A Mechanism of Resistance and Mode of Action for Drugs Against Plasmodium falciparum

The need for new drugs to control widespread malaria caused by Plasmodium falciparum is critical. Parasite resistance to currently used drugs is rampant and, in many cases, the drug's mode of action and/or mechanism of resistance is unknown. The three objectives of this dissertation address issues associated with resistance to the currently used antimalarial drugs, in addition to elucidating the mechanism of action of a novel antimalarial compound in development. First, a real time PCR method was developed to distinguish parasite genotypes associated with mefloquine resistance in vitro. Single nucleotide point mutations in the Plasmodium falciparum multi-drug resistance-1 (pfmdr1) gene are associated with mefloquine resistance in vitro. This method may be applied to clinical malaria samples and used to predict treatment outcome as well as for surveillance of drug resistance. In addition, the mechanism of action for the novel compound, [2,5-bis(4-amidinophenyl) furan], (DB75) was investigated. DB75, the active metabolite of the oral pro-drug DB289, is a broad spectrum antiparasitic agent with impressive antimalarial activity both in vitro and in vivo. It is currently in development for treatment of falciparum malaria, however the mode of action against falciparum parasites is unknown. Results from ultraviolet confocal microscopy showed DB75 localization exclusively in the nucleus of parasites in culture. Further, microscopy studies using blood smears to distinguish morphologies suggested DB75 has a life stage-specific mechanism. Parasites must be exposed during the ring stage for effective killing. Finally, real time PCR gene expression assays suggested high concentrations of DB75 may alter the expression pattern in a manner consistent with the delay in maturation. However, DB75 did not inhibit or enhance global nuclear transcription or developmental expression of six select genes. The third objective was to determine potential synergistic interactions of DB75 in combination with current antimalarial drugs to determine a mechanism of action for DB75 and to identify potential partner drugs for use in combination therapy with DB75. Taken together, this work contributes to the arsenal of tools for surveillance of falciparum malaria drug resistance and partially elucidates a mechanism of action for a novel antimalarial diamidine that may be used for malaria therapy

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

pfmdr1 GENOTYPING AND IN VIVO MEFLOQUINE RESISTANCE ON THE THAI-MYANMAR BORDER

Molecular markers have been proposed as a method of monitoring malaria drug resistance and could potentially be used to prolong the life span of antimalarial drugs. Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum gene pfmdr1 and increased gene copy number have been associated with in vitro drug resistance but have not been well studied in vivo. In a prospective cohort study of malaria patients receiving mefloquine treatment on the Thai-Myanmar border, there was no significant association between either pfmdr1 SNPs or in vitro drug sensitivity and mefloquine resistance in vivo. Increased pfmdr1 gene copy number was significantly associated with recrudescence (relative risk 2.30, 95% CI 1.27–4.15). pfmdr1 gene copy number may be a useful surveillance tool for mefloquine-resistant falciparum malaria in Thailand

A new method for detection of pfmdr1 mutations in Plasmodium falciparum DNA using real-time PCR

BACKGROUND: Surveillance for drug-resistant Plasmodium falciparum should be a component of malaria control programmes. Real-time PCR methods for the detection of parasite single-nucleotide polymorphisms (SNPs) and gene amplification could be useful survellance tools. METHODS: A real-time PCR assay has been developed that identifies single nucleotide polymorphisms (SNPs) at amino acids 86, 184, 1034 and 1042 in the P. falciparum multi-drug resistant (pfmdr 1) gene that may be associated with anti-malarial drug resistance. RESULTS: This assay has a sensitivity and specificity of 94% and 100% when compared to traditional PCR methods for genotyping. Only 54 of 68 (79%) paired pre- and post-culture DNA samples were concordant at all four loci. CONCLUSION: Real-time PCR is a sensitive and specific method to detect SNP's in pfmdr 1. Genotypes of parasites after in vitro culture may not reflect that seen in vivo

Meta-analysis of genome-wide association studies for cattle stature identifies common genes that regulate body size in mammals

