The Effects of Malaria in Pregnancy on Neurocognitive Development in Children at 1 and 6 Years of Age in Benin: A Prospective Mother-Child Cohort.

BACKGROUND: Malaria in pregnancy (MiP) contributes significantly to infant mortality rates in sub-Saharan Africa and has consequences on survivors, such as preterm birth and low birth weight. However, its impact on long-term neurocognitive development in children remains unknown. METHODS: Our prospective cohort included pregnant women and their live-born singletons from the Malaria in Pregnancy Preventive Alternative Drugs clinical trial. MiP was assessed using microscopy and real-time quantitative polymerase chain reaction (qPCR). Neurocognitive development in children was assessed using the Mullen Scales of Early Learning and the Kaufman Assessment Battery for Children, 2nd edition (KABC-II), at 1 and 6 years of age, respectively. RESULTS: Of 493 pregnant women, 196 (40%) were infected with malaria at least once: 121 (31%) with placental malaria diagnosed by qPCR. Multiple linear regression B-coefficients showed that impaired gross motor scores were associated with MiP at least once (-2.55; confidence interval [95% CI]: -5.15, 0.05), placental malaria by qPCR (-4.95; 95% CI: -7.65, -2.24), and high parasite density at delivery (-1.92; 95% CI: -3.86, 0.02) after adjustment. Malaria and high parasite density at the second antenatal care visit were associated with lower KABC-II Non-Verbal Index scores at 6 years (-2.57 [95% CI: -4.86, -0.28] and -1.91 [-3.51, -0.32]), respectively. CONCLUSIONS: This prospective cohort study provides evidence that MiP, particularly late term, could have important negative consequences on child development at 1 and 6 years of age. Mechanisms behind this association must be further investigated and diagnostic methods in low-income countries should be strengthened to provide adequate treatment. CLINICAL TRIALS REGISTRATION: NCT00811421.

Parasites Causing Cerebral Falciparum Malaria Bind Multiple Endothelial Receptors and Express EPCR and ICAM-1-Binding PfEMP1.

Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPCR); however, cytoadhesion patterns typical for pediatric malaria syndromes and the associated PfEMP1 members are still undefined. Methods: In a cohort of 94 hospitalized children with malaria, we characterized the binding properties of IE collected on admission, and var gene transcription using quantitative polymerase chain reaction. Results: IE from patients with cerebral malaria were more likely to bind EPCR and ICAM-1 than IE from children with uncomplicated malaria (P = .007). The level of transcripts encoding CIDRα1.4 and CIDRα1.5 domain subclasses was higher in patients with severe disease (P < .05). IE populations exhibiting binding to all 3 receptors had higher levels of transcripts encoding PfEMP1 with CIDRα1.4 and Duffy binding-like (DBL)-ß3 domains than parasites, which only bound CD36. Conclusions: These results underpin the significance of EPCR binding in pediatric malaria patients that require hospital admission, and support the notion that complementary receptor interactions of EPCR binding PfEMP1with ICAM-1 amplifies development of severe malaria symptoms.

Infected erythrocytes and plasma proteomics reveal a specific protein signature of severe malaria.

Cerebral malaria (CM), the most lethal complication of Plasmodium falciparum severe malaria (SM), remains fatal for 15-25% of affected children despite the availability of treatment. P. falciparum infects and multiplies in erythrocytes, contributing to anemia, parasite sequestration, and inflammation. An unbiased proteomic assessment of infected erythrocytes and plasma samples from 24 Beninese children was performed to study the complex mechanisms underlying CM. A significant down-regulation of proteins from the ubiquitin-proteasome pathway and an up-regulation of the erythroid precursor marker transferrin receptor protein 1 (TFRC) were associated with infected erythrocytes from CM patients. At the plasma level, the samples clustered according to clinical presentation. Significantly, increased levels of the 20S proteasome components were associated with SM. Targeted quantification assays confirmed these findings on a larger cohort (n = 340). These findings suggest that parasites causing CM preferentially infect reticulocytes or erythroblasts and alter their maturation. Importantly, the host plasma proteome serves as a specific signature of SM and presents a remarkable opportunity for developing innovative diagnostic and prognostic biomarkers.

[Delivery in free position perhaps a solution to change delivery in traditional position in delivery rooms in Africa: results of a prospective study in Cotonou in Bénin]. / L'accouchement en position libre peut-il être une solution de rechange à l'accouchement en position classique dans les salles de naissance en Afrique : résultats d'une étude. prospective à Cotonou au Bénin

INTRODUCTION: In most of Africa's birthing units, women are often forbidden to stand during labour and delivery. This study aimed to determine the effect of a freely vertical position (standing,sitting, crouching) compared with the traditional supine position on the course of delivery and on perinatal and maternal outcomes(including the satisfaction level of mothers).Methods: We compared a group of 490 women who chose to deliver in a freely vertical position with 490 women who delivered in a traditional position, at the Hopital de Ia mere et de l'enfant Lagune of Cotonou, from January 1, 2009, to December 31,2011. Variables studied include length of labour, type of delivery,frequency of episiotomy, fetal and neonatal well-being indices(fetal heart rate, Apgar score, resuscitation), and postnatal maternal outcomes (perineal tears, hemorrhagic complications),including maternal satisfaction. Descriptive analysis includes comparisons between those two groups.Results: Both groups had similar maternal (25 years old) and gestational (39 weeks) ages. The "Choice of position" group had a greater number of nulliparous women (53% vs. 40%,P < 0.001) and a higher educational level (high-school diploma and postgraduate training: 77.5% vs. 45.1 %; P < 0.001 ). The average length of the active phase was shortened by 20 minutes in women who freely chose their delivery position (P < 0.01 ).Moreover, these women experienced spontaneous delivery in higher numbers, with 10 times fewer assisted deliveries (0.4%vs. 4.3%; P < 0.01) and two times fewer episiotomy interventions(3.5% vs. 8.0%; P < 0.01) than their counterparts. When women freely choose their delivery position, there are close to three times fewer cases of fetal heart rate anomalies and meconium liquor (2.9% vs. 8.9%; P < 0.01 and 0.4% vs. 1.4%; P < 0.01 ).Perineal and cervical tears are rare and occur in similar numbers in both groups, and the same can be said of cases of postpartum hemorrhage. The number of satisfied mothers is higher in the"Choice of position" group (87.0% vs. 61.2%; P < 0.01 ).Conclusion: The choice of a freely vertical position seems to be an efficient and safe option during labour and delivery, and is more satisfying for mothers.

ICAM-1-binding Plasmodium falciparum erythrocyte membrane protein 1 variants elicits opsonic-phagocytosis IgG responses in Beninese children.

Members of the highly polymorphic Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLß domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLß-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLß from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLß of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.

[Formula: see text]Neurodevelopmental assessment at one year of age predicts neuropsychological performance at six years in a cohort of West African Children.

Rural children from Benin, west Africa were evaluated with the Mullen Scales of Early Learning (MSEL) at one year of age and then at six years with the Kaufman Assessment Battery for Children (KABC-II), the visual computerized Tests of Variables of Attention (TOVA), and the Bruininks-Oseretsky Test (BOT-2) of motor proficiency (N = 568). Although both the MSEL and KABC-II were available to the assessors in French, instructions to the mother/child were in local language of Fon. Mothers were evaluated with the Edinburgh Postpartum Depression Scale (EPDS), Caldwell HOME Scale, educational level and literacy, and a Socio-Economic Scale - also in their local language (Fon). After adjusting for maternal factors, MSEL cognitive composite was correlated with KABC-II with moderate effect sizes, but not with TOVA scores. Overall eta-squared effect for the multivariate models were moderately to strongly correlated (.07 to .37). Neurodevelopmental assessments in early childhood adapted cross-culturally are predictive of school-age neuropsychological cognitive ability.

Soil-transmitted helminth infection in pregnancy and long-term child neurocognitive and behavioral development: A prospective mother-child cohort in Benin.

BACKGROUND: An estimated 30% of women in Sub-Saharan Africa suffer from soil-transmitted helminth infection during pregnancy (SHIP), which has been shown to increase risk of pre-term birth, low birth weight, and maternal anemia. A previous study in Benin found that SHIP was associated with impaired cognitive and gross motor development scores in 635 one-year-old children. The objective of the present study was to follow children prospectively to investigate whether the association between SHIP and child neurocognitive and behavioral development persisted at age six. PRINCIPAL FINDINGS: Our prospective child cohort included 487 live-born singletons of pregnant women enrolled in the Malaria in Pregnancy Preventive Alternative Drugs clinical trial in Allada, Benin. SHIP was assessed at three antenatal visits (ANVs) through collection and testing of stool samples. Neurocognitive and behavioral development was assessed in six-year-old children by trained investigators using the Kaufman Assessment Battery for Children 2nd edition and the parent-reported Strengths and Difficulties Questionnaire (SDQ). Multiple linear regression models generated coefficients and 95% confidence intervals and potential mediating factors were tested. Prevalence of SHIP was 13% at the 1st ANV, 9% at the 2nd ANV, and 1% at delivery. 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 (95% CI 0.15, 2.00) and at least once during pregnancy 0.79 (95% CI 0.12, 1.46) in adjusted models. Mediation analysis did not reveal significant indirect effects of several mediators on this association. CONCLUSIONS: Our study shows that while SHIP is not associated with impaired long-term neurocognitive development, infections may have significant negative impacts on emotional development in six-year-old children. SHIP remains a critical public health issue, and adequate prevention and treatment protocols should be enforced in low- and middle-income countries.

Comparison of growth models to describe growth from birth to 6 years in a Beninese cohort of children with repeated measurements.

OBJECTIVE: To select a growth model that best describes individual growth trajectories of children and to present some growth characteristics of this population. SETTINGS: Participants were selected from a prospective cohort conducted in three health centres (Allada, Sekou and Attogon) in a semirural region of Benin, sub-Saharan Africa. PARTICIPANTS: Children aged 0 to 6 years were recruited in a cohort study with at least two valid height and weight measurements included (n=961). PRIMARY AND SECONDARY OUTCOME MEASURES: This study compared the goodness-of-fit of three structural growth models (Jenss-Bayley, Reed and a newly adapted version of the Gompertz growth model) on longitudinal weight and height growth data of boys and girls. The goodness-of-fit of the models was assessed using residual distribution over age and compared with the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC). The best-fitting model allowed estimating mean weight and height growth trajectories, individual growth and growth velocities. Underweight, stunting and wasting were also estimated at age 6 years. RESULTS: The three models were able to fit well both weight and height data. The Jenss-Bayley model presented the best fit for weight and height, both in boys and girls. Mean height growth trajectories were identical in shape and direction for boys and girls while the mean weight growth curve of girls fell slightly below the curve of boys after neonatal life. Finally, 35%, 27.7% and 8% of boys; and 34%, 38.4% and 4% of girls were estimated to be underweight, wasted and stunted at age 6 years, respectively. CONCLUSION: The growth parameters of the best-fitting Jenss-Bayley model can be used to describe growth trajectories and study their determinants.

SEPSIS project: a protocol for studying biomarkers of neonatal sepsis and immune responses of infants in a malaria-endemic region.

INTRODUCTION: Neonatal sepsis outreaches all causes of neonatal mortality worldwide and remains a major societal burden in low and middle income countries. In addition to limited resources, endemic morbidities, such as malaria and prematurity, predispose neonates and infants to invasive infection by altering neonatal immune response to pathogens. Nevertheless, thoughtful epidemiological, diagnostic and immunological evaluation of neonatal sepsis and the impact of gestational malaria have never been performed. METHODS AND ANALYSIS: A prospective longitudinal multicentre follow-up of 580 infants from birth to 3 months of age in urban and suburban Benin will be performed. At delivery, and every other week, all children will be examined and clinically evaluated for occurrence of sepsis. At delivery, cord blood systematic analysis of selected plasma and transcriptomic biomarkers (procalcitonin, interleukin (IL)-6, IL-10, IP10, CD74 and CX3CR1) associated with sepsis pathophysiology will be evaluated in all live births as well as during the follow-up, and when sepsis will be suspected. In addition, whole blood response to selected innate stimuli and extensive peripheral blood mononuclear cells phenotypic characterisation will be performed. Reference intervals specific to sub-Saharan neonates will be determined from this cohort and biomarkers performances for neonatal sepsis diagnosis and prognosis tested. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Comité d'Ethique de la Recherche - Institut des Sciences Biomédicales Appliquées (CER-ISBA 85 - 5 April 2016, extended on 3 February 2017). Results will be disseminated through international presentations at scientific meetings and publications in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov registration number: NCT03780712.

