RESUMO
Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60 percent of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.
Assuntos
Humanos , Animais , Masculino , Adulto , Pessoa de Meia-Idade , Di-Hidropteroato Sintase/genética , Mutação , Plasmodium falciparum/enzimologia , Tetra-Hidrofolato Desidrogenase/genética , Aminoácidos/genética , Antimaláricos/uso terapêutico , Brasil , Resistência a Medicamentos , Genótipo , Malária/tratamento farmacológico , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da PolimeraseRESUMO
Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.
Assuntos
Di-Hidropteroato Sintase/genética , Mutação , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Adulto , Idoso , Aminoácidos/genética , Animais , Antimaláricos/uso terapêutico , Brasil , Resistência a Medicamentos , Genótipo , Humanos , Malária/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da PolimeraseRESUMO
Malaria parasites are genetically diverse at all levels of endemicity. In contrast, the merozoite surface protein (MSP) alleles in samples from 2 isolated populations of Yanomami Amerindians during an epidemic of Plasmodium falciparum were identical. The nonvariable restriction fragment length polymorphism patterns further suggested that the sequential outbreak comprised only a single P. falciparum genotype. By examination of serial samples from single human infections, the MSP characteristics were found to remain constant throughout the course of infection. An apparent clonal population structure of parasites seemed to cause outbreaks in small isolated villages. The use of standard molecular epidemiologic methods to measure genetic diversity in malaria revealed the occurrence of a genetically monomorphic population of P. falciparum within a human community.
Assuntos
Surtos de Doenças , Indígenas Sul-Americanos , Malária Falciparum/etnologia , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Animais , Antígenos de Protozoários/genética , Southern Blotting , DNA de Protozoário/análise , Eletroforese em Gel de Ágar , Genes de Protozoários , Humanos , Proteína 1 de Superfície de Merozoito/genética , Epidemiologia Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , População Rural , Venezuela/epidemiologiaRESUMO
The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Antimaláricos/farmacologia , DNA de Protozoário/análise , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Animais , Brasil/epidemiologia , Resistência a Medicamentos , Resistência a Múltiplos Medicamentos/genética , Quimioterapia Combinada , Genes de Protozoários/genética , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/metabolismo , Mefloquina/farmacologia , Plasmodium falciparum/classificação , Plasmodium falciparum/isolamento & purificação , Mutação Puntual , Quinina/farmacologiaRESUMO
Leishmaniavirus is a double-stranded RNA virus that persistently infects some strains of the protozoan parasite Leishmania. There is considerable interest in the possibility that the presence of this virus alters parasite phenotype and may affect disease pathogenesis. If so, the virus marker could provide a valuable prognostic indicator for human leishmaniasis, particularly in those cases caused by New World parasite strains. The virus has been detected in cultured L. braziliensis, L. b. guyanensis, and L. major. To date there has been no information as to the extent of infection in samples prior to culturing in the laboratory. This study demonstrates, through the reverse transcription-polymerase chain reaction, that Leishmaniavirus exists in human biopsy samples of leishmaniasis prior to manipulation in culture.
Assuntos
Leishmaniose Cutânea/virologia , Leishmaniavirus/isolamento & purificação , Pele/virologia , Animais , Sequência de Bases , Biópsia por Agulha , Sequência Consenso , DNA Viral/análise , DNA Viral/química , Humanos , Leishmaniose Cutânea/etiologia , Leishmaniose Cutânea/patologia , Leishmaniavirus/genética , Leishmaniavirus/fisiologia , Dados de Sequência Molecular , Peru , Reação em Cadeia da Polimerase , RNA Viral/genética , Análise de Sequência de DNARESUMO
A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.
Assuntos
Antiprotozoários/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/genética , Leishmania/efeitos dos fármacos , Verapamil/farmacologia , Vimblastina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , DNA de Protozoário , Eletroforese em Gel de Campo Pulsado , Amplificação de Genes , Genes de Protozoários , Leishmania/genética , Técnicas de Sonda Molecular , Fenótipo , RNA de Protozoário , Rodamina 123 , Rodaminas/metabolismoRESUMO
We have isolated and characterized an Entamoeba histolytica alpha-tubulin (alpha Tub)-encoding gene (Eh alpha tub). A 700-bp DNA fragment was amplified by PCR using primers derived from consensus alpha- and beta-Tub amino acid (aa) sequences from different organisms and E. histolytica DNA as the template. These PCR fragments were used to screen both genomic DNA and cDNA libraries in order to isolate an Eh alpha tub structural gene. Two overlapping clones containing the complete alpha tub ORF (1392 bp) were isolated from the genome and cDNA libraries. The deduced aa sequence of Eh alpha Tub has 55.5, 50 and 52% identity to Plasmodium falciparum alpha Tub 2, Saccharomyces cerevisiae alpha Tub 2 and human alpha Tub, respectively. Interestingly, the predicted Eh alpha Tub protein lacks a poly-acidic motif at its C terminus which is involved in Tub polymerization and microtubule-associated protein binding in other organisms. This fact may indicate a difference in tubule assembly in this organism and could provide a potential key for the development of therapeutic agents. According to Southern blot experiments and the sequences of several clones, at least two non-adjacent copies of alpha tub are present in the E. histolytica genome. A 1.5-kb transcript corresponding to the alpha tub mRNA was detected in mRNA from asynchronous E. histolytica trophozoites.
Assuntos
Entamoeba histolytica/genética , Genes de Protozoários/genética , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Fases de Leitura Aberta/genética , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
We used the polymerase chain reaction (PCR) to study the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica in a rural community in Mexico. Formalin-fixed stool samples were used for extraction of DNA. The PCR amplifications were performed using two sets of primers that discriminate between pathogenic or non-pathogenic E. histolytica. A total of 201 randomly selected individuals were studied. Among them, 25 (12%) were diagnosed to be infected with E. histolytica by microscopy; PCR identified 24 of these as positive (sensitivity = 0.96) and of 176 negative individuals, only three were identified as positive (specificity = 0.98). The PCR analysis defined three populations: 14 cases were positive for both pathogenic and nonpathogenic E. histolytica, nine cases were positive for pathogenic and negative for nonpathogenic E. histolytica, and only one case was negative for pathogenic and positive for nonpathogenic E. histolytica. Infection by E. histolytica was strongly associated to infection with Entamoeba coli (odds ratio [OR] = 9.41, 95% confidence interval [CI] = 3.09, 28.65, P < 0.0004) and Endolimax nana (OR = 6.15, 95% CI = 2.03, 18.17, P < 0.0004). This new technique has high specificity and sensitivity; it is simple, reproducible, fast, avoids the need to culture trophozoites, and can be applied in the field for epidemiologic studies.
Assuntos
DNA de Protozoário/análise , Disenteria Amebiana/epidemiologia , Entamoeba histolytica/patogenicidade , Adolescente , Adulto , Amebíase/complicações , Animais , Sequência de Bases , Criança , Pré-Escolar , DNA de Protozoário/química , Disenteria Amebiana/complicações , Disenteria Amebiana/parasitologia , Endolimax/isolamento & purificação , Entamoeba histolytica/genética , Fezes/parasitologia , Feminino , Humanos , Lactente , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , População Rural , Sensibilidade e EspecificidadeAssuntos
Colchicina/farmacologia , Emetina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Verapamil/farmacologia , Animais , DNA de Protozoário/genética , Interações Medicamentosas , Resistência a Medicamentos , Entamoeba histolytica/genética , Entamoeba histolytica/crescimento & desenvolvimento , Amplificação de Genes , Hibridização de Ácido NucleicoRESUMO
In order to understand the mechanisms which generate minicircle sequence diversity, we sequenced three minicircles belonging to the same or closely related sequence classes from the kinetoplast DNA of Leishmania mexicana amazonensis strains, PH8, Raimundo, and Josefa. Closely related minicircles from PH8 and Raimundo were unexpectedly found to differ at 11% of positions within the evolutionarily conserved region, but at only 3.9% of positions in the variable region. It thus appears that accumulation of point mutations will not account for the wide intra-strain and intra-subspecies divergence of the variable region. Comparison of more distantly related minicircles from PH8 and Josefa revealed only two short stretches of 70% homology within the variable region. These stretches of homology are not located in the same positions relative to the conserved regions in their respective minicircles. They may represent vestiges of recombinational events responsible for the rapid divergence of minicircle variable regions.