumbricoides species.

Figure 3 .

 3 Figure 3. World map showing malaria transmission around the world.

Figure 4 .

 4 Figure 4. Life cycle and transmission of Plasmodium parasites in mosquito vector and human host.

  WHO, there is currently only one vaccine against malaria that has been proven in clinical trials to be effective in reducing malaria illness, specifically severe, lifethreatening malaria, in children in medium-and high-transmission areas (117). The RTS,S malaria vaccine acts against P. falciparum malaria infection and has been shown to prevent 4 in 10 malaria cases in children over a 4-year period(2). Pilot programmes for the administration of the vaccine has begun in some SSA countries. More research and clinical trials are needed to continue the search for effective vaccines to decrease the burden of malaria around the world and especially in endemic areas, particularly for high-risk populations like children under five and pregnant women.

Figure 5 .

 5 Figure 5. Effects of malaria in pregnancy through the life course.

Figure 6 .

 6 Figure 6. Fetal brain development over the course of pregnancy.

Figure 7 .

 7 Figure 7. Cognitive development in early and middle childhood.

Figure 8 .

 8 Figure 8. DAG showing pathways between parasitic infection during pregnancy and child neurocognitive development.

  described for each study, as well as interventions given to participants. Statistical analyses used for each thesis objective are detailed individually in subsequent chapters and in attached publications.Data sourcesInformation on biological, sociodemographic, and anthropometric data from pregnant women and their offspring was collected during two parallel studies: the Malaria in Pregnancy Preventive Alternative Drugs (MiPPAD) randomized clinical trial (NCT00811421) and the Anaemia in Pregnancy: Aetiologies and Consequences (APEC) nested-cohort study. Additional biological data of a sub-cohort of infants was collected within the APEC study at birth, three, six, nine, and twelve months of age. Parasitic infection and haematological factors were measured in a sub-population of infants from APEC between birth and twelve months of age in the Tolérance Immunitaire et Paludisme (TOLIMMUNPAL) prospective cohort study. The TOVI ('Child from the country' in Fon) cross-sectional study assessed postnatal characteristics and neurocognitive development in one-year-old children born to mothers from the MiPPAD and APEC cohorts. Six-year-old children were further assessed for long-term neurocognitive development in the EXposition au PLomb et au manganèse et Risques pour l'Enfant (EXPLORE) cross-sectional study.

(

  219). Single-dose MQ has been associated with mild secondary effects after administration and rare psychological and neurological effects (220); however, these effects were attenuated when MQ was given to individuals in a split dose in one previous study(221). Given the lack of adequate information on the efficacy of IPTp-MQ as a replacement for IPTp-SP, apart from two promising clinical trials inBenin (222,223), researchers conducted MiPPAD open-label randomized clinical trial comparing the safety and efficacy of IPTp-SP with IPTp-MQ full-dose and split-dose in HIV-negative pregnant women in the context of LLITNs across four countries in SSA. Researchers also recruited HIV-positive women from three other countries for the same comparison of IPTp; however, these women were not part of the cohorts described in this thesis.The clinical trial recruited eligible pregnant women from four countries in SSA (Benin, Gabon, Mozambique, and Tanzania); only data from the women participating at the Benin study site is discussed within the remainder of this thesis. The trial was conducted from 2009 until 2013 in Allada, Benin, a semirural district about 50 kilometers north of the economic capital and major port city, Cotonou. The district is comprised of 84 villages with a total population of 127, 512 inhabitants according to a 2013 report by the Institut National de la Statistique et de l'Analyse Economique (224). Figure9shows a map of the study location in Benin.

Figure 9 .

 9 Figure 9. Study site of Allada, Benin. Source: https://www.actualitix.com/carte-benin.html

(

  BMI) was determined based on the weight, height, and gestational age assessed at recruitment by subtracting the expected weight gain during pregnancy (roughly 1kg per month from the end of the first trimester until delivery (225)) and calculating BMI in kg/m 2 .

  funded through a grant from the Bill and Melinda Gates Foundation to the Liverpool School of Tropical Medicine. Ethical consideration for the APEC nested cohort was approved by the French Comité Consultatif de Déontologie et d'Ethique and the Ethics Committee of the University of Abomey-Calavi in Benin. Informed consent was obtained from all human adult participants and from the parents or legal guardians of minors.

