Humoral antimalaria immune response in Nigerian children exposed to helminth and malaria parasites.

Background: Malaria and helminthic parasites are endemic in tropical countries, and co-infections might influence host-parasite interactions. In this community-based cross-sectional study, the effect that the presence of soil-transmitted helminths (STH) (Hookworm, Hymenolepis nana) and Schistosoma haematobium infections could have on the immunoglobulin (Ig) candidate protein of the malaria vaccine GMZ2 levels was evaluated. Methods: Blood, stool, and urine samples were collected from 5-15-year-old children to diagnose P. falciparum (Pf), STH, and Schistosoma haematobium, respectively. Identification and quantification of the parasite load of STH and S. haematobium were achieved by light microscopy. A polymerase chain reaction was carried out to detect submicroscopic infections of P. falciparum. Plasma levels of GMZ2 specific IgG and its subclasses were quantified by ELISA. Results: The median level of total IgG in individuals with co-infection with Pf/H. nana was significantly lower in the mono-infected group with Pf (p = 0.0121) or study participants without infection (p=0.0217). Similarly, the median level of IgG1 was statistically lower in Pf/H. nana group compared to Pf-group (p=0.0137). Equally, the Pf/H. nana infected individuals posted a lower level of IgG1 compared to Pf-group (p=0.0137) and IgG4 compared to the Pf-group (p=0.0144). Spearman rank correlation analyses indicated positive relationships between the densities of H. nana (ρ=0.25, p=0.015) and S. haematobium (ρ=0.36, p<0.0001). Conclusions: Hookworm and H. nana infections are associated with reduced GMZ2 specific IgG levels. This study shows the possible manipulation of immune responses by helminths for their survival and transmission, which may have serious implications for vaccine development and deployment in helminth-endemic regions.

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.

Antiplasmodial and Genotoxic Study of Selected Ghanaian Medicinal Plants.

RESULTS: Five out of the eight plants, A. boonei stem bark, S; siamea Lam root, M. lucida Benth leaves, P. niruri, and A. hispidum DC whole plants, showed varying degrees of antiplasmodial activity against the asexual stage of the parasite. The most active extract against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. falciparum strains is the A. hispidum extract which yielded a mean inhibitory concentration at 50% (IC50) of 3.66 µg/ml and 3.71 µg/ml for 3D7 and Dd2, respectively. This was followed by S. siamea Lam with 3.95 µg/ml for 3D7 and 4.47 µg/ml for Dd2. The IC50 values of the A. boonei extract against 3D7 and Dd2 P. falciparum parasites were 5.13 µg/ml and 3.62 µg/ml, respectively. For the M. lucida Benth extract, the least IC50 value was 6.46 µg/ml. All five extracts exhibited dose-dependent antiplasmodial activity. Assessment of the genotoxic effects the A. hispidum extract by the comet assay revealed substantial damage to P. falciparum DNA. CONCLUSION: This study demonstrates that the crude extract of A. hispidum DC, one of the plants used traditionally to treat malaria, inhibits the growth of P. falciparum in vitro and could be a potential source of antimalarial drug. The report has highlighted genotoxic and cytotoxic effects of the selected plant extracts on human leukocytes as well.

Sub-microscopic Plasmodium falciparum infections in matched peripheral, placental and umbilical cord blood samples from asymptomatic Congolese women at delivery.

In malaria-endemic areas, most pregnant women are susceptible to asymptomatic Plasmodium falciparum infections. We present here the results of a cross-sectional study conducted in Madibou, a southern district of Brazzaville in the Republic of Congo, between March 2014 and April 2015. The main aim was to characterize P. falciparum infections. Blood samples corresponding to peripheral, placental and cord from 370 asymptomatic malaria women at delivery were diagnosed for plasmodium infection by thick blood smears (microscopic infection). Sub-microscopic infection was detected by PCR, using the MSP-2 gene as marker. Microscopic infections were detected in peripheral, placental and cord blood samples with a prevalence of respectively 7.3% (27/370), 2.7% (10/370) and 0%. The negative samples were submitted to sub-microscopic detection, with respective prevalence of 25.4% (87/343), 16.7% (60/360) and 9.4% (35/370) (P < 0.001). We further investigated the genetic diversity of the parasite by characterizing MSP2 allelic families 3D7 (24 distinct alleles) and FC27 (20 distinct alleles). The total number of alleles for these two families were 31, 25 and 19 in peripheral, placental and cord samples respectively. The 3D7 MSP-2 was the predominant allelic family. The multiplicity of infections (MOI) in peripheral (mean 1.4 ± 0.01; range 1-4), placental (mean 1.2 ± 0.01; range 1-3) and cord samples (1.4 ± 0.01; range 1-3) were similar (P = 0.9) and are unaffected by age, gravidity or sulfadoxine-pyrimethamine. These results shown a high prevalence of sub-microscopic infection and a high genetic diversity of Plasmodium falciparum strains in Congo. Age, gravidity and doses of preventive treatment based on sulfadoxine-pyrimethamine do not interfere with the multiplicity of infections.

