West African Centre for Cell Biology of Infectious Pathogens
Permanent URI for this collectionhttp://197.255.125.131:4000/handle/123456789/30108
Browse
18 results
Search Results
Item Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana(nature microbiology, 2023) Girgis, S.T.; Adika, E.; Ngoi, J.M.; et al.Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide important information for malaria control policies, including detection of antimalarial drug resistance. However, genome sequencing capacity in malaria-endemic countries is limited. We designed and implemented an end-to-end workfow to detect Plasmodium falciparum antimalarial resistance markers and diversity in the vaccine target circumsporozoite protein (csp) using nanopore sequencing in Ghana. We analysed 196 clinical samples and showed that our method is rapid, robust, accurate and straightforward to implement. Importantly, our method could be applied to dried blood spot samples, which are readily collected in endemic settings. We report that P. falciparum parasites in Ghana are mostly susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine resistance and no evidence of artemisinin resistance. Multiple single nucleotide polymorphisms were identified in csp, but their significance is uncertain. Our study demonstrates the feasibility of nanopore sequencing for malaria genomic surveillance in endemic countries.Item Stepwise in vitro screening of MMV pathogen box compounds against Plasmodium falciparum to identify potent antimalarial candidates(International Journal for Parasitology: Drugs and Drug Resistance, 2023) Mbye, H.; Bojang, F.; Jaiteh, F.K.; et al.Development of resistance to deployed antimalarial drugs is inevitable and needs prompt and continuous dis covery of novel candidate drugs. Therefore, the antimalarial activity of 125 compounds from the Medicine for Malaria Ventures (MMV) pathogen box was determined. Combining standard IC50 and normalised growth rate inhibition (GR50) analyses, we found 16 and 22 compounds had higher potencies than CQ respectively. Seven compounds with relatively high potencies (low GR50 and IC50) against P. falciparum 3D7 were further analysed. Three of these were tested on 10 natural P. falciparum isolates from The Gambia using our newly developed parasite survival rate assay (PSRA). According to the IC50, GR50 and PSRA analyses, compound MMV667494 was most potent and highly cytotoxic to parasites. MMV010576 was slow acting but more potent than dihydroartemisinin (DHA) 72 h after exposure. MMV634140 was potent against the laboratory-adapted 3D7 isolate, but 4 out of 10 natural Gambian isolates survived and replicated slowly despite 72 h of exposure to the compound, suggesting potential drug tolerance and risk of resistance development. These results emphasise the usefulness of in vitro testing as a starting point for drug discovery. Improved approaches to data analyses and the use of natural isolates will facilitate the prioritisation of compounds for further clinical development.Item Stepwise in vitro screening of MMV pathogen box compounds against Plasmodium falciparum to identify potent antimalarial candidates(Elsevier Ltd, 2023) Mbye, H.; Bojang, F.; Amambua-Ngwa, A.; et al.ABSTRACT Development of resistance to deployed antimalarial drugs is inevitable and needs prompt and continuous dis covery of novel candidate drugs. Therefore, the antimalarial activity of 125 compounds from the Medicine for Malaria Ventures (MMV) pathogen box was determined. Combining standard IC50 and normalised growth rate inhibition (GR50) analyses, we found 16 and 22 compounds had higher potencies than CQ respectively. Seven compounds with relatively high potencies (low GR50 and IC50) against P. falciparum 3D7 were further analysed. Three of these were tested on 10 natural P. falciparum isolates from The Gambia using our newly developed parasite survival rate assay (PSRA). According to the IC50, GR50 and PSRA analyses, compound MMV667494 was most potent and highly cytotoxic to parasites. MMV010576 was slow acting but more potent than dihydroartemisinin (DHA) 72 h after exposure. MMV634140 was potent against the laboratory-adapted 3D7 isolate, but 4 out of 10 natural Gambian isolates survived and replicated slowly despite 72 h of exposure to the compound, suggesting potential drug tolerance and risk of resistance development. These results emphasise the usefulness of in vitro testing as a starting point for drug discovery. Improved approaches to data analyses and the use of natural isolates will facilitate the prioritisation of compounds for further clinical developmentItem Marmesin isolated from Celtis durandii Engl. root bioactive fraction inhibits β-hematin formation and contributes to antiplasmodial activity(Elsevier Ireland Ltd, 2023) Chirawurah, J.D.; Ezenyi, I.C.; Sahal, D.; et al.ABSTRACT Ethnopharmacological relevance: Malaria is a leading cause of death in many developing countries, especially in sub-Saharan Africa. Nigeria is endowed with an abundance of medicinal plants, many of which are used to treat malaria. Celtis durandii Engl. is one such plant used as a traditional antimalarial remedy in southeast Nigeria. However, its antiplasmodial potential is poorly explored. Aim of the study: The study aimed at identifying the antiplasmodial components of C. durandii root extract through antiplasmodial activity-guided fractionation. Materials and methods: Dichloromethane/methanol mixture extract (1:1 v/v) of C. durandii root was prepared and partitioned against water to obtain the organic phase, which was further separated by column chromatography into nine (C1 – C9) fractions. The antiplasmodial activity was evaluated by in vitro screening of the different fractions against drug-sensitive and drug-resistant Plasmodium falciparum strains. Further purification of the active column fractions resulted in a potent anti-Plasmodial compound that was subsequently investigated for its effect on β-hematin formation. Additionally, the isolated compound was characterized and identified as mar mesin using mass spectrometry and nuclear magnetic resonance spectroscopy. Results: Celtis durandii root extract exhibited promising antiplasmodial activity {IC50 (μg/ml) 5.92, 6.04, and 6.92} against PfW2mef, PfINDO, and Pf3D7 respectively. Pooled fractions with good antiplasmodial activity {IC50 (μg/ml) Pf3D7: 3.99; PfINDO: 2.24} and selectivity for the parasites (SI: 21) yielded a compound that was fourteen-fold potent in antiplasmodial activity against Pf3D7(IC50: 0.28 μg/ml). It also inhibited β-hematin formation with an IC50 = 150 μM. Further studies using spectral data, literature, and chemical databases identified the purified compound as marmesin. Conclusion: This work has demonstrated that Celtis durandii root extract has good antiplasmodial activity against drug-sensitive and drug-resistant P. falciparum. The inhibition of β-hematin formation by marmesin accounts in part for this activity.Item Individual-level variations in malaria susceptibility and acquisition of clinical protection(Wellcome Open Research, 2022) Valletta, J.J.; Addy, J.W.G.; Reid, A.J.; Ndungu, F.M.; Bediako, Y.; Mwacharo, J.; Mohammed, K.S.; Musyoki, J.; Ngoi, J.M.; Wambua, J.; Otieno, E.; Berriman, M.; Bejon, P.; Marsh, K.; Langhorne, J.; Newbold, C.I.; Recker, M.After decades of research, our understanding of when and why individuals infected with Plasmodium falciparum develop clinical malaria is still limited. Correlates of immune protection are often sought through prospective cohort studies, where measured host factors are correlated against the incidence of clinical disease over a set period of time. However, robustly inferring individual-level protection from these population-level findings has proved difficult due to small effect sizes and high levels of variance underlying such data. In order to better understand the nature of these interindividual variations, we analysed the long-term malaria epidemiology of children ≤12 years old growing up under seasonal exposure to the parasite in the sub-location of Junju, Kenya. Despite the cohort’s limited geographic expanse (ca. 3km x 10km), our data reveal a high degree of spatial and temporal variability in malaria prevalence and incidence rates, causing individuals to experience varying levels of exposure to the parasite at different times during their life. Analysing individual-level infection histories further reveal an unexpectedly high variability in the rate at which children experience clinical malaria episodes. Besides exposure to the parasite, measured as disease prevalence in the surrounding area, we find that the birth time of year has an independent effect on the individual’s risk of experiencing a clinical episode. Furthermore, our analyses reveal that those children with a history of an above average number of episodes are more likely to experience further episodes during the upcoming transmission season. These findings are indicative