RESUMO
The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Artemisininas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Quinina/farmacologia , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , DNA Complementar , Fases de Leitura Aberta/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76. METHODS: Small-volume samples (≤ 500 µL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences. RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination. CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt.
Assuntos
Processamento Alternativo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Pseudogenes , Precursores de RNA/metabolismo , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Humanos , Lactente , Masculino , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Polimorfismo Genético , Conformação Proteica , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Análise de Sequência de DNA , Sudão , TanzâniaRESUMO
BACKGROUND: Persistent digestive disorders account for considerable disease burden in the tropics. Despite advances in understanding acute gastrointestinal infections, important issues concerning epidemiology, diagnosis, treatment and control of most persistent digestive symptomatologies remain to be elucidated. Helminths and intestinal protozoa are considered to play major roles, but the full extent of the aetiologic spectrum is still unclear. We provide an overview of pathogens causing digestive disorders in the tropics and evaluate available reference tests. METHODS: We searched the literature to identify pathogens that might give rise to persistent diarrhoea, chronic abdominal pain and/or blood in the stool. We reviewed existing laboratory diagnostic methods for each pathogen and stratified them by (i) microscopy; (ii) culture techniques; (iii) immunological tests; and (iv) molecular methods. Pathogen-specific reference tests providing highest diagnostic accuracy are described in greater detail. RESULTS: Over 30 pathogens may cause persistent digestive disorders. Bacteria, viruses and parasites are important aetiologic agents of acute and long-lasting symptomatologies. An integrated approach, consisting of stool culture, microscopy and/or specific immunological techniques for toxin, antigen and antibody detection, is required for accurate diagnosis of bacteria and parasites. Molecular techniques are essential for sensitive diagnosis of many viruses, bacteria and intestinal protozoa, and are increasingly utilised as adjuncts for helminth identification. CONCLUSIONS: Diagnosis of the broad spectrum of intestinal pathogens is often cumbersome. There is a need for rapid diagnostic tests that are simple and affordable for resource-constrained settings, so that the management of patients suffering from persistent digestive disorders can be improved.
Assuntos
Gastroenteropatias/diagnóstico , Gastroenteropatias/etiologia , Medicina Tropical , Animais , Bactérias/patogenicidade , Técnicas de Laboratório Clínico/métodos , Humanos , Parasitos/patogenicidade , Vírus/patogenicidadeRESUMO
The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (-)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (-)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC(50) ratio of (-)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (-) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.
Assuntos
Antimaláricos/farmacologia , Alcaloides de Cinchona/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Protozoários/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Quinidina/análogos & derivados , Quinidina/farmacologia , Quinolinas/farmacologiaRESUMO
BACKGROUND: Parasite resistance to the anti-malarial drug chloroquine is common in eastern Sudan. Dynamic within-host changes in the relative abundance of both sensitive and resistant Plasmodium falciparum parasites were examined in a cohort of chloroquine-treated patients presenting with uncomplicated falciparum malaria, using a novel allele-specific quantitative approach. METHODS: Treatment outcomes were determined for 93 patients of all ages in a per protocol cohort using a modified 14-day WHO protocol. Parasite DNA samples at days 0, 1, 2, 3, 7 and 14 following treatment were analysed using real-time quantitative PCR methods that distinguished resistant and sensitive genotypes at amino acids 72-76 of the pfcrt locus. RESULTS: Chloroquine treatment was not efficacious, and of 93 assessable patients, only 10 individuals (10.7%; 95% C.I. 4.34-17.2%) enjoyed an adequate clinical and parasitological response. Resistant parasites