Differential contribution of interleukin-10 promoter variants in malaria and schistosomiasis mono- and co-infections among Nigerian children.

OBJECTIVE: Interleukin-10 (IL-10) is an anti-inflammatory cytokine produced by Th1 cells and macrophages. The rationale of this study was to examine and validate possible contributions of IL-10 promoter polymorphisms in sub-Saharan Africa in children infected with either Plasmodium falciparum or Schistosoma haematobium and in children co-infected with both parasites. MATERIALS AND METHODS: A total of 309 Nigerian children aged 4-15 years were recruited. The study group consisted of individuals infected either with P. falciparum (n = 76) or S. haematobium (n = 94) in mono-infections, a group of children co-infected with both P. falciparum and S. haematobium (n = 62) and matched healthy controls (n = 77). The IL-10 promoter polymorphisms -1082G/A, -819C/T and -592C/A were genotyped by direct sequencing. RESULTS: The frequencies of the IL-10 -1082GG genotype, the -1082G allele and haplotype GCC (positions -1082, -819 and -592) were higher in children infected with P. falciparum than in healthy controls, indicating that the -1082GG genotype and the -1082G allele and the GCC haplotype are associated with increased susceptibility to malaria infection (OR = 3.4, 95% CI = 1.2-10.8, P = 0.02; OR = 2.5, 95% CI = 1.1-3.4, P = 0.02; OR = 3.8, 95% CI = 2.0-7.2, P = 0.0001, respectively). Children with the -1082GG genotype had a higher parasitaemia than children with the -1082AA or -1082AG genotypes (P = 0.0017). Haplotype GCC occurred more frequently in children infected with S. haematobium, while haplotype GTA was less frequent than in controls (OR = 2.2, 95% CI = 1.2-4.4, P = 0.017 and OR = 0.1, 95% CI = 0.02-0.5, P = 0.0004, respectively). No differences in the frequencies of IL-10 promoter polymorphisms were observed between children with P. falciparum-S. haematobium co-infections and healthy controls. CONCLUSION: Although IL-10 promoter polymorphisms are not associated with P. falciparum and S. haematobium co-infection, variant -1082G/A and haplotype GCC are associated with malaria, whereas the IL-10 haplotypes GCC and GTA are associated with schistosomiasis.

Geographical distribution of complement receptor type 1 variants and their associated disease risk.

BACKGROUND: Pathogens exert selective pressure which may lead to substantial changes in host immune responses. The human complement receptor type 1 (CR1) is an innate immune recognition glycoprotein that regulates the activation of the complement pathway and removes opsonized immune complexes. CR1 genetic variants in exon 29 have been associated with expression levels, C1q or C3b binding and increased susceptibility to several infectious diseases. Five distinct CR1 nucleotide substitutions determine the Knops blood group phenotypes, namely Kna/b, McCa/b, Sl1/Sl2, Sl4/Sl5 and KCAM+/-. METHODS: CR1 variants were genotyped by direct sequencing in a cohort of 441 healthy individuals from Brazil, Vietnam, India, Republic of Congo and Ghana. RESULTS: The distribution of the CR1 alleles, genotypes and haplotypes differed significantly among geographical settings (p≤0.001). CR1 variants rs17047660A/G (McCa/b) and rs17047661A/G (Sl1/Sl2) were exclusively observed to be polymorphic in African populations compared to the groups from Asia and South-America, strongly suggesting that these two SNPs may be subjected to selection. This is further substantiated by a high linkage disequilibrium between the two variants in the Congolese and Ghanaian populations. A total of nine CR1 haplotypes were observed. The CR1*AGAATA haplotype was found more frequently among the Brazilian and Vietnamese study groups; the CR1*AGAATG haplotype was frequent in the Indian and Vietnamese populations, while the CR1*AGAGTG haplotype was frequent among Congolese and Ghanaian individuals. CONCLUSION: The African populations included in this study might have a selective advantage conferred to immune genes involved in pathogen recognition and signaling, possibly contributing to disease susceptibility or resistance.

Triggering receptor expressed on myeloid cells 1 (TREM-1) and cytokine gene variants in complicated and uncomplicated malaria.

