Browsing by Subject "Plasmodium falciparum"

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    Absence of kelch13 artemisinin resistance markers but strong selection for lumefantrine-tolerance molecular markers following 18 years of artemisinin-based combination therapy use in Mpumalanga Province, South Africa (2001–2018)
    (2019-08-22) Raman, Jaishree; Kagoro, Frank M; Mabuza, Aaron; Malatje, Gillian; Reid, Anthony; Frean, John; Barnes, Karen I
    Abstract Background The ability of Plasmodium falciparum parasites to develop resistance to widely used anti-malarials threatens malaria control and elimination efforts. Regular drug efficacy monitoring is essential for ensuring effective treatment policies. In low transmission settings where therapeutic efficacy studies are often not feasible, routine surveillance for molecular markers associated with anti-malarial resistance provides an alternative for the early detection of emerging resistance. Such a longitudinal survey of changes in the prevalence of selected molecular markers of resistance was conducted in the malaria-endemic regions of Mpumalanga Province, South Africa, where malaria elimination at a district-level is being pursued. Methods Molecular analyses to determine the prevalence of alleles associated with resistance to lumefantrine (mdr86N, crt76K and mdr1 copy number variation) and sulfadoxine–pyrimethamine (dhfr triple, dhps double, SP quintuple) were conducted between 2001 and 2018, while artemisinin resistance markers (kelch13 mutations) were assessed only in 2018. Results Parasite DNA was successfully amplified from 1667/2393 (70%) of malaria-positive rapid diagnostic tests routinely collected at primary health care facilities. No artemisinin resistance-associated kelch13 mutations nor amplification of the mdr1 gene copy number associated with lumefantrine resistance were observed. However, prevalence of both the mdr86N and crt76K alleles increased markedly over the study period, with all isolates collected in 2018 carrying these markers. SP quintuple mutation prevalence increased steadily from 14% in 2001 to 96% in 2018. Mixed alleles at any of the codons assessed were rare by 2018. Conclusion No kelch13 mutations confirmed or suspected to be associated with artemisinin resistance were identified in 2018. Although parasites carrying the mdr86N and crt76K alleles associated with reduced lumefantrine susceptibility were strongly selected for over the study period, nearing fixation by 2018, the marker for lumefantrine resistance, namely increased mdr1 copy number, was not observed in this study. The increase in mdr86N and crt76K allele prevalence together with intense regional artemether–lumefantrine drug pressure, raises concern regarding the sustained artemether–lumefantrine efficacy. Regular, rigorous anti-malarial resistance marker surveillance across all three South African malaria-endemic provinces to inform case management is recommended.
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    Acquired immune responses to three malaria vaccine candidates and their relationship to invasion inhibition in two populations naturally exposed to malaria
    (BioMed Central, 2016-02-05) Addai-Mensah, Otchere; Seidel, Melanie; Amidu, Nafiu; Maskus, Dominika J; Kapelski, Stephanie; Breuer, Gudrun; Franken, Carmen; Owusu-Dabo, Ellis; Frempong, Margaret; Rakotozandrindrainy, Raphaël; Schinkel, Helga; Reimann, Andreas; Klockenbring, Torsten; Barth, Stefan; Fischer, Rainer; Fendel, Rolf
    Background: Malaria still represents a major cause of morbidity and mortality predominantly in several developing countries, and remains a priority in many public health programmes. Despite the enormous gains made in control and prevention the development of an effective vaccine represents a persisting challenge. Although several para site antigens including pre-erythrocytic antigens and blood stage antigens have been thoroughly investigated, the identification of solid immune correlates of protection against infection by Plasmodium falciparum or clinical malaria remains a major hurdle. In this study, an immuno-epidemiological survey was carried out between two populations naturally exposed to P. falciparum malaria to determine the immune correlates of protection. Methods: Plasma samples of immune adults from two countries (Ghana and Madagascar) were tested for their reactivity against the merozoite surface proteins MSP1-19, MSP3 and AMA1 by ELISA. The antigens had been selected on the basis of cumulative evidence of their role in anti-malarial immunity. Additionally, reactivity against crude P. falciparum lysate was investigated. Purified IgG from these samples were furthermore tested in an invasion inhibition assay for their antiparasitic activity. Results: Significant intra- and inter- population variation of the reactivity of the samples to the tested antigens were found, as well as a significant positive correlation between MSP1-19 reactivity and invasion inhibition (p < 0.05). Interestingly, male donors showed a significantly higher antibody response to all tested antigens than their female counterparts. In vitro invasion inhibition assays comparing the purified antibodies from the donors from Ghana and Madagascar did not show any statistically significant difference. Although in vitro invasion inhibition increased with breadth of antibody response, the increase was not statistically significant. Conclusions: The findings support the fact that the development of semi-immunity to malaria is probably con tingent on the development of antibodies to not only one, but a range of antigens and that invasion inhibition in immune adults may be a function of antibodies to various antigens. This supports strategies of vaccination including multicomponent vaccines as well as passive vaccination strategies with antibody cocktails.
