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

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

Co-infection of Plasmodium falciparum and Schistosoma mansoni is associated with anaemia.

BACKGROUND: Malaria and schistosomiasis persist as major public health challenge in sub-Saharan Africa. These infections have independently and also in polyparasitic infection been implicated in anaemia and nutritional deficiencies. This study aimed at assessing asymptomatic malaria, intestinal Schistosoma infections and the risk of anaemia among school children in the Tono irrigation area in the Kassena Nankana East Municipal (KNEM) in the Upper East Region of Northern Ghana. METHODS: A cross sectional survey of 326 school children was conducted in the KNEM. Kato Katz technique was used to detect Schistosoma eggs in stool. Finger-prick capillary blood sample was used for the estimation of haemoglobin (Hb) concentration and blood smear for malaria parasite detection by microscopy. RESULTS: The average age and Hb concentration were 10.9 years (standard deviation, SD: ± 2.29) and 11.2 g/dl (SD: ± 1.39) respectively with 58.9% (n = 192) being females. The overall prevalence of infection with any of the parasites (single or coinfection) was 49.4% (n = 161, 95% confidence interval, CI [44.0-54.8]). The prevalence of malaria parasite species or Schistosoma mansoni was 32.0% (n = 104) and 25.2% (n = 82), respectively with 7.7% (n = 25) coinfection. The prevalence of anaemia in the cohort was 40.5% (95%CI [35.3-45.9]), of which 44.4% harboured at least one of the parasites. The prevalence of anaemia in malaria parasite spp or S. mansoni mono-infections was 41.8% and 38.6%, respectively and 64.0% in coinfections. There was no statistically significant difference in the odds of being anaemic in mono-infection with malaria (OR = 1.22, 95% CI 0.71-2.11, p = 0.47) or S. mansoni (OR = 1.07, 95% CI 0.58-1.99, p = 0.83) compared to those with no infection. However, the odds of being anaemic and coinfected with malaria parasite species and S. mansoni was 3.03 times higher compared to those with no infection (OR = 3.03, 95% CI 1.26-7.28, p = 0.013). Conclusion The data show a high burden of malaria, S. mansoni infection and anaemia among school children in the irrigation communities. The risk of anaemia was exacerbated by coinfections with malaria parasite(s) and S. mansoni. Targeted integrated interventions are recommended in this focal area of KNEM.

The temporal dynamics of Plasmodium species infection after artemisinin-based combination therapy (ACT) among asymptomatic children in the Hohoe municipality, Ghana.

BACKGROUND: The routine surveillance of asymptomatic malaria using nucleic acid-based amplification tests is essential in obtaining reliable data that would inform malaria policy formulation and the implementation of appropriate control measures. METHODS: In this study, the prevalence rate and the dynamics of Plasmodium species among asymptomatic children (n = 1697) under 5 years from 30 communities within the Hohoe municipality in Ghana were determined. RESULTS AND DISCUSSION: The observed prevalence of Plasmodium parasite infection by polymerase chain reaction (PCR) was 33.6% (571/1697), which was significantly higher compared to that obtained by microscopy [26.6% (451/1697)] (P < 0.0001). Based on species-specific analysis by nested PCR, Plasmodium falciparum infection [33.6% (570/1697)] was dominant, with Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections accounting for 0.1% (1/1697), 0.0% (0/1697), and 0.0% (0/1697), respectively. The prevalence of P. falciparum infection among the 30 communities ranged from 0.0 to 82.5%. Following artesunate-amodiaquine (AS + AQ, 25 mg/kg) treatment of a sub-population of the participants (n = 184), there was a substantial reduction in Plasmodium parasite prevalence by 100% and 79.2% on day 7 based on microscopy and nested PCR analysis, respectively. However, there was an increase in parasite prevalence from day 14 to day 42, with a subsequent decline on day 70 by both microscopy and nested PCR. For parasite clearance rate analysis, we found a significant proportion of the participants harbouring residual Plasmodium parasites or parasite genomic DNA on day 1 [65.0% (13/20)], day 2 [65.0% (13/20)] and day 3 [60.0% (12/20)] after initiating treatment. Of note, gametocyte carriage among participants was low before and after treatment. CONCLUSION: Taken together, the results indicate that a significant number of individuals could harbour residual Plasmodium parasites or parasite genomic DNA after treatment. The study demonstrates the importance of routine surveillance of asymptomatic malaria using sensitive nucleic acid-based amplification techniques.

