RESUMO
The immunogenicity and protective efficacy of various antigen-adjuvant formulations derived either from the merozoite-surface protein-3 (MSP-3) or the glutamate-rich protein (GLURP) of Plasmodium falciparum were evaluated in Saimiri sciureus monkeys. These proteins were selected for immunogenicity studies based primarily on their capacity of inducing an antibody-dependent cellular inhibition effect on parasite growth. Some of the S. sciureus monkeys immunized with MSP-3(212-380)-AS02 or GLURP(27-500)-alum were able to fully or partially control parasitaemia upon an experimental P. falciparum [Falciparum Uganda Palo Alto (FUP-SP) strain] blood-stage infection, and this protection was related to the prechallenge antibody titres induced. The data are indicative that MSP-3 and GLURP can induce protective immunity against an experimental P. falciparum infection using adjuvants that are acceptable for human use and this should trigger further studies with those new antigens.
Assuntos
Anticorpos/sangue , Antígenos de Protozoários/farmacologia , Vacinas Antimaláricas/farmacologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/farmacologia , Animais , Anticorpos/imunologia , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/imunologia , Imunofluorescência , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Peptídeos/administração & dosagem , Peptídeos/imunologia , Peptídeos/farmacologia , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/imunologia , SaimiriRESUMO
The Neotropical malaria vector Anopheles aquasalis Curry is distributed predominantly along the Atlantic and Pacific Coasts because of its tolerance for breeding in salt water. We tested the hypothesis that the freshwater Amazon River acts as a barrier to gene flow in northeastern Brazil, by examining variation at a 588-nucleotide fragment of the mitochondrial cytochrome oxidase Igene from five populations. We identified 15 haplotypes of which 5 were shared both (1) between sample localities and (2) across the Amazon River Delta. Sequence divergence ranged from 0.0017-0.0272 (average = 0.0137). Estimates of genetic subdivision based on the presence of the Amazon Riverwere greatest within localities (phi = 0.029) and among regions (phi = 0.018), followed by among localities (phi = 0.011), but none were significant. Parsimony, neighbor-joining, and Nested Clade Analyses were used to estimate relationships among populations and infer evolutionary processes. Two phylogenetically distinct clusters of populations were moderately supported by parsimony. Neighbor-joining trees were poorly resolved, thus providing no geographical resolution and no support for the Amazon River as a barrier to migration. Phylogeographic structure as detected by the Nested Clade Analysis was consistent with restricted gene flow coupled with isolation by distance. Taken together, these analyses suggest that the localities within this region of northeastern Brazil constitute a single large population of An. aquasalis that spans the Amazon Delta.
Assuntos
Anopheles/genética , Animais , Anopheles/classificação , Anopheles/patogenicidade , Brasil , Água Doce , Variação Genética , Geografia , Haplótipos , Insetos Vetores , Malária/parasitologia , Modelos Genéticos , FilogeniaAssuntos
Transportadores de Cassetes de Ligação de ATP , Antimaláricos/uso terapêutico , Malária Falciparum/tratamento farmacológico , Mefloquina/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Animais , Brasil/epidemiologia , Resistência a Múltiplos Medicamentos , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility.
Assuntos
Variação Genética/genética , Malária Falciparum/epidemiologia , Glicoproteínas de Membrana/genética , Repetições de Microssatélites/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , África/epidemiologia , Alelos , Animais , Brasil/epidemiologia , Frequência do Gene , Genética Populacional , Haplótipos , Humanos , Malária Falciparum/parasitologia , Malásia/epidemiologia , Plasmodium falciparum/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo ÚnicoRESUMO
We report the evaluation of four techniques for Giardia lamblia diagnosis in children's stool. The Iron haematoxilin staining and direct examination with lugol showed lower positivity, while the method of Faust et al. Continues to be a good option for G. lamblia diagnosis and Immunoenzymatic assay increases the detection of this parasite.
