Molecular diagnosis of Chagas disease in seronegative patients with megaesophagus

Orientador: Sandra Cecilia Botelho CostaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: A doença de Chagas, cujo agente etiológico é o protozoário Trypanosoma cruzi, completa 100 anos de descoberta. Apesar da implementação de programas visando ao controle da transmissão vetorial, a infecção chagásica segue como um importante problema de saúde pública na América Latina. A globalização da doença de Chagas, consequente à migração de pessoas infectadas para países onde a doença não é endêmica, traz à tona a necessidade de instituição de medidas de controle e vigilância em algumas áreas da Europa e da América do Norte. A patologia caracteriza-se por uma fase aguda geralmente assintomática com elevada parasitemia e uma fase crônica, em que os parasitos dificilmente são detectados no sangue periférico por métodos parasitológicos convencionais. O curso clínico da infecção por T. cruzi é variável, sendo que boa parte dos infectados permanece na formaindeterminada da doença enquanto outros desenvolvem a forma cardíaca, digestiva ou nervosa. A prevalência do comprometimento do trato digestivo na doença de Chagas varia conforme a área endêmica, podendo alcançar até 14% no Brasil central. A forma digestiva caracteriza-se por lesão dos plexos nervosos intramurais e disfunção motora principalmente do esôfago e do cólon, levando à dilatação progressiva desses órgãos. Em nosso país, a doença de Chagas é o único fator etiológico comprovado para o megaesôfago. Devido à parasitemia baixa e intermitente, o diagnóstico na fase crônica baseia-se na detecção de anticorpos específicos anti-T.cruzi por metodologias sorológicas convencionais. Apesar de serem considerados métodos sensíveis e que apresentam boa especificidade, há relatos de resultados falso-positivos devido à reação cruzada com outros tripanosomatídeos que circulam na mesma área geográfica de T. cruzi. Por outro lado, existem relatos de imunodiagnóstico inconclusivo ou negativo em pacientes com antecedentes epidemiológicos e manifestações clínicas sugestivas de infecção chagásica. Nestes casos, métodos diagnósticos não convencionais devem ser empregados com o intuito de esclarecer a etiologia da doença. Neste estudo, determinou-se a soroprevalência para doença de Chagas em uma população com esofagopatia. Dos 518 pacientes com resultados de exames sorológicos convencionais, 409 (79%) apresentaram-se positivos, 31 (6%) inconclusivos e 78 (15%) negativos. A presença de DNA de T. cruzi foi avaliada pela N-PCR (nested-PCR) em amostras de sangue de pacientes portadores de megas digestivos com sorologia inconclusiva ou negativa para doença de Chagas. De 41 amostras analisadas, a N-PCR foi positiva em 31 (75,6%) dos casos. Como todos os pacientes incluídos no estudo apresentavam manifestações clínicas compatíveis com doença de Chagas e a maioria deles nasceu em áreas endêmicas, enfatizamos a importância da busca por métodos diagnósticos mais eficazes. Nossos resultados indicam que a N-PCR é uma ferramenta adequada para detecção de DNA de T. cruzi em pacientes soronegativos e pode ser utilizada para determinar a etiologia do megaesôfago.Abstract: After 100 years of research and despite control programs launched in endemic areas, Chagas disease, caused by Trypanosoma cruzi, still remains an important public health problem in Latin America. Immigration of infected persons from endemic to nonendemic areas leads to Chagas disease spread and it increases the need for establishment of control programs and surveillance in North America and some European countries. Acute phase of Chagas disease is usually free of symptoms and a high parasitemia is observed. Following the acute phase, the chronic stage is characterized by low and intermittent parasitemia. Presentation of chronic chagasic infection is pleomorphic ranging from absence of symptoms to severe cardiac involvement. Symptomatic chronic phase is usually characterized by cardiac or digestive commitment and its current diagnosis relies on the measurement of T. cruzi-specific antibodies produced in response to the infection. Motility disorders and enlargement of digestive organs (commonly esophagus and colon) are consequences of neuronal destruction caused by the persistence of T. cruzi. Since idiopathic megaesophagus is a rare condition in Brazil, Chagas disease is considered the main etiology of megaesophagus. Despite its high sensitivity, conventional serology may present false-positive results due to cross-reactivity to other trypanosomes. On the other hand, false-negative results have been described in patients from endemic areas with suggestive signs of chagasic infection. In order to clarify this controversial situation, alternative methods such as PCR (polymerase chain reaction) have been employed for diagnostic purposes. In this study, prevalence of Chagas disease in a population with esophageal disorders was assessed by conventional serology. From 518 patients, 409 (79%) presented positive results, 31 (6%) and 78 (15%) presented inconclusive and negative results, respectively. For the following step, we recruited 41 patients with megaesophagus and negative or inconclusive serology for Chagas disease. T. cruzi DNA was detected by NPCR (nested-PCR) in 31/41 (75.6%) cases. As all patients included in our study presented suggestive clinical signs of digestive form of Chagas disease and most of them were born in endemic areas, we highlight the importance of diagnosis improvement and its implication in blood banks screening. Our data suggest that N-PCR is an effective tool for detection of T. cruzi DNA in patients with inconclusive or negative serology and, eventually, it may be useful to clarify megaesophagus etiology.MestradoCiências BásicasMestre em Clinica Medic

