Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay.

BACKGROUND: The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. METHODS: Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. RESULTS: The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. CONCLUSIONS: The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could improve upon the sensitivity and specificity of currently available diagnostic tests. Overall, the results provide further evidence of the usefulness of identifying specific linear B-cell epitopes for improving diagnostic tools.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats.

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosome-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T. cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Trypanosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia.

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1 percent of CDt patients (23/28) remained clinically stable and 95.4 percent of the INDt (21/22) and 33.3 percent of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2 percent/year and was especially low in INDt patients (0.5 percent/year) relative to CARD/DIGt patients (7.4 percent/year). Positive haemoculture in treated patients was observed in 7.1 percent of the cases. None of the INDt (0/21) and 33.3 percent of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2 percent, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7 percent of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution ...

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment.

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1% of CDt patients (23/28) remained clinically stable and 95.4% of the INDt (21/22) and 33.3% of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2%/year and was especially low in INDt patients (0.5%/year) relative to CARD/DIGt patients (7.4%/year). Positive haemoculture in treated patients was observed in 7.1% of the cases. None of the INDt (0/21) and 33.3% of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2%, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7% of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution were selectively observed in the group of INDt patients and did not occur for CARD/DIGt patients.

Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission.

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosoma cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TCI presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TCI and TCIIa from wild primates (16 TCI and five TCIIa), Rhodnius spp. (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TCI and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T. cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region.

Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria.

BACKGROUND: Malaria is one of the most significant infectious diseases in the world and is responsible for a large proportion of infant deaths. Toll-like receptors (TLRs), key components of innate immunity, are central to countering infection. Variants in the TLR-signaling pathway are associated with susceptibility to infectious diseases. METHODS: We genotyped single nucleotide polymorphisms (SNPs) of the genes associated with the TLR-signaling pathway in patients with mild malaria and individuals with asymptomatic Plasmodium infections by means of polymerase chain reaction. RESULTS: Genotype distributions for the TLR-1 I602S differed significantly between patients with mild malaria and persons with asymptomatic infection. The TLR-1 602S allele was associated with an odds ratio (OR) of 2.2 (P= .003; P(corrected)= .015) for malaria among patients with mild malaria due to any Plasmodium species and 2.1 (P= .015; P(corrected)= .75) among patients with mild malaria due to Plasmodium falciparum only. The TLR-6 S249P SNP showed an excess of homozygotes for the TLR-6 249P allele in asymptomatic persons, compared with patients with mild malaria due to any Plasmodium species (OR 2.1; 95% confidence interval [CI], 1.1- 4.2; P= .01; P(corrected)= .05), suggesting that the TLR-6 249S allele may be a risk factor for malaria (OR, 2.0; 95% CI, 1.1-3.7; P=0.01; P(corrected)= .05). The TLR-9 -1486C allele showed a strong association with high parasitemia (P< .001). CONCLUSIONS: Our findings indicate that the TLR-1 and TLR-6 variants are significantly associated with mild malaria, whereas the TLR-9-1486C/T variants are associated with high parasitemia. These discoveries may bring additional understanding to the pathogenesis of malaria.

An improved serodiagnostic test for Chagas' disease employing a mixture of Trypanosoma cruzi recombinant antigens.

BACKGROUND: Blood transfusion is one of the most important transmission routes of Chagas' disease, a major parasitic infection in Latin America. Therefore, screening for antibodies to Trypanosoma cruzi is mandatory in blood banks in South America. Most of the commercial serologic tests employ epimastigote antigens and show a high number of inconclusive and false-positive results, with high economic and social costs. STUDY DESIGN AND METHODS: An ELISA using a mixture of three T. cruzi recombinant antigens, B13, 1F8, and H49 (mix-ELISA), was evaluated, first with a panel of well-characterized sera from 617 patients with Chagas' disease and 277 nonchagasic individuals, living in nine countries of South and Central America. Subsequently, the mix-ELISA was evaluated with 451 samples, from an endemic area of Brazil (Goiás), that were rejected from several blood banks because they presented discrepant results by two commercially available kits (indirect immunofluorescence assay, indirect hemagglutination assay, and/or ELISA). RESULTS: The mix-ELISA exhibited 99.7 percent sensitivity and 98.6 percent specificity in the first evaluation with the 894 samples. In the second evaluation, 451 sera that had discrepant results in the first screening for Chagas' disease were further analyzed with the mix-ELISA. Upon consideration of the consensus results obtained with the trypomastigote excreted-secreted antigens blot test, a confirmatory test for Chagas' disease, the mix-ELISA led to a reduction in 99.6 percent in the number of discordant sera. CONCLUSION: The combination of three T. cruzi recombinant antigens in a multiantigen immunoassay was highly sensitive and specific for Chagas' disease diagnosis. It is proposed that it can be applicable in blood bank screening in conjunction with the conventional serologic tests.