BACKGROUND: Malaria elicits inflammatory responses, which, if not well regulated, may exert detrimental effects. When activated, triggering receptor expressed on myeloid cells 1 (TREM-1) enhances inflammatory responses by increasing secretion of IL-8 and other Th1 cytokines. In contrast, TREM-like transcript 1 (TREML-1) promotes anti-inflammatory responses by binding to TREM-1 ligands and competing with TREM-1, thus antagonizing TREM-1 activation to reduce inflammation. Endothelial protein C receptor (EPCR) also mediates anti-inflammatory responses by activating endothelial protein C (PC). Upon microbial stimulation, soluble forms of TREM-1 (sTREM-1) and soluble EPCR (sEPCR) are released. Their plasma levels reflect the degree of inflammation and the severity of infection. METHODS: In a cross-sectional study comparing patients with severe with uncomplicated malaria, sTREM-1, soluble TREML-1 (sTREML-1) and sEPCR plasma levels as well as plasma levels of sEPCR derived from convalescent patients were quantified. Samples were collected on admittance of paediatric patients infected with Plasmodium falciparum to hospitals in Accra, Ghana. Distinct genetic regions of the genes encoding TREM-1, EPCR, interleukin (IL)-8 and IL-18 encompassing known genetic polymorphisms that influence plasma levels underwent DNA sequencing. RESULTS: Higher sTREM-1 levels were observed among children suffering from severe malaria compared to those with uncomplicated malaria (P = 0.049). Low TREM-1 to TREML-1 ratios were associated with uncomplicated malaria (P = 0.033). The TREM1 rs2234237T variant causing the amino acid exchange Thr25Ser, which has been associated with higher TREM-1 plasma levels, was significantly more frequent among patients with severe malaria than in those with uncomplicated malaria (P = 0.036). Low levels of sEPCR were observed in severe and uncomplicated malaria, while variant genotypes of IL8, IL18 and EPCR did not show any association. CONCLUSION: Higher plasma levels of sTREM-1 alone or relative to sTREML-1 during malaria predispose to the phenotype of severe malaria. Carriage of the TREM1 rs2234237T allele appears to be a risk factor for the development of severe malaria.

A STAT6 Intronic Single-Nucleotide Polymorphism is Associated with Clinical Malaria in Ghanaian Children.

Malaria pathogenesis may be influenced by IgE responses and cytokine cross-regulation. Several mutations in the IL-4/STAT6 signaling pathway can alter cytokine cross-regulation and IgE responses during a Plasmodium falciparum malarial infection. This study investigated the relationship between a STAT6 intronic single-nucleotide polymorphism (rs3024974), total IgE, cytokines, and malaria severity in 238 Ghanaian children aged between 0.5 and 13 years. Total IgE and cytokine levels were measured by ELISA, while genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Compared with healthy controls, heterozygosity protected against clinical malaria: uncomplicated malaria (odds ratios [OR] = 0.13, P < 0.001), severe malarial anemia (OR = 0.18, P < 0.001), and cerebral malaria (OR = 0.39, P = 0.022). Levels of total IgE significantly differed among malaria phenotypes (P = 0.044) and rs3024974 genotypes (P = 0.037). Neither cytokine levels nor IL-6/IL-10 ratios were associated with malaria phenotypes or rs3024974 genotypes. This study suggests a role for rs3024974 in malaria pathogenesis and offers further insights into an IL-4/STAT6 pathway mutation in malaria pathogenesis.

A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification.

BACKGROUND: Improved living conditions together with appropriate diagnosis can reduce avoidable malarial deaths substantially. Microscopy remains the gold standard in the diagnosis of malaria. However, rapid molecular diagnostic tests (RmDT) are becoming increasingly important and will, most likely, be the diagnostic techniques of choice in the next years. METHODS: In this study, a rapid and reliable nucleic acid extraction procedure from human blood and malarial parasites using microwave irradiation as a promising platform is described. In addition, a tailored loop mediated isothermal amplification (LAMP) methodology that utilizes hydroxynaphthol blue (HNB) and Bst 2.0 DNA polymerases in molecular detection of malarial parasites is described. RESULTS: Following microwave irradiation for DNA isolation, conventional PCR assays were able to detect up to five malaria parasites/µl. The LAMP methodology described here was capable to detect as low as one Plasmodium falciparum parasite/µl after DNA extraction by microwave irradiation. A turnover time of 45 minutes from nucleic acid extraction to final visual read-out was achieved. CONCLUSIONS: The described procedure offers a cheap, simple and fast method of molecular detection of malaria parasites. This test can easily be performed in basic laboratories. The methodology has been validated as a proof of concept and has specifically be developed for use at low-resource settings. Such RmDTs may aid health providers to make timely therapeutic interventions in malaria endemic regions.

