Association of Endothelial Protein C Receptor (EPCR) rs867186 Gene Polymorphism With Increased Levels of Soluble EPCR and High Risk of Severe Malaria and Fatality in Beninese Children.

The endothelial protein C receptor (EPCR)-rs867186 G allele has been linked to high plasma levels of soluble EPCR (sEPCR) and controversially associated with either susceptibility or resistance to severe and cerebral malaria. In this study, quantitative enzyme-linked immunosorbent assay and sequencing were used to assess sEPCR levels and EPCR-rs867186 polymorphism in blood samples from Beninese children with different clinical presentations of malaria. Our findings show that sEPCR levels were higher at hospital admission than during convalescence and that EPCR-rs867186 G allele was associated with increased sEPCR plasma levels, malaria severity, and mortality rate (P < .001, P = .03, and P = .04, respectively), suggesting a role of sEPCR in the pathogenesis of severe malaria.

ICAM-1 Kilifi variant is not associated with cerebral and severe malaria pathogenesis in Beninese children.

BACKGROUND: Cytoadhesion and sequestration of Plasmodium falciparum infected red blood cells (iRBC) in the microvasculature of vital organs are a major cause of malaria pathology. Several studies have provided evidence on the implication of the human host intercellular adhesion molecule-1 (ICAM-1) as a major receptor for iRBCs binding to P. falciparum erythrocyte membrane protein 1 (PfEMP1) in the development of severe and cerebral malaria. The genetic polymorphism K29M in the immunoglobulin-like domain of ICAM-1, known as ICAM-1Kilifi, has been associated with either increased or decreased risk of developing cerebral malaria. METHODS: To provide more conclusive results, the genetic polymorphism of ICAM-1Kilifi was assessed by PCR and sequencing in blood samples from 215 Beninese children who presented with either mild or severe malaria including cerebral malaria. RESULTS AND CONCLUSIONS: The results showed that in this cohort of Beninese children, the ICAM-1kilifi variant is present at the frequencies of 0.27, similar to the frequency observed in other African countries. This ICAM-1kilifi variant was not associated with disease severity in agreement with other findings from the Gambia, Tanzania, Malawi, Gabon, and Thailand, suggesting no evidence of a direct link between this polymorphism and the pathogenesis of severe and cerebral malaria.

High plasma levels of soluble endothelial protein C receptor are associated with increased mortality among children with cerebral malaria in Benin.

Loss of endothelial protein C receptor (EPCR) occurs at the sites of Plasmodium falciparum-infected erythrocyte sequestration in patients with or who died from cerebral malaria. In children presenting with different clinical syndromes of malaria, we assessed the relationships between endogenous plasma soluble EPCR (sEPCR) levels and clinical presentation or mortality. After adjustment for age, for treatment before admission, and for a known genetic factor, sEPCR level at admission was positively associated with cerebral malaria (P = .011) and with malaria-related mortality (P = .0003). Measuring sEPCR levels at admission could provide an early biological marker of the outcome of cerebral malaria.

Increased systemic exposures of artemether and dihydroartemisinin in infants under 5 kg with uncomplicated Plasmodium falciparum malaria treated with artemether-lumefantrine (Coartem®).

BACKGROUND: Artemether-lumefantrine (AL) dispersible formulation was developed for the treatment of uncomplicated Plasmodium falciparum malaria in infants and children weighing 5 to <35 kg. However, there are no clinical studies with artemisinin-based combination therapy in infants <5 kg. METHODS: This multicentre, open-label, single-arm study evaluated the efficacy, safety and pharmacokinetics of AL dispersible in infants aged >28 days and <5 kg of body weight, who were treated with one AL dispersible tablet (20 mg artemether/120 mg lumefantrine) given twice-daily for three days and followed up for six weeks (core follow-up) and at 12 months of age (long-term follow-up). RESULTS: A total of 20 patients were enrolled and completed the six-week core study follow-up. In the per protocol population, PCR-corrected cure rate at days 28 and 42 was 100% (95% CI: 79.4, 100). AL dispersible was well tolerated with reported adverse events of mild to moderate severity. Pharmacokinetic data showed that lumefantrine levels were similar, however, artemether and dihydroartemisinin levels were on average two- to three-fold greater than historical values in infants and children ≥5 kg. CONCLUSIONS: A three-day regimen of AL dispersible formulation was efficacious and generally well tolerated in infants weighing <5 kg with uncomplicated P. falciparum malaria, but artemether and dihydroartemisinin exposures could not be supported by the preclinical safety margins for neurotoxicity. Hence, dosing recommendations cannot be made in infants <5 kg as implications for toxicity are unknown. TRIAL REGISTRATION: Clinicaltrials.gov NCT01619878.

Cytoadherence phenotype of Plasmodium falciparum-infected erythrocytes is associated with specific pfemp-1 expression in parasites from children with cerebral malaria.