Epidemiological aspects of the brazilian spotted fever: serological survey of dogs and horses in an endemic area in the state of Sao Paulo, Brazil

Com o objetivo de obter informacoes sobre a febre maculosa brasileira, um estudo em animais domesticos foi conduzido no municipio de Pedreira, Sao Paulo, Brasil, onde 17 casos humanos foram notificados. Amostras de soro obtidas de animais foram testadas pelo teste de imunofluorescencia indireta para deteccao de anticorpos para rickettsia do grupo da febre maculosa. Soro reatividade foi observada em 12 (36,4 por cento) dos 33 caes e sete (77,8 por cento) dos nove equinos procedentes da area endemica. Para comparacao, amostras de sangue de caes e de equinos procedentes de area nao endemica foram testadas e quatro (12,9 por cento) dos 31 caes e tres dos 10 equinos foram positivos...

Aspectos epidemiológicos, sociais e sanitários de uma área no Rio Negro, estado do Amazonas, com especial referência às parasitoses intestinais e à infecçäo chagásica / Epidemiological, social, and sanitary aspects in a area of the Rio Negro, state of Amazonas, with special reference to intestinal parasites and Chagas'Disease

Estudo seccional realizado na populaçäo residente em um de cada quatro domicílios habitados na cidade de Barcelos (no norte do estado do Amazonas), visando avaliar as condiçoes sociais, sanitárias e os indicadores específicos para as parasitoses intestinais e para infecçäo chagásica. Foram aplicados dois questionários, um domiciliar para avaliar os aspectos sociais e sanitários, e outro individual, para a avaliaçäo das condiçoes sociais e epidemiológicas da populaçäo.

O falso dilema sobre a luta antivetorial e as perspectivas de controle da doença de chagas no Brasil: BHC ou BNH? / The false dilemma about antivectorial strategies and possibilities for controlling chagas' disease in Brazil: BHC or BNH

As bases técnicas para o controle da doença de Chagas no Brasil foram estabelecidas com a criaçao do posto avançado de pesquisa do Instituto Oswaldo Cruz em Bambuí no oeste de Minas Gerais, no começo da década de 40, sob a liderança de Emmanuel Dias. Entretanto, somente com a criaçao do Departamento Nacional de Endemias Rurais (DNERu), em março de 1956, sob a direçao de Mario Pinotti, no governo de Juscelino Kubitschek, as medidas de controle foram implementadas. Das "campanhas" de controle das 12 endemias rurais estabelecidas pelo DNERu, a malária, pelo seu caráter de doença aguda e explosiva, sempre teve a maior parte orçamentaria. A doença de Chagas e as outras endemias foram sempre relegadas a um plano secundário de prioridade. Por outro lado, a partir da década de 60, os novos ecologistas passaram a criticar o uso de inseticidas, com o slogan de que para controlar a doença de Chagas era necessário o BNH (construçao de casas), e nao o BHC (uso de inseticidas). Esta opiniao, embora equivocada para o controle a curto prazo, teve uma enorme influência negativa sobre o controle dos vetores domiciliados. Apesar disso, algum progresso foi feito neste sentido. Na década de 70, a epidemia de meningite meningocócica e a priorizaçao do Programa Especial de Controle da Esquistossomose (PECE), pelo Ministro Almeida Machado, com deslocamento de verbas e de pessoal da Superintendência de Campanhas (SUCAM0) para esses programas, atrasaram ainda mais o controle da doença de Chagas. Somente na década de 80, com a decisao política do Banco Nacional de Desenvolvimento Econômico e Social (BNDES) de alocar 10 bilhoes de cruzeiros do Finsocial, o programa de controle da doença de Chagas teve um importante desenvolvimento em 2.000 municípios de 19 Estados brasileiros. Mais uma vez o programa sofre um atraso nesta década, com o deslocamento do pessoal da SUCAM para a campanha contra a epidemia da dengue. Finalmente, concluímos que, a curto prazo, o uso de inseticidas é a medida mais efetiva para o controle da transmissao natural da doença de Chagas. Discutimos também, nesta revisao, as outras medidas de controle e a urbanizaçao da doença

Endemias e meio ambiente no século XXI / Endemics and the environment in the 21st. century

Na introduçao do trabalho, o autor define saúde como a "adaptaçao do homem ao meio, preservando sua integridade física, funcional, mental e social". Por outro lado, amplifica o conceito de desenvolvimento como uma "boa qualidade de vida em termos de alimentaçao, habitaçao, educaçao e saúde, seguridade social, recreaçao, segurança e liberdade". A relaçao entre desenvolvimento e saúde está associada com os recursos e meios naturais e artificiais e com os comportamentos da populaçao humana, tais como crescimento populacional, aglomeraçao e migraçoes. O homem é "hóspede da natureza e vítima de si próprio". Embora a Conferência de Alma-Ata de 1978 tenha previsto "Saúde para todos no ano 2000", a saúde do mundo piorou na última década devido à crise econômica na América Latina, Åsia e Åfrica. Ao lado da crise econômica, o crescimento da populaçao, de cerca de um bilhao de pessoas por década, nao pôde ser acompanhado pelos serviços de saúde, educaçao, transporte, alimentaçao, habitaçao e seguridade social. As doenças infecciosas sao responsáveis por 34% das mortes no mundo subdesenvolvido e somente 1% nos países desenvolvidos. Anualmente, 15 milhoes de crianças morrem por doenças infecciosas e/ou má nutriçao, e 93% das mortes evitáveis ocorrem exatamente nos países em desenvolvimento. Nao há uma correlaçao direta entre desenvolvimento econômico e qualidade de saúde; ela depende da organizaçao dos serviços sociais e de saúde