Identificação do sangue ingerido por Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912) e Lutzomyia (Lutzomyia) almerioi (Galati & Nunes, 1999) pela técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina

Lutzomyia longipalpis e Lutzomyia almerioi, espécies integrantes da fauna flebotomínea da Serra da Bodoquena, no Estado de Mato Grosso do Sul, têm sido objeto de estudo devido às suas elevadas abundâncias no Assentamento Guaicurus, foco de leishmaniose tegumentar humana e visceral canina. Em pesquisas que vem sendo realizadas neste acampamento para a identificação de vetores destas parasitoses, foram capturados no período de 2002 a 2004, com armadilhas automáticas luminosas, instaladas em ambiente peridoméstico (galinheiro), 83 exemplares ingurgitados de Lutzomyia longipalpis e Lutzomyia almerioi. O presente estudo teve como objetivo a investigação do hábito alimentar para ave das fêmeas de ambas as espécies de flebotomíneos, mediante o emprego da técnica imunoenzimática de captura,comparando-se a reatividade durante os anos de 2002 a 2004. Dentre 57 amostras de Lutzomyia longipalpis e 26 de Lutzomyia almerioi, foram encontradas 72% reagentes para ave em Lutzomyia longipalpis e 96% em Lutzomyia almerioi, o que justifica o estudo do hábito alimentar na região, como medida de prevenção e instituição de vigilância epidemiológica.

Comportamento do método quimioluminescente-ELISA em relação a resultados considerados discordantes por meio de três técnicas convencionais para diagnóstico da doença de Chagas

Quando utilizadas, em conjunto, a hemaglutinação indireta, a imunofluorescência indireta e ELISA para diagnóstico sorológico da doença de Chagas por vezes ocorrem resultados considerados discordantes, por não haver concordância entre o que indicam essas técnicas. A disponibilidade do método quimioluminescente-ELISA permitiu executá-lo com 200 soros que examinados pelos três testes citados que motivaram a obtenção de resultados discordantes. Com o método quimioluminescente-ELISA sucederam 193 negativos e sete positivos. O emprego desse novo procedimento trouxe mais um subsídio para compreensão do assunto, mas avanço mais concreto dependerá de documentação com soros de pessoas infectadas ou não pelo Trypanosoma cruzi conforme comprovação parasitológica.

Evaluation of Taenia solium and Taenia crassiceps cysticercal antigens for immunodiagnosis of neurocysticercosis using ELISA on cerebrospinal fluid samples

The efficacy of whole parasite and vesicular fluid antigen extracts from Taenia solium and Taenia crassiceps cysticerci for immunodiagnosis of neurocysticercosis was evaluated using ELISA on cerebrospinal fluid samples. Anticysticercal IgG antibodies were assayed in cerebrospinal fluid samples from 23 patients with neurocysticercosis and 35 patients with other neurological disorders. The ELISA reaction for the whole Taenia solium cysticercal extract showed 91.3% sensitivity and 94.3% specificity, whereas the sensitivity and specificity of the ELISA for the whole Taenia crassiceps cysticercal extract were 87% and 94.3%, respectively. The ELISA reactions for vesicular fluid from Taenia solium or Taenia crassiceps showed 91.3% sensitivity and 97.1% specificity. Considering the results obtained from the four antigen preparations, vesicular fluid from Taenia solium and Taenia crassiceps cysticerci may be useful as a source of antigens for immunological reactions that are used for detecting specific antibodies in cerebrospinal fluid samples from patients with neurocysticercosis.

Estudo de validação comparativo entre as técnicas Elisa e RIFI para diagnosticar Leishmania sp em cães errantes apreendidos no município de Campos dos Goytacazes, Estado do Rio de Janeiro

Foi realizada uma pesquisa objetivando-se verificar a eficácia do teste ELISA, para detecção de anticorpos contra Leishmania sp em cães, comparando-o com o RIFI, padrão em humanos, e investigar a situação sorológica desta zoonose na microrregião. Os testes tiveram uma concordância de 97,6%, classificada como forte.

Detecção de sífilis por ensaios de ELISA e VDRL em doadores de sangue do Hemonúcleo de Guarapuava, Estado do Paraná

O objetivo desse estudo foi avaliar a soroprevalência de sífilis em 5.752 doadores de sangue atendidos no Hemonúcleo de Guarapuava-PR, em 2006. As taxas de positividade foram de 2,1% pelo teste de ensaio imunoenzimático e 0,2% pelo Veneral Disease Research Laboratory, mostrando baixa prevalência de sífilis nos indivíduos que procuraram este banco de sangue.

Cohesion decay: quantitative analysis of partial sister chromatid cohesion

Cell division is a highly dynamic process where sister chromatids remain associated with each other from the moment of DNA replication until the later stages of mitosis, giving rise to two daughter cells with equal genomes. The “molecular glue” that links sister DNA molecules is called cohesin, a tripartite ring-like protein complex composed of two Structural Maintenance of Chromosome proteins (Smc1 and Smc3) bridged by a kleisin subunit Rad21/Scc1, that together prevent precocious sister chromatid separation. Accumulating evidence has suggested that cohesion decay may be the cause of segregation errors that underlie certain human pathologies. However it remains to be determined how much cohesin loss abolishes functional sister chromatid cohesion. To answer these questions, we have developed different experimental conditions aiming to titrate the levels of cohesin on mitotic chromosomes in a precise manner. Using these tools, we will determine the minimal amount of cohesin needed to confer functional cohesion. The approaches described here take advantage of a system in Drosophila melanogaster where the Tobacco Etch Virus (TEV) protease can cleave the Rad21 subunit of cohesin leading to precocious sister chromatid separation. Firstly, we tried to express different levels of TEV protease to obtain partial loss of cohesion. However, this approach has failed to produce systematic different levels of sister chromatid separation. Most of the work was therefore focused on a second strategy, for which we established strains with different levels of cohesin sensitive/cohesin resistant to TEV protease. Strains containing different amounts of functional cohesin (TEV resistant) were tested by in vitro cleavage and by in vivo injections in embryos for their ability to promote sister chromatid cohesion. Our results reveal that removal of half of the cohesin complexes does not impair chromosome segregation, implying that chromosome cohesion is less sensitive to cohesin amounts than previously anticipated.

Comportamento dos testes sorológicos ML Flow e ELISA (PGL-I) em áreas endêmica e não endêmica de hanseníase

O ML Flow e o ELISA PGL-I são testes sorológicos que detectam anticorpos IgM contra o glicolipídio fenólico I específico do Mycobacterium leprae. Para avaliar o comportamento destes testes em áreas endêmica e não endêmica para hanseníase foram estudados 351 voluntários no Brasil e no Chile, incluindo pacientes com hanseníase, controles sadios, portadores de outras doenças infecciosas, não infecciosas e dermatoses que fazem diagnóstico diferencial com hanseníase. O ponto de corte do ELISA foi estabelecido pelo método da Curva ROC (> 0,157). Em área endêmica, o ML Flow apresentou resultados positivos em 70% dos pacientes com hanseníase; o ELISA foi positivo em 53,3%. Em área não endêmica, o ML Flow foi negativo em todos os voluntários testados; o ELISA foi positivo em 4 voluntários. O ML Flow é um ensaio mais rápido, facilmente aplicável e, portanto, mais adequado para ser utilizado na Atenção Básica; o ELISA necessita, alem de uma infra-estrutura de laboratório adequada, pessoal treinado e especializado em sua execução.

Pesquisa de anticorpos anti PGL-I através de ELISA em tatus selvagens do Brasil

Tatus têm sido envolvidos na transmissão da hanseníase e considerados como fonte de Mycobacterium leprae em muitas publicações. Médicos de partes dos EUA consideram o contato com tatus um fator de risco para hanseníase. Entretanto, há um desafio associado ao papel do tatu na perpetuação da hanseníase no Continente Americano. Foi pesquisada a presença de anticorpos anti-PGL-I em tatus selvagens de áreas endêmicas em hanseníase do Estado do Espírito Santo, Brasil, através de ELISA realizado em amostras de soro de 47 animais. Elisa positivo foi encontrado em 5 (10.6%) tatus. Tatus infectados podem ter algum papel na transmissão da hanseníase disseminando bacilos no meio ambiente, talvez tornando mais difícil a interrupção da cadeia de transmissão e redução do número de casos novos de hanseníase. A técnica de ELISA é um eficiente método para investigação soroepidemiológica da presença do Mycobacterium leprae em tatus.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

Automated quantitative analysis of MCC-IMS spectra

Ion Mobility Spectrometry coupled with Multi Capillary Columns (MCC -IMS) is a fast analytical technique working at atmospheric pressure with high sensitivity and selectivity making it suitable for the analysis of complex biological matrices. MCC-IMS analysis generates its information through a 3D spectrum with peaks, corresponding to each of the substances detected, providing quantitative and qualitative information. Sometimes peaks of different substances overlap, making the quantification of substances present in the biological matrices a difficult process. In the present work we use peaks of isoprene and acetone as a model for this problem. These two volatile organic compounds (VOCs) that when detected by MCC-IMS produce two overlapping peaks. In this work it’s proposed an algorithm to identify and quantify these two peaks. This algorithm uses image processing techniques to treat the spectra and to detect the position of the peaks, and then fits the data to a custom model in order to separate the peaks. Once the peaks are separated it calculates the contribution of each peak to the data.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

Marketing plan for Book-a-Cook

This thesis studies the marketing plan for the new brand Book-a-Cook. Book-a-Cook is an online platform connecting well-to-do urban Germans with professional chefs that prepare meals at consumer's homes. By analyzing the external and internal audit, it was possible to develop an appropriate strategy to launch the service in Germany. The main goals are stimulating demand and creating a strong brand in order to establish a successful business model. A quantitative research was made to identify potential customers, their needs and how Book-a-Cook can satisfy them.

Effects of quantitative easing on the USA economy: A test for policy effectiveness

The catastrophic disruption in the USA financial system in the wake of the financial crisis prompted the Federal Reserve to launch a Quantitative Easing (QE) programme in late 2008. In line with Pesaran and Smith (2014), I use a policy effectiveness test to assess whether this massive asset purchase programme was effective in stimulating the economic activity in the USA. Specifically, I employ an Autoregressive Distributed Lag Model (ARDL), in order to obtain a counterfactual for the USA real GDP growth rate. Using data from 1983Q1 to 2009Q4, the results show that the beneficial effects of QE appear to be weak and rather short-lived. The null hypothesis of policy ineffectiveness is not rejected, which suggests that QE did not have a meaningful impact on output growth.

Clinical practice guidelines in Portugal : quantitative and qualitative evaluation

RESUMO: Introdução As normas de orientação clínica são ferramentas úteis na translação de conhecimentos desde a investigação para a prática clínica diária. Estratégias ativas de implementação de normas de orientação clínica requerem elevado esforço organizacional e financeiro. Quando os recursos são escassos, as estratégias passivas podem ser a única opção de disseminação. Desde 2011 a Direção Geral da Saúde publicou cento e cinquenta e nove normas de orientação clínica. Nesta Tese é feita uma avaliação do impacto que estratégias de disseminação de normas de orientação clínica têm no padrão de prescrição dos médicos e uma avaliação qualitativa do processo das normas de orientação clínica em Portugal. Métodos: O primeiro artigo é um estudo quasi experimental usando uma série de análises temporais interrompida para comparar os níveis observados e esperados de prescrição de inibidores da ciclooxigenasa-2, antes e depois da publicação da norma de orientação clínica sobre a utilização de anti-inflamatórios não esteroides. O segundo estudo é um artigo de opinião e debate no qual numa primeira parte contextualiza o processo das normas da Direcção Geral da Saúde, na segunda parte aponta virtudes e defeitos no processo e a terceira parte constitui uma contribuição com vista à melhoria do processo. Discussão A produção de normas de orientação clínica requer metodologia rigorosa e complexa. A literatura médica revela que a translação de conhecimento é uma tarefa árdua. Estratégias de implementação ativas requerem recursos financeiros e organizacionais sólidos. Estratégias de implementação passivas podem representar uma solução aceitável se os recursos financeiros e organizacionais escasseiam. Pouco é conhecido sobre a eficácia destas estratégias fora do contexto de investigação. Com esta Tese pretendo contribuir para a clarificação desta resposta, outros países e instituições podem ver utilidade nesta informação, bem como pretendo contribuir para a discussão e melhoria do processo das normas de orientação clínica em Portugal. ------------------ ABSTRACT: Introduction Clinical practice guidelines can help address the failure to translate research findings into clinical practice. Active clinical practice guidelines implementation strategies require active efforts from organizations and are resource and financially demanding. Passive implementation strategies may represent the only option if resources are scarce. Out of research environment, real world efficacy of passive implementation strategies is still undetermined. Since 2011 the Portuguese General Health Directorate published one hundred and fifty nine guidelines. In this Thesis I evaluate the impact of passive dissemination of clinical practice guideline in clinician’s prescription behavior and review, from a qualitative point of view, the Portuguese clinical practice guideline process. Methods The first study is a quasi-experimental study using a retrospective interrupted time-series analysis design to compare the observed and expected prescription of cyclooxygenase-2 before and after the non steroidal antiinflammatory guideline publication. The second study is an opinion and debate article in which I firstly review the General Health Directorate guideline process. The second part states positive and negative aspects in the process and the third part is a contribution aimed at improving the process in the future. Discussion Clinical practice guidelines production demands a rigorous and complex methodology. medical iterature reveals that knowledge translation is a difficult task. Active implementation strategies demand solid financial and organizational resources. Passive implementation strategies may represent an acceptable solution if financial and organizational resources are scarce. Little is known about the efficacy of these strategies out of the research context. With this Thesis I intend to contribute to clarify this question, other countries and institutions with similar conditions may find this information useful, and also to contribute for the discussion and general improvement of national clinical practice guidelines process.