Comparison and Optimization of Different Methods for the In Vitro Production of Plasmodium falciparum Gametocytes

The generation of sexually committed parasites (gametocytogenesis) is poorly understood in malaria. If the mechanisms regulating this process were elucidated, new opportunities for blocking malaria transmission could be revealed. Here we compare several methods described previously for the in vitro production of Plasmodium falciparum gametocytes. Our approach relies on the combination of several factors that we demonstrated as impacting on or being critical to gametocytogenesis. An improved method has been developed for the in vitro production of P. falciparum gametocytes as the first step toward obtaining adequate numbers of pure gametocytes for in vitro studies, such as, for example, the identification of transmission blocking drugs

Bat flies (Diptera: Nycteribiidae and Streblidae) infesting cave-dwelling bats in Gabon: Diversity, dynamics and potential role in Polychromophilus melanipherus transmission

Background Evidence of haemosporidian infections in bats and bat flies has motivated a growing interest in characterizing their transmission cycles. In Gabon (Central Africa), many caves house massive colonies of bats that are known hosts of Polychromophilus Dionisi parasites, presumably transmitted by blood-sucking bat flies. However, the role of bat flies in bat malaria transmission remains under-documented. Methods An entomological survey was carried out in four caves in Gabon to investigate bat fly diversity, infestation rates and host preferences and to determine their role in Polychromophilus parasite transmission. Bat flies were sampled for 2–4 consecutive nights each month from February to April 2011 (Faucon and Zadie caves) and from May 2012 to April 2013 (Kessipoughou and Djibilong caves). Bat flies isolated from the fur of each captured bat were morphologically identified and screened for infection by haemosporidian parasites using primers targeting the mitochondrial cytochrome b gene. Results Among the 1,154 bats captured and identified as Miniopterus inflatus Thomas (n = 354), Hipposideros caffer Sundevall complex (n = 285), Hipposideros gigas Wagner (n = 317), Rousettus aegyptiacus Geoffroy (n = 157, and Coleura afra Peters (n = 41), 439 (38.0 %) were infested by bat flies. The 1,063 bat flies recovered from bats belonged to five taxa: Nycteribia schmidlii scotti Falcoz, Eucampsipoda africana Theodor, Penicillidia fulvida Bigot, Brachytarsina allaudi Falcoz and Raymondia huberi Frauenfeld group. The mean infestation rate varied significantly according to the bat species (ANOVA, F (4,75) = 13.15, P < 0.001) and a strong association effect between bat fly species and host bat species was observed. Polychromophilus melanipherus Dionisi was mainly detected in N. s. scotti and P. fulvida and less frequently in E. africana, R. huberi group and B. allaudi bat flies. These results suggest that N. s. scotti and P. fulvida could potentially be involved in P. melanipherus transmission among cave-dwelling bats. Sequence analysis revealed eight haplotypes of P. melanipherus. Conclusions This work represents the first documented record of the cave-dwelling bat fly fauna in Gabon and significantly contributes to our understanding of bat fly host-feeding behavior and their respective roles in Polychromophilus transmission. (Résumé d'auteur

Anti-malarial drugs: how effective are they against Plasmodium falciparum gametocytes?

<p>Abstract</p> <p>Background</p> <p>Recent renewed emphasis on the eradication of malaria has highlighted the need for more tools with which to achieve this ambitious goal. One high priority area is the need to determine the gametocytocidal activity of both currently used anti-malarial drugs and those in the development pipeline. However, testing the activity of compounds against <it>Plasmodium falciparum </it>gametocytes is technically challenging both <it>in vivo </it>and <it>in vitro</it>.</p> <p>Methods</p> <p>Here the use of a simple robust assay to screen a panel of currently used and experimental anti-malarial drugs against mature <it>P. falciparum </it>gametocytes is described.</p> <p>Results</p> <p>Eight of 44 compounds tested reduced gametocyte viability by at least 50% and three showed IC₅₀values in nM range.</p> <p>Conclusions</p> <p>There is a need to identify new compounds with activity against late stage gametocytes and the information provided by this <it>in vitro </it>assay is a valuable first step, which can guide future clinical studies.</p

Modulation of drug sensitivity in yeast cells by the ATP‐binding domain of human DNA topoisomerase IIα

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIα (htopoIIα), one in the catalytic core of the enzyme and one in the ATP‐binding N‐terminal domain. Here we have tested how N‐terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full‐length enzymes, mutations that lower the drug binding capacity of the N‐terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIα N‐terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N‐terminal ATP‐binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non‐equivalent drug‐binding site

Activity of clinically relevant antimalarial drugs on plasmodium falciparum mature gametocytes in an atp bioluminescence >transmission blocking> assay

Background: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. Methods and Findings: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. Conclusions: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways. © 2012 Lelièvre et al.Peer Reviewe

Transmission-blocking drugs for malaria elimination

Preventing human-to-mosquito transmission of malaria parasites provides possible solutions to interrupt the malaria parasite life cycle for malaria elimination. The development of validated routine assays enabled the discovery of such transmission-blocking compounds. Currently, one development priority remains on combinations of dual-active compounds with equipotent activity against both the disease-causing asexual and transmissible, sexual erythrocytic stages. Additionally, transmission-blocking compounds that target gametocyte-specific biology could be used in combination with compounds against asexual parasites. In either case, preventing transmission will reduce the risk of reinfection and, if different processes are targeted, also curb the spread of drug resistance. Here, we provide an updated roadmap to the discovery and development of new antimalarials with transmission-blocking activity to guide drug discovery for malaria elimination.The South African Department of Science and Innovation (DSI); the National Research Foundation (SA NRF) South African Research Chair Initiative; drug discovery funding from the Italy/South Africa Joint Research Program (ISARP) from the SA DSI and NRF and the Italian Ministries of Health and of Foreign Affairs and International Cooperation.http://www.journals.elsevier.com/trends-in-parasitologyhj2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC

Zika virus in Gabon (Central Africa) - 2007 : a new threat from Aedes albopictus ?