Trypanosoma Cruzi: Parasite Persistence In Tissues In Chronic Chagasic Brazilian Patients.

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.10685-9

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Detection and quantitation of Trypanosoma cruzi DNA in patients with megaesophagus

Orientadores: Sandra Cecília Botelho Costa, Eros Antonio de AlmeidaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Atualmente, o diagnóstico laboratorial da doença de Chagas crônica baseia-se em métodos sorológicos convencionais. Entretanto, resultados falso-negativos e falso-positivos podem ocorrer. A hemocultura também pode ser utilizada como método diagnóstico, mas sua sensibilidade é limitada na fase crônica. Métodos diagnósticos baseados em princípios de biologia molecular vêm sendo estudados e a Reação em Cadeia da Polimerase (PCR) tem sido a técnica mais utilizada ultimamente. Este estudo teve como objetivo principal avaliar o desempenho da PCR qualitativa e quantitativa no diagnóstico da doença de Chagas em pacientes portadores de megaesôfago. Foram estudadas amostras de DNA de sangue de 26 pacientes com sorologia não reagente ou inconclusiva e de 33 pacientes soropositivos para doença de Chagas. O alvo genético mais adequado para PCR qualitativa, que apresentou maior positividade, foi o Sat-DNA de T. cruzi. Quando comparada com os resultados de PCR quantitativa, houve concordância de 72,72% em ambos os grupos. Além disso, observou-se que todos os pacientes com hemocultura positiva apresentaram PCR qualitativa positiva. A metodologia quantitativa, embora considerada altamente sensível, não detectou DNA de T. cruzi em uma amostra de paciente com hemocultura positiva. Nossos resultados indicam um possível subdiagnóstico da doença de Chagas em pacientes com megaesôfago devido aos exames sorológicos negativos ou inconclusivos. A PCR qualitativa mostra-se uma ferramenta útil para esclarecimento da etiologia do megaesôfago e, por apresentar positividade semelhante à qPCR, não se justifica o uso de metodologia quantitativa para fins exclusivamente diagnósticosAbstract: Current laboratory diagnosis of chronic Chagas disease relies on conventional serological tests but false-positive and false-negative results may occur. Hemoculture can also be used as a diagnostic method but its sensitivity in chronic phase is limited due to the low/intermittent parasitemia. Molecular diagnosis methods have been studied and the main technique that has been extensively tested is the Polymerase Chain Reaction (PCR). This study aimed to evaluate the qualitative and quantitative PCR performance for diagnostic purposes in patients with megaesophagus. We studied DNA blood samples from 26 patients with negative or inconclusive conventional serology for Chagas disease and from 33 patients with positive serology. The most suitable target for qualitative PCR presenting high positivity was the Sat-DNA of T. cruzi. When compared to the quantitative results, the concordance between the two molecular methods in both groups was 72.72%. In addition, we observed that all patients with positive results of hemoculture had positive qualitative PCR. The qPCR methodology although highly sensitive could not detect minimal amounts of T. cruzi DNA in one patient with positive hemoculture. Our results indicate a possible misdiagnosis of patients with megaesophagus given by the negative or inconclusive serology for Chagas disease. Due to its good performance, the qualitative PCR is a useful tool to determine the megaesophagus etiologyDoutoradoClinica MedicaDoutora em Clínica Médic

Trypanosoma cruzi : parasite persistence in tissues in chronic chagasic Brazilian patients

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis