Profiling the Plasmodium falciparum Erythrocyte Membrane Protein 1-Specific Immununoglobulin G Response Among Ghanaian Children With Hemoglobin S and C.

Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are important targets for protective immunity. Abnormal display of PfEMP1 on the surfaces of infected erythrocytes (IEs) and reduced cytoadhesion have been demonstrated in hemoglobin (Hb) AS and HbAC, inherited blood disorders associated with protection against severe P. falciparum malaria. We found that Ghanaian children with HbAS had lower levels of immunoglobulin G against several PfEMP1 variants and that this reactivity increased more slowly with age than in their HbAA counterparts. Moreover, children with HbAS have lower total parasite biomass than those with HbAA at comparable peripheral parasitemias, suggesting impaired cytoadhesion of HbAS IEs in vivo and likely explaining the slower acquisition of PfEMP1-specific immunoglobulin G in this group. In contrast, the function of acquired antibodies was comparable among Hb groups and appears to be intact and sufficient to control parasitemia via opsonization and phagocytosis of IEs.

Antibody response to malaria vaccine candidates in pregnant women with Plasmodium falciparum and Schistosoma haematobium infections.

Malaria in pregnancy has severe consequences for the mother and foetus. Antibody response to specific malaria vaccine candidates (MVC) has been associated with a decreased risk of clinical malaria and its outcomes. We studied Plasmodium falciparum (Pf) and Schistosoma haematobium (Sh) infections and factors that could influence antibody responses to MVC in pregnant women. A total of 337 pregnant women receiving antenatal care (ANC) and 139 for delivery participated in this study. Pf infection was detected by qPCR and Sh infection using urine filtration method. Antibody levels against CSP, AMA-1, GLURP-R0, VAR2CSA and Pfs48/45 MVC were quantified by ELISA. Multivariable linear regression models identified factors associated with the modulation of antibody responses. The prevalence of Pf and Sh infections was 27% and 4% at ANC and 7% and 4% at delivery. Pf infection, residing in Adidome and multigravidae were positively associated with specific IgG response to CSP, AMA-1, GLURP-R0 and VAR2CSA. ITN use and IPTp were negatively associated with specific IgG response to GLURP-R0 and Pfs48/45. There was no association between Sh infection and antibody response to MVC at ANC or delivery. Pf infections in pregnant women were positively associated with antibody response to CSP, GLURP-R0 and AMA-1. Antibody response to GLURP-R0 and Pfs48/45 was low for IPTp and ITN users. This could indicate a lower exposure to Pf infection and low malaria prevalence observed at delivery.

The Rapid and Spontaneous Postpartum Clearance of Plasmodium falciparum Is Related to Expulsion of the Placenta.

Parasitemia among pregnant women with protective immunity to Plasmodium falciparum malaria is often dominated by VAR2CSA-positive infected erythrocytes (IEs). VAR2CSA mediates sequestration of IEs in the placenta. We hypothesized that the previously observed spontaneous postpartum clearance of parasitemia in such women is related to the expulsion of the placenta, which removes the sequestration focus of VAR2CSA-positive IEs. We assessed parasitemias and gene transcription before and shortly after delivery in 17 Ghanaian women. The precipitous decline in parasitemia postpartum was accompanied by selective reduction in transcription of the gene encoding VAR2CSA. Our findings provide a mechanistic explanation for the earlier observation.

High burden of asymptomatic malaria and anaemia despite high adherence to malaria control measures: a cross-sectional study among pregnant women across two seasons in a malaria-endemic setting in Ghana.

PURPOSE: Anaemia remains a serious concern among pregnant women, and thus, it is closely monitored from the onset of pregnancy through to delivery to help prevent adverse maternal and neonatal outcomes. In malaria-endemic settings, continuous low-level carriage of P. falciparum parasites is common and its contribution to maternal anaemia should not be underestimated. In this study, we evaluated the impact of adherence to malaria control measures [number of antenatal clinics (ANC) attended, supervised intake of sulphadoxine pyrimethamine (SP), and use of insecticide treated bed nets (ITNs)] on asymptomatic malaria and anaemia outcomes among pregnant women on ANC in hospitals in the Central region of Ghana. METHODS: The study was conducted during two seasons; October-November 2020 (dry season, n = 124) and May-June 2021 (rainy season, n = 145). Among the women, there was a high adherence to the control measures for both seasons (ANC ≥ 3 visits; ~ 82.0%, intake of SP; ~ 80.0% and ITNs use; ~ 75.0%). RESULTS: Asymptomatic P. falciparum carriage was high for both seasons (44.4% for the dry season; 46.9% for the rainy season). Correspondingly, the occurrence of anaemia was high for both seasons (57.3% for the dry season; 68.3% for the rainy season) and was strongly predicted by carriage of P. falciparum parasites. Despite the high adherence to ANC protocols, asymptomatic P. falciparum infection was common and contributed to the high burden of maternal anaemia. CONCLUSIONS: Our findings emphasize the need for improved control measures that can clear asymptomatic/sub-microscopic P. falciparum infection and protect against malaria-induced anaemia among pregnant women attending ANC in malaria endemic-settings.

Intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine and parasite resistance: cross-sectional surveys from antenatal care visit and delivery in rural Ghana.

BACKGROUND: Despite decades of prevention efforts, the burden of malaria in pregnancy (MiP) remains a great public health concern. Sulfadoxine-pyrimethamine (SP), used as intermittent preventive treatment in pregnancy (IPTp-SP) is an important component of the malaria prevention strategy implemented in Africa. However, IPTp-SP is under constant threat from parasite resistance, thus requires regular evaluation to inform decision-making bodies. METHODS: In two malaria endemic communities in the Volta region (Adidome and Battor), a cross-sectional hospital-based study was conducted in pregnant women recruited at their first antenatal care (ANC) visit and at delivery. Basic clinical and demographic information were documented and their antenatal records were reviewed to confirm IPTp-SP adherence. Peripheral and placental blood were assayed for the presence of Plasmodium falciparum parasites by quantitative polymerase chain reaction (qPCR). One hundred and twenty (120) positive samples were genotyped for mutations associated with SP resistance. RESULTS: At first ANC visit, P. falciparum prevalence was 28.8% in Adidome and 18.2% in Battor. At delivery, this decreased to 14.2% and 8.2%, respectively. At delivery, 66.2% of the women had taken at least the recommended 3 or more doses of IPTp-SP and there was no difference between the two communities. Taking at least 3 IPTp-SP doses was associated with an average birth weight increase of more than 360 g at both study sites compared to women who did not take treatment (p = 0.003). The Pfdhfr/Pfdhps quintuple mutant IRNI-A/FGKAA was the most prevalent (46.7%) haplotype found and the nonsynonymous Pfdhps mutation at codon A581G was higher at delivery among post-SP treatment isolates (40.6%) compared to those of first ANC (10.22%). There was also an increase in the A581G mutation in isolates from women who took 3 or more IPTp-SP. CONCLUSIONS: This study confirms a positive impact following the implementation of the new IPTp-SP policy in Ghana in increasing the birth weight of newborns. However, the selection pressure exerted by the recommended 3 or more doses of IPTp-SP results in the emergence of parasites carrying the non-synonymous mutation on codon A581G. This constant selective pressure calls into question the time remaining for the clinical utility of IPTp-SP treatment during pregnancy in Africa.

Comparison of the impact of allelic polymorphisms in PfAMA1 on the induction of T Cell responses in high and low malaria endemic communities in Ghana.

BACKGROUND: Malaria eradication requires a combined effort involving all available control tools, and these efforts would be complemented by an effective vaccine. The antigen targets of immune responses may show polymorphisms that can undermine their recognition by immune effectors and hence render vaccines based on antigens from a single parasite variant ineffective against other variants. This study compared the influence of allelic polymorphisms in Plasmodium falciparum apical membrane antigen 1 (PfAMA1) peptide sequences from three strains of P. falciparum (3D7, 7G8 and FVO) on their function as immunodominant targets of T cell responses in high and low malaria transmission communities in Ghana. METHODS: Peripheral blood mononuclear cells (PBMCs) from 10 subjects from a high transmission area (Obom) and 10 subjects from a low transmission area (Legon) were tested against 15 predicted CD8 + T cell minimal epitopes within the PfAMA1 antigen of multiple parasite strains using IFN-γ ELISpot assay. The peptides were also tested in similar assays against CD8 + enriched PBMC fractions from the same subjects in an effort to characterize the responding T cell subsets. RESULTS: In assays using unfractionated PBMCs, two subjects from the high transmission area, Obom, responded positively to four (26.7%) of the 15 tested peptides. None of the Legon subject PBMCs yielded positive peptide responses using unfractionated PBMCs. In assays with CD8 + enriched PBMCs, three subjects from Obom made positive recall responses to six (40%) of the 15 tested peptides, while only one subject from Legon made a positive recall response to a single peptide. Overall, 5 of the 20 study subjects who had positive peptide-specific IFN-γ recall responses were from the high transmission area, Obom. Furthermore, while subjects from Obom responded to peptides in PfAMA1 from multiple parasite strains, one subject from Legon responded to a peptide from 3D7 strain only. CONCLUSIONS: The current data demonstrate the possibility of a real effect of PfAMA1 polymorphisms on the induction of T cell responses in malaria exposed subjects, and this effect may be more pronounced in communities with higher parasite exposure.