peer-reviewedH.D.D., A.J.C., P.J.B. and B.J.H. would like to acknowledge the Dairy Futures Cooperative Research Centre for funding. H.P. and R.F. acknowledge funding from the German Federal Ministry of Education and Research (BMBF) within the AgroClustEr ‘Synbreed—Synergistic Plant and Animal Breeding’ (grant 0315527B). H.P., R.F., R.E. and K.-U.G. acknowledge the Arbeitsgemeinschaft Süddeutscher Rinderzüchter, the Arbeitsgemeinschaft Österreichischer Fleckviehzüchter and ZuchtData EDV Dienstleistungen for providing genotype data. A. Bagnato acknowledges the European Union (EU) Collaborative Project LowInputBreeds (grant agreement 222623) for providing Brown Swiss genotypes. Braunvieh Schweiz is acknowledged for providing Brown Swiss phenotypes. H.P. and R.F. acknowledge the German Holstein Association (DHV) and the Confederación de Asociaciones de Frisona Española (CONCAFE) for sharing genotype data. H.P. was financially supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (DFG) (grant PA 2789/1-1). D.B. and D.C.P. acknowledge funding from the Research Stimulus Fund (11/S/112) and Science Foundation Ireland (14/IA/2576). M.S. and F.S.S. acknowledge the Canadian Dairy Network (CDN) for providing the Holstein genotypes. P.S. acknowledges funding from the Genome Canada project entitled ‘Whole Genome Selection through Genome Wide Imputation in Beef Cattle’ and acknowledges WestGrid and Compute/Calcul Canada for providing computing resources. J.F.T. was supported by the National Institute of Food and Agriculture, US Department of Agriculture, under awards 2013-68004-20364 and 2015-67015-23183. A. Bagnato, F.P., M.D. and J.W. acknowledge EU Collaborative Project Quantomics (grant 516 agreement 222664) for providing Brown Swiss and Finnish Ayrshire sequences and genotypes. A.C.B. and R.F.V. acknowledge funding from the public–private partnership ‘Breed4Food’ (code BO-22.04-011- 001-ASG-LR) and EU FP7 IRSES SEQSEL (grant 317697). A.C.B. and R.F.V. acknowledge CRV (Arnhem, the Netherlands) for providing data on Dutch and New Zealand Holstein and Jersey bulls.Stature is affected by many polymorphisms of small effect in humans1. In contrast, variation in dogs, even within breeds, has been suggested to be largely due to variants in a small number of genes2,3. Here we use data from cattle to compare the genetic architecture of stature to those in humans and dogs. We conducted a meta-analysis for stature using 58,265 cattle from 17 populations with 25.4 million imputed whole-genome sequence variants. Results showed that the genetic architecture of stature in cattle is similar to that in humans, as the lead variants in 163 significantly associated genomic regions (P < 5 × 10−8) explained at most 13.8% of the phenotypic variance. Most of these variants were noncoding, including variants that were also expression quantitative trait loci (eQTLs) and in ChIP–seq peaks. There was significant overlap in loci for stature with humans and dogs, suggesting that a set of common genes regulates body size in mammals

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries

Interactions of DB75, a Novel Antimalarial Agent, with Other Antimalarial Drugs In Vitro▿

Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the in vitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline. None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine

The diamidine DB75 targets the nucleus of Plasmodium falciparum

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Resistance to Antimalarials in Southeast Asia and Genetic Polymorphisms in pfmdr1

Resistance to antimalarial drugs is a public health problem worldwide. Molecular markers for drug-resistant malaria, such as pfcrt and pfmdr1 polymorphisms, could serve as useful surveillance tools. To evaluate this possibility, sequence polymorphisms in pfcrt (position 76) and pfmdr1 (positions 86, 184, 1034, 1042, and 1246) and in vitro drug sensitivities were measured for 65 Plasmodium falciparum isolates from Thailand, Myanmar, Vietnam, and Bangladesh. The pfcrt Thr76 polymorphism was present in 97% of samples, consistent with observations that chloroquine resistance is well established in this region. Polymorphisms in pfmdr1 clustered into four specific patterns: the wild type (category I), a Tyr86 polymorphism only (category II), a Phe184 polymorphism only (category III), and Phe184 in combination with Cys1034 and/or Asp1042 (category IV). Isolates in categories I and III were more sensitive to chloroquine and more resistant to mefloquine, artesunate, and artemisinin than isolates in categories II and IV (P ≤ 0.01). Mefloquine resistance was significantly more common in category I and III isolates than in category II and IV isolates, with a prevalence ratio of 14.95 (95% confidence interval, 3.88 to 57.56). These categories identified mefloquine resistance with a sensitivity and a specificity of 94 and 91%, respectively. The pfmdr1 gene copy number was measured by real-time PCR as a ratio of the amount of pfmdr1 DNA to the amount of lactate dehydrogenase (ldh) DNA. Eight samples had pfmdr1 DNA/ldh DNA ratios ≥3. The isolates in all 8 samples fell into categories I and III and were significantly more resistant to mefloquine, quinine, artemisinin, and artesunate and more sensitive to chloroquine than the isolates in the 57 samples with <3 copies of the gene (P ≤ 0.001). Thus, measurement of pfmdr1 mutations and gene copy number may be useful for surveillance of mefloquine-resistant malaria in Southeast Asia