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

Rapid Diagnostic Test Performance Assessed Using Latent Class Analysis for the Diagnosis of Plasmodium falciparum Placental Malaria.

Placental malaria causes low birth weight and neonatal mortality in malaria-endemic areas. The diagnosis of placental malaria is important for program evaluation and clinical care, but is compromised by the suboptimal performance of current diagnostics. Using placental and peripheral blood specimens collected from delivering women in Malawi, we compared estimation of the operating characteristics of microscopy, rapid diagnostic test (RDT), polymerase chain reaction, and histopathology using both a traditional contingency table and a latent class analysis (LCA) approach. The prevalence of placental malaria by histopathology was 13.8%; concordance between tests was generally poor. Relative to histopathology, RDT sensitivity was 79.5% in peripheral and 66.2% in placental blood; using LCA, RDT sensitivities increased to 93.7% and 80.2%, respectively. Our results, if replicated in other cohorts, indicate that RDT testing of peripheral or placental blood may be suitable approaches to detect placental malaria for surveillance programs, including areas where intermittent preventive therapy in pregnancy is not used

Maternal–Fetal Microtransfusions and HIV-1 Mother-to-Child Transmission in Malawi

Background: Between 25% and 35% of infants born to HIV-infected mothers become HIV-1 infected. One potential route of mother-to-child transmission (MTCT) could be through a breakdown in the placental barrier (i.e., maternal–fetal microtransfusions). Methods and Findings: Placental alkaline phosphatase (PLAP) is a 130-kD maternal enzyme that cannot cross the intact placental barrier. We measured PLAP activity in umbilical vein serum as an indicator of maternal–fetal microtransfusion, and related this to the risk of HIV-1 MTCT. A case-cohort study was conducted of 149 women randomly selected from a cohort of HIV-1-infected pregnant Malawians; these women served as a reference group for 36 cases of in utero MTCT and 43 cases of intrapartum (IP) MTCT. Cord PLAP activity was measured with an immunocatalytic assay. Infant HIV status was determined by real-time PCR. The association between cord PLAP activity and HIV-1 MTCT was measured with logistic regression using generalized estimating equations. Among vaginal deliveries, PLAP was associated with IP MTCT (risk ratio, 2.25 per $log_{10}$ ng/ml PLAP; 95% confidence interval, 0.95–5.32) but not in utero MTCT. In a multivariable model adjusted for HIV-1 RNA load, chorioamnionitis, and self-reported fever, the risk of IP MTCT almost tripled for every $log_{10}$ increase in cord PLAP activity (risk ratio, 2.87; 95% confidence interval, 1.05–7.83). Conclusion: These results suggest that during vaginal deliveries, placental microtransfusions are a risk factor for IP HIV-1 MTCT. Future studies are needed to identify factors that increase the risk for microtransfusions in order to prevent IP HIV-1 MTCT

Low prevalence of Plasmodium malariae and Plasmodium ovale mono-infections among children in the Democratic Republic of the Congo: a population-based, cross-sectional study

Abstract Background In an effort to improve surveillance for epidemiological and clinical outcomes, rapid diagnostic tests (RDTs) have become increasingly widespread as cost-effective and field-ready methods of malaria diagnosis. However, there are concerns that using RDTs specific to Plasmodium falciparum may lead to missed detection of other malaria species such as Plasmodium malariae and Plasmodium ovale. Methods Four hundred and sixty six samples were selected from children under 5 years old in the Democratic Republic of the Congo (DRC) who took part in a Demographic and Health Survey (DHS) in 2013–14. These samples were first tested for all Plasmodium species using an 18S ribosomal RNA-targeted real-time PCR; malaria-positive samples were then tested for P. falciparum, P. malariae and P. ovale using a highly sensitive nested PCR. Results The prevalence of P. falciparum, P. malariae and P. ovale were 46.6, 12.9 and 8.3 %, respectively. Most P. malariae and P. ovale infections were co-infected with P. falciparum—the prevalence of mono-infections of these species were only 1.0 and 0.6 %, respectively. Six out of these eight mono-infections were negative by RDT. The prevalence of P. falciparum by the more sensitive nested PCR was higher than that found previously by real-time PCR. Conclusions Plasmodium malariae and P. ovale remain endemic at a low rate in the DRC, but the risk of missing malarial infections of these species due to falciparum-specific RDT use is low. The observed prevalence of P. falciparum is higher with a more sensitive PCR method

Efficacy of DB289 in Thai Patients with Plasmodium vivax or Acute, Uncomplicated Plasmodium falciparum Infections

BackgroundDB289 is the orally active prodrug of the diamidine DB75, which was developed for the treatment of human African trypanosomiasis MethodsWe tested the safety and efficacy of DB289 for the treatment of Plasmodium vivax and acute, uncomplicated P. falciparum infections in an open-label pilot study at the Hospital for Tropical Diseases in Bangkok. Nine patients with P. vivax infections and 23 patients with P. falciparum infections were admitted and treated with 100 mg of DB289 given orally twice a day for 5 days and were followed for 28 days. Patients with P. vivax infections were also treated with primaquine on days 10-23 ResultsAll patients cleared parasites by day 7, with a mean±SD clearance time of 43±41 h. One patient with a P. vivax infection had a recurrence of parasitemia on day 9. Of the 23 patients with P. falciparum infections, 3 had recurrences of parasitemia caused by P. vivax and 2 had recurrences of parasitemia caused by P. falciparum. In only 1 of 2 recurrences of parasitemia caused by P. falciparum were the parasites genotypically distinct from the infecting parasites the patient had at enrollment, which means there was a 96% cure rate ConclusionsDB289 is a promising new antimalarial compound that could become an important component of new antimalarial combination

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

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

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

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