BACKGROUND: Cytoadherence of Plasmodium falciparum-infected erythrocytes (IEs) in deep microvasculature endothelia plays a major role in the pathogenesis of cerebral malaria (CM). This biological process is thought to be mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP-1) and human receptors such as CD36 and ICAM-1. The relationship between the expression of PfEMP-1 variants and cytoadherence phenotype in the pathology of malaria is not well established. METHODS: Cytoadherence phenotypes of IEs to CD36, ICAM-1, CSPG and the transcription patterns of A, B, var2csa, var3, var gene groups and domain cassettes DC8 and DC13 were assessed in parasites from children with CM and uncomplicated malaria (UM) to determine if cytoadherence is related to a specific transcription profile of pfemp-1 variants. RESULTS: Parasites from CM patients bind significantly more to CD36 than those from UM patients, but no difference was observed in their binding ability to ICAM-1 and CSPG. CM isolates highly transcribed groups A, B, var2csa, var3, DC8 and DC13 compared to UM parasites. The high transcription levels of var genes belonging to group B positively correlated with increased binding level to CD36. CONCLUSION: CM isolates bind significantly more to CD36 than to ICAM-1, which was correlated with high transcription level of group B var genes, supporting their implication in malaria pathogenesis.

Plasmodium falciparum malaria in infants under 5 kg: retrospective surveillance of hospital records in five sub-saharan African countries.

OBJECTIVE: To investigate the disease burden, clinical features, treatment and outcomes of Plasmodium falciparum malaria in neonates and infants weighing <5 kg in five sub-Saharan African countries. METHODS: Pediatric hospital records were retrospectively reviewed for relevant cases. Details of clinical features, treatment and clinical outcomes were collected, and a descriptive analysis of data was carried out. RESULTS: The annual number of malaria cases ranged from 12 to 120 cases across hospitals and calendar years. The most frequent reason for seeking care was fever. Parasite density was low. Quinine was the most common treatment, followed by artemisinin-based combination therapy. The majority of patients recovered from their illness following treatment. CONCLUSION: Plasmodium falciparum malaria exists in this subpopulation. Further epidemiological data are needed to estimate malaria morbidity and mortality in young infants. Moreover, clinical evidence on the efficacy and safety of artemisinin-based combination therapies in this subpopulation is warranted.

High Plasma Levels of Neopterin Are Associated with Increased Mortality among Children with Severe Malaria in Benin.

Among the barriers to accessing adequate treatment and high-level monitoring for malaria febrile patients is the lack of effective prognostic markers. Neopterin, which is a marker of monocyte/macrophage activation, was found have increased during severe malaria. In this study, we used quantitative ELISA in order to assess the levels of plasma soluble neopterin in 151 patients from a cohort of Beninese children with severe malaria. We evaluated the prognostic accuracy of this molecule in order to predict the outcome of the disease. Our results show that neopterin levels were not significantly different between patients with different forms of severe malaria, including severe non-cerebral malaria (SNCM) and cerebral malaria (CM). However, the levels of this molecule were found to be higher in patients with severe malarial anemia (SMA) among both CM and SNCM cases (p-value = 0.02). Additionally, the levels of this molecule were found to be higher in patients who died from these pathologies compared to those who survived among the two clinical groups (p-value < 0.0001) and within the same group (p-value < 0.0001 for the CM group, p-value = 0.0046 for the SNCM group). The AUC-ROC for fatality among all the severe cases was 0.77 with a 95%CI of (0.69-0.85). These results suggest that plasma neopterin levels constitute a potential biomarker for predicting fatality among severe falciparum malaria patients.

Specific Combinations of Inflammatory, Angiogenesis and Vascular Integrity Biomarkers Are Associated with Clinical Severity, Coma and Mortality in Beninese Children with Plasmodium Falciparum Malaria.

Malaria-related deaths could be prevented if powerful diagnostic and reliable prognostic biomarkers were available to allow rapid prediction of the clinical severity allowing adequate treatment. Using quantitative ELISA, we assessed the plasma concentrations of Procalcitonin, Pentraxine-3, Ang-2, sTie-2, suPAR, sEPCR and sICAM-1 in a cohort of Beninese children with malaria to investigate their potential association with clinical manifestations of malaria. We found that all molecules showed higher levels in children with severe or cerebral malaria compared to those with uncomplicated malaria (p-value < 0.005). Plasma concentrations of Pentraxine-3, Procalcitonin, Ang-2 and the soluble receptors were significantly higher in children with coma as defined by a Blantyre Coma Score < 3 (p < 0.001 for Pentraxine-3, suPAR, and sTie-2, p = 0.004 for PCT, p = 0.005 for sICAM-1, p = 0.04 for Ang-2). Moreover, except for the PCT level, the concentrations of Pentraxine-3, suPAR, sEPCR, sICAM-1, sTie-2 and Ang-2 were higher among children who died from severe malaria compared to those who survived (p = 0.037, p = 0.035, p < 0.0001, p= 0.0008, p = 0.01 and p = 0.02, respectively). These findings indicate the ability of these molecules to accurately discriminate among clinical manifestations of malaria, thus, they might be potentially useful for the early prognostic of severe and fatal malaria, and to improve management of severe cases.