1 .

 1 The MSEL was translated and back-translated between English and French by two separate psychologists and both English versions (translated and backtranslated) were compared by study personnel and modified if needed. The backtranslation was validated by Pearson Assessments, the copy-right holder of the official MSEL. An oral translation of the MSEL in the local Beninese language, Fon, was used for assessments. 2. Research nurses and the coordinating psychologist of the study were trained to administer the MSEL and other assessments used in the TOVI study by psychologist Michael Boivin, specialized in child development in SSA, for a twoweek period. 3. During the two-week training period, a pilot study was conducted and trained staff administered the MSEL and other assessments to fifteen participating children and their mothers. Later, thirty-two additional children were assessed prior to the onset of the study. Administration and scoring of the MSEL by research nurses were audited and reviewed prior to child enrollment. 4. During the training and pilot study, research coordinators and staff reviewed and adapted assessments accordingly. Three items were adapted for use in this population: a potato chip was used instead of a cheerio to utilize local materials, children received one point if they handed the ball to their mother/guardian instead of throwing it underhand, and one question was changed from 'Why do we have refrigerators?' to 'Why do we have lanterns?' since most participants did not have refrigerators.

(

  ANR). Ethical approval was obtained from the Consultative Committee on Ethics and Deontology (CCDE) of the Institute of Research for Development (France) and the Ethics Committee of the Faculty of Health Sciences, University of Abomey Calavi, Benin (43/11/2010/CE/FSS/UAC).

  Investigator and study psychologist to administer neurocognitive assessments to children, measure anthropometric factors, collect biological samples, and administer a questionnaire to mothers to evaluate maternal depression and socio-demographic factors. Validation of assessments and quality control was assured prior to the onset of the study. A common translation of oral instructions from French to Fon was validated by trained assessors and study nurses for all assessments given to mothers and their children. Participating children were treated for anemia, soil-transmitted helminth infection, and malaria infection if present according to existing national guidelines in Benin.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

  Plasmodium falciparum a été déterminée à chaque CPN, y compris lors de l'accouchement, par des tests de gouttes épaisses et fins à partir d'échantillons de sang collectés en utilisant la technique de Lambaréné. Lors de l'accouchement, le sang périphérique maternel et le sang placentaire ont été déposés sur des papiers filtres pour une évaluation parasitologique. Les papiers filtres ont été testés pour la présence et la densité de P. falciparum par un test quantitatif en temps réel Des échantillons de sang et de selles ont été prélevés chez les enfants pour évaluer l'infection par les helminthes et le paludisme au moment des évaluations neurocognitives, et les enfants infectés ont été traités conformément aux directives nationales. La batterie d'évaluation Kaufman pour enfants, 2e édition (KABC-II), a

	Les femmes enceintes de cette cohorte prospective mère-enfant ont été recrutées dans
	l'essai clinique Malaria in Pregnancy Preventive Alternative Drugs (MiPPAD)
	(NCT00811421) au cours du deuxième trimestre de grossesse. L'objectif de l'essai était de Résultats
	comparer l'efficacité de deux traitements préventifs intermittents du paludisme pendant la La prévalence du SHIP dans notre population était de 12,6% à la 1ère CPN, 8,5% à la
	grossesse (TPIp), la sulfadoxine-pyriméthamine (SP) et la méfloquine, dans trois centres de mesuré les processus simultanés et séquentiels, ainsi que les capacités de planification et 2ème CPN et 1,2% à l'accouchement. Toutes les femmes ont reçu un total de 600 mg de
	santé du district d'Allada au Bénin (Sékou, Allada et Attogon). Les informations sur les de réaction en chaîne par polymérase (qPCR) ciblant l'ADN ribosomal 18S (19). Une sous-d'apprentissage des enfants. L'indice des processus mentaux (IPM), une mesure de la capacité mébendazole (100 mg deux fois par jour pendant 3 jours) à prendre après la 1ère CPN ;
	critères d'inclusion et d'exclusion sont détaillées dans une publication précédente (16). cohorte de 400 enfants a été choisie pour participer à deux études auxiliaires, APEC et cognitive globale, et l'indice non verbal (INV), une évaluation non verbale du traitement et cependant, la prise n'a pas été directement observée. La prévalence de la MIP périphérique par
	Brièvement, les critères d'inclusion des femmes étaient les suivants : résidence permanente Tolérance Immunitaire et Paludisme (TOLIMMUNPAL), qui ont suivi les enfants de la des capacités cognitives de l'enfant, tous deux générés par les scores KABC-II, ont été utilisés goutte épaisse était de 15% à la 1ère CPN, de 3% à la 2ème CPN et de 10% à l'accouchement.
	dans la zone d'étude, âge gestationnel ≤28 semaines, séronégativité au moment du naissance jusqu'à l'âge de 24 mois pour l'infection parasitaire et les facteurs hématologiques et dans les analyses. La Bruininks-Oseretsky Test of Motor Proficiency Edition 2 (BOT-2) a été A l'accouchement, 31% des femmes testées avaient un paludisme placentaire selon les
	recrutement, absence d'allergies connues aux sulfamides ou à la méfloquine, absence de environnementaux associés. A chaque visite entre la naissance et 12 mois, 8 ml de sang utilisée pour l'évaluation des habiletés motrices dans cette population ; les scores composites analyses qPCR. Le SHIP diagnostiqué à chaque CPN et l'infection helminthique globale ainsi
	domicile, mesurées par l'inventaire HOME (Home Observation for Measurement of the
	Ces études préliminaires ont permis d'étudier les rôles potentiels de confusion de la Environment), ont été recueillies après son adaptation pour être utilisées dans cette
	plombémie, de la dépression maternelle et des interactions parent-enfant dans la relation entre population. Les mères ont également été évaluées pour les symptômes de dépression du post-
	les infections par helminthes et le paludisme et le développement de l'enfant. partum au moyen de la version française validée de l'EPDS (Edinburgh Postnatal Depression
	Scale).
	Méthodes

Objectifs secondaires a. Étudier l'association entre la dépression maternelle et les interactions parentenfant et le risque d'infection par helminthes et le paludisme. b. Étudier l'association entre la plombémie de l'enfant et le risque d'infection palustre. traitement du paludisme par ces médicaments dans les quatre semaines précédant le recrutement, et absence d'antécédents de maladie rénale, hépatique, psychiatrique ou neurologique grave. Les femmes enceintes incluses dans la population d'étude de cette thèse ont été également suivies dans le cadre du projet Anemia in Pregnancy : Etiologies and Consequences (APEC) afin d'être suivies pour l'infection parasitaire pendant la grossesse.

Dans le cadre de l'essai clinique, 1,005 femmes enceintes ont été vues à deux consultations prénatales (CPNs) dans l'une des trois maternités à au moins un mois d'intervalle, ainsi qu'à l'accouchement, pour un total de trois CPNs programmées. Dans le cadre des directives de soins prénatals standard au Bénin, les femmes ont reçu des moustiquaires imprégnées et 600 mg de mébendazole (100 mg à prendre deux fois par jour pendant trois jours) pour la prévention des infections helminthiques au moment du recrutement, ainsi que des suppléments de sulfate ferreux et d'acide folique à chaque CPN pour la prévention de l'anémie. Les épisodes de paludisme clinique ont été traités par quinine par voie orale au premier trimestre ou par artéméther-luméfantrine aux trimestres suivants pour le paludisme non compliqué ; le paludisme grave a été traité par quinine parentale

[START_REF] Ouédraogo | Placental impression smears is a good indicator of placental malaria in sub-Saharan Africa[END_REF]

.

