The mode of action of antimalarial endoperoxides

The mechanism of action of artemisinin appears to involve two steps. In the first step, activation, intra-parasitic iron catalyses the cleavage of the endoperoxide bridge and the generation of free radicals. In the second step, alkylation, the artemisinin-derived free radical forms covalent bonds with parasite proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31511/1/0000433.pd

Alkylation of proteins by artemisinin : Effects of heme, pH, and drug structure

Artemisinin and its derivatives are a promising new class of antimalarial agents containing an endoperoxide bridge. [14C]Artemisinin alkylated various proteins in vitro. Between 5 and 18% of added drug bound to hemoproteins such as catalase, cytochrome c, and hemoglobin. However, it did not react with heme-free globin. For catalase and hemoglobin, most of the drug reacted with the protein moiety rather than the heme. Artemisinin bound to human serum albumin (HSA) more efficiently at pH 8.6 than 7.4, more efficiently in Dulbecco's PBS than in Tris-HCl buffer, and better when HSA had been made fatty acid-free. Dihydroartemisinin also bound to HSA, whereas deoxyartemisinin, an inactive derivative, did not. There was no binding between DNA and artemisinin. These data provide insight into the mechanism of the reaction between artemisinin and proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31394/1/0000308.pd

Quinine localizes to a non-acidic compartment within the food vacuole of the malaria parasite Plasmodium falciparum

Abstract Background The naturally fluorescent compound quinine has long been used to treat malaria infections. Although some evidence suggests that quinine acts in the parasite food vacuole, the mechanism of action of quinine has not yet been resolved. The Plasmodium falciparum multidrug resistance (pfmdr1) gene encodes a food vacuolar membrane transporter and has been linked with parasite resistance to quinine. The effect of multiple pfmdr1 copies on the subcellular localization of quinine was explored. Methods Fluorescence microscopy was used to evaluate the subcellular localization of quinine in parasites containing different pfmdr1 copy numbers to determine if copy number of the gene affects drug localization. The acidotropic dye LysoTracker Red was used to label the parasite food vacuole. Time-lapse images were taken to determine quinine localization over time following quinine exposure. Results Regardless of pfmdr1 copy number, quinine overlapped with haemozoin but did not colocalize with LysoTracker Red, which labeled the acidic parasite food vacuole. Conclusions Quinine localizes to a non-acidic compartment within the food vacuole possibly haemozoin. Pfmdr1 copy number does not affect quinine subcellular localization

The diamidine DB75 targets the nucleus of Plasmodium falciparum

Abstract Background DB289, [2,5-bis(4-amidinophenyl)furan bis-O-methylamidoxime], is a broad spectrum anti-parasitic compound which has been shown to be effective against malaria in recent clinical trials. DB75, [2,5-bis(4-amidinophenyl)furan], is the active metabolite of this drug. The objective of this study was to determine the mechanism of action of DB75 in Plasmodium falciparum. Methods Live parasites were observed by confocal microscopy after treatment with organelle specific dyes and DB75, an inherently fluorescent compound. Parasites were exposed to DB75 and assessed for growth and morphological changes over time using blood smears and light microscopy. Also, to determine if DB75 affects gene transcription, real time PCR was used to monitor transcript levels over time for six developmentally expressed genes, including trophozoite antigen R45-like (PFD1175w), lactate dehydrogenase (PF13_0141), DNA primase (PFI0530c), isocitrate dehydrogenase (PF13_0242), merozoite surface protein-1 (PFI1475w), and merozoite surface protein-7 (PF13_0197). Results The results show that DB75 localizes in the parasite nucleus but not in other organelles. Once rings are exposed, parasites mature to the trophozoite stage and stall. No stage-dependent or gene-specific inhibition of transcription was seen. However, DB75 delayed peak transcription of trophozoite-stage genes. Conclusion Taken together, DB75 appears to concentrate in the nucleus and delay parasite maturation

The role of submicroscopic parasitemia in malaria transmission: what is the evidence?

Achieving malaria elimination requires targeting the human reservoir of infection, including those with asymptomatic infection. Smear-positive asymptomatic infections detectable by microscopy are an important reservoir because they often persist for months and harbor gametocytes, the parasite stage infectious to mosquitoes. However, many asymptomatic infections are submicroscopic and can only be detected by molecular methods. While there is some evidence that persons with submicroscopic malaria can infect mosquitoes, transmission is much less likely to occur at submicroscopic gametocyte levels. As malaria elimination programs pursue mass screening and treatment of asymptomatic individuals, further research should strive to define the degree to which submicroscopic malaria contributes to the infectious reservoir, and in turn, what diagnostic detection threshold is needed to effectively interrupt transmission

Duration of Effectiveness of Permethrin-Treated Clothing to Prevent Mosquito Bites Under Simulated Conditions

Presented for World Environmental Health Day, September 26, 2016 in Greenville, North Carolina.Biological hazards such as exposure to ticks and mosquitoes can affect worker health. Permethrin is a repellant/insecticide approved for human use by the Environmental Protection Agency. Permethrin-treated clothing is commercially available to the public. Permethrin-treated clothing (50% cotton/50% nylon) has been shown to retain repellency through 70 washings. Work attire differs between state and consulting foresters, park rangers, etc.; hence, variation in protection from vector borne disease may existThis study was funded by the Southeast Center for Agricultural Health and Injury Prevention (# 3049025288-14-060)

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

Human African trypanosomiasis (HAT) is a fatal tropical disease caused by infection with protozoans of the species Trypanosoma brucei gambiense and T. b. rhodesiense. An oral prodrug, DB289, is a promising new therapy undergoing phase III clinical trials for early-stage HAT. DB289 is metabolically converted to the active trypanocidal diamidine DB75 [2,5-bis(4-amidinophenyl)furan]. We previously determined that DB75 inhibits yeast mitochondrial function (C. A. Lanteri, B. L. Trumpower, R. R. Tidwell, and S. R. Meshnick, Antimicrob. Agent Chemother. 48:3968-3974, 2004). The purpose of this study was to investigate if DB75 targets the mitochondrion of T. b. brucei bloodstream forms. DB75 rapidly accumulates within the mitochondria of living trypanosomes, as indicated by the fluorescent colocalization of DB75 with a mitochondrion-specific dye. Fluorescence-activated cell sorting analysis of rhodamine 123-stained living trypanosomes shows that DB75 and other trypanocidal diamidines (pentamidine and diminazene) collapse the mitochondrial membrane potential. DB75 inhibits ATP hydrolysis within T. brucei mitochondria and appears to inhibit the oligomycin-sensitive F1F0-ATPase and perhaps other ATPases. DB75 is most likely not an inhibitor of electron transport within trypanosome mitochondria, since DB75 fails to inhibit mitochondrial respiration when glycerol-3-phosphate is used as the respiratory substrate. However, DB75 inhibits whole-cell respiration (50% inhibitory concentration, 20 μM) at drug concentrations and incubation durations that also result in the dissipation of the mitochondrial membrane potential. Taken together, these findings suggest that the mitochondrion is a target of the trypanocidal action of DB75

Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals

A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia -specific PCR targets ( ompA , gltA , and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “ Candidatus Rickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia . The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. “ Candidatus Rickettsia amblyommii,” R. montanensis , R. felis , and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii

Short Report: Detection of the Dihydrofolate Reductase–164L Mutation in Plasmodium falciparum Infections from Malawi by Heteroduplex Tracking Assay

Standard polymerase chain reaction methods often cannot detect drug-resistance mutations in Plasmodium falciparum infections if the mutation is present in ≤ 20% of the parasites. A heteroduplex tracking assay was developed that can detect dihydrofolate reductase 164-L mutations in variants representing 1% of the parasites in an individual host. Using this assay, we confirmed the presence of the mutation in P. falciparum infections in Malawi

Cost-Effectiveness of Adding Bed Net Distribution for Malaria Prevention to Antenatal Services in Kinshasa, Democratic Republic of the Congo

We evaluated the cost-effectiveness of distributing insecticide-treated bed nets (ITNs) for malaria prevention at antenatal clinics in Kinshasa, Democratic Republic of the Congo. A decision tree model was used to estimate costs, outcomes, and incremental cost-effectiveness for 17,893 pregnant women attending 28 antenatal clinics who received long-lasting ITNs free of charge. Costs including purchase, transportation, storage, and distribution of ITNs were derived from program records. The ITN efficacy and other parameters were derived from peer-reviewed literature. Outcomes modeled included low birth weight (LBW) deliveries, infant deaths averted, life-years saved (LYs), and disability-adjusted life-years (DALYs) averted. Deterministic and probabilistic sensitivity analyses were conducted. For the 17,893 women in our program, ITN distribution would be expected to avert 587 LBW deliveries and 414 infant deaths. The incremental cost-effectiveness was US $17.22 per DALY averted (95$8.54-$30.90), US$15.70 per LY saved (95% CI = US $7.65-$27.68), and US $411.13 per infant death averted (95$353.95-$1,085.89). If resources were constrained, the greatest benefit would be among women in their first through fourth pregnancies. Thus, ITN distribution is a cost-effective addition to antenatal services