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1.
Antimicrob Agents Chemother ; 58(12): 7390-7, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25267670

ABSTRACT

Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.


Subject(s)
Drug Resistance, Multiple/genetics , INDEL Mutation , Multidrug Resistance-Associated Proteins/genetics , Plasmodium falciparum/genetics , Polymorphism, Single Nucleotide , Amino Acid Sequence , Antimalarials/pharmacology , Artemisinins/pharmacology , Biological Transport , Chloroquine/pharmacology , Chromosomes/chemistry , Erythrocytes/drug effects , Erythrocytes/parasitology , Humans , Malaria, Falciparum/parasitology , Mefloquine/pharmacology , Molecular Sequence Data , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Parasitic Sensitivity Tests , Plasmodium falciparum/drug effects , Plasmodium falciparum/isolation & purification , Plasmodium falciparum/metabolism , Quinolines/pharmacology , Thailand
2.
Antimicrob Agents Chemother ; 58(1): 1-10, 2014.
Article in English | MEDLINE | ID: mdl-24100489

ABSTRACT

Artemisinin-based combination therapies (ACTs) are the main option to treat malaria, and their efficacy and susceptibility must be closely monitored to avoid resistance. We assessed the association of Plasmodium falciparum polymorphisms and ex vivo drug susceptibility with clinical effectiveness. Patients enrolled in an effectiveness trial comparing artemether-lumefantrine (n = 96), fixed-dose artesunate-amodiaquine (n = 96), and sulfadoxine-pyrimethamine (n = 48) for the treatment of uncomplicated malaria 2007 in Benin were assessed. pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps polymorphisms were analyzed pretreatment and in recurrent infections. Drug susceptibility was determined in fresh baseline isolates by Plasmodium lactate dehydrogenase enzyme-linked immunosorbent assay (ELISA). A majority had 50% inhibitory concentration (IC50) estimates (the concentration required for 50% growth inhibition) lower than those of the 3D7 reference clone for desethylamodiaquine, lumefantrine, mefloquine, and quinine and was considered to be susceptible, while dihydroartemisinin and pyrimethamine IC50s were higher. No association was found between susceptibility to the ACT compounds and treatment outcome. Selection was observed for the pfmdr1 N86 allele in artemether-lumefantrine recrudescences (recurring infections) (4/7 [57.1%] versus 36/195 [18.5%]), and of the opposite allele, 86Y, in artesunate-amodiaquine reinfections (new infections) (20/22 [90.9%] versus 137/195 [70.3%]) compared to baseline infections. The importance of pfmdr1 N86 in lumefantrine tolerance was emphasized by its association with elevated lumefantrine IC50s. Genetic linkage between N86 and Y184 was observed, which together with the low frequency of 1246Y may explain regional differences in selection of pfmdr1 loci. Selection of opposite alleles in artemether-lumefantrine and artesunate-amodiaquine recurrent infections supports the strategy of multiple first-line treatment. Surveillance based on clinical, ex vivo, molecular, and pharmacological data is warranted.


Subject(s)
Antimalarials/pharmacology , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Polymorphism, Single Nucleotide/genetics , Protozoan Proteins/genetics , Amodiaquine/analogs & derivatives , Amodiaquine/pharmacology , Artemisinins/pharmacology , Drug Combinations , Ethanolamines/pharmacology , Female , Fluorenes/pharmacology , Humans , Inhibitory Concentration 50 , Lumefantrine , Male , Mefloquine/pharmacology , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Pyrimethamine/pharmacology , Quinine/pharmacology , Sulfadoxine/pharmacology
3.
Antimicrob Agents Chemother ; 57(7): 3121-30, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23612201

ABSTRACT

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.


