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Existing tools (including GeneXpert) for diagnosis of multidrug resistant TB (MDR-TB) have limited utility when sputum samples for microbiological analyses cannot be obtained. There is the need for immunological biomarkers which could serve as putative diagnostic markers of MDR-TB. We measured and compared the serum cytokine levels of inflammatory cytokines (IFN-γ, TNF-α, IL12p70, IL-17A, granzyme B) and anti-inflammatory cytokines (IL-10, IL-6, IL-4) among MDR-TB, drug-susceptible (DS)-TB and healthy controls (no-TB) using the Human Magnetic Luminex Multiplex Immunoassay. Levels of IFN-γ and IL-4 were respectively 1.5 log lower and 1.9 log higher in MDR-TB compared to DS-TB cases. Moreover, IFN-γ, TNF-α, IL-10, IL-6, and IL-4 levels were significantly higher in individuals with MDR-TB and DS-TB cases compared to healthy controls. Pairs of cytokines, IL-4 and IFN-γ (p = 0.019), IL-4 and TNF (p = 0.019), and Granzyme B and TNF-α (p = 0.019), showed significant positive correlation in MDR-TB. Serum cytokine profiles can be exploited for immunodiagnostics, as made evident by the Interferon Gamma Release Assays (IGRAs) for TB infection. Using area under the curve values, no single or multiple cytokine combinations could discriminate between DS- and MDR-TB in this study. Studies with a larger sample size and more cytokines could better address the issue.

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.

Betty Bandoh,1,2 Eric Kyei-Baafour,2 Belinda Aculley,2 William van der Puije,2 Bernard Tornyigah,2 Kwadwo Akyea-Mensah,2 Lars Hviid,3,4 Robert A Ngala,1 Margaret T Frempong,1 Michael F Ofori2 1Department of Molecular Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; 2Department of Immunology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Accra, Ghana; 3Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; 4Department of Infectious Diseases, Copenhagen University Hospital Rigshospitalet, Copenhagen, DenmarkCorrespondence: Michael F Ofori, Department of Immunology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Post Office Box LG581, Legon, Accra, Ghana, Tel +233 244 715975, Fax +233 302 502182, Email mofori@noguchi.ug.edu.ghPurpose: The severity of Plasmodium falciparum infections is associated with the ability of the infected red blood cells to cytoadhere to host vascular endothelial surfaces and to uninfected RBCs. Host blood group antigens and two serum proteins α2-macroglobulin (α2M) and IgM have been implicated in rosette formation in laboratory-adapted P. falciparum. However, there is only limited information about these phenotypes in clinical isolates.Methods: This was a hospital-based study involving children under 12 years-of-age reporting to the Hohoe Municipal Hospital with different clinical presentations of malaria. Parasite isolates were grown and rosette capabilities and characteristics were investigated by fluorescence microscopy. α2M and IgM were detected by ELISA.Results: Rosette formation was observed in 46.8% (75/160) of the parasite isolates from all the blood groups tested. Rosettes were more prevalent (55%) among isolates from patients with severe malaria compared to isolates from patients with uncomplicated malaria (45%). Rosette prevalence was highest (30%) among patients with blood group O (30%) and B (29%), while the mean rosette frequency was higher in isolates from patients with blood group A (28.7). Rosette formation correlated negatively with age (r = − 0.09, P= 0.008). Participants with severe malaria had a lower IgM concentration (3.683± 3.553) than those with uncomplicated malaria (5.256± 4.294) and the difference was significant (P= 0.0228). The mean concentrations of anti-parasite IgM measured among the clinical isolates which formed rosettes was lower (4.2 ± 3.930 mg/mL), than that in the non rosetting clinical isolates (4.604 ± 4.159 mg/mL) but the difference was not significant (P=0.2733). There was no significant difference in plasma α2M concentration between rosetting and non rosetting isolates (P=0.442).Conclusion: P. falciparum parasite rosette formation was affected by blood group type and plasma concentration of IgM. A lower IgM concentration was associated with severe malaria whilst a higher α2M concentration was associated with uncomplicated malaria.Keywords: Plasmodium falciparum, severe malaria, uncomplicated malaria, rosettes, blood group, alpha-2 macroglobulin, immunoglobulin M

Background The pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected 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.

Plasmodium falciparum causes the most severe form of malaria in humans. The adhesion of the infected erythrocytes (IEs) to endothelial receptors (sequestration) and to uninfected erythrocytes (rosetting) are considered major elements in the pathogenesis of the disease. Both sequestration and rosetting appear to involve particular members of several IE variant surface antigens (VSAs) as ligands, interacting with multiple vascular host receptors, including the ABO blood group antigens. In this study, we subjected genetically distinct P. falciparum parasites to in vitro selection for increased IE adhesion to ABO antigens in the absence of potentially confounding receptors. The selection resulted in IEs that adhered stronger to pure ABO antigens, to erythrocytes, and to various human cell lines than their unselected counterparts. However, selection did not result in marked qualitative changes in transcript levels of the genes encoding the best-described VSA families, PfEMP1 and RIFIN. Rather, overall transcription of both gene families tended to decline following selection. Furthermore, selection-induced increases in the adhesion to ABO occurred in the absence of marked changes in immune IgG recognition of IE surface antigens, generally assumed to target mainly VSAs. Our study sheds new light on our understanding of the processes and molecules involved in IE sequestration and rosetting.

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.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria., Here, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to differences in the ability to induce FcγRIIIa-dependent natural killer cell degranulation.

The current global malaria control and elimination agenda requires development of additional effective disease intervention tools. Discovery and characterization of relevant parasite antigens is important for the development of new diagnostics and transmission monitoring tools and for subunit vaccine development. This study assessed the natural antibody response profile of seven novel Plasmodium falciparum pre-erythrocytic antigens and their potential association with protection against clinical malaria. Antigen-specific antibody levels in plasma collected at six time points from a longitudinal cohort of one-to-five year old children resident in a seasonal malaria transmission area of northern Ghana were assessed by ELISA. Antibody levels were compared between parasite-positive and parasite-negative individuals and the association of antibody levels with malaria risk assessed using a regression model. Plasma antibody levels against five of the seven antigens were significantly higher in parasite-positive children compared to parasite-negative children, especially during low transmission periods. None of the antigen-specific antibodies showed an association with protection against clinical malaria. The study identified five of the seven antigens as markers of exposure to malaria, and these will have relevance for the development of disease diagnostic and monitoring tools. The vaccine potential of these antigens requires further assessment.

Detection of antibody reactivity to appropriate, specific parasite antigens may constitute a sensitive and cost-effective alternative to current tools to monitor malaria transmission across different endemicity settings. This study aimed to determine the suitability of IgG responses to a number of P. falciparum antigens as markers of transmission intensity and pattern. Antibody responses to multiple malaria antigens were determined in 905 participants aged 1–12 years from three districts with low (Keta), medium (Hohoe) and high (Krachi) transmission intensity in the Volta region of Ghana. Blood film microscopy slides and dry blood spots (DBS) were obtained for parasitaemia detection and antibody measurement, respectively. Sera were eluted from DBS and levels of IgG specific for 10 malaria antigens determined by a multiplex assay. Results were compared within and among the districts. Total IgG responses to MSPDBL1, MSPDBLLeucine, MSP2-FC27, RAMA, and PfRh2a and PfRh2b were higher in Krachi than in Hohoe and Keta. Seroprevalence of IgG specific for MSPDBLLeucine, RON4, and PfRh2b were also highest in Krachi. Responses to RALP-1, PfRh2a and PfRh2b were associated with patent but asymptomatic parasitaemia in Keta, while responses to MSPDBL1, MSPDBLLeucine, MSP2-FC27, RAMA, Rh2-2030, and PfRh2b were associated with parasite carriage in Hohoe, but not in Krachi. Using ROC analysis, only PfRh2b was found to predict patent, but asymptomatic, parasitaemia in Keta and Hohoe. Antibody breadth correlated positively with age (r = 0.29, pPfRh2b has potential as a marker of malaria transmission intensity and pattern. This could have implications for malaria control programs and vaccine trials.

Preeclampsia (PE) is a placental disorder with different phenotypic presentations. In malaria-endemic regions, high incidence of PE is reported, with debilitating foeto-maternal effects, particularly among primigravid women. However, the relationship between placental pathology and Plasmodium falciparum infection in the placenta with PE is underexplored. Placentas from 134 pregnant women were examined after delivery for pathological lesions and placental malaria (PM). They comprised of 69 women without PE (non-PE group) and 65 women diagnosed with PE (PE group). The presence of placental pathology increased the risk of PE, with particular reference to syncytial knots. Placental malaria was 64 (48.1%) and 21 (15.8%) respectively for active and past infections and these proportions were significantly higher in the PE group compared to the non-PE group. Further multivariate analyses showed placental pathology (adjusted (aOR) 3.0, 95% CI = 1.2–7.5), active PM (aOR 6.7, 95% CI = 2.3–19.1), past PM (aOR 12.4, 95% CI = 3.0–51.0) and primigravidity (aOR 6.6, 95% CI 2.4–18.2) to be associated with PE. Our findings suggest that placental histological changes and PM are independent risk factors for PE particularly in primigravida. These findings might improve the management of PE in malaria-endemic regions.

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.

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.

Despite the clinically proven advantages of intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP), utilisation has been low in many African countries. To increase uptake and achieve the desired effect, the World Health Organization revised the policy to a monthly administration. Assessing the coverage and impact of the revised policy on pregnancy and neonatal outcomes is, therefore, a necessity. A 2-parallel cross-sectional hospital-based study was carried out among pregnant women attending first antenatal care (ANC) and delivery. Maternal and cord blood samples were assayed for malaria parasites by quantitative PCR targeting both the 18S rDNA and the acidic terminal segment of Plasmodium falciparum var genes, and plasma SP levels were measured by liquid chromatography coupled to tandem mass spectrometry. Parasite prevalence was similar between the two study sites but decreased significantly between the first ANC (9% or 43%) and delivery (4% or 11%) based on the qPCR target. At delivery, 64.5% of women received ≥3 IPTp-SP dose, 15.5% received 2 doses and 6% had 1 dose. Taking ≥3 IPTp-SP doses was associated with an average birth weight increase of more than 0.165 kg. IPTp-SP uptake was associated with plasma SP level at delivery (OR = 32.3, p ≤ 0.005, 95% CI (13.3;78.4) for those that reported ≥3 IPTp-SP doses) while the same trend of improved birth weight was observed with high plasma SP levels. The new IPTp policy is well implemented and well utilised by women in the sites considered in this study and translates to the improved birth weight observed. This study confirms the interest and the clinical benefit expected from this policy change.

BackgroundMost 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.MethodsConventional 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.ResultsThere 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.ConclusionMalaria 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.

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.

The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from a pool of donors (e.g., HBEC-5i). Here we demonstrate, for the first time, the successful isolation of blood outgrowth endothelial cells (BOECs) from frozen stabilates of peripheral blood mononuclear cells obtained from small-volume peripheral blood samples from paediatric malaria patients. BOECs are a sub-population of human endothelial cells, found within the peripheral blood. We demonstrate that these cells express receptors such as Intercellular Adhesion Molecule 1 (ICAM-1/CD54), Endothelial Protein C Receptor (EPCR/CD201), platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), Thrombomodulin (CD141), and support adhesion of P. falciparum IEs.

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−1po) 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.