Effect of Drug Pressure on Promoting the Emergence of Antimalarial-Resistant Parasites among Pregnant Women in Ghana.

The continuous spread of antimalarial drug resistance is a threat to current chemotherapy efficacy. Therefore, characterizing the genetic diversity of drug resistance markers is needed to follow treatment effectiveness and further update control strategies. Here, we genotyped Plasmodium falciparum resistance gene markers associated with sulfadoxine-pyrimethamine (SP) and artemisinin-based combination therapy (ACT) in isolates from pregnant women in Ghana. The prevalence of the septuple IRN I- A/FG K GS/Tpfdhfr/pfdhps haplotypes, including the pfdhps A581G and A613S/T mutations, was high at delivery among post-SP treatment isolates (18.2%) compared to those of first antenatal care (before initiation of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine [IPTp-SP]; 6.1%; P = 0.03). Regarding the pfk13 marker gene, two nonsynonymous mutations (N458D and A481C) were detected at positions previously related to artemisinin resistance in isolates from Southeast Asia. These mutations were predicted in silico to alter the stability of the pfk13 propeller-encoding domain. Overall, these findings highlight the need for intensified monitoring and surveillance of additional mutations associated with increased SP resistance as well as emergence of resistance against artemisinin derivatives.

Binding of human serum proteins to Plasmodium falciparum-infected erythrocytes and its association with malaria clinical presentation.

BACKGROUND: The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite­infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α2-macroglobulin (α2M), to increase the avidity of PfEMP1-mediated binding to erythrocyte receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here. METHODS: Children 1-12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α2M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α2M was also evaluated. Experimental results were analysed according to the clinical presentation of the patients. RESULTS: Clinical data from 108 children classified as UM (n = 54) and SM cases (n = 54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from UM (n = 14) and SM (n = 20) children were analysed. Most of the field isolates bound non-immune IgM (33/34), whereas the α2M-binding was less common (23/34). Binding of both non-immune IgM and α2M was higher but not significant in IEs from children with SM than from children with UM. In combination, IgM and α2M supported rosette formation at levels similar to that observed in the presence of 10% human serum. CONCLUSIONS: The results support the hypothesis that binding of non-immune IgM and/or α2M to IEs facilitates rosette formation and perhaps contributes to P. falciparum malaria severity.

Prevalence of sickle cell disorders and malaria infection in children aged 1-12 years in the Volta Region, Ghana: a community-based study.

BACKGROUND: Alterations in the structure of haemoglobin (Hb) are usually brought about by point mutations affecting one or, in some cases, two codons encoding amino acids of the globin chains. One in three Ghanaians are said to have sickle cell disorders, whereas malaria continues to be one of the leading causes of mortality among children. This study determined the prevalence of sickle cell disorders and malaria infection among children aged 1-12 years in the Volta Region. METHODS: This was a community-based cross-sectional survey that involved 938 children aged 1-12 years selected from three districts, one each from the 3 geographical zones of the Volta Region using a multistage sampling method. Demographic information was collected using a standard questionnaire and anthropometric indices were measured. Isoelectric focusing (IEF) electrophoresis was used to determine the Hb genotypes and sub-microscopic parasites were determined by PCR. RESULTS: The prevalence of sickling screening positive was 16.0% with an overall prevalence of sickle cell disorders being 2.0%. Among the individual genotypes making up the sickle cell disorders, genotype HbSF was the highest (0.9% as compared to 0.2%; HbSS, 0.6%; HbSC and 0.3%; HbSCF). Microscopic Plasmodium falciparum parasitaemia was detected among 5.5% of the children and 14.2% sub-microscopic prevalence by PCR. Children with sickle cell disorders were more likely to have sub-microscopic parasitaemia (AOR = 5.51 95%CI (2.15, 14.10), p < 0.001) as well as anaemia (AOR = 3.03 95% CI (1.04, 8.82), p = 0.042), compared to those with normal genotypes. There was no significant difference observed between sickle cell disorders and growth and development of the children screened. CONCLUSIONS: Sickle cell disorders were significantly associated with sub-microscopic parasitaemia as well as anaemia in this study. Establishment of sickle cell clinics in the district and regional hospitals will help in the management of children with the disorder and also generate a national database on sickle cell disorders. National neonatal screening policies must also be put in place to help in early detection and management of these disorders.