Assuntos
Fezes/parasitologia , Giardia lamblia/isolamento & purificação , Giardíase/diagnóstico , Adolescente , Animais , Brasil , Criança , Humanos , Parasitologia/métodosRESUMO
We conducted a survey to determine the vectors of malaria in six localities of Serra do Navio municipality, State of Amapá, from 1990 to 1991. Malaria infection rates of 29.3%, 6.2% and 20.4% were detected by human blood smears in Colônia Agua Branca, Porto Terezinha and Arrependido, respectively. There was no malaria infection detected in Serra do Navio. Fifteen species were identified among 3,053 anopheline mosquitoes collected by human bait and 64.4% were identified as Anopheles albitarsis s.l., 16.7% An. braziliensis, 9.5% An. nuneztovari and 5.8% An. triannulatus. An. darlingi, the main vector of malaria in the Amazon region of Brazil, was scare. Using enzyme-linked immunosorbent assay (ELISA), a total positive rate of 0.8% (23/2876) was found for six species: fifteen An. albitarsis s.l., four An. nuneztovari, and one of each: An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Nine of 23 positive mosquitoes were infected with Plasmodium malariae, eight with P. vivax VK210, three with P. vivax VK247 and three with P. falciparum. Since An. albitarsis s.l. was collected feeding on humans, was present in the highest density and was positive by ELISA for malaria sporozoites, it probably plays an important role in malaria transmission in this area.
Assuntos
Anopheles/parasitologia , Insetos Vetores/parasitologia , Malária/transmissão , Plasmodium/isolamento & purificação , Animais , Brasil , Ensaio de Imunoadsorção Enzimática , Humanos , Estações do AnoRESUMO
We conducted a survey to determine the vectors of malaria in six localities of Serra do Navio municipality, State of Amapá, from 1990 to 1991. Malaria infection rates of 29.3 percent, 6.2 percent and 20.4 percent were detected by human blood smears in Colônia ægua Branca, Porto Terezinha and Arrependido, respectively. There was no malaria infection detected in Serra do Navio. Fifteen species were identified among 3,053 anopheline mosquitoes collected by human bait and 64.4 percent were identified as Anopheles albitarsis s.l., 16.7 percent An. braziliensis, 9.5 percent An. nuneztovari and 5.8 percent An. triannulatus. An. darlingi, the main vector of malaria in the Amazon region of Brazil, was scare. Using enzyme-linked immunosorbent assay (ELISA), a total positive rate of 0.8 percent (23/2876) was found for six species: fifteen An. albitarsis s.l., four An. nuneztovari, and one of each: An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Nine of 23 positive mosquitoes were infected with Plasmodium malariae, eight with P. vivax VK210, three with P. vivax VK247 and three with P. falciparum. Since An. albitarsis s.l. was collected feeding on humans, was present in the highest density and was positive by ELISA for malaria sporozoites, it probably plays an important role in malaria transmission in this area
Assuntos
Humanos , Animais , Anopheles/parasitologia , Insetos Vetores/parasitologia , Malária/transmissão , Plasmodium/isolamento & purificação , Brasil , Ensaio de Imunoadsorção Enzimática , Estações do AnoRESUMO
We report the evaluation of four techniques for Giardia lamblia diagnosis in children's stool. The Iron haematoxilin staining and direct examination with lugol showed lower positivity, while the method of Faust et al. Continues to be a good option for G. lamblia diagnosis and Immunoenzymatic assay increases the detection of this parasite.
Relatamos a comparação de quatro metodologias para o diagnóstico da Giardia lamblia em material fecal de crianças, Belém/PA. A Hematoxilina Férrica e o método direto apresentaram menor positividade, enquanto que o Método de Faust continua uma boa escolha para o diagnóstico e o Ensaio imunoenzimático melhora a qualidade da detecção deste parasito.
Assuntos
Adolescente , Animais , Criança , Humanos , Fezes/parasitologia , Giardia lamblia/isolamento & purificação , Giardíase/diagnóstico , Brasil , Parasitologia/métodosRESUMO
The present study evaluated the glass fibre membrane (GFM)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique for genotyping the Plasmodium vivax variants, to verify the distribution of P. vivax variants (VK210, VK247 and P. vivax-like) in parts of Brazil and their correlation with levels of parasitaemia, previous malaria experience and clearance of parasitaemia linked to different treatment schedules. The samples were taken from individuals living in Macapá, Porto Velho and Belém, all of which are endemic areas of vivax malaria in the Amazon region of Brazil. Blood samples were collected on GFMs. The gene that codes for the circumsporozoite proteins of P. vivax variants was amplified by PCR and the amplified fragments were hybridized to variant-specific, digoxigenin-labelled oligonucleotide probes by ELISA. The GFM-PCR-ELISA technique was shown to be accurate for epidemiological surveys of the vivax complex. All variants were detected in all 3 areas, but only P. vivax VK210 was found as a single agent of infection, while the other 2 occurred as mixed infections. The P. vivax-like variant was found to be associated with low parasitaemia and VK210 with the highest parasitaemia levels; none of the P. vivax variants was linked with a previous malaria experience. In all cases parasitaemia clearance was identical regarding the type of treatment and consequently it is not possible to confirm the previously reported correlation between P. vivax genotype and response to chloroquine.
Assuntos
Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Malária Vivax/tratamento farmacológico , Plasmodium vivax/genética , Animais , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Malária Vivax/epidemiologia , Parasitemia/tratamento farmacológico , Parasitemia/epidemiologia , Reação em Cadeia da Polimerase/métodosRESUMO
The origin and geographical spread of Plasmodium falciparum is here determined by analysis of mitochondrial DNA sequence polymorphism and divergence from its most closely related species P. reichenowi (a rare parasite of chimpanzees). The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (pi=0.0004) contrasts with the divergence at such sites between the two species (kappa=0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population. To survey the geographical distribution of mitochondrial haplotypes in P. falciparum, 104 isolates from several endemic areas were typed for each of the identified single nucleotide polymorphisms. The haplotypes show a radiation out of Africa, with unique types in Southeast Asia and South America being related to African types by single nucleotide changes. This indicates that P. falciparum originated in Africa and colonised Southeast Asia and South America separately.
Assuntos
DNA Mitocondrial/genética , Genoma de Protozoário , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium/genética , África , Animais , Sudeste Asiático , DNA de Protozoário/genética , Evolução Molecular , Haplótipos , Humanos , Dados de Sequência Molecular , Plasmodium falciparum/classificação , Polimorfismo de Nucleotídeo Único , Seleção Genética , América do SulRESUMO
We compare diagnostic methods for Entamoeba histolytica in fecal samples from the city of Belém, Pará, Brazil. We analyze stool samples from children and adults (Group I); stool and serum samples from adults (Group II); and stool samples from children (Group III). In groups I and III, we used direct examination with lugol (DM), Faust et al (FM), and ELISA (detection of E. histolytica anti-GIAP coproantigen) and in group II, DM, iron hematoxylin staining (IHS), FM, ELISA, and the indirect immunofluorescence test (IFAT) for detection of IgG antibodies. Positivity was 10.50% by DM plus FM and 28.99% by ELISA. There was no correlation between positivity and age group. In Group II (n = 87), the positive rate was 4.59% by DM plus FM, 8.04% by IHS, 4.59% by IFAT, and 21.83% by ELISA. The ELISA test was the most sensitive for all groups. IFAT alone is still not a useful tool for diagnosis of E. histolytica infection. The ELISA test is simple, performed in one-third of cases used for IHS and IFAT, and greatly improves quality of diagnosis. We recommend this as the method of choice for diagnosis of suspected E. histolytica infection.
