Comparison of the susceptibility of Plasmodium knowlesi and Plasmodium falciparum to antimalarial agents.

BACKGROUND: The simian malaria parasite Plasmodium knowlesi is now a well-recognized pathogen of humans in South-East Asia. Clinical infections appear adequately treated with existing drug regimens, but the evidence base for this practice remains weak. The availability of P. knowlesi cultures adapted to continuous propagation in human erythrocytes enables specific studies of in vitro susceptibility of the species to antimalarial agents, and could provide a surrogate system for testing investigational compounds against Plasmodium vivax and other non-Plasmodium falciparum infections that cannot currently be propagated in vitro. OBJECTIVES: We sought to optimize protocols for in vitro susceptibility testing of P. knowlesi and to contrast outputs with those obtained for P. falciparum under comparable test conditions. METHODS: Growth monitoring of P. knowlesi in vitro was by DNA quantification using a SYBR Green fluorescent assay or by colorimetric detection of the lactate dehydrogenase enzyme. For comparison, P. falciparum was tested under conditions identical to those used for P. knowlesi. RESULTS: The SYBR Green I assay proved the most robust format over one (27 h) or two (54 h) P. knowlesi life cycles. Unexpectedly, P. knowlesi displays significantly greater susceptibility to the dihydrofolate reductase inhibitors pyrimethamine, cycloguanil and trimethoprim than does P. falciparum, but is less susceptible to the selective agents blasticidin and DSM1 used in parasite transfections. Inhibitors of dihydroorotate dehydrogenase also demonstrate lower activity against P. knowlesi. CONCLUSIONS: The fluorescent assay system validated here identified species-specific P. knowlesi drug susceptibility profiles and can be used for testing investigational compounds for activity against non-P. falciparum malaria

A dual colour fluorescence in situ hybridization (FISH) assay for identifying the zoonotic malaria parasite Plasmodium knowlesi with a potential application for the specific diagnosis of knowlesi malaria in peripheral-level laboratories of Southeast Asia.

BACKGROUND: Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast Asian countries. Precise identification of the parasite in the blood of patients presently relies on an expensive and elaborate PCR procedure because microscopic examination of blood and other available field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P. knowlesi. RESULTS: A P. knowlesi 18S rDNA sequence-based DNA probe was used to test thin blood smears of P. knowlesi by FISH, and fluorescence viewed in a light microscope fitted with a light emitting diode light source and appropriate emission and barrier filters. The limit of detection in the P. knowlesi FISH assay was 84 parasites per μl in infected monkey blood and 61 parasites per μl for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species. CONCLUSIONS: To our knowledge, this is the first description of a FISH assay for P. knowlesi that is potentially useful for diagnosing infections in remote laboratories in endemic countries

Regulation and Essentiality of the StAR-related Lipid Transfer (START) Domain-containing Phospholipid Transfer Protein PFA0210c in Malaria Parasites.

StAR-related lipid transfer (START) domains are phospholipid- or sterol-binding modules that are present in many proteins. START domain-containing proteins (START proteins) play important functions in eukaryotic cells, including the redistribution of phospholipids to subcellular compartments and delivering sterols to the mitochondrion for steroid synthesis. How the activity of the START domain is regulated remains unknown for most of these proteins. The Plasmodium falciparum START protein PFA0210c (PF3D7_0104200) is a broad-spectrum phospholipid transfer protein that is conserved in all sequenced Plasmodium species and is most closely related to the mammalian START proteins STARD2 and STARD7. PFA0210c is unusual in that it contains a signal sequence and a PEXEL export motif that together mediate transfer of the protein from the parasite to the host erythrocyte. The protein also contains a C-terminal extension, which is very uncommon among mammalian START proteins. Whereas the biochemical properties of PFA0210c have been characterized, the function of the protein remains unknown. Here, we provide evidence that the unusual C-terminal extension negatively regulates phospholipid transfer activity. Furthermore, we use the genetically tractable Plasmodium knowlesi model and recently developed genetic technology in P. falciparum to show that the protein is essential for growth of the parasite during the clinically relevant asexual blood stage life cycle. Finally, we show that the regulation of phospholipid transfer by PFA0210c is required in vivo, and we identify a potential second regulatory domain. These findings provide insight into a novel mechanism of regulation of phospholipid transfer in vivo and may have important implications for the interaction of the malaria parasite with its host cell

Farm-Tractor-Related Fatalities -- Kentucky, 1994

Fatalities associated with farm tractors are the most common cause of work-related death in the U.S. agricultural industry (1). To characterize farm-tractor-related fatalities in Kentucky, the Kentucky Fatality Assessment and Control Evaluation (KY FACE) Project studied all fatal farm injuries occurring among persons in that state during 1994, the initial year of operation for FACE in Kentucky. This report summarizes the results of that study

Novel Endochin-Like Quinolones Exhibit Potent In Vitro Activity against Plasmodium knowlesi but Do Not Synergize with Proguanil.

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc1 complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown. We screened several of these novel endochin-like quinolones (ELQs) for their activity against P. knowlesiin vitro and compared this with their activity against P. falciparum tested under identical conditions. We demonstrated that ELQs are potent against P. knowlesi (50% effective concentration, <117?nM) and equally effective against P. falciparum We then screened selected quinolones and partner drugs using a longer exposure (2.5 life cycles) and found that proguanil is 10-fold less potent against P. knowlesi than P. falciparum, while the quinolones demonstrate similar potency. Finally, we used isobologram analysis to compare combinations of the ELQs with either proguanil or atovaquone. We show that all quinolone combinations with proguanil are synergistic against P. falciparum However, against P. knowlesi, no evidence of synergy between proguanil and the quinolones was found. Importantly, the combination of the novel quinolone ELQ-300 with atovaquone was synergistic against both species. Our data identify potentially important species differences in proguanil susceptibility and in the interaction of proguanil with quinolones and support the ongoing development of novel quinolones as potent antimalarials that target multiple species

Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi.

Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species ( Pacheco et al., 2018 ) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions

An Orientation Program for Vertical Transfers in Engineering and Engineering Technology

This paper reports on a scholarship program funded by the National Science Foundation that focuses on students who transfer at the 3rd -year level from 2-year schools to the engineering and engineering technology BS programs at our university. The objectives of the program are to: (i) expand and diversify the engineering/technology workforce of the future, (ii) develop linkages and articulations with 2-year schools and their S-STEM (Scholarships in Science, Technology, Engineering and Mathematics) programs, (iii) provide increased career opportunities and job placement rates through mandatory paid co-op experiences, and (iv) serve as a model for other universities to provide vertical transfer students access to the baccalaureate degree. The program is in its third year. It recruited its first group of 25 students in Fall 2017, and another group of 27 students in Fall 2018. We hope to recruit 26 more students in Fall 2019 for a total of 78 vertical transfers. The goal is to retain and graduate at least 95% of these scholars. To enhance the success of these scholars, a zero-credit six-week orientation course was developed in Fall 2017 focusing on four dimensions of student wellness: academic, financial, social, and personal. This paper describes the development of this course, its content, and the modifications that were made to the course for Fall 2018. The paper will also address the research conducted in order to generate knowledge about the program elements that will be essential for the success of vertical transfer programs at other universities. Two research instruments are described: an online survey and a focus group interview that were developed, and administered to the transfer scholars in their first year. Initial findings concerning students’ experiences at their 2-year schools, their reason for transferring, their experience in transferring as well as their initial conceptions of what life at a 4-year institution will be like are presented

Scholarship Programs for Vertical Transfers in Engineering and Engineering Technology

This paper introduces two scholarship projects funded by the National Science Foundation that focus on students who transfer at the 3rd year level from 2-year schools to the engineering and engineering technology BS programs at our university. The objectives of both the projects are: (i) to expand and diversify the engineering/technology workforce of the future, (ii) to develop linkages and articulations with 2-year schools and their S-STEM programs, (iii) to provide increased career opportunities and job placement rates through mandatory paid co-op experiences, and (iv) to serve as a model for other universities to provide vertical transfer students access to the baccalaureate degree. The Transfer Pipeline (TiPi) project awarded 25 new scholarships per year from 2012 to 2014 to a total of 75 engineering and engineering technology transfer students. By the end of Fall 2017, 66 (88%) scholars have graduated, 5 (7%) are in process of completing their degrees, and only 4 (5%) left our university, for a 95% retention rate. The paper describes our successes and challenges. The Vertical Transfer Access to the Baccalaureate (VTAB) project recruited its first group of 25 students in Fall 2017 with the goal of recruiting a total of 78 vertical transfers over the next three years. An additional goal of the VTAB project is to conduct research and generate knowledge about the VTAB project elements that will be essential for the success of vertical transfer programs at other universities. The paper describes the research instruments, and the results from an online survey and a focus group interview of the first cohort of VTAB scholars