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    Evaluation of Plasmodium falciparum gametocyte detection in different patient material
    (BioMed Central Ltd, 2013) Kast, Katharina; Berens-Riha, Nicole; Zeynudin, Ahmed; Abduselam, Nuredin; Eshetu, Teferi; Loscher, Thomas; Wieser, Andreas; Shock, Jonathan; Pritsch, Michael
    BACKGROUND:For future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. Sensitive, non-invasive methods to detect gametocytes under field conditions can play a role in monitoring transmission potential. METHODS: Microscopically Plasmodium falciparum-positive patients from Jimma, Ethiopia donated finger-prick blood, venous blood, saliva, oral mucosa and urine samples that were spotted on filter paper or swabs. All samples were taken and stored under equal, standardized conditions. RNA was extracted from the filter paper and detected by real-time QT-NASBA. Pfs16-mRNA and Pfs25-mRNA were measured with a time to positivity to detect gametocyte specific mRNA in different gametocyte stages. They were compared to 18S-rRNA, which is expressed in all parasite stages. Results were quantified via a known dilution series of artificial RNA copies. RESULTS: Ninety-six samples of 16 uncomplicated malaria patients were investigated. 10 (66.7%) of the slides showed gametocyte densities between 0.3-2.9 gametocytes/mul. For all RNA-targets, molecular detection in blood samples was most sensitive; finger-prick sampling required significantly smaller amounts of blood than venous blood collection. Detection of asexual 18S-rRNA in saliva and urine showed sensitivities of 80 and 67%, respectively. Non-invasive methods to count gametocytes proved insensitive. Pfs16-mRNA was detectable in 20% of urine samples, sensitivities for other materials were lower. Pfs25-mRNA was not detectable in any sample. CONCLUSIONS: The sensitivity of non-invasively collected material such as urine, saliva or mucosa seems unsuitable for the detection of gametocyte-specific mRNA. Sensitivity in asymptomatic carriers might be generally even lower. Finger-prick testing revealed the highest absolute count of RNA copies per muL, especially for Pfs25-mRNA copies. The method proved to be the most effective and should preferably be applied in future transmission control and eradication plans. A rapid test for gametocyte targets would simplify efforts.
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    Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum
    (BioMed Central Ltd, 2013) Le Manach, Claire; Scheurer, Christian; Sax, Sibylle; Schleiferbock, Sarah; Cabrera, Diego; Younis, Yassir; Paquet, Tanya; Street, Leslie; Smith, Peter; Ding, Xavier; Waterson, David; Witty, Michael; Leroy, Didier; Chibale, Kelly; Wittlin, Sergio
    BACKGROUND: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results. METHODS: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay. RESULTS: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting. CONCLUSION: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.