with the haplotype CVIET at codons 72-76 of the pfcrt locus were dominant in the starting population. Chloroquine sensitive parasites with the haplotype CVMNK were detected in 19 individuals prior to treatment (20.43%; 95% C.I. 5.14-18.5%). In these patients, CQ treatment rapidly selected CVIET parasites, and this haplotype overwhelmingly dominated the parasite population in each individual by day 2 after treatment. CONCLUSIONS: Such rapid intra-host selection of particular genotypes after the introduction of drug will cause frequent misidentification of parasite genotypes present in the starting population. This will have a potentially serious confounding effect on clinical trials which employ PCR-corrected estimates of treatment failure, as resistant parasites below the detection threshold in the pre-treatment sample can be erroneously classified as "new" infections during follow-up, over-estimating drug efficacy.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Adolescente , Adulto , Idoso , Alelos , Antimaláricos/uso terapêutico , População Negra , Criança , Pré-Escolar , Cloroquina/uso terapêutico , Ensaios Clínicos como Assunto , Estudos de Coortes , DNA de Protozoário/genética , Feminino , Seguimentos , Genótipo , Haplótipos , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Sudão/epidemiologia , Resultado do Tratamento , Adulto JovemRESUMO
Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, have been proposed as drug candidates for Chagas disease, a neglected infectious tropical disease caused by the protozoan parasite Trypanosoma cruzi. To understand better the mechanism of action and resistance to these inhibitors, a clone of the T. cruzi Y strain was cultured under intermittent and increasing concentrations of ravuconazole until phenotypic stability was achieved. The ravuconazole-selected clone exhibited loss in fitness in vitro when compared to the wild-type parental clone, as observed in reduced invasion capacity and slowed population growth in both mammalian and insect stages of the parasite. In drug activity assays, the resistant clone was above 300-fold more tolerant to ravuconazole than the sensitive parental clone, when the half-maximum effective concentration (EC50) was considered. The resistant clones also showed reduced virulence in vivo, when compared to parental sensitive clones. Cross-resistance to posaconazole and other CYP51 inhibitors, but not to other antichagasic drugs that act independently of CYP51, such as benznidazole and nifurtimox, was also observed. A novel amino acid residue change, T297M, was found in the TcCYP51 gene in the resistant but not in the sensitive clones. The structural effects of the T297M, and of the previously described P355S residue changes, were modelled to understand their impact on interaction with CYP51 inhibitors.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Resistência a Múltiplos Medicamentos/genética , Esterol 14-Desmetilase/genética , Trypanosoma cruzi , Animais , Linhagem Celular , Doença de Chagas/tratamento farmacológico , Genes de Protozoários , Mutação , Nitroimidazóis/farmacologia , Tiazóis/farmacologia , Triazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
OBJECTIVES: To assess the extent of drug resistance in Uige through molecular genetic analysis and to test whether the dhfr triple mutant alleles present in Angola are of southeast Asian origin. METHODS: Seventy-one samples of blood from children admitted to the Pediatric Emergency Unit of Uige Provincial Hospital in 2004 were screened for resistance mutations at pfcrt, pfmdr1, pfdhfr, pfdhps and pfATPase6. RESULTS: Mutations in pfcrt (codon76), pfmdr1 (codon86), pfdhfr (codons 51, 59, 108) and pfdhps (codons 436, 437) were common. Among the 66 isolates for which we were able to determine complete genetic information 13.7% carried all seven of these mutations. Flanking microsatellite analysis revealed the triple mutant pfdhfr was derived from the southeast Asian lineage, while the N51I+S108N double mutant pfdhfr alleles are a local origin. pfATPase6 mutations were rare and S769N was not found. CONCLUSION: The parasite population of Uige Angola has high frequency mutations in pfcrt, dhfr and dhps associated with resistance to chloroquine and sulphadoxine pyrimethamine, reflecting past reliance on these two drugs which were the mainstay of treatment until recently. Our findings show that drug resistance in Uige has occurred through a combination of local drug pressure and the regional and international dispersal of resistance mutant alleles.
Assuntos
Antimaláricos/uso terapêutico , Resistência a Múltiplos Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Protozoários/genética , Pirimetamina/uso terapêutico , Angola , Criança , Genótipo , Humanos , Malária Falciparum/genética , Repetições de Microssatélites/genética , Mutação/genéticaRESUMO
BACKGROUND: Malaria reduction and future elimination in China is made more difficult by the importation of cases from neighboring endemic countries, particularly Myanmar, Laos, and Vietnam, and increased travel to Africa by Chinese nationals. The increasing prevalence of artemisinin resistant parasites across Southeast Asia highlights the importance of monitoring the parasite importation into China. Artemisinin resistance in the Mekong region is associated with variants of genes encoding the K13 kelch domain protein (pf13k), found in specific genetic backgrounds, including certain alleles of genes encoding the chloroquine resistance transporter (pfcrt) and multidrug resistance transporter PgH1 (pfmdr1). METHODS: In this study we investigated the prevalence of drug resistance markers in 72 P. falciparum samples from uncomplicated malaria infections in Tengchong and Yingjiang, counties on the Yunnan-Myanmar border. Variants of pf13k, pfcrt and pfmdr1 are described. RESULTS: Almost all parasites harboured chloroquine-resistant alleles of pfcrt, whereas pfmdr1 was more diverse. Major mutations in the K13 propeller domain associated with artemisinin resistance in the Mekong region (C580Y, R539T and Y493H) were absent, but F446I and two previously undescribed mutations (V603E and V454I) were identified. Protein structural modelling was carried out in silico on each of these K13 variants, based on recently published crystal structures for the K13 propeller domain. Whereas F446I was predicted to elicit a moderate destabilisation of the propeller structure, the V603E substitution is likely to lead to relatively high protein instability. We plotted these stability estimates, and those for all previously described variants, against published values for in vivo parasitaemia half-life, and found that quadratic regression generates a useful predictive algorithm. CONCLUSION: This study provides a baseline of P. falciparum resistance-associated mutations prevalent at the China-Myanmar border. We also show that protein modelling can be used to generate testable predictions as to the impact of pfk13 mutations on in vivo (and potentially in vitro) artemisinin susceptibility.
Assuntos
Artemisininas/uso terapêutico , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Algoritmos , Alelos , Antimaláricos/uso terapêutico , China , Cloroquina/uso terapêutico , Variação Genética , Humanos , Malária Falciparum/epidemiologia , Proteínas de Membrana Transportadoras/genética , Mutação , Mianmar , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Prevalência , Proteínas de Protozoários/genética , Análise de Regressão , Análise de Sequência de DNA , MigrantesRESUMO
Chloroquine (CQ), a 4-aminoquinoline, accumulates in acidic digestive vacuoles of the malaria parasite, preventing conversion of toxic haematin to beta-haematin. We examine how bis 4-aminoquinoline piperaquine (PQ) and its hydroxy-modification (OH-PQ) retain potency on chloroquine-resistant (CQ-R) Plasmodium falciparum. For CQ, PQ, OH-PQ and 4 and 5, representing halves of PQ, beta-haematin inhibitory activity (BHIA) was assayed, while potency was determined in CQ-sensitive (CQ-S) and CQ-R P. falciparum. From measured pK(a)s and the pH-modulated distribution of base between water and lipid (logD), the vacuolar accumulation ratio (VAR) of charged drug from plasma water (pH 7.4) into vacuolar water (pH 4.8) and lipid accumulation ratio (LAR) were calculated. All agents were active in BHIA. In CQ-S, PQ, OH-PQ and CQ were equally potent while 4 and 5 were 100 times less potent. CQ with two basic centres has a VAR of 143,482, while 4 and 5, with two basic centres of lower pK(a)s have VARs of 1287 and 1966. In contrast PQ and OH-PQ have four basic centres and achieve VARs of 104,378 and 19,874. This confirms the importance of VAR for potency against CQ-S parasites. Contrasting results were seen in CQ-R. 5, PQ and OH-PQ with LARs of 693; 973,492 and 398,118 (compared with 8.25 for CQ) showed similar potency in CQ-S and CQ-R. Importance of LAR for potency against CQ-R parasites probably reflects ability to block efflux by hydrophobic interaction with PfCRT but may relate to beta-haematin inhibition in vacuolar lipid.
Assuntos
Aminoquinolinas/farmacologia , Antimaláricos/farmacologia , Hemeproteínas/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Quinolinas/farmacologia , Vacúolos , Aminoquinolinas/síntese química , Aminoquinolinas/química , Animais , Antimaláricos/síntese química , Antimaláricos/química , Cloroquina/farmacologia , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Estágios do Ciclo de Vida/efeitos dos fármacos , Lipídeos/química , Modelos Moleculares , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Vacúolos/química , Vacúolos/efeitos dos fármacos , Água/químicaRESUMO
We report the preparation and antiparasitic activity in vitro and in vivo of a series of isoflavone derivatives related to genistein. These analogues retain the 5,7-dihydroxyisoflavone core of genistein: direct genistein analogues (2-H isoflavones), 2-carboethoxy isoflavones, and the precursor deoxybenzoins were all evaluated. Excellent in vitro activity against Cryptosporidium parvum was observed for both classes of isoflavones in cell cultures, and the lead compound 19, RM6427, shows high in vivo efficacy against an experimental infection.
Assuntos
Coccidiostáticos/síntese química , Cryptosporidium parvum/efeitos dos fármacos , Isoflavonas/síntese química , Animais , Bovinos , Linhagem Celular Tumoral , Coccidiostáticos/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/isolamento & purificação , Feminino , Genisteína/análogos & derivados , Genisteína/síntese química , Genisteína/farmacologia , Gerbillinae , Humanos , Hospedeiro Imunocomprometido , Isoflavonas/farmacologia , Masculino , Relação Estrutura-AtividadeRESUMO
Antimalarial chloroquine (CQ) prevents haematin detoxication when CQ-base concentrates in the acidic digestive vacuole through protonation of its p-aminopyridine (pAP) basic aromatic nitrogen and sidechain diethyl-N. CQ export through the variant vacuolar membrane export channel, PFCRT, causes CQ-resistance in Plasmodium falciparum but 3-methyl CQ (sontochin SC), des-ethyl amodiaquine (DAQ) and bis 4-aminoquinoline piperaquine (PQ) are still active. This is determined by changes in drug accumulation ratios in parasite lipid (LAR) and in vacuolar water (VAR). Higher LAR may facilitate drug binding to and blocking PFCRT and also aid haematin in lipid to bind drug. LAR for CQ is only 8.3; VAR is 143,482. More hydrophobic SC has LAR 143; VAR remains 68,523. Similarly DAQ with a phenol substituent has LAR of 40.8, with VAR 89,366. In PQ, basicity of each pAP is reduced by distal piperazine N, allowing very high LAR of 973,492, retaining VAR of 104,378. In another bis quinoline, dichlorquinazine (DCQ), also active but clinically unsatisfactory, each pAP retains basicity, being insulated by a 2-carbon chain from a proximal nitrogen of the single linking piperazine. While LAR of 15,488 is still high, the lowest estimate of VAR approaches 4.9 million. DCQ may be expected to be very highly lysosomotropic and therefore potentially hepatotoxic. In 11 pAP antimalarials a quadratic relationship between logLAR and logResistance Index (RI) was confirmed, while log (LAR/VAR) vs logRI for 12 was linear. Both might be used to predict the utility of structural modifications.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Hemina/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Amodiaquina/análogos & derivados , Amodiaquina/química , Amodiaquina/metabolismo , Amodiaquina/farmacologia , Antimaláricos/metabolismo , Transporte Biológico , Cloroquina/análogos & derivados , Cloroquina/química , Cloroquina/metabolismo , Cloroquina/farmacologia , Desenho de Fármacos , Resistência a Medicamentos , Heme/antagonistas & inibidores , Heme/metabolismo , Hemina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Plasmodium falciparum/metabolismo , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacologia , Relação Estrutura-Atividade , Vacúolos/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0160091.].
RESUMO
BACKGROUND: Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance. Expressed in yeast, PfCRT resembles an anion channel. Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin. The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel. It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity. Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own. METHODS: Changes in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined. RESULTS AND CONCLUSIONS: The results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole. Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76. This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT.