Impact of helminth infection during pregnancy on cognitive and motor functions of one-year-old children.

OBJECTIVE: To determine the effect of helminth infection during pregnancy on the cognitive and motor functions of one-year-old children. METHODS: Six hundred and thirty five singletons born to pregnant women enrolled before 29 weeks of gestation in a trial comparing two intermittent preventive treatments for malaria were assessed for cognitive and motor functions using the Mullen Scales of Early Learning, in the TOVI study, at twelve months of age in the district of Allada in Benin. Stool samples of pregnant women were collected at recruitment, second antenatal care (ANC) visit (at least one month after recruitment) and just before delivery, and were tested for helminths using the Kato-Katz technique. All pregnant women were administered a total of 600 mg of mebendazole (100 mg two times daily for 3 days) to be taken after the first ANC visit. The intake was not directly observed. RESULTS: Prevalence of helminth infection was 11.5%, 7.5% and 3.0% at first ANC visit, second ANC visit and at delivery, respectively. Children of mothers who were infected with hookworms at the first ANC visit had 4.9 (95% CI: 1.3-8.6) lower mean gross motor scores compared to those whose mothers were not infected with hookworms at the first ANC visit, in the adjusted model. Helminth infection at least once during pregnancy was associated with infant cognitive and gross motor functions after adjusting for maternal education, gravidity, child sex, family possessions, and quality of the home stimulation. CONCLUSION: Helminth infection during pregnancy is associated with poor cognitive and gross motor outcomes in infants. Measures to prevent helminth infection during pregnancy should be reinforced.

Ultrasound diagnosis of varicocele in the adolescent: our experience from Benin.

BACKGROUND: The diagnosis of varicocèle is clinical. In order to improve diagnosis of varicocele, we compared the clinical with the ultrasound findings in schoolboys with the condition. This is because the conditions can affect testicular growth. PATIENTS AND METHODS: It was a cross-sectional, descriptive study of schoolboys aged from 10 to 19 years who had varicocele. Among 2724 boys examined, 149 had varicocele and only 81 had scrotal (18 with Doppler) and renal ultrasound examination. RESULTS: Among the 81 adolescents who were clinically diagnosed with varicocele and also with the aid of ultrasound scan, 25, 36 and 20 had grade 1, 2 and 3, respectively. Testicular hypotrophy (TH) was clinically noticed in 17 cases. At ultrasonography, varicocele was bilateral in 87.66% and unilateral in 12.34% (P = 0.01) with 32 adolescents (39.51%) showing TH compared with 20.99% being diagnosed with TH using clinical examination alone (P = 0.01). In 50 schoolboys (61.73%) with unilateral varicocele, a subclinical type was discovered at other side. Renal ultrasound revealed abnormalities in 4.93% of cases. Doppler ultrasound helped in finding varicoceles along the top edge of the testis (n = 15) and under tunica albuginea (n = 3). CONCLUSION: TH due to varicocele is better studied by ultrasound.

Usefulness of child development assessments for low-resource settings in francophone Africa.

OBJECTIVE: Few tools are available to screen or assess infant's cognitive development, especially in French-speaking Africa. This study evaluated the use of the French translation of the Mullen Scales of Early Learning (MSEL), and the "Ten Questions" questionnaire (TQ) in 1-year-old children in Benin, a francophone country. METHODS: A cross-sectional study was conducted in 3 health centers serving a semirural area in Benin. Three hundred fifty-seven children aged 12 months and their mothers were enrolled in 2011. Infant development was assessed at local health centers followed by a home visit to collect information on socioeconomic status, maternal Raven score, maternal depressive symptoms, and mother-child interactions (Home Observation for the Measurement of the Environment [HOME] Inventory), and to administer the TQ. RESULTS: The infant's gender (female), the HOME, and maternal education were associated with a higher Early Learning Composite score in multivariate analyses (p = .02, p = .004, p = .007, respectively). The HOME and family wealth were also associated with the Gross Motor Scale (p = .03 and p = .03, respectively). Mothers were more likely to report difficulties on the TQ when the child presented lower score on the MSEL. When considering the Gross Motor Scale as the gold standard to define moderate delays, the 2 combined motor-related questions on the TQ showed good sensitivity and specificity (76.5 and 75.7). CONCLUSION: In a low-resource rural setting in Africa, the TQ effectively identified 3 quarters of 1-year-old infants with delayed development. After this screening, the MSEL may be useful for further assessment as it showed good feasibility and sensitivity to known risk factors for poor child development.

Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains ("domain cassettes" (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection. METHODS: Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS. RESULTS: Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples. CONCLUSION: This is the first study to show association between PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis.