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.

Atypical memory B cell frequency correlates with antibody breadth and function in malaria immune adults.

Clinical immunity to malaria develops slowly after repeated episodes of infection and antibodies are essential in naturally acquired immunity against malaria. However, chronic exposure to malaria has been linked to perturbation in B-cell homeostasis with the accumulation of atypical memory B cells. It is unclear how perturbations in B cell subsets influence antibody breadth, avidity, and function in individuals naturally exposed to malaria. We show that individuals living in high malaria transmission regions in Ghana have higher Plasmodium falciparum merozoite antigen-specific antibodies and an increased antibody breadth score but lower antibody avidities relative to low transmission regions. The frequency of circulating atypical memory B cells is positively associated with an individual's antibody breadth. In vitro growth inhibition is independent of the ability to bind to free merozoites but associated with the breadth of antibody reactivity in an individual. Taken together, our data shows that repeated malaria episodes hamper the development of high avid antibodies which is compensated for by an increase in antibody breadth. Our results provide evidence to reinforce the idea that in regions with high malaria prevalence, repeated malaria infections lead to the broadening of antibody diversity and the continued presence of atypical memory B cell populations.

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.

Clinical and laboratory characteristics of children with sickle cell disease on hydroxyurea treated with artemether-lumefantrine for acute uncomplicated malaria.

Introduction: Limited information exists on any interactions between hydroxyurea (HU) and antimalarials in sickle cell disease (SCD). We evaluated changes in clinical and laboratory parameters among children with SCD on HU therapy treated with artemether-lumefantrine (AL) for acute uncomplicated malaria (UM). Methods: A prospective, non-randomized, pilot study of 127 children with SCD (23, UM; 104, steady state) were recruited from three hospitals in Accra. UM participants were treated with standard doses of AL and followed up, on days 1, 2, 3, 7, 14, and 28. Venous blood was collected at baseline and follow-up days in participants with UM for determination of malaria parasitaemia, full blood count, reticulocytes, and clinical chemistry. Further, Plasmodium falciparum identification of rapid diagnostic test (RDT) positive samples was done using nested polymerase chain reaction (PCR). Results: Among SCD participants with UM, admission temperature, neutrophils, alanine-aminotransferase, gamma-glutamyl-transferase, and haemoglobin significantly differed between HU recipients (HU+) and steady state, while white blood cell, neutrophils, reticulocytes, bilirubin, urea, and temperature differed significantly between non-HU recipients (no-HU), and steady state. Mean parasitaemia (HU+, 2930.3 vs. no-HU, 1,060, p = 0.74) and adverse events (HU+, 13.9% vs. no-HU, 14.3%), were comparable (p = 0.94). Day 28 reticulocyte count was higher in the HU+ (0.24) (0.17 to 0.37) vs. no-HU, [0.15 (0.09 to 0.27), p = 0.022]. Significant differences in lymphocyte [HU+ 2.74 95% CI (-5.38 to 58.57) vs. no-HU -0.34 (-3.19 to 4.44), p = 0.024]; bilirubin [HU+, -4.44 (-16.36 to 20.74) vs. no-HU -18.37 (-108.79 to -7.16)]; and alanine aminotransferase, [HU+, -4.00 (-48.55 to 6.00) vs. no-HU, 7.00 (-22.00 to 22.00)] were observed during follow up. Conclusion: Parasite clearance and adverse event occurrence were comparable between SCD children treated with AL irrespective of HU status. However, distinct patterns of changes in laboratory indices suggest the need for larger, more focused studies.

Knowledge, attitudes, and practices of caregivers on childhood immunization in Okaikoi sub-metro of Accra, Ghana.

Background: Immunization remains one of the most cost-effective health interventions. However, there are still issues of vaccine hesitancy especially in caregivers who are required to protect their children from vaccine-preventable diseases. This thwarts the overall vaccine coverage in disease-endemic areas such as sub-Saharan Africa. Therefore, to determine the factors that promote vaccine hesitancy in caregivers, this study sought to assess the knowledge, attitude, and practices of caregivers on childhood immunization in Okaikoi, a sub-metro of Accra in Ghana. Methods: A cross-sectional study on childhood immunization was conducted to determine the knowledge, attitudes, and practices of caregivers. A total of 120 caregivers with infants aged 12 months to 23 months were interviewed with a structured questionnaire containing open-ended and closed-ended queries. Results: From the community, infants whose caregivers had adhered completely to immunization constituted 53.3% while the rest were partially immunized. The two main deterrents to complete immunization were time constraints (25.8%) and forgetfulness (17.5%). It was observed that vaccination uptake and maternal level of education, as well as vaccination adverse reaction, did not impact the completion of the EPI program by these caregivers. Unfortunately, it was noted that caregivers with higher education levels were unable to complete their vaccination schedules due to their busy work schedules. Nonetheless, the main deterrent to adhering to complete childhood immunization was poor maternal knowledge (58%). Conclusion: The study revealed that, the caregivers in the community had poor knowledge on vaccination and its benefits, and therefore, with no strict adherence to vaccination schedules. This promoted the incomplete immunization of children in the community by their caregivers. Also, since the main source of information with regard to immunization in the sub-metro was through the antenatal and postnatal child welfare clinics and the media, we recommend that the health workers collaborate with media personnel to ensure that standardized information is disseminated.

Malaria, Urogenital Schistosomiasis, and Anaemia in Pregnant Ghanaian Women.

Background: Anaemia is common in sub-Saharan Africa, and parasitic infections could worsen its burden during pregnancy. Moreover, women become susceptible to malaria during pregnancy. We investigated Plasmodium falciparum (P. falciparum) and Schistosoma haematobium (S. haematobium) infections and determined their association with anaemia during pregnancy. Methods: A cross-sectional study involving 707 pregnant women attending antenatal care visits (ANC) and 446 at delivery was conducted in Battor and Adidome hospitals. Pregnant women were screened by microscopy and qPCR for P. falciparum and S. haematobium infections. Haemoglobin (Hb) levels were determined, and most participants received intermittent preventive treatment during pregnancy (IPTp) during ANC till delivery. Regression analyses were performed for associations between parasite infection and anaemia. Results: P. falciparum microscopy prevalence at ANC and delivery was 8% and 2%, respectively, and by PCR 24% at ANC and 12% at delivery. Anaemia prevalence at ANC was 52% and 49% at delivery. There was an increased risk of anaemia with P. falciparum infection (aOR = 1.92; p = 0.04). IPTp (p = 0.003) and age (p = 0.004) were associated with increased Hb levels at delivery. S. haematobium prevalence by microscopy was 4% at ANC and 2% at delivery. No significant correlation between S. haematobium and Hb levels was observed (coef. = -0.62 g/dl; p = 0.07). Conclusion: High anaemia prevalence was observed during pregnancy, and P. falciparum infection was associated with anaemia at ANC. Low S. haematobium prevalence could be attributed to previous praziquantel treatment during mass drug administration. Routine diagnosis and treatment of S. haematobium infections in endemic areas could be initiated to reduce schistosomiasis during pregnancy.

Plasmodium falciparum infection and naturally acquired immunity to malaria antigens among Ghanaian children in northern Ghana.

Background: The surge in malaria cases and deaths in recent years, particularly in Africa, despite the widespread implementation of malaria-control measures could be due to inefficiencies in malaria control and prevention measures in malaria-endemic communities. In this context, this study provides the malaria situation report among children in three Municipalities in Northern Ghana, where Seasonal Malaria Chemotherapy (SMC) is implemented by Ghana Health Service (GHS). Methods: A cross-sectional household survey was carried out to assess the malaria knowledge, attitudes, and practices (KAP) and malaria prevalence in 394 households in 13 rural communities in the Kumbugu, Nanton and Tolon Municipalities, Northern Region, Ghana. This was followed by screening for P. falciparum infection with anti-HRP2 RDT and PCR among children 1-17 years in the households. Plasma levels of IgG specific for crude P. falciparum antigen (3D7) and four recombinant malaria antigens (CSP, GLURP, MSP3, and Pfs230) were assessed by ELISA. The malaria and parasitaemia data were converted into frequency and subgroup proportions and disaggregated by study sites and demographic information of the participants. The ELISA data was converted to arbitrary units (AU) and similarly compared across study sites and demographic information. Results: The P. falciparum infection rate and frequency of malaria were high in the study areas with significant age-dependent and inter-community differences, which were reflected by differences in plasma levels of P. falciparum-specific IgG. Over 60% of households reported the use of bed nets and indoor insecticide sprays/coils, and 14% mentioned bush clearing around homes (14%) as malaria preventive measures. Community health centres were the preferred place for households (88%) to seek malaria treatment but over-the-counter drug stores were the major source (66%) of their antimalarials. Overall, malaria preventive and treatment practices were sub-optimal. Conclusions: P. falciparum infection and malaria are still high in the studied communities, indicating that preventive and control measures against the disease in the region remain inadequate. Efforts to ensure high SMC compliance and to improve preventative and treatment practices thus seem cost-beneficial "low-hanging fruits" in the fight against malaria in the Northern Region of Ghana.