Changes in monocyte subsets are associated with clinical outcomes in severe malarial anaemia and cerebral malaria.

Monocytes are plastic heterogeneous immune cells involved in host-parasite interactions critical for malaria pathogenesis. Human monocytes have been subdivided into three populations based on surface expression of CD14 and CD16. We hypothesised that proportions and phenotypes of circulating monocyte subsets can be markers of severity or fatality in children with malaria. To address this question, we compared monocytes sampled in children with uncomplicated malaria, severe malarial anaemia, or cerebral malaria. Flow cytometry was used to distinguish and phenotype monocyte subsets through CD14, CD16, CD36 and TLR2 expression. Data were first analysed by univariate analysis to evaluate their link to severity and death. Second, multinomial logistic regression was used to measure the specific effect of monocyte proportions and phenotypes on severity and death, after adjustments for other variables unrelated to monocytes. Multivariate analysis demonstrated that decreased percentages of non-classical monocytes were associated with death, suggesting that this monocyte subset has a role in resolving malaria. Using univariate analysis, we also showed that the role of non-classical monocytes involves a mostly anti-inflammatory profile and the expression of CD16. Further studies are needed to decipher the functions of this sub-population during severe malaria episodes, and understand the underlying mechanisms.

From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases.

BACKGROUND: PfEMP1 is the major protein from parasitic origin involved in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability is endless and current publicly available protein repositories do not reflect the high diversity of the sequences of PfEMP1 proteins. The identification of PfEMP1 protein sequences expressed with samples remains challenging. The aim of our study is to identify the different PfEMP1 proteins variants expressed within patient samples, and therefore identify PfEMP1 proteins domains expressed by patients presenting uncomplicated malaria or severe malaria in malaria endemic setting in Cotonou, Benin. METHODS: We performed a multi-omic approach to decipher PfEMP1 expression at the patient's level in different clinical settings. Using a combination of whole genome sequencing approach and RNA sequencing, we were able to identify new PfEMP1 sequences and created a new custom protein database. This database was used for protein identification in mass spectrometry analysis. RESULTS: The differential expression analysis of RNAsequencing data shows an increased expression of the var domains transcripts DBLα1.7, DBLα1.1, DBLα2 and DBLß12 in samples from patients suffering from Cerebral Malaria compared to Uncomplicated Malaria. Our approach allowed us to attribute PfEMP1 sequences to each sample and identify new peptides associated to PfEMP1 proteins in mass spectrometry. CONCLUSION: We highlighted the diversity of the PfEMP1 sequences from field sample compared to reference sequences repositories and confirmed the validity of our approach. These findings should contribute to further vaccine development strategies based on PfEMP1 proteins.

Proteomic analysis of Plasmodium falciparum parasites from patients with cerebral and uncomplicated malaria.

Plasmodium falciparum is responsible of severe malaria, including cerebral malaria (CM). During its intra-erythrocytic maturation, parasite-derived proteins are expressed, exported and presented at the infected erythrocyte membrane. To identify new CM-specific parasite membrane proteins, we conducted a mass spectrometry-based proteomic study and compared the protein expression profiles between 9 CM and 10 uncomplicated malaria (UM) samples. Among the 1097 Plasmodium proteins identified, we focused on the 499 membrane-associated and hypothetical proteins for comparative analysis. Filter-based feature selection methods combined with supervised data analysis identified a subset of 29 proteins distinguishing CM and UM samples with high classification accuracy. A hierarchical clustering analysis of these 29 proteins based on the similarity of their expression profiles revealed two clusters of 15 and 14 proteins, respectively under- and over-expressed in CM. Among the over-expressed proteins, the MESA protein is expressed at the erythrocyte membrane, involved in proteins trafficking and in the export of variant surface antigens (VSAs), but without antigenic function. Antigen 332 protein is exported at the erythrocyte, also involved in protein trafficking and in VSAs export, and exposed to the immune system. Our proteomics data demonstrate an association of selected proteins in the pathophysiology of CM.