Lors de la première CPN, des informations sociodémographiques et anthropométriques ont été recueillies, notamment l'âge maternel, la gravidité, l'indice de masse corporelle (IMC) avant la grossesse, l'état civil, la profession et le niveau d'éducation de la mère et du père, la zone de résidence, et des informations relatives à l'utilisation des moustiquaires imprégnées la nuit précédente. Dans chaque CPN, des gouttes épaisses d'échantillons de selles ont été préparés et évalués à l'aide de la technique de Kato-Katz pour obtenir une estimation quantitative de l'intensité de l'infection par les helminthes selon les normes de l'OMS

[START_REF] Organization | Bench aids for the diagnosis of intestinal parasites[END_REF]

.

Les oeufs d'helminthes des espèces

Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale et Necator americanus ont été identifiés. L'infection palustre par veineux étaient prélevés chez les nourrissons et la technique de Lambaréné était utilisée pour diagnostiquer le paludisme sur des gouttes épaisses. De 12 à 24 mois, l'infection palustre a été diagnostiquée au cours du suivi de 12 mois en utilisant à la fois des tests gouttes épaisses et des tests de diagnostic rapide (TDR) lors des visites systématiques mensuelles et bihebdomadaires, ainsi que lors des visites d'urgence. Les 863 singletons survivants à l'âge d'un an et nés de femmes enceintes de l'essai MiPPAD étaient éligibles pour participer à l'étude TOVI (enfant en Fon). Le développement neurocognitif et moteur du nourrisson a été mesuré par une infirmière de recherche qualifiée lors d'une visite programmée un an après l'accouchement dans une maternité locale, à l'aide de l'édition de l'American Guidance Service des Mullen Scales of Early Learning (MSEL) dont la traduction française réalisée lors de l'étude a été approuvée par Pearson assessment (20). Les scores bruts de chacune des cinq sous-échelles ont été calculés puis convertis en scores t normatifs ; les scores t pour les échelles de motricité fine, de langage réceptif, de langage expressif et de perception visuelle ont été convertis en score composite d'apprentissage précoce (ELC), indicatif du fonctionnement cognitif global des enfants. La plombémie des enfants a été évaluée en analysant des échantillons de sang veineux par spectrométrie de masse à plasma à couplage inductif. Des informations sur l'environnement d'apprentissage à À l'âge de six ans, 578 enfants ont été suivis dans le cadre de l'étude EXPLORE (EXposition au PLomb et au manganèse et Risques pour l'Enfant) pour des évaluations neurocognitives supplémentaires. finaux pour la motricité fine et la motricité globale ont été normalisés selon l'âge et le sexe et utilisés aux fins de cette thèse. Le Test of Variables of Attention (TOVA), un test de performance non basé sur le langage, a été administré pour identifier le déficit d'attention et l'hyperactivité chez les enfants. Enfin, les mères ont répondu au Strengths and Difficulties Questionnaire (SDQ), un bref outil de dépistage de 25 questions visant à évaluer le développement comportemental des enfants en fonction de cinq sous-échelles : problèmes émotionnels, problèmes de comportement, hyperactivité, problèmes avec les pairs et comportements pro-sociaux ; les quatre premières sous-échelles étant utilisées pour générer un score global de difficultés. Les caractéristiques démographiques de la population incluse dans les analyses ont d'abord été décrites et comparées à la population perdue de vue entre la naissance et l'âge de six ans à l'aide de tests univariés appropriés (test du chi deux, Fisher exact, Kruskal-Wallis ou Wilcoxon ranksum). Des modèles de régression linéaire multiple avec des intervalles de confiance bootstrapped à 95 % ont testé les principales associations entre l'exposition à l'infection parasitaire pendant la grossesse et les scores neurocognitifs et comportementaux continus des enfants à un et six ans. Diverses analyses de sensibilité, dont l'imputation multiple et les analyses de médiation, ont été menées pour garantir la robustesse des résultats de la régression. Les analyses portant sur les facteurs de confusion potentiels de la plombémie de l'enfant et de la dépression maternelle ont fait appel à des modèles de régression binomiale négative et logistique simple, dont les méthodes sont précisées dans chaque publication spécifique incluse dans cette thèse. Les analyses statistiques ont été réalisées à l'aide de STATA 13.1 (StataCorp. 2013. Logiciel statistique Stata : Release 13. College Station, TX : StataCorp LP) pour Windows et STATA 16 (StataCorp. 2019. Logiciel statistique Stata : Release 16. College Station, TX : StataCorp LLC) pour Mac. Tous les tests statistiques étaient bilatéraux avec un risque alpha de 5%, sauf indication contraire dans le manuscrit ou dans les publications. que la densité d'ankylostomes pendant la grossesse ne se sont pas avérés être associés au développement neurocognitif, à la fonction motrice ou au trouble de l'attention/hyperactivité chez les enfants à l'âge de six ans. Cependant, le SHIP a été associé à une altération du développement émotionnel, mesurée par le score d'intériorisation du SDQ. L'infection à helminthes lors de la 2e CPN/l'accouchement et à n'importe quel moment de la grossesse était associée à des scores d'intériorisation plus élevés, indiquant davantage de problèmes émotionnels, coefficient bêta [1,07 intervalle de confiance (IC) à 95 % (0,15, 2,00)] et [0,79