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

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

Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis.

Bacillus stearothermophilus large fragment (BSTLF) DNA polymerase is reported, isolated on silica via a fused R5 silica-affinity peptide and used in nucleic acid diagnostics. mCherry (mCh), included in the fusion construct, was shown as an efficient fluorescent label to follow the workflow from gene to diagnostic. The R5 immobilisation on silica from cell lysate was consistent with cooperative R5-specific binding of R52-mCh-FL-BSTLF or R52-mCh-H10-BSTLF fusion proteins followed by non-specific protein binding (including E. coli native proteins). Higher R5-binding could be achieved in the presence of phosphate, but phosphate residue reduced loop-mediated isothermal amplification (LAMP) performance, possibly blocking sites on the BSTLF for binding of ß- and γ-phosphates of the dNTPs. Quantitative assessment showed that cations (Mg2+ and Mn2+) that complex the PPi product optimised enzyme activity. In malaria testing, the limit of detection depended on Plasmodium species and primer set. For example, 1000 copies of P. knowlesi 18S rRNA could be detected with the P.KNO-LAU primer set with Si-R52-mCh-FL-BSTLF , but 10 copies of P. ovale 18S rRNA could be detected with the P.OVA-HAN primer set using the same enzyme. The Si-immobilised BSTLF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R52-mCh-FL-BSTLF production was transferred from Cambridge to Accra and set up de novo for a trial with clinical samples. Different detection limits were found, targeting the mitochondrial DNA or the 18S rRNA gene for P. falciparum. The results are discussed in comparison with qPCR and sampling protocol and show that the Si-BSTLF polymerase can be optimised to meet the WHO recommended guidelines.

Ultrasensitive electrochemical genosensors for species-specific diagnosis of malaria.

The absence of reliable species-specific diagnostic tools for malaria at point-of-care (POC) remains a major setback towards effective disease management. This is partly due to the limited sensitivity and specificity of the current malaria POC diagnostic kits especially in cases of low-density parasitaemia and mixed species infections. In this study, we describe the first label-free DNA-based genosensors based on electrochemical impedance spectroscopy (EIS) for species-specific detection of P. falciparum, P. malariae and P. ovale. The limits of detection (LOD) for the three species-specific genosensors were down in attomolar concentrations ranging from 18.7 aM to 43.6 aM, which is below the detection limits of previously reported malaria genosensors. More importantly, the diagnostic performance of the three genosensors were compared to quantitative real-time polymerase chain reaction (qPCR) assays using purified genomic DNA and the paired whole blood lysates from clinical samples. Remarkably, all the qPCR-positive purified genomic DNA samples were correctly identified by the genosensors indicating 100% sensitivity for each of the three malaria species. The specificities of the three genosensors ranged from 66.7% to 100.0% with a Therapeutic Turnaround Time (TTAT) within 30 min, which is comparable to the TTAT of current POC diagnostic tools for malaria. This work represents a significant step towards the development of accurate and rapid species-specific nucleic acid-based toolkits for the diagnosis of malaria at the POC.

Plasmodium malariae and Plasmodium falciparum comparative susceptibility to antimalarial drugs in Mali.

OBJECTIVES: To evaluate Plasmodium malariae susceptibility to current and lead candidate antimalarial drugs. METHODS: We conducted cross-sectional screening and detection of all Plasmodium species malaria cases, which were nested within a longitudinal prospective study, and an ex vivo assessment of efficacy of a panel of antimalarials against P. malariae and Plasmodium falciparum, both PCR-confirmed mono-infections. Reference compounds tested included chloroquine, lumefantrine, artemether and piperaquine, while candidate antimalarials included the imidazolopiperazine GNF179, a close analogue of KAF156, and the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. RESULTS: We report a high frequency (3%-15%) of P. malariae infections with a significant reduction in ex vivo susceptibility to chloroquine, lumefantrine and artemether, which are the current frontline drugs against P. malariae infections. Unlike these compounds, potent inhibition of P. malariae and P. falciparum was observed with piperaquine exposure. Furthermore, we evaluated advanced lead antimalarial compounds. In this regard, we identified strong inhibition of P. malariae using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drug currently in clinical Phase IIb testing. Finally, in addition to GNF179, we demonstrated that the Plasmodium PI4K-specific inhibitor KDU691 is highly inhibitory against P. malariae and P. falciparum. CONCLUSIONS: Our data indicated that chloroquine, lumefantrine and artemether may not be suitable for the treatment of P. malariae infections and the potential of piperaquine, as well as new antimalarials imidazolopiperazines and PI4K-specific inhibitor, for P. malariae cure.

Recent Advances in the Development of Biosensors for Malaria Diagnosis.

The impact of malaria on global health has continually prompted the need to develop more effective diagnostic strategies that could overcome deficiencies in accurate and early detection. In this review, we examine the various biosensor-based methods for malaria diagnostic biomarkers, namely; Plasmodium falciparum histidine-rich protein 2 (PfHRP-2), parasite lactate dehydrogenase (pLDH), aldolase, glutamate dehydrogenase (GDH), and the biocrystal hemozoin. The models that demonstrate a potential for field application have been discussed, looking at the fabrication and analytical performance characteristics, including (but not exclusively limited to): response time, sensitivity, detection limit, linear range, and storage stability, which are first summarized in a tabular form and then described in detail. The conclusion summarizes the state-of-the-art technologies applied in the field, the current challenges and the emerging prospects for malaria biosensors.

tRNA epitranscriptomics and biased codon are linked to proteome expression in Plasmodium falciparum.

Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites.

Functional Characterization of Plasmodium falciparum Surface-Related Antigen as a Potential Blood-Stage Vaccine Target.

Plasmodium falciparum erythrocyte invasion is a multistep process that involves a spectrum of interactions that are not well characterized. We have characterized a 113-kDa immunogenic protein, PF3D7_1431400 (PF14_0293), that possesses coiled-coil structures. The protein is localized on the surfaces of both merozoites and gametocytes, hence the name Plasmodium falciparum surface-related antigen (PfSRA). The processed 32-kDa fragment of PfSRA binds normal human erythrocytes with different sensitivities to enzyme treatments. Temporal imaging from initial attachment to internalization of viable merozoites revealed that a fragment of PfSRA, along with PfMSP119, is internalized after invasion. Moreover, parasite growth inhibition assays showed that PfSRA P1 antibodies potently inhibited erythrocyte invasion of both sialic acid-dependent and -independent parasite strains. Also, immunoepidemiological studies show that malaria-infected populations have naturally acquired antibodies against PfSRA. Overall, the results demonstrate that PfSRA has the structural and functional characteristics of a very promising target for vaccine development.

P. falciparum RH5-Basigin interaction induces changes in the cytoskeleton of the host RBC.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.

PfRH2b specific monoclonal antibodies inhibit merozoite invasion.

Erythrocyte invasion by merozoite is a multistep process involving multiple ligand-receptor interactions. The Plasmodium falciparum reticulocyte binding protein homologues (PfRHs) consists of five functional members. The differential expression of PfRHs has been linked to the utilization of different invasion pathways by the merozoites as well as a mechanism of immune evasion. PfRHs are expressed at the apical end of merozoite and form interactions with distinct red blood cell (RBC) surface receptors that are important for successful invasion. Here we show that PfRH2b undergoes processing before and during merozoite invasion. The different processed fragments bind to chymotrypsin sensitive RBC surface receptors. We also show that PfRH2b follows the merozoite tight junction during invasion. Monoclonal antibodies (mAbs) inhibit merozoites invasion by blocking tight junction formation. mAbs binding to PfRH2b block merozoites intracellular Ca2+ signal necessary for EBA175 surface expression. The data suggests that a conserved function of PfRHs, where their interaction with RBC surface receptors facilitated recruitment of EBA175 and other tight junction proteins necessary for merozoite invasion by modulating merozoite intracellular Ca2+ signals.

Patterns of inflammatory responses and parasite tolerance vary with malaria transmission intensity.

BACKGROUND: In individuals living in malaria-endemic regions, parasitaemia thresholds for the onset of clinical symptoms vary with transmission intensity. The mechanisms that mediate this relationship are however, unclear. Since inflammatory responses to parasite infection contribute to the clinical manifestation of malaria, this study investigated inflammatory cytokine responses in children with malaria from areas of different transmission intensities (ranging from low to high). METHODS: Blood samples were obtained from children confirmed with malaria at community hospitals in three areas with differing transmission intensities. Cytokine levels were assessed using the Luminex®-based magnetic bead array system, and levels were compared across sites using appropriate statistical tests. The relative contributions of age, gender, parasitaemia and transmission intensity on cytokine levels were investigated using multivariate regression analysis. RESULTS: Parasite density increased with increasing transmission intensity in children presenting to hospital with symptomatic malaria, indicating that the parasitaemia threshold for clinical malaria increases with increasing transmission intensity. Furthermore, levels of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-6, IL-8, and IL-12, decreased with increasing transmission intensity, and correlated significantly with parasitaemia levels in the low transmission area but not in high transmission areas. Similarly, levels of anti-inflammatory cytokines, including IL-4, IL-7, IL-10 and IL-13, decreased with increasing transmission intensity, with IL-10 showing strong correlation with parasitaemia levels in the low transmission area. Multiple linear regression analyses revealed that transmission intensity was a stronger predictor of cytokine levels than age, gender and parasitaemia. CONCLUSION: Taken together, the data demonstrate a strong relationship between the prevailing transmission intensity, parasitaemia levels and the magnitude of inflammatory responses induced during clinical malaria.

A Disposable Amperometric Sensor Based on High-Performance PEDOT:PSS/Ionic Liquid Nanocomposite Thin Film-Modified Screen-Printed Electrode for the Analysis of Catechol in Natural Water Samples.

A conducting polymer-based composite material of poly(3,4-ethylenedioxythiophene) (PEDOT): poly(4-styrenesulfonate) (PSS) doped with different percentages of a room temperature ionic liquid (IL), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]), was prepared and a very small amount of the composite (2.0 µL) was drop-coated on the working area of a screen-printed carbon electrode (SPCE). The SPCE, modified with PEDOT:PSS/IL composite thin-film, was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), profilometry and sessile contact angle measurements. The prepared PEDOT:PSS/IL composite thin-film exhibited a nano-porous microstructure and was found to be highly stable and conductive with enhanced electrocatalytic properties towards catechol, a priority pollutant. The linear working range for catechol was found to be 0.1 µM-330.0 µM with a sensitivity of 18.2 mA·mM·cm-2 and a calculated limit of detection (based on 3× the baseline noise) of 23.7 µM. When the PEDOT:PSS/IL/SPCE sensor was used in conjunction with amperometry in stirred solution for the analysis of natural water samples, the precision values obtained on spiked samples (20.0 µM catechol added) (n = 3) were 0.18% and 0.32%, respectively, with recovery values that were well over 99.0%.

Cellular immune response to SARS-CoV-2 and clinical presentation in individuals exposed to endemic malaria.

Ghana and other parts of West Africa have experienced lower COVID-19 mortality rates than other regions. This phenomenon has been hypothesized to be associated with previous exposure to infections such as malaria. This study investigated the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the influence of previous malaria exposure. Blood samples were collected from individuals with asymptomatic or symptomatic COVID-19 (n = 217). A variety of assays were used to characterize the SARS-CoV-2-specific immune response, and malaria exposure was quantified using Plasmodium falciparum ELISA. The study found evidence of attenuated immune responses to COVID-19 among asymptomatic individuals, with elevated proportions of non-classical monocytes and greater memory B cell activation. Symptomatic patients displayed higher P. falciparum-specific T cell recall immune responses, whereas asymptomatic individuals demonstrated elevated P. falciparum antibody levels. Summarily, this study suggests that P. falciparum exposure-associated immune modulation may contribute to reduced severity of SARS-CoV-2 infection among people living in malaria-endemic regions.