Subject(s)
Antimalarials/pharmacology , Artemisinins/pharmacology , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Amodiaquine/analogs & derivatives , Amodiaquine/pharmacology , Automation, Laboratory , Chloroquine/pharmacology , Drug Resistance , Drug Therapy, Combination , High-Throughput Screening Assays , Internet , Malaria, Falciparum/parasitology , Mefloquine/pharmacology , Parasitic Sensitivity Tests , Quinine/pharmacology
4.
Emerg Infect Dis ; 18(8): 1346-9, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22840888

ABSTRACT

We investigated chloroquine sensitivity to Plasmodium falciparum in travelers returning to France and Canada from Haiti during a 23-year period. Two of 19 isolates obtained after the 2010 earthquake showed mixed pfcrt 76K+T genotype and high 50% inhibitory concentration. Physicians treating malaria acquired in Haiti should be aware of possible chloroquine resistance.


Subject(s)
Chloroquine/pharmacology , Drug Resistance/genetics , Earthquakes , Plasmodium falciparum/drug effects , Travel , Adolescent , Adult , Aged , Animals , Antimalarials/pharmacology , Antimalarials/therapeutic use , Canada/epidemiology , Child , Child, Preschool , Chloroquine/therapeutic use , Disasters , Female , France/epidemiology , Genotype , Haiti , Humans , Infant , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Male , Membrane Transport Proteins/genetics , Middle Aged , Parasitic Sensitivity Tests , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Protozoan Proteins/genetics , Young Adult
5.
Malar J ; 10: 380, 2011 Dec 20.
Article in English | MEDLINE | ID: mdl-22185672

ABSTRACT

BACKGROUND: This study aimed to explore Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment with artemisinin-based combination therapy in children with clinical malaria in a high transmission area in Africa. METHODS: A total of 50 children aged 1-10 years with acute uncomplicated P. falciparum malaria in Bagamoyo District, Tanzania, were enrolled. Participants were hospitalized and received supervised standard treatment with artemether-lumefantrine according to body weight in six doses over 3 days. Blood samples were collected 11 times, i.e. at time of diagnosis (-2 h) and 0, 2, 4, 8, 16, 24, 36, 48, 60 and 72 h after initiation of treatment. Parasite population dynamics were assessed using nested polymerase chain reaction (PCR)-genotyping of merozoite surface protein (msp) 1 and 2. RESULTS: PCR-analyses from nine sequential blood samples collected after initiation of treatment identified 20 and 21 additional genotypes in 15/50 (30%) and 14/50 (28%) children with msp1 and msp2, respectively, non-detectable in the pre-treatment samples (-2 and 0 h combined). Some 15/20 (75%) and 14/21 (67%) of these genotypes were identified within 24 h, whereas 17/20 (85%) and 19/21 (90%) within 48 h for msp1 and msp2, respectively. The genotype profile was diverse, and varied considerably over time both within and between patients, molecular markers and their respective families. CONCLUSION: PCR analyses from multiple blood samples collected during the early treatment phase revealed a complex picture of parasite sub-populations. This underlines the importance of interpreting PCR-outcomes with caution and suggests that the present use of PCR-adjustment from paired blood samples in anti-malarial drug trials may overestimate assessment of drug efficacy in high transmission areas in Africa.The study is registered at http://www.clinicaltrials.gov with identifier NCT00336375.


Subject(s)
Antimalarials/administration & dosage , Artemisinins/therapeutic use , Ethanolamines/therapeutic use , Fluorenes/therapeutic use , Malaria, Falciparum/parasitology , Plasmodium falciparum/drug effects , Antigens, Protozoan/analysis , Antigens, Protozoan/genetics , Antimalarials/therapeutic use , Artemether, Lumefantrine Drug Combination , Artemisinins/administration & dosage , Body Weight , Child , Child, Preschool , Drug Combinations , Ethanolamines/administration & dosage , Female , Fluorenes/administration & dosage , Genetic Markers , Genetic Variation , Genotype , Genotyping Techniques , Humans , Infant , Malaria, Falciparum/drug therapy , Malaria, Falciparum/epidemiology , Male , Merozoite Surface Protein 1/analysis , Merozoite Surface Protein 1/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Polymerase Chain Reaction , Protozoan Proteins/analysis , Protozoan Proteins/genetics , Tanzania/epidemiology , Time Factors , Treatment Outcome
6.
Antimicrob Agents Chemother ; 54(11): 4780-8, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20713675

ABSTRACT

The combination of artemether (ARM) and lumefantrine is currently the first-line treatment of uncomplicated falciparum malaria in mainland Tanzania. While the exposure to lumefantrine has been associated with the probability of adequate clinical and parasitological cure, increasing exposure to artemether and the active metabolite dihydroartemisinin (DHA) has been shown to decrease the parasite clearance time. The aim of this analysis was to describe the pharmacokinetics and pharmacodynamics of artemether, dihydroartemisinin, and lumefantrine in African children with uncomplicated malaria. In addition to drug concentrations and parasitemias from 50 Tanzanian children with falciparum malaria, peripheral parasite densities from 11 asymptomatic children were included in the model of the parasite dynamics. The population pharmacokinetics and pharmacodynamics of artemether, dihydroartemisinin, and lumefantrine were modeled in NONMEM. The distribution of artemether was described by a two-compartment model with a rapid absorption and elimination through metabolism to dihydroartemisinin. Dihydroartemisinin concentrations were adequately illustrated by a one-compartment model. The pharmacokinetics of artemether was time dependent, with typical oral clearance increasing from 2.6 liters/h/kg on day 1 to 10 liters/h/kg on day 3. The pharmacokinetics of lumefantrine was sufficiently described by a one-compartment model with an absorption lag time. The typical value of oral clearance was estimated to 77 ml/h/kg. The proposed semimechanistic model of parasite dynamics, while a rough approximation of the complex interplay between malaria parasite and the human host, adequately described the early effect of ARM and DHA concentrations on the parasite density in malaria patients. However, the poor precision in some parameters illustrates the need for further data to support and refine this model.


Subject(s)
Antimalarials/pharmacokinetics , Antimalarials/therapeutic use , Artemisinins/pharmacokinetics , Artemisinins/therapeutic use , Ethanolamines/pharmacokinetics , Ethanolamines/therapeutic use , Fluorenes/pharmacokinetics , Fluorenes/therapeutic use , Malaria, Falciparum/drug therapy , Artemether , Body Temperature , Child , Child, Preschool , Female , Humans , Infant , Lumefantrine , Male , Tanzania , Treatment Outcome
7.
J Infect Dis ; 200(9): 1456-64, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19807279

ABSTRACT

Plasmodium falciparum response mechanisms to the major artemisinin-based combination therapies (ACTs) are largely unknown. Multidrug-resistance protein (MRP)-like adenosine triphosphate (ATP)-binding cassette transporters are known to be related to multidrug resistance in many organisms. Therefore, we hypothesized that sequence variation in pfmrp1 can contribute to decreased parasite sensitivity to ACT. Through sequencing of the pfmrp1 open reading frame for 103 geographically diverse P. falciparum infections, we identified 27 single-nucleotide polymorphisms (SNPs), of which 21 were nonsynonymous and 6 synonymous. Analyses of clinical efficacy trials with artesunate-amodiaquine and artemether-lumefantrine detected a specific selection of the globally prevalent I876V SNP in recurrent infections after artemether-lumefantrine treatment. Additional in silico studies suggested an influence of variation in amino acid 876 on the ATP hydrolysis cycle of pfMRP1 with potential impact on protein functionality. Our data suggest for the first time, to our knowledge, the involvement of pfMRP1 in P. falciparum in vivo response to ACT.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Drug Resistance/genetics , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Africa , Animals , Clinical Trials as Topic , Drug Therapy, Combination , Humans , Open Reading Frames/genetics , Polymorphism, Single Nucleotide/genetics , Selection, Genetic , Sweden , Travel
8.
Antimicrob Agents Chemother ; 53(6): 2553-6, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19364873