  μg/L, au-dessus du seuil d'intervention des Centers for Disease Control (CDC). Nos analyses ont révélé que la plombémie dans cette population d'enfants n'était pas associée à un plus grand nombre d'épisodes de paludisme entre

	développement des enfants à un et six ans, comme le KABC-II dont la validité de
	construction a été validé dans une étude pilote et le SDQ et BOT-2 qui ont été utilisés dans
	des études antérieures dans des populations similaires (21,22). Des analyses de sensibilité ont
	permis d'étudier d'autres facteurs intermédiaires et de confusion potentiels, tels que le groupe
	de traitement TPIp dans l'essai clinique, la carence en fer pendant la grossesse, la plombémie
	de l'enfant, l'inflammation pendant la grossesse, la naissance prématurée et le faible poids de
	naissance ; aucun de ces facteurs n'a modifié les résultats de manière significative.
	L'imputation multiple a confirmé nos résultats, excluant ainsi tout biais potentiel lié à
	l'absence de données. Enfin, il n'y avait pas de différences en termes d'exposition aux
	infections parasitaires chez les femmes incluses et exclues des analyses, le biais de sélection
	est donc peu probable. Nos analyses présentent plusieurs limites, notamment la taille limitée
	de l'échantillon dans certaines analyses et la multitude de tests effectués dans cette population,
	ce qui augmente la probabilité que les résultats soient le fruit du hasard. Les femmes enceintes
	et les enfants ont été suivis de manière extensive et traités rapidement en cas d'infection
	parasitaire, ce qui pourrait sous-estimer les associations entre l'infection et le développement
	neurocognitif de l'enfant.
	Au moment de l'essai clinique, seules deux doses d'TPIp-SP étaient recommandées
	pour prévenir le paludisme dans les zones de forte transmission de l'ASS, comme le Bénin.
	Nous avons vu dans notre suivi qu'il y avait une prévalence plus élevée d'infection par
	helminthes et de paludisme chez les femmes enceintes à l'accouchement par rapport à la 2e
	CPN, ce qui suggère un traitement préventif inadéquat pour ces femmes qui sont
	régulièrement exposées aux parasites responsables de ces infections. Le paludisme placentaire
	et la densité parasitaire à l'accouchement, diagnostiqués par qPCR, se sont avérés
	particulièrement préjudiciables aux scores neurocognitifs et moteurs des enfants. Cela
	pourrait s'expliquer par le fait que les infections palustres placentaires submicroscopiques
	peuvent être particulièrement délétères pour la croissance et le développement du foetus
	(4,23,24). Alors que la technologie qPCR est capable d'identifier les infections palustres
	tels que la plombémie et la dépression maternelle, qui pourraient finalement avoir un impact submicroscopiques indétectables par microscopie, les ressources de laboratoire ne sont pas
	sur le développement neurocognitif de l'enfant. facilement disponibles dans les pays à faible revenu comme le Bénin. Par conséquent, les
	La nature prospective de cette cohorte mère-enfant et les évaluations biologiques et femmes présentant des infections submicroscopiques dans notre population n'ont pas été
	neurocognitives complètes réalisées au sein de cette population sont des caractéristiques immédiatement diagnostiquées et traitées au moment de l'accouchement et ces infections
	déterminantes des méthodes utilisées. Ces analyses sont les premières du genre à étudier les pourraient refléter des infections plus chroniques qui ne sont pas facilement diagnostiquées
	conséquences neurocognitives à long terme de l'infection parasitaire pendant la grossesse. par les méthodes de microscopie et traitées rapidement.
	Parmi les autres points forts de cette thèse, citons les différents outils utilisés pour évaluer le