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    Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data
    (2016)
    : Gametocytes are responsible for transmission of malaria from human to mosquito. Artemisinin combination therapy (ACT) reduces post-treatment gametocyte carriage, dependent upon host, parasite and pharmacodynamic factors. The gametocytocidal properties of antimalarial drugs are important for malaria elimination efforts. An individual patient clinical data meta-analysis was undertaken to identify the determinants of gametocyte carriage and the comparative effects of four ACTs: artemether-lumefantrine (AL), artesunate/amodiaquine (AS-AQ), artesunate/mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DP). : Factors associated with gametocytaemia prior to, and following, ACT treatment were identified in multivariable logistic or Cox regression analysis with random effects. All relevant studies were identified through a systematic review of PubMed. Risk of bias was evaluated based on study design, methodology, and missing data. : The systematic review identified 169 published and 9 unpublished studies, 126 of which were shared with the WorldWide Antimalarial Resistance Network (WWARN) and 121 trials including 48,840 patients were included in the analysis. Prevalence of gametocytaemia by microscopy at enrolment was 12.1 % (5887/48,589), and increased with decreasing age, decreasing asexual parasite density and decreasing haemoglobin concentration, and was higher in patients without fever at presentation. After ACT treatment, gametocytaemia appeared in 1.9 % (95 % CI, 1.7-2.1) of patients. The appearance of gametocytaemia was lowest after AS-MQ and AL and significantly higher after DP (adjusted hazard ratio (AHR), 2.03; 95 % CI, 1.24-3.12; P = 0.005 compared to AL) and AS-AQ fixed dose combination (FDC) (AHR, 4.01; 95 % CI, 2.40-6.72; P < 0.001 compared to AL). Among individuals who had gametocytaemia before treatment, gametocytaemia clearance was significantly faster with AS-MQ (AHR, 1.26; 95 % CI, 1.00-1.60; P = 0.054) and slower with DP (AHR, 0.74; 95 % CI, 0.63-0.88; P = 0.001) compared to AL. Both recrudescent (adjusted odds ratio (AOR), 9.05; 95 % CI, 3.74-21.90; P < 0.001) and new (AOR, 3.03; 95 % CI, 1.66-5.54; P < 0.001) infections with asexual-stage parasites were strongly associated with development of gametocytaemia after day 7. : AS-MQ and AL are more effective than DP and AS-AQ FDC in preventing gametocytaemia shortly after treatment, suggesting that the non-artemisinin partner drug or the timing of artemisinin dosing are important determinants of post-treatment gametocyte dynamics.
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    Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity
    (2021-01-11) Seidel-Greven, Melanie; Addai-Mensah, Otchere; Spiegel, Holger; Chiegoua Dipah, Gwladys N; Schmitz, Stefan; Breuer, Gudrun; Frempong, Margaret; Reimann, Andreas; Klockenbring, Torsten; Fischer, Rainer; Barth, Stefan; Fendel, Rolf
    Abstract Background Plasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirixâ„¢ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates. Material and methods Using antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1–3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays. Results The selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2. Conclusion We have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.
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    Making data map-worthy—enhancing routine malaria data to support surveillance and mapping of Plasmodium falciparum anti-malarial resistance in a pre-elimination sub-Saharan African setting: a molecular and spatiotemporal epidemiology study
    (2022-06-29) Kagoro, Frank M.; Allen, Elizabeth; Mabuza, Aaron; Workman, Lesley; Magagula, Ray; Kok, Gerdalize; Davies, Craig; Malatje, Gillian; Guérin, Philippe J; Dhorda, Mehul; Maude, Richard J; Raman, Jaishree; Barnes, Karen I
    Background Independent emergence and spread of artemisinin-resistant Plasmodium falciparum malaria have recently been confirmed in Africa, with molecular markers associated with artemisinin resistance increasingly detected. Surveillance to promptly detect and effectively respond to anti-malarial resistance is generally suboptimal in Africa, especially in low transmission settings where therapeutic efficacy studies are often not feasible due to recruitment challenges. However, these communities may be at higher risk of anti-malarial resistance. Methods From March 2018 to February 2020, a sequential mixed-methods study was conducted to evaluate the feasibility of the near-real-time linkage of individual patient anti-malarial resistance profiles with their case notifications and treatment response reports, and map these to fine scales in Nkomazi sub-district, Mpumalanga, a pre-elimination area in South Africa. Results Plasmodium falciparum molecular marker resistance profiles were linked to 55.1% (2636/4787) of notified malaria cases, 85% (2240/2636) of which were mapped to healthcare facility, ward and locality levels. Over time, linkage of individual malaria case demographic and molecular data increased to 75.1%. No artemisinin resistant validated/associated  Kelch-13 mutations were detected in the 2385 PCR positive samples. Almost all 2812 samples assessed for lumefantrine susceptibility carried the wildtype mdr86ASN and crt76LYS alleles, potentially associated with decreased lumefantrine susceptibility. Conclusion Routine near-real-time mapping of molecular markers associated with anti-malarial drug resistance on a fine spatial scale provides a rapid and efficient early warning system for emerging resistance. The lessons learnt here could inform scale-up to provincial, national and regional malaria elimination programmes, and may be relevant for other antimicrobial resistance surveillance.