Acquisition of IgG to ICAM-1-Binding DBLß Domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigen Family Varies between Groups A, B, and C.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant subfamilies. ICAM-1-binding group A PfEMP1 proteins also bind endothelial protein C receptor and have been associated with cerebral malaria in children. IgG to these PfEMP1 proteins is acquired later in life than that to group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here. Gene sequences encoding group B and C PfEMP1 with DBLß domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBLß domains from group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria. Seven new ICAM-1-binding DBLß domains from group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBLß domains from group A than from groups B and C. However, the opposite pattern was found in children with malaria, particularly among young patients. Acquisition of IgG specific for DBLß domains binding ICAM-1 differs between PfEMP1 groups.

Reliable cell and tissue morphology-based diagnosis of endemic Burkitt lymphoma in resource-constrained settings in Ghana.

BACKGROUND: Endemic Burkitt lymphoma (eBL) is an aggressive B-cell lymphoma, which is a common childhood cancer in areas with intense transmission of Plasmodium falciparum parasites. Early and accurate diagnosis is a prerequisite for successful therapy, but it optimally involves advanced laboratory investigations. These are technologically demanding, expensive, and often difficult to implement in settings where eBL is prevalent. Diagnosis is thus generally based on clinical assessment and morphological examination of tumour biopsies or fine-needle aspirates (FNAs). METHODS: The purpose of the present study was to assess the accuracy of eBL diagnosis at two tertiary hospitals in Ghana. To that end, we studied FNAs from 29 eBL patients and 21 non-eBL lymphoma patients originally diagnosed in 2018. In addition, we examined 111 archival formalin-fixed and paraffin-embedded (FFPE) biopsies from Ghanaian patients originally diagnosed as eBL (N = 55) or non-eBL (N = 56) between 2010 and 2017. Availability-based subsets of samples were subjected to haematoxylin-eosin or Giemsa staining, C-MYC immunohistochemistry, and fluorescence in situ hybridisation (FISH) analysis of c-myc rearrangements. RESULTS: We found a good correlation between original diagnosis and subsequent retrospective assessment, particularly for FNA samples. However, evidence of intact c-myc genes and normal C-MYC expression in samples from some patients originally diagnosed as eBL indicates that morphological assessment alone can lead to eBL over-diagnosis in our study area. In addition, several FFPE samples could not be assessed retrospectively, due to poor sample quality. Therefore, the simpler FNA method of obtaining tumour material is preferable, particularly when careful processing of biopsy specimens cannot be guaranteed. CONCLUSION: We conclude that the accuracy of eBL diagnostic tools available in Ghana is generally adequate, but could be improved by implementation of additional pathology laboratory investigations. Improved attention to adequate preservation of archival samples is recommended.

Etiology of Placental Plasmodium falciparum Malaria in African Women.

Plasmodium falciparum parasites causing placental malaria express the VAR2CSA type of the clonally variant antigen family erythrocyte membrane protein 1 (PfEMP1). This enables evasion of preexisting immunity and results in placental accumulation of infected erythrocytes. We present data on seasonal variation in levels of VAR2CSA-specific immunoglobulin G (IgG) and IgG specific for a placental malaria-unrelated PfEMP1 protein among Ghanaian women at their first antenatal visit. Our results indicate that placental malaria does not require recent exposure to infected mosquitoes, in contrast to malaria in general. This has implications for the impact of insecticide-treated bed nets on placental malaria incidence and for antenatal care in woman with preexisting immunity.

Natural and Vaccine-Induced Acquisition of Cross-Reactive IgG-Inhibiting ICAM-1-Specific Binding of a Plasmodium falciparum PfEMP1 Subtype Associated Specifically with Cerebral Malaria.

Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded erythrocyte membrane protein 1 (PfEMP1) that facilitates dual binding to host intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR). The PfEMP1 subtype is characterized by the presence of a particular motif (DBLß_motif) in the constituent ICAM-1-binding DBLß domain. The rate of natural acquisition of DBLß_motif-specific IgG antibodies and the ability to induce such antibodies by vaccination are unknown, and the aim of this study was to provide such data. We used an enzyme-linked immunosorbent assay (ELISA) to measure DBLß-specific IgG in plasma from Ghanaian children with malaria. The ability of human immune plasma and DBLß-specific rat antisera to inhibit the interaction between ICAM-1 and DBLß was assessed using ELISA and in vitro assays of IE adhesion under flow. The acquisition of DBLß_motif-specific IgG coincided with age-specific susceptibility to CM. Broadly cross-reactive antibodies inhibiting the interaction between ICAM-1 and DBLß_motif domains were detectable in immune plasma and in sera of rats immunized with specific DBLß_motif antigens. Importantly, antibodies against the DBLß_motif inhibited ICAM-1-specific in vitro adhesion of erythrocytes infected by four of five P. falciparum isolates from cerebral malaria patients. We conclude that natural exposure to P. falciparum as well as immunization with specific DBLß_motif antigens can induce cross-reactive antibodies that inhibit the interaction between ICAM-1 and a broad range of DBLß_motif domains. These findings raise hope that a vaccine designed specifically to prevent CM is feasible.

Malaria causes long-term effects on markers of iron status in children: a critical assessment of existing clinical and epidemiological tools.

BACKGROUND: Most epidemiological studies on the interplay between iron deficiency and malaria risk classify individuals as iron-deficient or iron-replete based on inflammation-dependent iron markers and adjustment for inflammation by using C-reactive protein (CRP) or α-1-acid glycoprotein (AGP). The validity of this approach and the usefulness of fibroblast growth factor 23 (FGF23) as a proposed inflammation-independent iron marker were tested. METHODS: Conventional iron markers and FGF23 were measured in children with acute falciparum malaria and after 1, 2, 4, and 6 weeks. Children, who were transfused or received iron supplementation in the follow-up period, were excluded, and iron stores were considered to be stable throughout. Ferritin levels 6 weeks after admission were used as a reference for admission iron status and compared with iron markers at different time points. RESULTS: There were long-term perturbations in iron markers during convalescence from acute malaria. None of the tested iron parameters, including FGF23, were independent of inflammation. CRP and AGP normalized faster than ferritin after malaria episodes. CONCLUSION: Malaria may bias epidemiological studies based on inflammation-dependent iron markers. Better markers of iron status during and after inflammation are needed in order to test strategies for iron supplementation in populations at risk of malaria.

Effect of IPTp on Plasmodium falciparum antibody levels among pregnant women and their babies in a sub-urban coastal area in Ghana.

BACKGROUND: Women exposed to Plasmodium infection develop antibodies and become semi-immune. This immunity is suppressed during pregnancy making both the pregnant woman and the foetus vulnerable to the adverse effects of malaria, particularly by Plasmodium falciparum. Intermittent preventive treatment of malaria in pregnancy (IPTp) with Sulfadoxine-pyrimethamine (SP) tablets is one of the current interventions to mitigate the effects of malaria on both the pregnant woman and the unborn child. The extent to which IPTp may interfere with the acquisition of protective immunity against pregnancy-associated malaria (PAM) is undefined in Ghana. METHODS: Three-hundred-and-twenty pregnant women were randomly enrolled at the antenatal clinic (ANC) in Madina, Accra. Venous blood samples were obtained at first ANC registration and at 4-week intervals (post-IPTp administration). Placental and cord blood samples were obtained at delivery and the infants were followed monthly for 6 months after birth. Anti-IgG and IgM antibodies against a crude antigen preparation and the glutamate-rich protein (GLURP) of P. falciparum were quantified by the enzyme-linked immunosorbent assay (ELISA). RESULTS: There was a general decline in the trend of mean concentrations of all the antibodies from enrolment to delivery. The levels of antibodies in cord blood and placenta were well correlated. Children did not show clinical signs of malaria at 6 months after birth. CONCLUSIONS: IgG against both crude antigen and GLURP were present in placenta and cord blood and it is therefore concluded that there is a trend of declining antibody from enrolment to delivery and IPTp-SP may have reduced malaria exposure, however, this does not impact on the transfer of antibodies to the foetus in utero. The levels of maternal and cord blood antibodies at delivery showed no adverse implications on malaria among the children at 6 months. However, the quantum and quality of the antibody transferred needs further investigation to ensure that the infants are protected from severe episodes of malaria.