12 et 24 mois, même après ajustement sur d'autres facteurs de risque pertinents. Nos analyses de régression n'ont pas trouvé que la dépression maternelle un an après l'accouchement avait un impact sur le risque d'infection parasitaire chez les enfants. Cependant, nos analyses ont confirmé que la dépression maternelle était associée à une qualité et à une quantité moindre d'interactions parents-enfants, telles que mesurées par l'échelle HOME, comme l'ont montré des recherches antérieures pour avoir un impact sur le développement de l'enfant.

Discussion

Les études menées dans le cadre de cette thèse sont les premières du genre à explorer les conséquences neurocognitives à long terme d'infections parasitaires pendant la grossesse, ajoutant ainsi une contribution significative à la recherche épidémiologique existante. Nos analyses ont montré que le SHIP à n'importe quel moment de la grossesse était associé à une altération du développement comportemental de l'enfant six ans après la naissance. Nos analyses montrent une tendance à une association entre une infection palustre tardive et des scores neurocognitifs plus faibles chez les enfants à un et six ans. Plus important encore, nos analyses suggèrent que les infections palustres submicroscopiques et asymptomatiques sont plus présentes chez les femmes enceintes dans notre population. Cette thèse a également exploré les relations complexes entre l'infection parasitaire et les facteurs de risque potentiels

Table 1 . WHO treatment guidelines for P. falciparum infection in high-transmission areas of SSA. Uncomplicated malaria Severe malaria Children over 25kg and adults (including pregnant women in 2 nd and 3 rd trimesters)

 1

	One of the following	Intravenous or intramuscular
	artemisinin-based combination	artesunate for at least 24 hours
	therapies (ACTs) for 3 days: Artemether + lumefrantrine	and until they can tolerate oral
	Artesunate + amodiaquine	medication, then complete
	Artesunate + mefloquine	treatment with 3 days of ACT
	Dihydroartemisinin +
	piperaquine
	Artesunate + sulfadoxine-
	pyrimethamine (SP)

  MiPPAD clinical trial was funded by the European and Developing CountriesThe Ethics Committee of the Hospital Clinic of Barcelona in Spain, the IRD Consultative Ethics Committee in France, and the regulatory authorities and the National Ethics Review Committee of Benin approved study protocol and informed consent forms. Written informed consent was obtained by pregnant women in the presence of a witness, with thumbprints provided for consent if women could not read and/or write.

	Clinical Trials Partnership (EDCTP; IP.2007.31080.002), the Malaria in Pregnancy
	Consortium, as well as the following national agencies: Instituto de Salud Carlos III
	(PI08/0564), Spain; Federal Ministry of Education and Research (BMBF FKZ: da01KA0803),
	Germany; Institut de Recherche pour le Developpement (IRD), France.

Table 2 . Infant-Toddler HOME inventory sub-scales (ages 0 to 3).