High plasma levels of soluble intercellular adhesion molecule (ICAM)-1 are associated with cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is responsible for most of the malaria-related deaths in children in sub-Saharan Africa. Although, not well understood, the pathogenesis of CM involves parasite and host factors which contribute to parasite sequestration through cytoadherence to the vascular endothelium. Cytoadherence to brain microvasculature is believed to involve host endothelial receptor, CD54 or intercellular adhesion molecule (ICAM)-1, while other receptors such as CD36 are generally involved in cytoadherence of parasites in other organs. We therefore investigated the contributions of host ICAM-1 expression and levels of antibodies against ICAM-1 binding variant surface antigen (VSA) on parasites to the development of CM. METHODOLOGY/PRINCIPAL FINDINGS: Paediatric malaria patients, 0.5 to 13 years were recruited and grouped into CM and uncomplicated malaria (UM) patients, based on well defined criteria. Standardized ELISA protocol was used to measure soluble ICAM-1 (sICAM-1) levels from acute plasma samples. Levels of IgG to CD36- or ICAM-1-binding VSA were measured by flow cytometry during acute and convalescent states. Wilcoxon sign rank-test analysis to compare groups revealed association between sICAM-1 levels and CM (p<0.0037). Median levels of antibodies to CD36-binding VSA were comparable in the two groups at the time of admission and 7 days after treatment was initiated (p>0.05). Median levels of antibodies to CD36-binding VSAs were also comparable between acute and convalescent samples within any patient group. Median levels of antibodies to ICAM-1-binding VSAs were however significantly lower at admission time than during recovery in both groups. CONCLUSIONS/SIGNIFICANCE: High levels of sICAM-1 were associated with CM, and the sICAM-1 levels may reflect expression levels of the membrane bound form. Anti-VSA antibody levels to ICAM-binding parasites was more strongly associated with both UM and CM than antibodies to CD36 binding parasites. Thus, increasing host sICAM-1 levels were associated with CM whilst antibodies to parasite expressing non-ICAM-1-binding VSAs were not.

Fc γ receptor IIIB (FcγRIIIB) polymorphisms are associated with clinical malaria in Ghanaian children.

Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p=0.0061; recessive: p=0.097; dominant: p=0.0076) of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p=0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p=0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.

Polymorphisms in the RNASE3 gene are associated with susceptibility to cerebral malaria in Ghanaian children.

BACKGROUND: Cerebral malaria (CM) is the most severe outcome of Plasmodium falciparum infection and a major cause of death in children from 2 to 4 years of age. A hospital based study in Ghana showed that P. falciparum induces eosinophilia and found a significantly higher serum level of eosinophil cationic protein (ECP) in CM patients than in uncomplicated malaria (UM) and severe malaria anemia (SA) patients. Single nucleotide polymorphisms (SNPs) have been described in the ECP encoding-gene (RNASE3) of which the c.371G>C polymorphism (rs2073342) results in an arginine to threonine amino acid substitution p.R124T in the polypeptide and abolishes the cytotoxicity of ECP. The present study aimed to investigate the potential association between polymorphisms in RNASE3 and CM. METHODOLOGY/PRINCIPAL FINDINGS: The RNASE3 gene and flanking regions were sequenced in 206 Ghanaian children enrolled in a hospital based malaria study. An association study was carried out to assess the significance of five SNPs in CM (n=45) and SA (n=56) cases, respectively. The two severe case groups (CM and SA) were compared with the non-severe control group comprising children suffering from UM (n=105). The 371G allele was significantly associated with CM (p=0.00945, OR=2.29, 95% CI=1.22-4.32) but not with SA. Linkage disequilibrium analysis demonstrated significant linkage between three SNPs and the haplotype combination 371G/*16G/*94A was strongly associated with susceptibility to CM (p=0.000913, OR=4.14, 95% CI=1.79-9.56), thus, defining a risk haplotype. The RNASE3 371GG genotype was found to be under frequency-dependent selection. CONCLUSIONS/SIGNIFICANCE: The 371G allele of RNASE3 is associated with susceptibility to CM and forms part of a risk associated haplotype GGA defined by the markers: rs2073342 (G-allele), rs2233860 (G-allele) and rs8019343 (A-allele) respectively. Collectively, these results suggest a hitherto unrecognized role for eosinophils in CM pathogenesis.