ABSTRACT

Sulfadoxine-pyrimethamine (SP) remains widely recommended for intermittent preventive treatment against Plasmodium falciparum malaria for pregnant women and infants in Africa. Resistance to SP is increasing and associated primarily with mutations in the P. falciparum dhfr (Pfdhfr) and Pfdhps genes. This study aimed to explore the hypothetical association of genetic alterations in the P. falciparum multidrug resistance protein gene (Pfmrp1) with the in vivo response to SP by detecting the selection of single nucleotide polymorphisms (SNPs) following standard single-dose treatment administered to children with acute uncomplicated P. falciparum malaria in Tanzania. We detected significant selection of parasites carrying the Pfmrp1 1466K allele in samples from children with recrudescent infections, with 12 (100%) of 12 such samples being positive for this allele, compared to 52 (67.5%) of 77 baseline samples (P = 0.017), in parallel with the selection of the Pfdhfr Pfdhps quintuple mutant haplotype in cases of recrudescence (P = 0.001). There was no association between the 1466K SNP and the Pfdhfr Pfdhps quintuple mutation, indicating independent selections. Our data point for the first time to a role for a P. falciparum multidrug resistance protein homologue in the antimalarial activity of SP. Moreover, they add to the growing evidence of the potential importance of Pfmrp1 in antimalarial drug resistance.


Subject(s)
Antimalarials/pharmacology , Multidrug Resistance-Associated Proteins/genetics , Plasmodium falciparum/drug effects , Polymorphism, Single Nucleotide , Pyrimethamine/pharmacology , Sulfadoxine/pharmacology , Animals , Child , Drug Combinations , Genotype , Haplotypes , Humans
9.
Infect Genet Evol ; 8(3): 340-5, 2008 May.
Article in English | MEDLINE | ID: mdl-18359278

ABSTRACT

The sarco/endoplasmic reticulum Ca(2+)-ATPase orthologue of Plasmodium falciparum (PfATP6) has been suggested to be involved in the mechanism of action and resistance to artemisinins, the main constituent of artemisinin-based combination therapy (ACT). In previous studies only six single-nucleotide polymorphisms (SNPs) have been described in clinical samples and field isolates. Our aim was to sequence a large number of clinical samples with different geographical origins to further explore the natural diversity of PfATP6. We sequenced three genetic regions of PfATP6 in 388 samples from 17 countries, mainly Zanzibar and Tanzania, and identified 33 SNPs, of which 29 were non-synonymous and 4 synonymous. To our knowledge 29 of these SNPs have not been described previously. Three mutations were found in high frequency in Zanzibar and Tanzania; E431K, N569K and A630S were present in respectively 31% (95% CI, 26-37%), 36% (95% CI, 30-42%), and 2% (95% CI, 1-5%) of Zanzibar samples and in 39% (95% CI, 29-51%), 29% (95% CI, 16-45%) and 7% (95% CI, 1-22%) of the Tanzania Mainland samples. No variation was found in position 263, suggested to be involved in artemisinin binding to PfATP6, or in position 769, proposed to be related to decreased sensitivity to artemether in vitro. A considerable difference in diversity was observed between the three genetic regions. In conclusion our findings show that PfATP6 is a more diverse gene than previously demonstrated. This natural variation may constitute a starting ground for artemisinin-driven progressive selection of resistant parasites.


Subject(s)
Calcium-Transporting ATPases/genetics , Plasmodium falciparum/genetics , Polymorphism, Single Nucleotide , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sequence Homology, Amino Acid , Animals , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Drug Resistance/genetics , Endoplasmic Reticulum/enzymology , Lactones/therapeutic use , Malaria, Falciparum/drug therapy , Models, Biological , Plasmodium falciparum/enzymology , Sesquiterpenes/therapeutic use , Tanzania
10.
Trop Med Int Health ; 12(6): 736-42, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17550470

ABSTRACT

OBJECTIVE: Artemether-lumefantrine (AL), presently the most favoured combination therapy against uncomplicated Plasmodium falciparum malaria in Africa, has recently shown to select for the pfmdr1 86N allele. The objective of this study was to search for the selection of other mutations potentially involved in artemether-lumefantrine tolerance and/or resistance, i.e. pfmdr1 gene amplification, pfmdr1 Y184F, S1034C, N1042D, D1246Y, pfcrt S163R and PfATP6 S769N. METHODS: The above mentioned SNPs were analysed by PCR-restriction fragment length polymorphism and pfmdr1 gene amplification by real-time PCR based protocols in parasites from 200 children treated with AL for uncomplicated P. falciparum malaria in Zanzibar. RESULTS: A statistically significant selection of pfmdr1 184F mostly in combination with 86N was seen in reinfections after treatment. No pfmdr1 gene amplification was found. CONCLUSION: The results suggest that different pfmdr1 alleles are involved in the development of tolerance/resistance to lumefantrine.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Ethanolamines/therapeutic use , Fluorenes/therapeutic use , Genes, MDR/genetics , Malaria, Falciparum/drug therapy , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Animals , Artemether, Lumefantrine Drug Combination , Child , Drug Combinations , Drug Resistance/genetics , Gene Amplification/genetics , Gene Frequency/genetics , Haplotypes/genetics , Humans , Malaria, Falciparum/epidemiology , Malaria, Falciparum/genetics , Mutation/genetics , Plasmodium falciparum/drug effects , Polymorphism, Restriction Fragment Length , Recurrence , Tanzania/epidemiology
11.
Proc Natl Acad Sci U S A ; 103(18): 6976-81, 2006 May 02.
Article in English | MEDLINE | ID: mdl-16636273

ABSTRACT

Deformylase inhibitors belong to a novel antibiotic class that targets peptide deformylase, a bacterial enzyme that removes the formyl group from N-terminal methionine in nascent polypeptides. Using the bacterium Salmonella enterica, we isolated mutants with resistance toward the peptide deformylase inhibitor actinonin. Resistance mutations were identified in two genes that are required for the formylation of methionyl (Met) initiator tRNA (tRNAi)(fMet): the fmt gene encoding the enzyme methionyl-tRNA formyltransferase and the folD gene encoding the bifunctional enzyme methylenetetrahydrofolate-dehydrogenase and -cyclohydrolase. In the absence of antibiotic, these resistance mutations conferred a fitness cost that was manifested as a reduced growth rate in laboratory medium and in mice. By serially passaging the low-fitness mutants in growth medium without antibiotic, the fitness costs could be partly ameliorated either by intragenic mutations in the fmt/folD genes or by extragenic compensatory mutations. Of the extragenically compensated fmt mutants, approximately one-third carried amplifications of the identical, tandemly repeated metZ and metW genes, encoding tRNAi. The increase in metZW gene copy number varied from 5- to 40-fold and was accompanied by a similar increase in tRNAi levels. The rise in tRNAi level compensated for the lack of methionyl-tRNA formyltransferase activity and allowed translation initiation to proceed with nonformylated methionyl tRNAi. Amplified units varied in size from 1.9 to 94 kbp. Suppression of deleterious mutations by gene amplification may be involved in the evolution of new gene functions.


Subject(s)
Drug Resistance/genetics , Gene Expression Regulation, Bacterial , RNA, Transfer, Met/genetics , Salmonella typhimurium , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/genetics , Amidohydrolases/metabolism , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Evolution, Molecular , Hydroxamic Acids/metabolism , Hydroxamic Acids/pharmacology , Mice , Mutation , RNA, Transfer, Met/metabolism , Salmonella typhimurium/drug effects , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics
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