Prevalence of Leucocytozoon infection in domestic birds in Ghana.

Leucocytozoon is a haemosporidian parasite known to cause leucocytozoonosis in domestic and wild birds in most parts of the world. It is an important pathogen, as some species can be pathogenic, especially in domestic birds. One of the factors affecting poultry health management worldwide is parasitism. However, the study of haemosporidian parasites in Ghana is still lacking. This study sought to assess the prevalence and diversity of Leucocytozoon parasites in domestic birds in Ghana. Blood samples were collected from domestic birds in Ghana's Bono and Eastern regions to screen for Leucocytozoon parasites. Thin blood smears were prepared for microscopy and DNA was extracted from whole blood kept in ethylenediaminetetraacetic acid (EDTA) tubes for PCR. Due to the large number of samples, real-time PCR was performed to amplify the conserved rDNA gene. Two different nested PCR protocols were performed on the positive samples obtained from real-time PCR results, to amplify a partial region of the mitochondrial cytochrome b gene and the amplicons were sequenced. Sequencing revealed six new lineages of Leucocytozoon sp. recovered in 976 individual domestic birds and these sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank. An overall Leucocytozoon prevalence of 11.6% was reported in all birds sampled. The most prevalent lineage LGHA146 (GenBank accession no. OM643346) (93.8%) was found infecting 3 bird species, Gallus gallus, Meleagris gallopavo, and Anas platyrhynchos. Phylogenetic analysis revealed that the new lineages (GenBank accession nos. OM643342, OM643343, OM643344, OM643345, OM643346, and OM643347), reported in this study were closely related to Leucocytozoon schoutedeni. We suggest that further studies be conducted to evaluate the effect of these parasite species on the general well-being of poultry in Ghana.

Comparative Susceptibility of Plasmodium ovale and Plasmodium falciparum Field Isolates to Reference and Lead Candidate Antimalarial Drugs in Ghana.

Malaria treatments resulted in the decline of the deadliest Plasmodium falciparum globally while species, such as P. ovale, infections have been increasingly detected across sub-Saharan Africa. Currently, no experimental drug sensitivity data are available to guide effective treatment and management of P. ovale infections, which is necessary for effective malaria elimination. We conducted a prospective study to evaluate P. ovale epidemiology over 1 year and determined ex vivo susceptibility of the field isolates to existing and lead advanced discovery antimalarial drugs. We report that while P. falciparum dominated both symptomatic and asymptomatic malaria cases, P. ovale in mono or co-infections caused 7.16% of symptomatic malaria. Frontline antimalarials artesunate and lumefantrine inhibited P. ovale as potently as P. falciparum. Chloroquine, which has been withdrawn in Ghana, was also highly inhibitory against both P. ovale and P. falciparum. In addition, P. ovale and P. falciparum displayed high susceptibility to quinine, comparable to levels observed with chloroquine. Pyrimethamine, which is a major drug for disease massive prevention, also showed great inhibition of P. ovale, comparable to effects on P. falciparum. Furthermore, we identified strong inhibition of P. ovale using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drugs currently in clinical phase II testing. We further demonstrated that the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor, KDU691, is highly inhibitory against P. ovale and P. falciparum field isolates. Our data indicated that existing and lead advanced discovery antimalarial drugs are suitable for the treatment of P. ovale infections in Ghana. IMPORTANCE Current malaria control and elimination tools such as drug treatments are not specifically targeting P.ovale. P. ovale can form hypnozoite and cause relapsing malaria. P. ovale is the third most dominant species in Africa and requires radical cure treatment given that it can form liver dormant forms called hypnozoites that escape all safe treatments. The inappropriate treatment of P. ovale would sustain its transmission in Africa where the medical need is the greatest. This is a hurdle for successful malaria control and elimination. Here, we provided experiment data that were lacking to guide P. ovale treatment and disease control policy makers using reference antimalarial drugs. We also provided key experimental data for 2 clinical candidate drugs that can be used for prioritization selection of lead candidate's identification for clinical development.