 2

	Subscale	Description	Example
	Responsivity	Verbal and affective interactions	Caregiver spontaneously vocalizes to
		between caregiver and child	child at least twice during visit
	Acceptance	How the caregiver disciplines the child	Caregiver does not express overt
			annoyance with or hostility towards the
			child
	Organization	How the child's time is organized	The child's play environment appears
		outside of the home environment and	safe and free of hazards
		what the child's personal space is like
	Learning	Availability of age-appropriate toys	Child is provided pieces of equipment
	Materials	and activities for the child	appropriate for their age (i.e. playpen,
			infant seat)
	Involvement	How the caregiver interacts physically	Caregiver tends to keep child within
		with the child	visual range and looks at them often
	Variety	How the child's daily routine	Family visits or receives visits from
		incorporates variety and social	relatives approximately once per month

Table 3 . Summary of all studies and variables collected during follow-up.

 3

	Timing		Pregnancy	Singleton	3	6	9	12	12	24	6 years
					Birth	months	months	months	months	months	months
		1 st	2 nd	Delivery						Systematic monthly
		ANV	ANV							follow-up
	Year		2009-2011			2009-2011		2011-2013	2011-2014	2016-2018
	Study		MiPPAD & APEC			APEC		TOVI	TOLIMMUNPAL	EXPLORE
	N	1,005	978	941	863	Sub-cohort of 400	747	Sub-cohort of 400	578
	Soil-transmitted
	helminth infection

  . Stata Statistical Software: Release 13. College Station, TX: StataCorp LP) for Windows and STATA 16 (StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC) for Mac. All statistical tests were two-tailed with an alpha risk of 5%, unless otherwise specified in the manuscript or in publications.

Acknowledgements

, I need to thank my thesis director, Dr. Florence Bodeau-Livinec, who is one of the primary reasons I decided to complete a PhD thesis at this point in my life.

After working with her for my M2 internship and thesis at Ecole des , Florence.

Thank you to Asia Maselko and Suzanne Maman from UNC Chapel Hill Gillings

Thank you for the funders of the work carried out for this thesis, Sorbonne Université

Research funding:

This thesis was funded by a grant awarded to Amanda Garrison from Sorbonne Université (doctoral contract n° 3228/2018). A grant was also received from the Programme de Recherche Interdisciplinaire sur les Crises et la Protection Sanitaires (PRINCEPS). Funding sources for the studies included in the work of this thesis are presented in the Materials and Methods.

List of publications and conferences

List of Abbreviations and Acronyms

Summary of PAIR Project:

"Treatment and prevention of parasitic infections and iron deficiency: a qualitative study on experiences, barriers, and the role of maternal depression among pregnant women in Benin"

In a previous project in the Allada area (TOVI project), we found high exposure of pregnant women and children to malaria, helminths and iron deficiency. We therefore propose to study the perceptions, uses and barriers to the use of malaria treatments, iron supplements and helminth treatments. We propose a qualitative study of the knowledge, experiences, perceptions, and barriers that women and their children face regarding preventive and treatment strategies for anemia and parasitic infections. This will allow for better interpretation of the findings and data collected from children at age six in the EXPLORE project.

Primary Objective:

1. To describe women's knowledge and perceptions of prevention and treatment of anemia and parasitic infections for themselves and their children.

Secondary Objectives: 1. To explore factors that influence adherence to these preventive strategies, including maternal depression. 2. To identify feasible strategies in Benin to deliver the drugs, adapt the best schedule and duration of prevention and treatment of anemia and parasitic infections.

The interview will be conducted by Amanda Garrison, PhD student, and Romeo Zoumenou, psychologist. The interview will last approximately one hour. The interview guide asks women about their knowledge, previous experiences, perceived barriers, and recommendations for improving prevention and treatment strategies for anemia and parasitic infections during pregnancy and childhood. The interviews will be conducted in the local language, Fon, and will be recorded and transcribed into French and English for analysis. The data analysis will have four steps: 1) Content reading: 2) Coding: 3) Data reduction and 4) Data display. No statistical analysis will be performed as this is a qualitative study.