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    Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding
    (2021-02-23) Makumire, Stanley; Dongola, Tendamudzimu Harmfree; Chakafana, Graham; Tshikonwane, Lufuno; Chauke, Cecilia Tshikani; Maharaj, Tarushai; Zininga, Tawanda; Shonhai, Addmore
    Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.
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    Nonlinear mixed effects modeling of gametocyte carriage in patients with uncomplicated malaria
    (BioMed Central Ltd, 2010) Distiller, Greg; Little, Francesca; Barnes, Karen
    BACKGROUND:Gametocytes are the sexual form of the malaria parasite and the main agents of transmission. While there are several factors that influence host infectivity, the density of gametocytes appears to be the best single measure that is related to the human host's infectivity to mosquitoes. Despite the obviously important role that gametocytes play in the transmission of malaria and spread of anti-malarial resistance, it is common to estimate gametocyte carriage indirectly based on asexual parasite measurements. The objective of this research was to directly model observed gametocyte densities over time, during the primary infection. METHODS: Of 447 patients enrolled in sulphadoxine-pyrimethamine therapeutic efficacy studies in South Africa and Mozambique, a subset of 103 patients who had no gametocytes pre-treatment and who had at least three non-zero gametocyte densities over the 42-day follow up period were included in this analysis. RESULTS: A variety of different functions were examined. A modified version of the critical exponential function was selected for the final model given its robustness across different datasets and its flexibility in assuming a variety of different shapes. Age, site, initial asexual parasite density (logged to the base 10), and an empirical patient category were the co-variates that were found to improve the model. CONCLUSIONS: A population nonlinear modeling approach seems promising and produced a flexible function whose estimates were stable across various different datasets. Surprisingly, dihydrofolate reductase and dihydropteroate synthetase mutation prevalence did not enter the model. This is probably related to a lack of power (quintuple mutations n = 12), and informative censoring; treatment failures were withdrawn from the study and given rescue treatment, usually prior to completion of follow up.
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    Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials
    (BioMed Central Ltd, 2015) Combrinck, Jill; Fong, Kim; Gibhard, Liezl; Smith, Peter; Wright, David; Egan, Timothy
    BACKGROUND: The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes. METHODS: All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5%v/v, pH7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method. RESULTS: A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem. CONCLUSIONS: This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.
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    Safety and tolerability of single low-dose primaquine in a low-intensity transmission area in South Africa: an open-label, randomized controlled trial
    (2019-06-24) Raman, Jaishree; Allen, Elizabeth; Workman, Lesley; Mabuza, Aaron; Swanepoel, Hendrik; Malatje, Gillian; Frean, John; Wiesner, Lubbe; Barnes, Karen I
    Abstract Background To reduce onward falciparum malaria transmission, the World Health Organization recommends adding single low-dose (SLD) primaquine to artemisinin-based combination treatment in low transmission areas. However, uptake of this recommendation has been relatively slow given concerns about whether individual risks justify potential community benefit. This study was undertaken to generate comprehensive local data on the risk–benefit profile of SLD primaquine deployment in a pre-elimination area in South Africa. Methods This randomized, controlled open-label trial investigated adding a single low primaquine dose on day 3 to standard artemether–lumefantrine treatment for uncomplicated falciparum malaria. Efficacy, safety and tolerability of artemether–lumefantrine and primaquine treatment were assessed on days 3, 7, 14, 28 and 42. Lumefantrine concentrations were assayed from dried blood spot samples collected on day 7. Results Of 217 patients screened, 166 were enrolled with 140 randomized on day 3, 70 to each study arm (primaquine and no primaquine). No gametocytes were detected by either microscopy or PCR in any of the follow-up samples collected after randomization on day 3, precluding assessment of