Background: The interplay between Epstein-Barr virus infection, malaria, and endemic Burkitt’s Lymphoma is not well understood. Reports show diminished EBV-specific Th1 responses in children living in malaria endemic areas and deficiency of EBNA1-specific IFN-γ T cell responses in children with endemic Burkitt’s Lymphoma (eBL). This study, therefore, examined some factors involved in the loss of EBNA-1-specific T cell responses in eBL. Methods: T-cell subset frequencies, activation, and IFN-γ- or IL-4-specific responses were analyzed by flow-cytometry. Plasma cytokine levels were measured by ELISA. Results: CD4+ and CD8+ cells in age- and sex-matched healthy controls (n = 3) expressed more IFN-γ in response to all immunostimulants than in pediatric endemic BL (eBL) patients (n = 4). In healthy controls, IFN-γ expression was higher than IL-4 expression, whereas in eBL patients the expression of IL-4 by CD4+ cells to EBNA-1 was slightly higher than IFN-γ. Moreover, the blood levels of TNF-α was significantly lower (p = 0.004) while IL-10 was significantly higher (p = 0.038), in eBL patients (n = 21) compared to controls (n = 16). Additionally, the frequency of CD4+CD25hi+ T cells was higher in both age-matched acute uncomplicated malaria (n = 26) and eBL (n = 14) patients compared to healthy controls (n = 19, p = 0.000 and p = 0.027, respectively). Conclusion: The data suggest that reduced Th1 response in eBL might be due to increased levels of IL-10 and T reg cells.

Background A malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum. Methods Ex vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control. Results Of the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP. Conclusions These results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine. Electronic supplementary material The online version of this article (doi:10.1186/s12936-014-0539-5) contains supplementary material, which is available to authorized users.

Naturally acquired protective immunity to Plasmodium falciparum malaria takes years to develop. It relies mainly on Abs, particularly IgG specific for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins on the infected erythrocyte surface. It is only partially understood why acquisition of clinical protection takes years to develop, but it probably involves a range of immune-evasive parasite features, not least of which are PfEMP1 polymorphism and clonal variation. Parasite-induced subversion of immunological memory and expansion of “atypical” memory B cells may also contribute. In this first, to our knowledge, longitudinal study of its kind, we measured B cell subset composition, as well as PfEMP1-specific Ab levels and memory B cell frequencies, in Ghanaian women followed from early pregnancy up to 1 y after delivery. Cell phenotypes and Ag-specific B cell function were assessed three times during and after pregnancy. Levels of IgG specific for pregnancy-restricted, VAR2CSA-type PfEMP1 increased markedly during pregnancy and declined after delivery, whereas IgG levels specific for two PfEMP1 proteins not restricted to pregnancy did not. Changes in VAR2CSA-specific memory B cell frequencies showed typical primary memory induction among primigravidae and recall expansion among multigravidae, followed by contraction postpartum in all. No systematic changes in the frequencies of memory B cells specific for the two other PfEMP1 proteins were identified. The B cell subset analysis confirmed earlier reports of high atypical memory B cell frequencies among residents of P. falciparum–endemic areas, and indicated an additional effect of pregnancy. Our study provides new knowledge regarding immunity to P. falciparum malaria and underpins efforts to develop PfEMP1-based vaccines against this disease.

This work was set to evaluate the antiplasmodial activities of some plants used in traditional malaria therapy in Kano State, Nigeria. Nine medicinal plants, distributed into eight families, used for treatment of malaria fever by the Hausa people of Kano-Nigeria were selected based on the traditional claims of the folks. These were collected and extracted using aqueous and organic solvents. A total of nineteen extracts and forty four extract-fractions were obtained. Thirty extracts (7 aqueous extracts, 12 ethanol extracts and 11 methanol extracts-fraction) were selected and screened for their in vitro anti-plasmodial activities against laboratory adapted chloroquine susceptible Plasmodium falciparu m strain 3D7. Seven (23.33%) of these extracts that exhibited 70% or more inhibition at 50μg/mL concentration were further evaluated to establish their potency levels. The respective IC 50 values obtained ranged between 0.361±0.15μg/ml and 37.67±4.20μg/ml. CALF 1 from the leaves of C. aurantifolia (Rutaceae) and CBLF 1 (a combination of the leaves of C. aurantifolia , Carica papaya, Mangifera indica and Psidium guajava ) were further screened against a clinical isolate of P. falciparum and recorded IC 50 values similar to those obtained earlier against the laboratory strain, 12.42±3.94 μg/ml for CBLF 1 , and 29.69±0.65μg/ml for CALF 1 respectively. It could be concluded that majority of the plants have demonstrated appreciable concentration dependent in vitro anti-plasmodial effects against the laboratory adapted and field strains P. falciparum . Keywords: Plants Extracts, Traditional Malaria Therapy, Hausa people, Kano, Nigeria

BackgroundCerebral 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 findingsPaediatric 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 (p0.05). 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/significanceHigh 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.

BackgroundThe virulence of Plasmodium falciparum malaria is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density.Methodology/principal findingsWe used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from ∼20 h to ∼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions.Conclusions/significanceThe density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host.

Evidence-based practice in medicine requires clinical signs and symptoms of malaria to be con-firmed by microscopic detection of the parasite in blood sample of patients. In some cases, how-ever, such signs and symptoms persist without microscopic detection of the parasite in blood smears, making it difficult for treatment decision to be made by medics. To help solve the above problem and to ensure that antimalarial drugs are given to patients who actually suffer from malaria infection, we designed this cross-sectional study to measure ESR, mIgG, Hb and parasi-taemia levels in 78 clinically diagnosed malaria patients attending the Elmina Urban Health Centre, in the Komenda-Edina-Eguafo-Abirem Municipal Assembly in the Central Region of Ghana, with the view to finding alternative biomarkers for malaria infection. Apart from the Hb levels which differed significantly (P=0.001) between the sexes, the levels of all the other meas-ured indicators of malaria infection were comparable between the various groups. ESR corre-lated positively with mIgG (r = 0.242, P =0.033) but negatively with Hb (r = -0.348, P = 0.002) irrespective of age, sex or percentage parasitaemia. Our results appear to suggest that an ESR level of twice the upper reference value of an individual may be diagnostic of malaria infection in the presence of the appropriate recognized signs and symptoms of malaria without micro-scopically detectable parasitaemia in the blood sample of patients. We propose that ESR and mIgG be considered as complementary markers of malaria infection in cases of unsuccessful microscopic detection of the malaria parasite in blood smears.

In Plasmodium falciparum–endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

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.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.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.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.

Background Rotaviruses with G6P[14] specificity are mostly isolated in cattle and have been established as a rare cause of gastroenteritis in humans. This study reports the first detection of G6P[14] rotavirus strain in Ghana from the stool of an infant during a hospital-based rotavirus surveillance study in 2010. Methods Viral RNA was extracted and rotavirus VP7 and VP4 genes amplified by one step RT-PCR using gene-specific primers. The DNA was purified, sequenced and genotypes determined using BLAST and RotaC v2.0. Phylogenetic tree was constructed using maximum likelihood method in MEGA v6.06 software and statistically supported by bootstrapping with 1000 replicates. Phylogenetic distances were calculated using the Kimura-2 parameter model. Results The study strain, GHA-M0084/2010 was characterised as G6P[14]. The VP7 gene of the Ghanaian strain clustered in G6 lineage-III together with artiodactyl and human rotavirus (HRV) strains. It exhibited the highest nucleotide (88.1 %) and amino acid (86.9 %) sequence identity with Belgian HRV strain, B10925. The VP8* fragment of the VP4 gene was closely related to HRV strains detected in France, Italy, Spain and Belgium. It exhibited the strongest nucleotide sequence identity (87.9 %) with HRV strains, PA169 and PR/1300 (Italy) and the strongest amino acid sequence identity (89.3 %) with HRV strain R2775/FRA/07 (France). Conclusion The study reports the first detection of G6P[14] HRV strain in an infant in Ghana. The detection of G6P[14], an unusual strain pre-vaccine introduction in Ghana, suggests a potential compromise of vaccine effectiveness and indicates the necessity for continuous surveillance in the post vaccine era. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0643-y) contains supplementary material, which is available to authorized users.