Assuntos
Amodiaquina/análogos & derivados , Amodiaquina/farmacologia , Antimaláricos/farmacologia , Cloroquina/farmacologia , Canais Iônicos/genética , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Verapamil/farmacologia , Animais , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Resistência a Medicamentos , Sinergismo Farmacológico , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/química , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/química , TransfecçãoRESUMO
BACKGROUND: The 8-amino and 9-hydroxy substituents of antimalarial cinchona alkaloids have the erythro orientation while their inactive 9-epimers are threo. From the X-ray structures a 90 degrees difference in torsion angle between the N1-H1 and C9-O12 bonds in the two series is believed to be important. In order to kill the malaria parasite, alkaloids must cross the erythrocyte and parasite membranes to accumulate in the acid digestive vacuole where they prevent detoxication of haematin produced during haemoglobin breakdown. METHODS: Ionization constants, octanol/water distribution and haematin interaction are examined for eight alkaloids to explain the influence of small structural differences on activity. RESULTS: Erythro isomers have a high distribution ratio of 55:1 from plasma to the erythrocyte membrane, while for the more basic threo epimers this is only 4.5:1. This gives an increased transfer rate of the erythro drugs into the erythrocyte and thence into the parasite vacuole where their favourable conformation allows interaction with haematin, inhibiting its dimerization strongly (90 +/- 7%) and thereby killing the parasite. The threo compounds not only enter more slowly but are then severely restricted from binding to haematin by the gauche alignment of their N1-H1 and C9-O12 bonds. Confirmatory molecular models allowed measurement of angles and bond lengths and computation of the electronic spectrum of a quinine-haematin complex. CONCLUSION: Differences in the antiplasmodial activity of the erythro and threo cinchona alkaloids may therefore be attributed to the cumulative effects of lipid/aqueous distribution ratio and drug-haematin interaction. Possible insights into the mechanism of chloroquine-resistance are discussed.
Assuntos
Antiprotozoários/química , Antiprotozoários/farmacocinética , Alcaloides de Cinchona/química , Alcaloides de Cinchona/farmacocinética , Relação Quantitativa Estrutura-Atividade , Animais , Antiprotozoários/farmacologia , Cloroquina/farmacocinética , Cloroquina/farmacologia , Alcaloides de Cinchona/farmacologia , Desenho de Fármacos , Resistência a Medicamentos/fisiologia , Hemeproteínas/antagonistas & inibidores , Modelos Moleculares , Testes de Sensibilidade Parasitária , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Quinina/análogos & derivados , Quinina/química , Quinina/farmacocinética , Quinina/farmacologia , EstereoisomerismoRESUMO
We report the first in vitro and genetic confirmation of Malarone (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone. Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Malária Falciparum/tratamento farmacológico , Naftoquinonas/farmacologia , Parasitemia/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Proguanil/farmacologia , Substituição de Aminoácidos , Animais , Antimaláricos/uso terapêutico , Atovaquona , Códon/genética , Citocromos b/antagonistas & inibidores , Citocromos b/genética , Combinação de Medicamentos , Humanos , Londres , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Naftoquinonas/uso terapêutico , Nigéria/etnologia , Parasitemia/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Proguanil/uso terapêutico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , ViagemRESUMO
Every year there are 270 million clinical attacks of malaria and 2 million deaths, caused by the protozoan Plasmodium falciparum. Most of these cases occur in Africa. Chloroquine-resistance has led to reliance on anti-malarial antifolates, in particular the synergistic combination sulfadoxine/pyrimethamine (S/P) which targets enzymatic synthesis of folate co-factors through dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). Resistance to S/P is now increasing and replacement antimalarials are needed. Crystal structures are not yet available for these key enzymes in the folate pathway. This review focuses on the activity of drugs on DHFR in malaria parasites, attempts to interpret differences in activity of pyrimethamine and its related drugs, and to clarify how residue changes due to point mutations determine the development of resistance. In homology-modelled P. falciparum DHFR (PfDHFR), the typical structure of four alpha-helices, 8-stranded beta-sheet, four Loops and eight Turns is clearly seen. Long polar sequences specific for Plasmodium are inserted in Turns 1 and 2. Structures immediately concerned in drug binding are beta-A, L1, alpha-B, alpha-C, T-3, beta-E, alpha-F, and beta-F. The roles of several mutations associated with resistance are discussed. In view of sequence differences in turn 3 in PfDHFR and in the human enzyme, and the marked interaction with residues of T3 of the experimental flexible antifolate WR99210 effective in pyrimethamine and cycloguanil resistance, further drug development in this area is indicated.