of phenotypic differences in the rates by which children acquire clinical protection to malaria and offer important insights into the natural variability underlying malaria epidemiology.Item 10-year longitudinal study of malaria in children: Insights into acquisition and maintenance of naturally acquired immunity(Wellcome Open Research, 2022) Addy, J.W.G.; Bediako, Y.; Ndungu, F.M.; Valetta, J.J.; Reid, A.J.; Mwacharo, J.; Ngoi, J.M.; Wambua, J.; Otieno, E.; Musyoki, J.; Mohammed, K.S.; Berriman, M.; Marsh, K.; Bejon, P.; Recker, M.; Langhorne, J.Studies of long-term malaria cohorts have provided essential insights into how Plasmodium falciparum interacts with humans, and influences the development of antimalarial immunity. Immunity to malaria is acquired gradually after multiple infections, some of which present with clinical symptoms. However, there is considerable variation in the number of clinical episodes experienced by children of the same age within the same cohort. Understanding this variation in clinical symptoms and how it relates to the development of naturally acquired immunity is crucial in identifying how and when some children stop experiencing further malaria episodes. Where variability in clinical episodes may result from different rates of acquisition of immunity, or from variable exposure to the parasite. Methods: Using data from a longitudinal cohort of children residing in an area of moderate P. falciparum transmission in Kilifi district, Kenya, we fitted cumulative episode curves as monotonic-increasing splines, to 56 children under surveillance for malaria from the age of 5 to 15. Results: There was large variability in the accumulation of numbers of clinical malaria episodes experienced by the children, despite being of similar age and living in the same general location. One group of children from a particular sub-region of the cohort stopped accumulating clinical malaria episodes earlier than other children in the study. Despite lack of further clinical episodes of malaria, these children had higher asymptomatic parasite densities and higher antibody titres to a panel of P. falciparum blood-stage antigens. Conclusions: This suggests development of clinical immunity rather than lack of exposure to the parasite, and supports the view that this immunity to malaria disease is maintained by a greater exposure to P. falciparum, and thus higher parasite burdens. Our study illustrates the complexity of anti-malaria immunity and underscores the need for analyses which can sufficiently reflect the heterogeneity within endemic populations.Item ICAM‑1 Kilifi variant is not associated with cerebral and severe malaria pathogenesis in Beninese children(BMC, 2022) Blankson, S.O.; Dadjé, D.S.; Traikia, N.; Alao, M.J.; Ayivi, S.; Amoussou, A.; Deloron, P.; Ndam, N.T.; Milet, J.; Basco, L.K.; Aniweh, Y.; Tahar, R.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.Item Plasmodium falciparum Malaria Parasites in Ghana Show Signatures of Balancing Selection at Artemisinin Resistance Predisposing Background Genes(SAGE, 2021) Tandoh, K.Z.; Amenga-Etego, L.; Quashie, N.B.; Awandare, G.; Wilson, M.; Duah-Quashie, N.O.Sub-Saharan Africa is courting the risk of artemisinin resistance (ARTr) emerging in Plasmodium falciparum malaria parasites. Current molecular surveillance efforts for ARTr have been built on the utility of P. falciparum kelch13 (pfk13) validated molecular markers. However, whether these molecular markers will serve the purpose of early detection of artemisinin-resistant parasites in Ghana is hinged on a pfk13 dependent evolution. Here, we tested the hypothesis that the background pfk13 genome may be present before the pfk13 ARTr-conferring variant(s) is selected and that signatures of balancing selection on these genomic loci may serve as an early warning signal of ARTr. We analyzed 12198 single nucleotide polymorphisms (SNPs) in Ghanaian clinical isolates in the Pf3K MalariaGEN dataset that passed a stringent filter ing regimen. We identified signatures of balancing selection in 2 genes (phosphatidylinositol 4-kinase and chloroquine resistance transporter) previously reported as background loci for ARTr. These genes showed statistically significant and high positive values for Tajima’s D, Fu and Li’s F, and Fu and Li’s D. This indicates that the biodiversity required to establish a pfk13 background genome may have been primed in clinical isolates of P. falciparum from Ghana as of 2010. Despite the absence of ARTr in Ghana to date, our finding supports the current use of pfk13 for molecular surveillance of ARTr in Ghana and highlights the potential utility of monitoring malaria parasite populations for balancing selection in ARTr precursor background genes as early warning molecular signatures for the emergence of ARTr.Item Genomic analysis reveals independent evolution of Plasmodium falciparum populations in Ethiopia(Malaria Journal, 2021) Abera, D.; Kibet, C.K.; Degefa, T.; Amenga‑Etego, L.; Bargul, J.L.; Golassa, L.Background: Plasmodium falciparum parasite populations in Ethiopia have been experiencing local selective pressures from drugs and immunity, leading to evolutionary adaptation. However, there was a paucity of data on genomic characterization and evolutionary adaptations of P. falciparum isolates from the central area of Ethiopia. Methods: Whole-genome analysis of 25 P. falciparum isolates from central Ethiopia, specifcally from West Arsi, were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles against global isolates from Cambodia, Thailand, DR Congo, and Malawi. Results: A total of 18,517 high-quality single-nucleotide polymorphisms (SNPs) were identifed in Ethiopian P. falciparum isolates. About 84% of the Ethiopian P. falciparum isolates had a FWS value>0.95 showing a dominant single genotype infection in most isolates at the time of collection with little potential for out-crossing as expected in areas with low transmission intensity. Within-host diversity of Ethiopian infections was signifcantly diferent from East African (p<0.001), but not Southeast Asian infections (P>0.05). A signifcant population structure has been observed by PCA and population diferentiation between Ethiopian parasites and East African (Fst~10%) and Southeast Asian populations (Fst~18%), suggesting limited gene fow and the independent evolution of the Ethiopian parasite population. Moreover, a total of 125 genes under balancing selection was found that include ama1, trap, eba175, and lsa3, previously identifed as targets of human host immunity. Recent directional selection analysis using integrated standardized haplotype score (IHS) did not detect any selection signatures in the Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. However, known drug resistance-conferring mutations analysis showed that at least one SNP marker was fIxed in these genes, but not in Pfdhps and PfK13. Conclusion: Plasmodium falciparum populations in the central region of Ethiopia was structurally diverged from both Southeast Asian and other East African populations. Malaria infections in Ethiopia had low within-host diversity, and parasites carry fIxed chloroquine resistance markers despite the withdrawal of this drug for the treatment of P. falciparum.Item Cell trace far-red is a suitable erythrocyte dye for multi-color Plasmodium falciparum invasion phenotyping assays(Experimental Biology and Medicine, 2020-02-05) Thiam, L.G.; Aniweh, Y.; Quansah, E.B.; Donkor, J.K.; Gwira, T.M.; Kusi, K.A.; Niang, M.; Awandare, G.A.Plasmodium falciparum erythrocyte invasion phenotyping assays are a very useful tool for assessing parasite diversity and virulence, and for characterizing the formation of ligand– receptor interactions. However, such assays need to be highly sensitive and reproducible, and the selection of labeling dyes for differentiating donor and acceptor erythrocytes is a critical factor. We investigated the suitability of cell trace far-red (CTFR) as a dye for P. falciparum invasion phenotyping assays. Using the dyes carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and dichloro dimethyl acridin one succinimidyl ester (DDAO-SE) as comparators, we used a dye-dilution approach to assess the limitations and specific staining procedures for the applicability of CTFR in P. falciparum invasion phenotyping assays. Our data show that CTFR effectively labels acceptor erythrocytes and provides a stable fluorescent intensity at relatively low concentrations. CTFR also yielded a higher fluorescence intensity relative to DDAO-SE and with a more stable fluorescence intensity over time. Furthermore, CTFR did not affect merozoites invasion of erythrocytes and was not toxic to the parasite’s intraerythrocytic development. Additionally, CTFR offers flexibility in the choice of combinations with several other DNA dyes, which broaden its usage for P. falciparum erythrocyte invasion assays, considering a wider range of flow cytometers with various laser settings.