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.

Minimising invasiveness in diagnostics: developing a rapid urine-based monoclonal antibody dipstick test for malaria.

OBJECTIVE: To generate monoclonal antibodies (MAbs) for developing a rapid malaria diagnostic urine-based assay (RUBDA), using Plasmodium-infected human urinary antigens. METHODS: Plasmodium-infected human urinary (PAgHU) and cultured parasite (CPfAg) antigens were used to generate mouse MAbs. The reactivity and accuracy of the MAbs produced were then evaluated using microplate ELISA, SDS-PAGE, Western blotting assay, microscopy and immunochromatographic tests. RESULTS: Ninety-six MAb clones were generated, of which 68.8% reacted to both PAgHU and CPfAg, 31.3% reacted to PAgHU only, and none reacted to CPfAg only. One promising MAb (UCP4W7) reacted in WBA, to both PAgHU and CPfAg, but not to Plasmodium-negative human urine and blood, Schistosoma haematobium and S. mansoni antigens nor measles and poliomyelitis vaccines. CONCLUSION: MAb UCP4W7 seems promising for diagnosing Plasmodium infection. Urine is a reliable biomarker source for developing non-invasive malaria diagnostic tests. SDS-PAGE and MAb-based WBA appear explorable in assays for detecting different levels of Plasmodium parasitaemia.

Synergistic anti-malarial action of cryptolepine and artemisinins.

BACKGROUND: Cryptolepine (CPE) is the major indoloquinoline isolated from the popular West African anti-malarial plant, Cryptolepis sanguinolenta. CPE possesses various pharmacological activities with potent anti-malarial activity against both chloroquine (CQ)-resistant and -sensitive strains. The search for safe and novel anti-malarial agents and combinations to delay resistance development to Plasmodium falciparum directed this work aimed at evaluating the anti-malarial interaction and safety of CPE in combination with some artemisinin derivatives. METHODS: The in vitro SYBR Green I, fluorescent-based, drug sensitivity assay using a fixed ratio method was carried out on the CQ-sensitive plasmodial strain 3D7 to develop isobolograms from three CPE-based combinations with some artemisinin derivatives. CPE and artesunate (ART) combinations were also evaluated using the Rane's test in ICR mice infected with Plasmodium berghei NK-65 strains in a fixed ratio combination (1:1) and fractions of their ED50s in order to determine the experimental ED50 (Zexp) of the co-administered compounds. Isobolograms were constructed to compare the Zexp to the Zadd. RESULTS: CPE exhibited promising synergistic interactions in vitro with ART, artemether and dihydroartemisinin. In vivo, CPE combination with ART again showed synergy as the Zexp was 1.02 ± 0.02, which was significantly less than the Zadd of 8.3 ± 0.31. The haematological, biochemical, organ/body weight ratio and histopathology indices in the rats treated with CPE at all doses (25, 50, 100 mg kg(-1) po) and in combination with ART (4 mg kg(-1)) showed no significant difference compared to the control group. CONCLUSION: The combination of CPE with the artemisinin derivatives were safe in the rodent model and showed a synergistic anti-malarial activity in vivo and in vitro. This study supports the basis for the selection of CPE as a prospective lead compound as the search for new anti-malarial combinations continues.

Measurement of ex vivo ELISpot interferon-gamma recall responses to Plasmodium falciparum AMA1 and CSP in Ghanaian adults with natural exposure to malaria.

BACKGROUND: Malaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays. METHODS: Thirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9-10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens. RESULTS: For CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9-10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9-10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous. CONCLUSIONS: These results generally demonstrate that CSP and AMA1 peptides recalled ELISpot IFN-γ responses from naturally exposed individuals and that both CSP and AMA1 contain diverse class 1-restricted epitopes that are HLA-promiscuous and are widely recognized in this population.