Inflammatory cytokines as potential biomarkers for early diagnosis of severe malaria in children in Ghana.

BACKGROUND: Severe malaria (SM) is a fatal multi-system disease which accounted for an estimated 619,000 deaths in 2021. Less than 30% of children presenting with SM are diagnosed and treated promptly, resulting in increased mortality and neurologic impairments in survivors. Studies have identified cytokine profiles that differentiate the various clinical manifestations of malaria (severe and uncomplicated). However, the diagnostic capability of these cytokines in differentiating between the disease states in terms of cut-off values has not yet been determined. METHODS: The plasma levels of 22 pro-inflammatory cytokines (Eotaxin/CCL 11, interferon-gamma (IFN-γ), interleukin (IL)- 2, IL-6, IL-1ß, IL-12p40/p70, IL-17A, RANTES, MCP-1, IL-15, IL-5, IL-1RA, IL-2R, IFN-α, IP-10, TNF, MIG, MIP-1α, MIP-1ß, IL-7, IL-8 and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), and 3 anti-inflammatory cytokines-(IL-4, IL-13 and IL-10) in patients with SM, uncomplicated malaria (UM) and other febrile conditions, were measured and compared using the Human Cytokine Magnetic 25-Plex Panel. The receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of these cytokines. RESULTS: The level of the pro-inflammatory cytokine, IL-17A, was significantly higher in the SM group as compared to the UM group. Levels of the anti-inflammatory cytokines however did not differ significantly among the SM and UM groups. Only IL-1ß and IL-17A showed good diagnostic potential after ROC curve analysis. CONCLUSION: The data show that levels of pro-inflammatory cytokines correlate with malaria disease severity. IL-1ß and IL-17A showed good diagnostic potentials and can be considered for use in clinical practice to target treatment.

Betulin and Crinum asiaticum L. bulbs extract attenuate pulmonary fibrosis by down regulating pro-fibrotic and pro-inflammatory cytokines in bleomycin-induced fibrosis mice model.

Background: Pulmonary fibrosis (PF) is a lung disease characterized by scaring of lung tissue that impairs lung functions. The estimated survival time of patients with pulmonary fibrosis is 3-5 years. Bleomycin (BLM) is used clinically in the treatment of Hodgkin lymphoma and testicular germ-cell tumors. Bleomycin's mechanism of action is the inhibition of DNA and protein synthesis. This happens when leukocytes induce the release of cytokines and chemokines which increase the pro-fibrotic and pro-inflammatory cytokines such as IL-6, TNF-alpha, IL-13, IL-1ß and transforming growth factor-beta 1 (TGF-ß). Crinum asiaticum L. bulbs (CAE) are widely found in parts of Africa, Asia and Indian Ocean Island. It is also prevalent in southern part of Ghana and traditionally used by the indigenes to treat upper respiratory tract infections, and for wound healing. Betulin (BET) is found in the bulbs of Crinum asiaticum L. but widely isolated from the external bark of birches and sycamore trees. Betulin as a lupine type triterpenes has been researched for their pharmacological and biological activities including anticancer, anti-inflammatory, antimicrobial activities and anti-liver fibrosis effects.Aim of the study: The aim was to study the anti-pulmonary fibrosis effect of Crinum asiaticum L. bulbs extract and betulin in bleomycin-induced pulmonary fibrosis in mice. Materials and method: There was a single oropharyngeal administration of bleomycin (80 mg/kg) in mice followed by the treatment of CAE and BET after 48 h of exposure to BLM. Results: There was increased survival rate in CAE and BET treatment groups compared to the BLM induced group. There was a marked decreased in the levels of hydroxyproline, collagen I and III in the CAE and BET treatment groups compared to BLM-treated group. The treatment groups of CAE and BET significantly down regulated the levels of pro-fibrotic and pro-inflammatory cytokines concentrations such as TGF-ß1, MMP9, IL-6, IL-1ß and TNF-alpha compared to an increased in the BLM treated groups. The histological findings of the lungs suggested the curative effects of CAE and BET following BLM induced pulmonary fibrosis in mice, the study showed improved lung functions with wide focal area of viable alveolar spaces and few collagen fibers deposition on lungs of treatment groups. Conclusion: CAE and BET attenuated pulmonary fibrosis by down regulating pro-fibrotic and pro-inflammatory cytokines as well as improving lung function. This could be a lead in drug discovery where compounds with anti-fibrotic effects could be developed for the treatment of lung injury.

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.

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.

Population-based sero-epidemiological investigation of the dynamics of SARS-CoV-2 infections in the Greater Accra Region of Ghana.

The coronavirus disease 2019 (COVID-19) pandemic devastated countries worldwide, and resulted in a global shutdown. Not all infections are symptomatic and hence the extent of SARS-CoV-2 infection in the community is unknown. The paper presents the dynamics of the SARS-CoV-2 epidemic in the Greater Accra Metropolis, describing the evolution of seroprevalence through time and by age group. Three repeated independent population-based surveys at 6-week intervals were conducted in from November 2020 to July 2021. The global and by age-groups weighted seroprevalences were estimated and the risk factors for SARS-CoV-2 antibody seropositivity were assessed using logistic regression. The overall age-standardized SARS-CoV-2 antibody seroprevalence for both spike and nucleocapsid increased from 13.8% (95% CI 11.9, 16.1) in November 2020 to 39.6% (95% CI 34.8, 44.6) in July 2021. After controlling for gender, marital status, education level, and occupation, the older age group over 40 years had a higher odds of seropositivity than the younger age group (OR 3.0 [95% CI 1.1-8.5]) in the final survey. Pupils or students had 3.3-fold increased odds of seropositivity (OR 3.2 [95% CI 1.1-8.5]) compared to the unemployed. This study reinforces that, SARS-CoV-2 infections have been significantly higher than reported.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Perturbations in the T cell receptor ß repertoire during malaria infection in children: A preliminary study.

The changes occurring in the T cell repertoire during clinical malaria infection in children remain unknown. In this study, we undertook the first detailed comparative study of the T cell repertoire in African children with and without clinical malaria to test the hypothesis that clonotypic expansions that occur during P. falciparum infection will contribute to the generation of a T cell repertoire that is unique to each disease state. We profiled the complementarity-determining region 3 (CDR3) of the TCRß chain sequences from children with Plasmodium falciparum infections (asymptomatic, uncomplicated and severe malaria) and compared these with sequences from healthy children. Interestingly, we discovered that children with symptomatic malaria have a lower TCR diversity and frequency of shared (or "public") TCR sequences compared to asymptomatic children. Also, TCR diversity was inversely associated with parasitemia. Furthermore, by clustering TCR sequences based on their predicted antigen specificities, we identified a specificity cluster, with a 4-mer amino acid motif, that is overrepresented in the asymptomatic group compared to the diseased groups. Further investigations into this finding may help in delineating important antigenic targets for vaccine and therapeutic development. The results show that the T cell repertoire in children is altered during malaria, suggesting that exposure to P. falciparum antigens disrupts the adaptive immune response, which is an underlying feature of the disease.

Cytokines as Potential Biomarkers for Differential Diagnosis of Sepsis and Other Non-Septic Disease Conditions.

Sepsis defined as a dysregulated immune response is a major cause of morbidity in children. In sub-Saharan Africa, the clinical features of sepsis overlap with other frequent infections such as malaria, thus sepsis is usually misdiagnosed in the absence of confirmatory tests. Therefore, it becomes necessary to identify biomarkers that can be used to distinguish sepsis from other infectious diseases. We measured and compared the plasma levels of 18 cytokines (Th1 [GM-CSF, IFN-γ, TNF-α, IL-1ß, 1L-2, IL-6, IL-8, IL-12/IL-23p40, IL-15], Th2[IL-4, IL-5, IL-13), Th17 [IL17A], Regulatory cytokine (IL-10) and 7 chemokines (MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, RANTES/CCL5, Eotaxin/CCL11, MIG/CXCL9 and IP-10/CXCL10 using the Human Cytokine Magnetic 25-Plex Panel in plasma samples obtained from children with sepsis, clinical malaria and other febrile conditions. Children with sepsis had significantly higher levels of IL-1ß, IL-12 and IL-17A compared to febrile controls but lower levels of MIP1-ß/CCL4, RANTES/CCL5 and IP10/CXCL10 when compared to children with malaria and febrile controls. Even though levels of most inflammatory responses were higher in malaria compared to sepsis, children with sepsis had a higher pro-inflammatory to anti-inflammatory ratio which seemed to be mediated by mostly monocytes. A principal component analysis and a receiver operator characteristic curve analysis, identified seven potential biomarkers; IL-1ß, IL-7, IL-12, IL-1RA, RANTES/CCL5, MIP1ß/CCL4 and IP10/CXCL10 that could discriminate children with sepsis from clinical malaria and other febrile conditions. The data suggests that sepsis is associated with a higher pro-inflammatory environment. These pro-inflammatory cytokines/chemokines could further be evaluated for their diagnostic potential to differentiate sepsis from malaria and other febrile conditions in areas burdened with infectious diseases.

The Phytochemistry and Pharmacology of Tulbaghia, Allium, Crinum and Cyrtanthus: 'Talented' Taxa from the Amaryllidaceae.

Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to develop new drugs. The genera Allium, Tulbaghia, Cyrtanthus and Crinum biosynthesize novel alkaloids and other phytochemicals with traditional and pharmacological uses. Amaryllidaceae biomolecules exhibit multiple pharmacological activities such as antioxidant, antimicrobial, and immunomodulatory effects. Traditionally, natural products from Amaryllidaceae are utilized to treat non-communicable and infectious human diseases. Galanthamine, a drug from this family, is clinically relevant in treating the neurocognitive disorder, Alzheimer's disease, which underscores the importance of the Amaryllidaceae alkaloids. Although Amaryllidaceae provide a plethora of biologically active compounds, there is tardiness in their development into clinically pliable medicines. Other genera, including Cyrtanthus and Tulbaghia, have received little attention as potential sources of promising drug candidates. Given the reciprocal relationship of the increasing burden of human diseases and limited availability of medicinal therapies, more rapid drug discovery and development are desirable. To expedite clinically relevant drug development, we present here evidence on bioactive compounds from the genera Allium, Tulgbaghia, Cyrtanthus and Crinum and describe their traditional and pharmacological applications.

Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay.

The issue of antibody cross-reactivity is of central importance in immunology, and not least in protective immunity to Plasmodium falciparum malaria, where key antigens show substantial allelic variation (polymorphism). However, serological analysis often does not allow the distinction between true cross-reactivity (one antibody recognizing multiple antigen variants) and apparent cross-reactivity (presence of multiple variant-specific antibodies), as it requires analysis at the single B-cell/monoclonal antibody level. ELISpot is an assay that enables that, and a recently developed multiplexed variant of ELISpot (FluoroSpot) facilitates simultaneous assessment of B-cell/antibody reactivity to several different antigens. In this study, we present a further enhancement of this assay that makes direct analysis of monoclonal antibody-level cross-reactivity with allelic variants feasible. Using VAR2CSA-type PfEMP1-a notoriously polymorphic antigen involved in the pathogenesis of placental malaria-as a model, we demonstrate the robustness of the assay and its applicability to analysis of true cross-reactivity of monoclonal VAR2CSA-specific antibodies in naturally exposed individuals. The assay is adaptable to the analysis of other polymorphic antigens, rendering it a powerful tool in studies of immunity to malaria and many other diseases.

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.

Identification and Enumeration of Plasmodium falciparum Parasites by Light Microscopy.

Estimating malaria parasite density is important for patient care and management in rural and urban health centers in endemic areas. Here, we describe methodologically the protocols and methods to identify and enumerate Plasmodium falciparum parasites from infected blood using light microscopy. We provide step-by-step protocols and evaluate any possible drawbacks that may limit the methods and prospects of using light microscopy in diagnosing malaria.

Collection and Cryopreservation of Plasmodium falciparum Clinical Isolates in the Field.

P. falciparum causes the most severe form of malaria in younger children and pregnant women. In vitro culture systems allow researchers to understand parasite biology, elucidate mechanism of host immunity and test efficacy of antimalarial agents or vaccines in preclinical studies. Most laboratory-adapted parasite strains predate the emergence of artemisinin-based drug combinations and mainly originate from Asia or Europe. To fully understand the biochemical and phenotypic characteristics of parasites, it is imperative that researchers are able to culture parasites circulating in an area to unravel any geographical differences at the population level. Ex vivo culturing of clinical isolates can be challenging when collecting samples in the field and requires technical expertise and equipment. To overcome this challenge, clinical isolates are cryopreserved in the field and transported to a laboratory for in vitro studies. In this protocol, we describe different methods of cryopreserving P. falciparum isolates in the field and thawing them for subsequent in vitro culture.