primaquine efficacy. Prevalence of the CYP2D6*4, CYP2D6*10 and CYP2D6*17 mutant alleles was low with allelic frequencies of 0.02, 0.11 and 0.16, respectively; none had the CYP2D6*4/*4 variant associated with null activity. Among 172 RDT-positive patients G6PD-genotyped, 24 (14%) carried the G6PD deficient (A−) variant. Median haemoglobin concentrations were similar between treatment arms throughout follow-up. A third of participants had a haemoglobin drop > 2 g/dL; this was not associated with primaquine treatment but may be associated with G6PD genotype [52.9% (9/17) with A− genotype vs. 31% (36/116) with other genotypes (p = 0.075)]. Day 7 lumefantrine concentrations and the number and nature of adverse events were similar between study arms; only one serious adverse event occurred (renal impairment in the no primaquine arm). The artemether–lumefantrine PCR-corrected adequate clinical and parasitological response rate was 100%, with only one re-infection found among the 128 patients who completed 42-day follow-up. Conclusions Safety, tolerability, CYP2D6 and G6PD variant data from this study support the deployment of the WHO-recommended SLD primaquine without G6PD testing to advance malaria elimination in South African districts with low-intensity residual transmission. Trial registration Pan African Clinical Trial Registry, PACTR201611001859416. Registered 11 November 2016, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1859
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    Single low-dose primaquine for blocking transmission of Plasmodium falciparum malaria – a proposed model-derived age-based regimen for sub-Saharan Africa
    (2018) Taylor, W Robert; Naw, Htee Khu; Maitland, Kathryn; Williams, Thomas N; Kapulu, Melissa; D’Alessandro, Umberto; Berkley, James A; Bejon, Philip; Okebe, Joseph; Achan, Jane; Amambua, Alfred Ngwa; Affara, Muna; Nwakanma, Davis; van Geertruyden, Jean-Pierre; Mavoko, Muhindo; Lutumba, Pascal; Matangila, Junior; Brasseur, Philipe; Piola, Patrice; Randremanana, Rindra; Lasry, Estrella; Fanello, Caterina; Onyamboko, Marie; Schramm, Birgit; Yah, Zolia; Jones, Joel; Fairhurst, Rick M; Diakite, Mahamadou; Malenga, Grace; Molyneux, Malcolm; Rwagacondo, Claude; Obonyo, Charles
    BACKGROUND: In 2012, the World Health Organization recommended blocking the transmission of Plasmodium falciparum with single low-dose primaquine (SLDPQ, target dose 0.25 mg base/kg body weight), without testing for glucose-6-phosphate dehydrogenase deficiency (G6PDd), when treating patients with uncomplicated falciparum malaria. We sought to develop an age-based SLDPQ regimen that would be suitable for sub-Saharan Africa. METHODS: Using data on the anti-infectivity efficacy and tolerability of primaquine (PQ), the epidemiology of anaemia, and the risks of PQ-induced acute haemolytic anaemia (AHA) and clinically significant anaemia (CSA), we prospectively defined therapeutic-dose ranges of 0.15-0.4 mg PQ base/kg for children aged 1-5 years and 0.15-0.5 mg PQ base/kg for individuals aged ≥6 years (therapeutic indices 2.7 and 3.3, respectively). We chose 1.25 mg PQ base for infants aged 6-11 months because they have the highest rate of baseline anaemia and the highest risks of AHA and CSA. We modelled an anthropometric database of 661,979 African individuals aged ≥6 months (549,127 healthy individuals, 28,466 malaria patients and 84,386 individuals with other infections/illnesses) by the Box-Cox transformation power exponential and tested PQ doses of 1-15 mg base, selecting dosing groups based on calculated mg/kg PQ doses. RESULTS: From the Box-Cox transformation power exponential model, five age categories were selected: (i) 6-11 months (n = 39,886, 6.03%), (ii) 1-5 years (n = 261,036, 45.46%), (iii) 6-9 years (n = 20,770, 3.14%), (iv) 10-14 years (n = 12,155, 1.84%) and (v) ≥15 years (n = 328,132, 49.57%) to receive 1.25, 2.5, 5, 7.5 and 15 mg PQ base for corresponding median (1st and 99th centiles) mg/kg PQ base of: (i) 0.16 (0.12-0.25), (ii) 0.21 (0.13-0.37), (iii) 0.25 (0.16-0.38), (iv) 0.26 (0.15-0.38) and (v) 0.27 (0.17-0.40). The proportions of individuals predicted to receive optimal therapeutic PQ doses were: 73.2 (29,180/39,886), 93.7 (244,537/261,036), 99.6 (20,690/20,770), 99.4 (12,086/12,155) and 99.8% (327,620/328,132), respectively. CONCLUSIONS: We plan to test the safety of this age-based dosing regimen in a large randomised placebo-controlled trial (ISRCTN11594437) of uncomplicated falciparum malaria in G6PDd African children aged 0.5 - 11 years. If the regimen is safe and demonstrates adequate pharmacokinetics, it should be used to support malaria elimination.
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    Soluble expression of plasmodium falciparum glutamine synthetase and three-dimensional structure by single particle reconstruction
    (2015) Patel, Satishkumar Ishverlal; Sewell, Trevor
    [No subject] Malaria infection caused by the apicomplexa pathogen Plasmodium falciparum has a high rate of resistance to existing anti-malarial drugs. The World Health Organisation recommended interventions are unlikely to eliminate the growth of resistance and it would therefore be prudent to continue the search for new drug targets for the continued combatting of malaria. Plasmodium falciparum is parasitic on the host for its metabolites and therefore inhibiting the transportation of glutamine from the host, has long been considered a potential strategy for combating the spread of infection. The recently sequenced Plasmodium falciparum genome has however shown that pathways for independent survival are also conserved. Therefore, combating the spread of Plasmodium falciparum in the human host, in addition to inhibiting the transportation of glutamine, will also require the inhibition of the de novo expression of essential amino acids within the Plasmodium falciparum cell. This could be achieved by inhibiting the glutamine synthetase gene, which is an essential step in the tri-carboxylic acid cycle.
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    Studies on in vitro antiplasmodial activity of cleome rutidosperma
    (2010) Bose, Anindya; Lategan, Carmen Abriette; Smith, Peter J; Gupta, Jayanta Kumar
    Malaria is a protozoal disease transmitted by the Anopheles mosquito, caused by minute parasitic protozoa of the genus Plasmodium, which infect human and insect hosts alternatively. It affects over 40% of the worldís population, with 120 million cases reported, and about 2 million deaths annually (1). The P. falciparum variety of the parasite accounts for 80% of cases and 90% of deaths caused by malaria. The declining efficacy of classical medication in relation to the rapid increase of parasite resistant strains, mainly of Plasmodium falciparum, as well as the greater resistance of vectors to insecticides, and the difficulty of creating efficient vaccines have led to an urgent need for new efficient antimalarial drugs (2, 3). Natural molecules may provide innovative strategies towards malarial control, hence active research groups are now working to develop new active compounds as an alternative to chloroquine, especially from artemisinin (4, 5), a plant-based antimalaria drug isolated from the Chinese plant Artemisia annua (6). Plants may well, therefore, prove to be the sources of new antimalarial in view of the success with the two important chemotherapeutic agents, quinine and artemisinin, both of which are derived from plants. Cleome rutidosperma (Capparidaceae) is a low-growing herb, up to 70 cm tall, found in waste grounds and grassy places with trifoliate leaves and small, violet-blue flowers, which turn pink as they age. The elongated capsules display the asymmetrical, dull black seeds. The plant is native to West Africa, although it has become naturalized in various parts of tropical America as well as Southeast Asia (7, 8). The diuretic, laxative, anthelmintic, antimicrobial, analgesic, anti-inflammatory, antipyretic, antioxidant and free radical scavenging activities of Cleome rutidosperma were reported earlier by the authors (9-13). The plant is used as antimalarial by the traditional healers in Cameroon and mild antiplasmodial activity of chloroform/methanol (1:1) extract of leaves of Cleome rutidosperma against chloroquine-sensitive (F32) laboratory strain of P. falciparum was reported earlier in Cameroon (14). The present study investigates the in vitro antiplasmodial activity of ethanolic extract and its fractions of aerial parts of Cleome rutidosperma against the chloroquine sensitive (CQS) D10 strain of the parasite, as well as their toxicity against a mammalian cell lines.
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    Open Access
    Synthesis and investigation of benzimidazole and carbazole ß-haematin inhibiting scaffolds with antimalarial activity
    (2018) L'abbate, Fabrizio P; Egan, Timothy John; Hunter, Roger
    Chloroquine was one of the main malarial treatments until the late 1960s when resistance began to emerge. This antimalarial targets haemozoin formation which causes a cytotoxic accumulation of free haem in the malaria parasite leading to parasite death. This is still one of the most promising pathways for treatment of the most prevalent species of malaria parasite, Plasmodium falciparum to date but, owing to growing resistance to chloroquine and other current antimalarial drugs, there is a dire need for new drugs. One strategy is to investigate non-chloroquine haemozoin inhibitors. High-throughput screening (HTS) was previously used to investigate novel β-haematin (synthetic haemozoin) inhibitors with promising P. falciparum growth inhibition activities. Of the 144 330 compounds screened, two hit compounds were selected for investigation in this project with two different scaffolds, namely benzimidazole and carbazole indole. In order to preselect benzimidazole derivatives for synthesis, Discovery Studio and Pipeline Pilot where used in tandem to enumerate 325 728 in silico compounds. These were filtered according to predicted β-haematin inhibition activities, followed by predicted malaria parasite growth activities using previously developed models based on Bayesian statistics. The predicted active compounds were further subjected to an in silico aqueous solubility model and separated according to predicted solubility values however, only 68 out of the 35 124 active compounds showed moderate solubility whilst the rest were poorly soluble. From this data, eighteen compounds were chosen for synthesis with varying functional groups. Using the same Bayesian models, biological activities for seven fragment compounds derived from the benzimidazole hit compound were predicted. Six out of seven were predicted to be β-haematin inhibitors while five out of seven were predicted active against the malaria parasite growth inhibition model. Similar Bayesian predictions were carried out on the seven proposed carbazole indole compounds with three compounds predicted to be β-haematin inhibitors while six compounds were predicted to be active against the malaria parasite growth inhibition model. The eighteen benzimidazole compounds were synthesized using a two-step synthesis, via a condensation reaction using polyphosphoric acid (PPA), 4-aminobenzoic acid and o-phenylenediamine to form the primary amine benzimidazole intermediate after which ani acylation reaction with the appropriate acid chloride furnished the desired compounds. β-haematin inhibition analysis revealed a 78% hit rate compared to the Bayesian predictions which resulted in a 24-fold enrichment compared to random screening. SAR analysis revealed an activity trend related to the position of substituents on the ring system as follows: para < ortho < meta. The type of ring system was also investigated, with a trend of phenyl < furan < pyrrole < thiophene < pyridyl found. The fragment compounds were either purchased or synthesized via standard acylation conditions using acid chlorides or acetic anhydride with primary amines as before. β-haematin inhibition analysis showed all these compounds to be inactive at the 100 µM cut-off but these compounds were still carried through to the next stage of testing in spite of these results. Molecular docking was carried out on all eighteen benzimidazole compounds in Materials Studio using the (001) and (011) β-haematin crystal faces for adsorption, together with a modified CVFF force-field. This showed a correlation between adsorption energies of the (011) β-haematin crystal face with the experimental β-haematin inhibition values. This indicated that the (011) β-haematin crystal face was the most important for β-haematin inhibition. Analysis of the benzimidazole compounds and their π-π and hydrogen bonding interactions was performed. The number of π-π interactions were found to be important for β-haematin inhibition activity. Both sets of benzimidazole compounds were tested against the NF54 chloroquine sensitive malaria parasite using growth inhibition assays with a 50% hit rate shown for the benzimidazole compounds and a 71% hit rate for the fragment study leading to a 26-fold and 36-fold enrichments compared to random screening. SAR analysis of the benzimidazole compounds revealed a trend for activity in relation to substituent position of para ≈ ortho < meta and a ring system trend of phenyl < pyridyl < thiophene < furan < pyrrole. The benzimidazole compounds were further tested against the chloroquine resistant Dd2 P. falciparum strain which showed that disubstituted compounds were more active against this strain. Cellular haem fractionation studies revealed an increase in free haem and decrease in haemozoin confirming that haemozoin inhibition is the mode of action for the benzimidazole compounds. QSAR analysis of these compounds revealed a correlation between the -Log(P. falciparum IC50) which is also known as pLog(P. falciparum IC50) and 1/βhaematin IC50, number of hydrogen bond donors and molecular depth with 1/β-haematin IC50 the most dominant term. iv The first four carbazole indole compounds were synthesized using a two-step synthesis via deprotonation of carbazole and reaction with epichlorohydrin or 1,3-dibromopropane to furnish the epoxide or alkylbromine intermediates. These intermediates underwent a further SN2 reaction using deprotonated indole to furnish four final compounds. Synthesis of another three derivatives required benzyl protection of 7-hydroxyindole alcohol first, followed by reaction with the epoxide intermediates via an SN2 mechanism to furnish the final three compounds. Analysis using the turbidimetric solubility assay revealed the best aqueous solubility range of this series of compounds to be 10-20 µM (moderately soluble). β-haematin inhibition studies were carried out on this series of compounds with a 100% hit rate found when compared to the Bayesian model data which lead to 30-fold enrichment when compared to random screening. SAR analysis showed an increase in the number of hydroxyl groups led to an increase in β-haematin inhibition activity. Docking studies were performed on these seven compounds and showed that hydrogen bonding played a role in anchoring the molecules in the binding pocket on the crystal surface with increased adsorption energies seen with an increase in the number of hydroxyl groups. Malaria parasite growth inhibition studies showed no compounds to be active against the NF54 and Dd2 strains at the 2 µM cut-off. Cellular haem fractionation studies on the carbazole indole compounds showed that this series of compounds acts via a mechanism that results in inhibition of haemoglobin uptake into the food vacuole and not via haemozoin inhibition.
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    Open Access
    Therapeutic efficacy of sulfadoxine-pyrimethamine for Plasmodium falciparum malaria : A study 5 years after implementation of combination therapy in Mpumalanga, South Africa
    (2005) Mabuza, Aaron; Govere, John; La Grange, Kobus; Mngomezulu, Nicros; Allen, Elizabeth; Zitha, Alpheus; Mbokazi, Frans; Durrheim, David; Barnes, Karen
    Objectives. To assess the therapeutic efficacy of sulfadoxine-pyrimethamine (SP) after 5 years of use as first-line treatment of uncomplicated Plasmodium falciparum malaria, and thus guide the selection of artemisinin-based combination therapy in Mpumalanga, South Africa. Design. An open-label, in vivo therapeutic efficacy study of patients with uncomplicated P. falciparum malaria treated with a single oral dose of SP, with response to treatment monitored clinically and parasitologically on days 1, 2, 3, 7, 14, 21, 28 and 42. Setting. Mangweni and Naas public health care clinics, Tonga district in rural Mpumalanga. Subjects, outcome measures and results. Of 152 patients recruited sequentially, 149 (98%) were successfully followed up for 42 days. One hundred and thirty-four patients (90%) demonstrated adequate clinical and parasitological response. Of the 15 patients (10%) who failed treatment, 2 (1.3%) had an early treatment failure, and polymerase chain reaction confirmed recrudescent infection in all 13 patients (8.7%) who had late parasitological (N = 11) or clinical (N = 2) failure. Gametocyte carriage was prevalent following SP treatment (84/152) and this has increased significantly since implementation in 1998 (relative risk 2.77 (confidence interval 1.65 - 4.66); p = 0.00004). Conclusion. Asexual P. falciparum parasites in Mpumalanga remain sensitive to SP, with no significant difference between the baseline cure rate (94.5%) at introduction in 1998, and the present 90% cure rate (p = 0.14). However, since gametocyte carriage has increased significantly we recommend that SP be combined with artesunate in Mpumalanga to reduce gametocyte carriage and thus decrease malaria transmission and potentially delay antimalarial resistance.