Assuntos
Resistência a Medicamentos/genética , Antagonistas do Ácido Fólico/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Animais , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/microbiologia , Mutação/efeitos dos fármacos , Plasmodium falciparum/metabolismoRESUMO
BACKGROUND: Cryptosporidium epidemiology is poorly understood, but infection is suspected of contributing to childhood malnutrition and diarrhea-related mortality worldwide. METHODS/FINDINGS: A prospective cohort of 108 women and their infants in rural/semi-rural Tanzania were followed from delivery through six months. Cryptosporidium infection was determined in feces using modified Ziehl-Neelsen staining. Breastfeeding/infant feeding practices were queried and anthropometry measured. Maternal Cryptosporidium infection remained high throughout the study (monthly proportionâ=â44 to 63%). Infection did not differ during lactation or by HIV-serostatus, except that a greater proportion of HIV-positive mothers were infected at Month 1. Infant Cryptosporidium infection remained undetected until Month 2 and uncommon through Month 3 however, by Month 6, 33% of infants were infected. There were no differences in infant infection by HIV-exposure. Overall, exclusive breastfeeding (EBF) was limited, but as the proportion of infants exclusively breastfed declined from 32% at Month 1 to 4% at Month 6, infant infection increased from 0% at Month 1 to 33% at Month 6. Maternal Cryptosporidium infection was associated with increased odds of infant infection (unadjusted ORâ=â3.18, 95% CI 1.01 to 9.99), while maternal hand washing prior to infant feeding was counterintuitively also associated with increased odds of infant infection (adjusted ORâ=â5.02, 95% CIâ=â1.11 to 22.78). CONCLUSIONS: Both mothers and infants living in this setting suffer a high burden of Cryptosporidium infection, and the timing of first infant infection coincides with changes in breastfeeding practices. It is unknown whether this is due to breastfeeding practices reducing pathogen exposure through avoidance of contaminated food/water consumption; and/or breast milk providing important protective immune factors. Without a Cryptosporidium vaccine, and facing considerable diagnostic challenges and ineffective treatment in young infants, minimizing the overall environmental burden (e.g. contaminated water) and particularly, maternal Cryptosporidium infection burden as a means to protect against early infant infection needs prioritization.
Assuntos
Criptosporidiose/epidemiologia , Animais , Aleitamento Materno , Estudos de Coortes , Criptosporidiose/etiologia , Feminino , Infecções por HIV/complicações , Humanos , Lactente , Recém-Nascido , Masculino , Mães , Prevalência , Estudos Prospectivos , Saúde Pública , Fatores de Risco , Tanzânia/epidemiologiaRESUMO
BACKGROUND: Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. METHODS/PRINCIPAL FINDINGS: We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF(+) CD4(+) T cells and multifunctional IFNgamma(+)TNF(+) CD4(+) and CD8(+) T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin. CONCLUSIONS/SIGNIFICANCE: oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN64793756.
Assuntos
Artemisininas/farmacocinética , Carvão Vegetal/uso terapêutico , Malária Cerebral/prevenção & controle , Administração Oral , Adulto , Animais , Antimaláricos , Artesunato , Carvão Vegetal/farmacologia , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Feminino , Humanos , Infusões Parenterais , Malária Cerebral/tratamento farmacológico , Malária Cerebral/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Plasmodium berghei/efeitos dos fármacos , Taxa de SobrevidaRESUMO
Piperaquine is being developed as a long-acting component in artemisinin combination therapies. It was highly active in vitro and drug interaction studies showed that dihydroartemisinin combinations with piperaquine, chloroquine, and amodiaquine were indifferent tending toward antagonism. Competitive uptake of radiolabeled chloroquine and dihydroartemisinin in combination with other antimalarials was observed.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Plasmodium falciparum/efeitos dos fármacos , Quinolinas , Amodiaquina/farmacologia , Animais , Antimaláricos/metabolismo , Artemisininas/metabolismo , Artemisininas/farmacologia , Cloroquina/metabolismo , Antagonismo de Drogas , Combinação de Medicamentos , Interações Medicamentosas , Testes de Sensibilidade Parasitária , Quinolinas/farmacologia , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Trítio/metabolismoRESUMO
It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.