Assuntos
Disenteria Amebiana/diagnóstico , Entamoeba histolytica/isolamento & purificação , Fezes/parasitologia , Adolescente , Adulto , Animais , Brasil , Criança , Pré-Escolar , Disenteria Amebiana/imunologia , Entamoeba histolytica/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunológicas , Lactente , Recém-NascidoRESUMO
The polymorphic, merozoite surface protein-1 (MSP-1) of Plasmodium falciparum, an antigen of the parasite's asexual blood-stages, is a major malaria-vaccine candidate. Nucleotide sequences of each variable domain or block of this antigen may be grouped into one of three possible allelic types (K1, MAD20 and RO33), and 24 major types of the msp-1 gene may be defined, as unique combinations of allelic types in these variable blocks. Isolates collected from the Brazilian Amazon, over a period of 14 years, have now been investigated, by PCR-based typing of the msp-1 gene. Thirteen of the 24 possible gene-types were identified, and 336 P. falciparum clones were fully typed among 239 isolates. Most parasites (87%) belonged to one of the seven most frequent gene-types. Marked temporal variation in the distribution of msp-1 variants was found when comparing parasites sampled in the same sites at intervals of at least 5 years. Spatial variations were also found when comparing parasites from both neighbouring and distant sites within the Amazon Basin. The between-population variance in the frequencies of msp-1 allelic types found in Brazil, as estimated by Wright's FST statistic, is of similar magnitude to that found in previous world-wide comparisons. The potential implications of these findings for the development of an MSP-1-based, multivalent malaria vaccine are discussed.
Assuntos
Proteína 1 de Superfície de Merozoito/genética , Plasmodium falciparum/genética , Alelos , Animais , Brasil , Variação Genética , Humanos , Plasmodium falciparum/classificação , Reação em Cadeia da Polimerase/métodos , Fatores de TempoRESUMO
Parasite resistance to antimalarial drugs, particularly chloroquine, is the most disturbing problem of malaria chemotherapy. There is evidence that the codon 86Tyr polymorphism of the Pfmdr1 gene is associated with chloroquine resistance in West African Plasmodium falciparum. The association of this and four other coding alterations of the Pfmdr1 gene with chloroquine resistance has not been extensively investigated in South American isolates. In this study, we examined 51 Brazilian P. falciparum isolates for the presence or absence of Asn86Tyr, Asn1042Asp, and Asp1246Tyr polymorphisms. While these isolates were all sensitive in vitro to mefloquine, amodiaquine, and quinine, only 2 (4%) were chloroquine-sensitive. The findings reported here provide the first observations of this kind on a large number of field parasite samples from South America. We show that in vitro chloroquine-resistant and -sensitive strains carry the Asn1042Asp and Asp1246Tyr polymorphisms and provide support for earlier suggestions that Asn86Tyr may be rare or absent in South American P. falciparum.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Polimorfismo de Fragmento de Restrição , Amodiaquina/farmacologia , Animais , Resistência a Medicamentos/genética , Humanos , Mefloquina/farmacologia , Reação em Cadeia da Polimerase , Quinina/farmacologiaRESUMO
We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.
Assuntos
Coleta de Amostras Sanguíneas/métodos , DNA de Protozoário/análise , Malária/diagnóstico , Reação em Cadeia da Polimerase , Animais , Coleta de Amostras Sanguíneas/economia , Ensaio de Imunoadsorção Enzimática , Plasmodium/genéticaRESUMO
To determine the nature and extent of variation in the T cell sites of the Plasmodium falciparum circumsporozoite (CS) protein, a candidate antigen in the development of a malaria vaccine, we cloned and sequenced 69 recombinant clones of the CS protein gene representing 18 and 17 P. falciparum isolates from infected individuals from Madang, Papua New Guinea (PNG), a holoendemic malaria region, and Paragaminos and Jacunda, Brazil, relatively low endemic regions, respectively. As previously known, the amino acid sequence polymorphism was restricted to the three immunodominant regions of the protein, Th1R-N1, Th2R, and Th3R. While some of the observed nonsilent mutations in the T cell determinants of the CS protein were similar to those previously identified, we have found new amino acid changes in each of the polymorphic sequences in parasites from PNG and Brazil. A comparison of the CS epitope sequences of parasites from PNG and Brazil with the previously known CS epitope sequences of parasites from Brazil and The Gambia showed the following: 1) polymorphism was found in the Th1R-N1, Th2R, and Th3R region; however, while amino acid substitutions in the Th1R-N1 and Th2R region tended to be conservative, the substitutions found in the Th3R region were not, suggesting that the Th3R epitope may be rapidly evolving to allow parasites to escape host antiparasite cytotoxic T cell-enforced immune responses, and 2) the CS proteins of P. falciparum from high malaria-transmission regions (PNG and The Gambia) appear more polymorphic than the CS proteins of parasites from relatively low malaria-endemic regions in Brazil, where P. falciparum infection has been recently established.
Assuntos
Antígenos de Protozoários/genética , DNA de Protozoário/química , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Variação Antigênica , Antígenos de Protozoários/química , Sequência de Bases , Brasil/epidemiologia , Criança , Pré-Escolar , Epitopos/química , Epitopos/genética , Humanos , Malária Falciparum/epidemiologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , Papua Nova Guiné/epidemiologia , Plasmodium falciparum/imunologia , Polimorfismo Genético , Proteínas de Protozoários/química , Sequências Repetitivas de Ácido NucleicoRESUMO
Passage of malaria-infected blood through a two-layered column composed of acid-washed glass beads and CF 11 cellulose removes white cells from parasitized blood. However, because use of glass beads and CF 11 cellulose requires filtration of infected blood separately through these two resins and the addition of ADP, the procedure is time-consuming and may be inappropriate for use in the field, especially when large numbers of blood samples are to be treated. Our modification of this process yields parasitized cells free of contaminating leukocytes, and because of its operational simplicity, large numbers of blood samples can be processed. Our procedure also compares well with those using expensive commercial Sepacell resins in its ability to separate leukocytes from whole blood. As a test of usefulness in molecular biologic investigations, the parasites obtained from the blood of malaria-infected patients using the modified procedure yield genomic DNA whose single copy gene, the circumsporozoite gene, efficiently amplifies by polymerase chain reaction.
Assuntos
Separação Celular/métodos , Celulose , Cromatografia/métodos , Vidro , Leucócitos , Malária Falciparum/sangue , Malária Vivax/sangue , Microesferas , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Difosfato de Adenosina/farmacologia , Adsorção , Animais , Sequência de Bases , Separação Celular/instrumentação , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Filtração , Genes de Protozoários , Humanos , Leucócitos/parasitologia , Malária Falciparum/parasitologia , Malária Vivax/parasitologia , Dados de Sequência Molecular , Plasmodium falciparum/genética , Plasmodium vivax/genética , Ativação Plaquetária , Proteínas de Protozoários/genéticaRESUMO
The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.
Assuntos
Antígenos de Protozoários/genética , Plasmodium vivax/genética , Polimorfismo Genético , Proteínas de Protozoários , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Variação Antigênica , Antígenos de Protozoários/química , Sequência de Bases , Brasil , DNA de Protozoário/química , Epitopos/química , Epitopos/genética , Humanos , Dados de Sequência Molecular , Papua Nova Guiné , Plasmodium vivax/imunologia , Reação em Cadeia da Polimerase , Linfócitos T/imunologiaRESUMO
Pyrimethamine resistance in cultivated laboratory isolates of Plasmodium falciparum is linked to the dihydrofolate reductase mutation Asn-108, a mutation that acts by interrupting drug binding within the active site of the enzyme. To determine the prevalence of this mutation in endemic regions harboring pyrimethamine-resistant malaria, we used a mutation-specific polymerase chain reaction assay to survey P. falciparum strains from a wide section of the Brazilian Amazon. Mutations were identified directly from blood samples without intervening steps of in vitro cultivation. Of 42 samples collected from four states in Brazil, 38 (90%) contained the Asn-108 codon AAC that confers pyrimethamine resistance, four samples contained only the wild-type Ser-108 codon AGC, and none contained the Thr-108 codon ACC found in cycloguanil-resistant pyrimethamine-sensitive strains. These findings indicate that a very high incidence of the Asn-108 DHFR mutation is responsible for pyrimethamine resistance in the Amazon, and they are consistent with recent failure rates reported for Fansidar (pyrimethamine-sulfadoxine). We suggest that limited use of proguanil be evaluated as an alternative to pyrimethamine.