Development of an Affordable ELISA Targeting the SARS-CoV-2 Nucleocapsid and Its Application to Samples from the Ongoing COVID-19 Epidemic in Ghana.

INTRODUCTION: The true nature of the population spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations is often not fully known as most cases, particularly in Africa, are asymptomatic. Finding the true magnitude of SARS-CoV-2 spread is crucial to provide actionable data about the epidemiological progress of the disease for researchers and policymakers. This study developed and optimized an antibody enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid antigen expressed in-house using a simple bacterial expression system. METHODS: Nucleocapsid protein from SARS-CoV-2 was expressed and purified from Escherichia coli. Plasma samples used for the assay development were obtained from Ghanaian SARS-CoV-2 seropositive individuals during the pandemic, while seronegative controls were plasma samples collected from blood donors before the coronavirus disease 2019 (COVID-19) pandemic. Another set of seronegative controls was collected during the COVID-19 pandemic. Antibody detection and levels within the samples were validated using commercial kits and Luminex. Analyses were performed using GraphPad Prism, and the sensitivity, specificity and background cut-off were calculated. RESULTS AND DISCUSSION: This low-cost ELISA (£0.96/test) assay has a high prediction of 98.9%, and sensitivity and specificity of 97% and 99%, respectively. The assay was subsequently used to screen plasma from SARS-CoV-2 RT-PCR-positive Ghanaians. The assay showed no significant difference in nucleocapsid antibody levels between symptomatic and asymptomatic, with an increase of the levels over time. This is in line with our previous publication. CONCLUSION: This study developed a low-cost and transferable assay that enables highly sensitive and specific detection of human anti-SARS-CoV-2 IgG antibodies. This assay can be modified to include additional antigens and used for continuous monitoring of sero-exposure to SARS-CoV-2 in West Africa.

Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population.

Background: Erythrocyte invasion by P. falciparum involves functionally overlapping interactions between the parasite's ligands and the erythrocyte surface receptors. While some P. falciparum isolates necessarily engage the sialic acid (SA) moieties of the erythrocytes during the invasion, others use ligands whose binding is independent of SA for successful invasion. Deciphering the major pathway used by P. falciparum clinical isolates represent a key step toward developing an efficient blood stage malaria vaccine. Methods: We collected a total of 156 malaria-infected samples from Ghanaian children aged 2 to 14 years and used a two-color flow cytometry-based invasion assay to assess the invasion phenotype diversity of Ghanaian P. falciparum clinical isolates. Anti-human CR1 antibodies were used to determine the relative contribution of the PfRh4-CR1 interaction in the parasites invasion phenotype and RT-qPCR was used to assess the expression levels of key invasion-related ligands. Results: Our findings show no clear association between demographic or clinical data and existing reports on the malaria transmission intensity. The complete invasion data obtained for 156 isolates, showed the predominance of SA-independent pathways in Ghanaian clinical isolates. Isolates from Hohoe and Navrongo had the highest diversity in invasion profile. Our data also confirmed that the PfRh4-CR1 mediated alternative pathway is important in Ghanaian clinical isolates. Furthermore, the transcript levels of ten invasion-related genes obtained in the study showed little variations in gene expression profiles within and between parasite populations across sites. Conclusion: Our data suggest a low level of phenotypic diversity in Ghanaian clinical isolates across areas of varying endemicity and further highlight its importance in the quest for new intervention strategies, such as the investigation of blood-stage vaccine targets, particularly those targeting specific pathways and able to trigger the stimulation of broadly neutralizing invasion antibodies.

Short-term cryopreservation and thawing have minimal effects on Plasmodium falciparum ex vivo invasion profile.

Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites' invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.