Avaliação de três métodos de extração de DNA obtidos de amostras de sangue coletadas em papel de filtro em infecções subpatentes de Plasmodium da região Amazônica no Brasil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.Infecção assintomática por Plasmodium é um novo desafio para a saúde pública no Brasil. A reação em cadeia da polimerase (PCR) é o melhor método para detectar baixas parasitemias presentes em pacientes com infecção assintomática. Nas áreas endêmicas, a coleta de sangue total é dificultada pela distancia geográfica, transporte e adequada armazenagem das amostras. A coleta de sangue em papel de filtro pode ser uma alternativa nessas áreas de difícil acesso. Neste estudo foram comparados três diferentes métodos de extração de ADN a partir de papel de filtro usando como controle extração a partir de sangue total. O protocolo Chelex®-Saponina foi o que obteve o melhor resultado quando comparado com os outros três protocolos. No entanto a sensibilidade foi de 66,7% para o P. falciparum e 31,6% para o P. vivax. Conclui-se que em caso de infecção assintomática o papel de filtro não é ainda uma boa alternativa para coleta de amostras

UTILIDAD DE LA PCR RECURSIVA PARA INSERTAR UN APTAMER DE ALTA AFINIDAD EN EL ESPACIADOR MENOR DE CALMODULINA de Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness in Panama. In this parasite, regulation of gene expression is mainly a post transcriptional process. Has been suggested that RNA-binding proteins have a key role in this gene regulation. Calmodulin is a highly conserved calcium sensor protein, encodes by three genes separated by two intergenic spacers (major and minor spacer). The study of molecular basis that led RNA-proteins interactions in T. cruzi could contribute to understand the biology of the parasite to develop novel strategies for disease control. We developed a methodology based on a recursive PCR to insert a streptavidin high affinity aptamer into the sequence of the minor spacer, as the basis for the development of a protein capture system. The complete calmodulin locus sequence was download from GenBank (AAHK01001263.1). Previous reported aptameric sequence (83bp) was manipulated for primer design. Sequences were edited with UGENE bioinformatic software. Calmodulin minor spacer (CMS) sequence was fragmented into two regions. Primers were designed to flank the two regions, the inner forward/reverse oligos attached to half aptamer sequences each. Final recursive PCR was able to amplify the Calmodulin minor spacer incorporating the aptamer. Sequence was confirmed by Sanger sequencing. Recursive PCR represent a useful tool to insert high affinity sequences as aptamers to study specific protein-RNA interactions.Trypanosoma cruzi es el parásito protozoario causante de la enfermedad de Chagas, una enfermedad endémica en Panamá. En este parásito, la regulación de la expresión génica es principalmente un proceso postranscripcional. Se ha sugerido que las proteínas de unión al ARN tienen un papel clave en esta regulación génica. La calmodulina es una proteína sensora de calcio altamente conservada, codificada por tres genes separados por dos espaciadores intergénicos (espaciador mayor y menor). El estudio de las bases moleculares que llevaron a las interacciones ARN-proteínas en T. cruzi podría contribuir a comprender la biología del parásito para desarrollar estrategias novedosas para el control de enfermedades. Desarrollamos una metodología basada en una PCR recursiva para insertar un aptámero de alta afinidad de estreptavidina en la secuencia del espaciador menor, como base para el desarrollo de un sistema de captura de proteínas. La secuencia completa del locus de calmodulina se descargó de GenBank (AAHK01001263.1). La secuencia del aptámero reportadas en publicaciones anteriores (83 pb) se manipuló para el diseño del cebador. Las secuencias se editaron con el software bioinformático UGENE. La secuencia del espaciador menor de calmodulina (CMS) se fragmentó en dos regiones. Los cebadores se diseñaron para flanquear las dos regiones, cada uno con los oligonucleótidos internos directo / inverso unidos a secuencias de medio aptámero. La PCR recursiva final pudo amplificar el espaciador menor de calmodulina incorporando el aptámero. La secuencia fue confirmada por secuenciación de Sanger. La PCR recursiva representa una herramienta útil para insertar secuencias de alta afinidad como aptámeros para estudiar interacciones proteína-ARN específicas

Generalidades do Trypanosoma cruzi, do Trypanosoma rangeli e do seu vetor (Rhodnius pallescens) no Panamá

The eco-epidemiology of T. cruzi infection was investigated in the Eastern border of the Panama Canal in Central Panama. Between 1999 and 2000, 1110 triatomines were collected: 1050 triatomines (94.6%) from palm trees, 27 (2.4%) from periurban habitats and 33 (3.0%) inside houses. All specimens were identified as R. pallescens. There was no evidence of vector domiciliation. Salivary glands from 380 R. pallescens revealed a trypanosome natural infection rate of 7.6%, while rectal ampoule content from 373 triatomines was 45%. Isoenzyme profiles on isolated trypanosomes demonstrated that 85.4% (n = 88) were T. cruzi and 14.6% (n = 15) were T. rangeli. Blood meal analysis from 829 R. pallescens demonstrated a zoophilic vector behavior, with opossums as the preferential blood source. Seroprevalence in human samples from both study sites was less than 2%. Our results demonstrate that T. cruzi survives in the area in balanced association with R. pallescens, and with several different species of mammals in their natural niches. However, the area is an imminent risk of infection for its population, consequently it is important to implement a community educational program regarding disease knowledge and control measures.A epidemiologia da infecção do T. cruzi foi investigada na margem oriental do canal do Panamá, na região central da Republica do Panamá. A informação obtida durante o estudo avaliou fatores de risco da doença de Chagas nesta área. Entre 1999 e 2000, 1110 triatomíneos foram coletados: 1050 triatomíneos (94,6%) em palmeiras, 27 (2,4%) em habitats periurbanos e 33 (3,0%) no interior de casas. Todos os espécimens foram identificados como R. pallescens. Não havia nenhuma evidência de domiciliação do vetor. O exame de glândulas salivares de 380 R. pallescens revelaram taxa de infecção natural por Trypanosoma de 7,6%, mas o conteúdo da ampola rectal de 373 triatomíneos mostrou 45% de positividade. Os perfis de isoenzimas em Trypanosomas isolados demonstraram que 85,4% (n = 88) eram T. cruzi e 14,6% (n = 15) eram T. rangeli. A análise da refeição de sangue de 829 R. pallescens demonstrou comportamento zoofílico do vetor, sendo os gambás a fonte preferencial de sangue. Soroprevalência nos seres humanos de ambos locais de estudo foi menos que 2%. Nossos resultados demonstram que T. cruzi sobrevive na área em associação equilibrada com R. pallescens e com diversas espécies diferentes de mamíferos em seus nichos naturais. Entretanto, a área é um risco eminente de infecção para sua população, pelo que é importante executar um programa educacional na comunidade a respeito das medidas, do conhecimento e do controle da doença

The calmodulin intergenic spacer as molecular target for characterization of Leishmania species

Background: Human leishmaniasis is a neglected disease caused by parasites of the genus Leishmania. Clinical aspects of this disease can vary significantly, reflecting the wide range of parasites in the genus Leishmania. Knowing accurately the Leishmania species infecting humans is important for clinical case management and evaluation of epidemiological risk. Calmodulin is an essential gene in trypanosomatids that modulates the calcium metabolism in various cellular activities. Despite its strong conservation in trypanosomatids, it has been recently observed that its untranslated regions (UTR) diverge among species. Methods: In this study we analyzed the sequences and the absolute dinucleotide frequency of the intergenic spacer of the calmodulin gene (containing both, 3′ and 5′UTR) in nine reference Leishmania species and ten clinical isolates obtained from patients with cutaneous leishmaniasis. Results: We show that the short calmodulin intergenic spacers exhibit features that make them interesting for applications in molecular characterization and phylogenetic studies of Leishmania. Dendrograms based on sequence alignments and on the dinucleotide frequency indicate that this particular region of calmodulin gene might be useful for species typing between the Leishmania and Viannia subgenera. Conclusions: Mutations and composition of the calmodulin intergenic spacer from Leishmania species might have taxonomic value as parameters to define if an isolate is identical to a certain species or belongs to one of the two current subgenera

Prevalencia de autoanticuerpos contra receptores autonómicos en pacientes panameños con cardiopatía chagásica crónica y con otras formas de cardiopatía