Background Chikungunya and dengue viruses emerged in Gabon in 2007, with large outbreaks primarily affecting the capital Libreville and several northern towns. Both viruses subsequently spread to the south-east of the country, with new outbreaks occurring in 2010. The mosquito species Aedes albopictus, that was known as a secondary vector for both viruses, recently invaded the country and was the primary vector involved in the Gabonese outbreaks. We conducted a retrospective study of human sera and mosquitoes collected in Gabon from 2007 to 2010, in order to identify other circulating arboviruses. Methodology/Principal Findings Sample collections, including 4312 sera from patients presenting with painful febrile disease, and 4665 mosquitoes belonging to 9 species, split into 247 pools (including 137 pools of Aedes albopictus), were screened with molecular biology methods. Five human sera and two Aedes albopictus pools, all sampled in an urban setting during the 2007 outbreak, were positive for the flavivirus Zika (ZIKV). The ratio of Aedes albopictus pools positive for ZIKV was similar to that positive for dengue virus during the concomitant dengue outbreak suggesting similar mosquito infection rates and, presumably, underlying a human ZIKV outbreak. ZIKV sequences from the envelope and NS3 genes were amplified from a human serum sample. Phylogenetic analysis placed the Gabonese ZIKV at a basal position in the African lineage, pointing to ancestral genetic diversification and spread. Conclusions/Significance We provide the first direct evidence of human ZIKV infections in Gabon, and its first occurrence in the Asian tiger mosquito, Aedes albopictus. These data reveal an unusual natural life cycle for this virus, occurring in an urban environment, and potentially representing a new emerging threat due to this novel association with a highly invasive vector whose geographic range is still expanding across the globe. Author Summary Not previously considered an important human arboviral pathogen, the epidemic capacity of Zika virus (ZIKV, a dengue-related flavivirus) was revealed by the Micronesia outbreak in 2007, which affected about 5000 persons. Widely distributed throughout tropical areas of Asia and Africa, ZIKV is transmitted by a broad range of mosquito species, most of which are sylvatic or rural, Aedes aegypti, an anthropophilic and urban species, being considered the main ZIKV epidemic vector. In a context of emerging arbovirus infections (chikungunya (CHIKV) and dengue (DENV)) in Gabon since 2007, we conducted a retrospective study to detect other, related viruses. In samples collected during the concurrent CHIKV/DENV outbreaks that occurred in the capital city in 2007, we detected ZIKV in both humans and mosquitoes, and notably the Asian mosquito Aedes albopictus that recently invaded the country and was the main vector responsible for these outbreaks. We found that the Gabonese ZIKV strain belonged to the African lineage, and phylogenetic analysis suggested ancestral diversification and spread rather than recent introduction. These findings, showing for the first time epidemic ZIKV activity in an urban environment in Central Africa and the presence of ZIKV in the invasive mosquito Aedes albopictus, raise the possibility of a new emerging threat to human health

Antimalarial drug discovery - the path towards eradication

Malaria is a disease that still affects a significant proportion of the global human population. Whilst advances have been made in lowering the numbers of cases and deaths, it is clear that a strategy based solely on disease control year on year, without reducing transmission and ultimately eradicating the parasite, is unsustainable. This article highlights the current mainstay treatments alongside a selection of emerging new clinical molecules from the portfolio of Medicines for Malaria Venture (MMV) and our partners. In each case, the key highlights from each research phase are described to demonstrate how these new potential medicines were discovered. Given the increased focus of the community on eradicating the disease, the strategy for next generation combination medicines that will provide such potential is explaine

Discovering new transmission-blocking antimalarial compounds : challenges and opportunities

The ability to target human-mosquito parasite transmission challenges global malaria elimination. However, it is not obvious what a transmission-blocking drug will look like; should it 1) target only parasite transmission stages; 2) be combined with a partner drug killing the pathogenic asexual stages or 3) kill both the sexual and asexual blood stages, preferably displaying polypharmacology. The development of transmission-blocking anti-malarials requires objective analyses of the current strategies. Here, pertinent issues and unanswered questions regarding the target candidate profile of a transmission-blocking compound, and its role in malaria elimination strategies are highlighted and novel perspectives proposed. The essential role of a test cascade that integrates screening and validation strategies to identify next generation transmission-blocking anti-malarials is emphasised.Koen Dechering (TROPIQ, The Netherlands), Francesco Silvestrini (Istituto Superiore di Sanità, Rome, Italy), Sarah D’Alessandro and Donatella Taramelli (University of Milan, Italy), Robert Sauerwein (University of Nijmegen, The Netherlands) and Omar Vandal (The Bill & Melinda Gates Foundation) are acknowledged for their contributions towards the screening cascade, which they developed in the course of the Bill and Melinda Gates Foundation Project OPP1040394 ‘Gametocyte Assays for Malaria (GAM) for novel transmission blocking drugs’, coordinated by PA. The South African Transmission-blocking Consortium is funded by the Medical Research Council of South Africa as a Strategic Health Innovation Partnership (MRC SHIP) project.http://www.journals.elsevier.com/trends-in-parasitology2017-09-30hb2016Biochemistr