Introduction. Chagas´ disease is the main cause of chronic myocardiopathy in Central America. The mechanisms proposed for this cardiac pathology during the chronic phase remain controversial. Several studies have detected the presence of circulating autoantibodies against β-adrenergic and cholinergic muscarinic receptors of the myocardium in patients with Chagas disease. These autoantibodies can trigger intracellular signals and modify the cardiac function during the progression of the disease.Objectives. The serological frequency of these autoantibodies was compared among patients with chronic Chagas disease, patients with other cardiopathies and healthy controls.Materials and methods. The prevalence of autoantibodies against β-adrenergic and cholinergic muscarinic receptors was determined in four groups of Panamenian patients: 53 chagasic patients, 25 serologically negative patients with cardiac insufficiency, 25 patients with cardiac arrhythmia and 25 healthy individuals.Results. The antibodies against autonomic receptors were more frequently observed in patients with chronic chagasic cardiomyopathy (24.5%) compared to the cardiac insufficiency group (20.0%) and the cardiac arrhythmia group (16.0%). The proportion of autoantibodies was significantly different between the groups with chronic chagasic cardiomyopathy and healthy controls (24.5% versus 0%; p=0.015). Of the 53 chronically infected chagasic patients, 48 (90%) showed some degree of cardiac dysfunction.Conclusions. The frequency of autoantibodies against autonomic receptors is significantly increased in patients with chronic Chagas disease and in patients with other cardiopathies.Introducción. La enfermedad de Chagas es la principal causa de cardiomiopatía crónica en Centroamérica. Existe controversia sobre los mecanismos causantes de la patología cardiaca observada durante la fase crónica de esta parasitosis. Varios estudios han detectado la presencia de autoanticuerpos circulantes dirigidos contra receptores beta-adrenérgicos y colinérgicos muscarínicos del miocardio en pacientes chagásicos, que pueden desencadenar señales intracelulares y alterar la función cardiaca durante el curso de la enfermedad.Objetivo. Nuestro objetivo principal fue comparar la frecuencia sérica de estos autoanticuerpos en pacientes chagásicos crónicos con la observada en pacientes con otras formas de cardiopatía y en controles sanos.Materiales y métodos. Se determinó la prevalencia de autoanticuerpos contra receptores beta-adrenérgicos y colinérgicos muscarínicos en cuatro grupos de pacientes panamelos: 53 pacientes chagásicos, 25 pacientes seronegativos con insuficiencia cardiaca, 25 pacientes con diferentes tipos de arritmia cardiaca y 25 controles sanos.Resultados. Los autoanticuerpos contra receptores autonómicos fueron más frecuentes en el grupo de pacientes con cardiopatía chagásica crónica (24,5%) comparados con el grupo de insuficiencia cardiaca (20,0%) y con el grupo con arritmias cardiacas (16,0%). Al comparar la proporción de autoanticuerpos entre el grupo de pacientes con cardiopatía chagásica crónica y los controles sanos, se detectaron diferencias muy significativas (24,5% versus 0%; p=0,0015). De los 53 pacientes con infección crónica, 48 (90,6%) presentaron algún grado de alteración cardiaca.Conclusiones. En comparación con el grupo de controles sanos, la frecuencia de los autoanticuerpos contra receptores autonómicos se encuentra significativamente aumentada en pacientes con enfermedad de Chagas crónica y con otras formas de cardiopatía

Análise do processamento de transcritos do locus de calmodulina nos diferentes estágios da cepa Y e clone CL-Brener de Trypanosoma cruzi

Made available in DSpace on 2016-04-15T13:05:47Z (GMT). No. of bitstreams: 2 franklyn_acosta_ioc_dout_2015.pdf: 13033375 bytes, checksum: 9f1b37e297e771e874ab4f5d3f7aed83 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilA transcrição dirigida pela RNA polimerase II nos tripanossomatídeos produz um RNA precursor que abriga dezenas a centenas de genes que são processados para formar unidades monocistrônicas. Os processos de trans-splicing e poliadenilação vinculados ao processamento do RNA precursor são os responsáveis pela formação dos extremos do mRNA e portanto representam pontos importantes de controle pós-transcricional e de geração de diversidade funcional em termos de regiões não traduzidas (UTRs). O genoma de T. cruzi abriga genes tanto multicópia como de poucas cópias, os quais são importantes para a interação com seus hospedeiros e para realizar processos biológicos fundamentais para a sua sobrevivência. Isto levanta questões relevantes em relação à transcrição e processamento de genes duplicados ou multicópia no T. cruzi. Para acrescentar o conhecimento sobre o processamento de genes de poucas cópias nós estudamos este processo em duas cepas de Trypanosoma cruzi: Y e clone CL-Brener Na cepa Y as formas epimastigota, amastigota e tripomastigota sanguínea da cepa Y foram estudadas usando uma abordagem baseada no pirosequencimento e em CL-Brener usamos RT-PCR, RACE, clonagem e sequenciamento pelo método de Sanger das UTRs para as formas epimastigotas e tripomastigotas metacíclicos. Os resultados indicam que a transcrição e processamento dos transcritos do locus de calmodulina nos estágios estudados de ambas cepas produz formas alternativas predominantes com 5'UTRs longas e 3'UTR curtas. Encontramos também, um marcado desequilíbrio alélico na frequência dos transcritos das cópias de calmodulina nas diferentes formas estudadas de CL-Brener. Finalmente, existe uma aparente frequência assimétrica das isoformas de calmodulina dentro de cada cópia e entre as cópias em todos os estágios de ambas cepas estudadas indicando que possivelmente o controle da abundância dos transcritos exerce um papel importante na regulação da expressão deste geneThe RNA pol II transcription in tripanosomatids produces a RNA precursor harboring ten to hundreds of genes that must be processed to produce monocistronic mRNAs. The trans-splicing and polyadenylation processing of the RNA precursor is responsibles for the mRNA ends formation, important points of post-transcriptional regulation and functional diversity around the UTRs. The T. cruzi genome harbor both multicopy and few copies genes that are important for the interaction with the parasite host and to carry out essential biological process for the T. cruzi survival. This fact rases relevant aspects about the transcription and processing of genes in T. cruzi. To increase the knowledge on the mRNA processing of genes with few copies we studied this process in two strains: Y and CL-Brener clone. We analyzed the final mRNA from the epimastigote, amastigote and blood trypomastigote of Y strain using amplicon pyrosequencing and for the CL-brener epimastigote and metacyclic stage we devised an RT-PCR and RACE to target calmodulin UTRs combined with amplicon cloning and Sanger sequencing. The results point out that the mRNA processing of the transcripts of the calmodulin locus in all T. cruzi stages from studied strains produced mostly calmodulin mRNA bearing long 5'UTR and short 3'UTR. Also, we found a remarkable allelic imbalance in the frequency of the transcripts from calmodulin copies in all stages of CL-Brener. Finally, there was an inter-copy and intra-copy asymmetric frequency of calmodulin mRNA in all stages of the T. cruzi strain studied here indicating that the control of transcript abundance may has an important role in the calmodulin gene expression regulatio

Minimum free energy predicted base pairing in the 39 nt spliced leader and 5' UTR of calmodulin mRNA from Trypanosoma cruzi: influence of the multiple trans-splicing sites

Submitted by Sandra Infurna ([email protected]) on 2020-03-04T20:06:36Z No. of bitstreams: 1 AdeiltonBrandao_FranklinSamudio_IOC_2018.pdf: 3192068 bytes, checksum: cbdda8612004c11eedaf6305d62b307c (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-04T20:14:58Z (GMT) No. of bitstreams: 1 AdeiltonBrandao_FranklinSamudio_IOC_2018.pdf: 3192068 bytes, checksum: cbdda8612004c11eedaf6305d62b307c (MD5)Made available in DSpace on 2020-03-04T20:14:58Z (GMT). No. of bitstreams: 1 AdeiltonBrandao_FranklinSamudio_IOC_2018.pdf: 3192068 bytes, checksum: cbdda8612004c11eedaf6305d62b307c (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil / Instituto Conmemorativo Gorgas de Estudios de la Salud. Panamá, República de Panamá.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil.We analyzed the compositional changes and the stable base pairs in the predicted secondary structure of the 5' UTR calmodulin mRNA in T. cruzi. The three copies of calmodulin in T. cruzi genome display variable position of the trans splicing sites and give rise to several mRNA that differs slightly on 5' UTR composition in the epimastigote stage. We show that the pattern of high probability base pairs in the minimum free energy predicted secondary structures of the calmodulin 5' UTR remains unchanged despite the nucleotide composition variation. However, the 39 nt spliced leader (mini-exon, the 5' exon sequence transferred to trypanosome mRNAs by the mechanism of trans splicing) shows a variable pattern of high and low probability base pairing as consequence of the altered composition of the 5' UTR

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium Subpatent infections from the Amazon region in Brazil

Made available in DSpace on 2015-09-21T17:25:14Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) renata_miguel_etal_IOC_2013.pdf: 228472 bytes, checksum: b5248b387ac39cd33b27970cd08c93a8 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Doenças Parasitárias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Doenças Parasitárias. Rio de Janeiro, RJ, Brasil.Instituto Conmemorativo Gorgas de Estudios de la Salud. Panamá / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Doenças Parasitárias. Rio de Janeiro, RJ, Brasil.Infecção assintomática por Plasmodium é um novo desafio para a saúde pública no Brasil. A reação em cadeia da polimerase (PCR) é o melhor método para detectar baixas parasitemias presentes em pacientes com infecção assintomática. Nas áreas endêmicas, a coleta de sangue total é dificultada pela distancia geográfica, transporte e adequada armazenagem das amostras. A coleta de sangue em papel de filtro pode ser uma alternativa nessas áreas de difícil acesso. Neste estudo foram comparados três diferentes métodos de extração de ADN a partir de papel de filtro usando como controle extração a partir de sangue total. O protocolo Chelex®-Saponina foi o que obteve o melhor resultado quando comparado com os outros três protocolos. No entanto a sensibilidade foi de 66,7% para o P. falciparum e 31,6% para o P. vivax. Conclui-se que em caso de infecção assintomática o papel de filtro não é ainda uma boa alternativa para coleta de amostras.Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection