Vector borne-diseases make up a significant portion of morbidity and mortality worldwide, being responsible for around 700,000 deaths annually according to the World Health Organization. Neglected, tropical diseases such as Chagas disease have a significant impact on people in Latin America, affecting millions, and especially those residing in rural areas. Chagas disease is the number one cause for heart disease in Latin America, and is caused by the Trypanosoma cruzi parasite, carried by Triatominae insect vectors. The intricate life cycle of the parasite, ecology and behavior of the vector, and lack of disease treatment options, make Chagas disease challenging to control. Prevention measures are highly sought after, and implementation science approaches such as Ecohealth management engage affected communities in disease prevention. Knowing what insect vectors are feeding on sheds light on vector ecology and behavior, aiding in vector management which is pivotal in disease prevention. While DNA-based methods have traditionally been used to study vector blood meals, they come with limitations and challenges, such as the need for fresh, high abundance blood meals. Therefore, the goal of this research was to evaluate Chagas vector blood meal sources using an innovative protein mass spectrometry-based approach. We demonstrate first the ability to utilize liquid chromatography tandem mass spectrometry (LC-MS/MS) to correctly identify hemoglobin protein peptides from mouse blood and subsequently identify Chagas vector blood meal sources from field-collected insect vectors where blood meal identification is compared with traditional DNA-based methods as a control. An experimental feeding study allowed us to then demonstrate the longevity of hemoglobin protein peptides for blood meal detection, showing LC-MS/MS-based blood meal identification outperforms DNA-based polymerase chain reaction (PCR) at least 4 weeks post-feeding and 12 weeks post-molting. This allowed us to test the limits of our innovative detection method experimentally and comparatively. Finally, we evaluated blood meals in field-caught insect vectors collected as part of a large collaborative Ecohealth project in Central America. LC-MS/MS identified two times as many blood meals in insect vectors, including those that did not have blood meals detected with DNA-based PCR. As single vectors often feed on multiple sources, we also validated our ability to decipher multiple blood meals from an individual vector and showed the ability to quantify a blood meal using synthetic AQUA (Absolute QUAntification) peptides, a first step in using quantification data for identifying blood meals not currently in our underlying database. Furthermore, we show that lower resolution mass spectrometers are able to identify blood meals from taxa correctly, an important and strong attribute of our LC-MS/MS-based method, opening the door to using proteomics in countries where Chagas disease is endemic and resources are limited. Even though expertise and resources of research labs differ in locations across the globe, herein is described how LC-MS/MS is a valuable additional tool for fighting neglected tropical diseases. Ultimately, hemoglobin-based LC-MS/MS vector blood meal identification is a complementary technique to available molecular methods and can confidently identify Chagas vector blood meal sources to aid in understanding vector biology and ecology, and aid in developing appropriate Ecohealth vector control measures.

Trypanosoma cruzi is endemic in the U.S., infecting humans, dogs, and wildlife. This study identified a new geographic distribution for triatomine species within Texas based on 2,449 records obtained from published data and new field studies. Triatomine vectors of T. cruzi were reported from 97 counties covering all ecological zones. Triatoma gerstaeckeri was the most commonly collected species followed by T. sanguisuga. New field collections resulted in 233 specimens from 37 counties and a 52% T. cruzi infection rate. A second trypanosome, Blastocrithida triatomae was found in two specimens from different locations. A habitat suitability model for T. gerstaeckeri was developed using GIS and remote sensing applications. Forest and rangeland were the predominant land cover classes found within T. gerstaeckeri habitat, where as water and agriculture proved to have little influence on habitat suitability. Genetic variation of seven triatomine species from Texas was evaluated using cytochrome b DNA sequences from 61 new specimens. This is the first study of the taxonomic status of T. gerstaeckeri, T. indictiva, and T. neotomae using molecular markers. Intraspecific variation for T. sanguisuga and T. gerstaeckeri suggests significant gene flow across their ranges within Texas. Genetic variation of T. cruzi isolates from Texas was evaluated using SSU rRNA gene sequences. Included were 63 new sequences from five triatomine species, canine, baboon, and human isolates. Sequences partitioned into two groups in agreement with previous studies on U.S. isolates. Genetic variation of T. cruzi did not occur according to host, geographic location, or collection site. The extent of Chagas disease in domestic canines of Texas is described by geographic distribution, signalment, and clinical presentation and histopathology. Based on data from 553 cases, the geographic distribution in Texas is widespread (46 counties) and closely matches the distribution of the Triatomine vectors. Chagas disease was diagnosed in 33 breeds, primarily sporting/working dogs. This study represents the most comprehensive characterization of components of the Chagas disease transmission cycle in the U.S. to date. These findings should raise awareness among physicians, veterinarians, and public health practitioners regarding T. cruzi, its vectors, canine infection, and human risk for Chagas disease in Texas.

Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 361-367).

Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 296-303).

Mestre -- Escola Nacional de Saude Publica, Rio de Janeiro, 2000.

Orientador: Cilmery Suemi Kurokawa / Resumo: O texto apresentado faz parte dos estudos sobre doença de Chagas (DC), suas formas clínicas e correlação com morbidades associadas à obesidade e envelhecimento e confeccionado para obtenção do título de Mestre em Doenças Tropicais. Ele é dividido em dois capítulos, no qual o primeiro texto compreende uma revisão bibliográfica sobre a doença de Chagas, vitamina D3 e catelicidina LL-37; e, no segundo capítulo, um artigo com dados descritivos da população de estudo intitulado de “FATORES DE RISCO CARDIOVASCULAR ASSOCIADOS À DOENÇA DE CHAGAS CRÔNICA” e um artigo intitulado de “VITAMINA D3, CATELICIDINA LL-37 E POLIMORFISMOS DO GENE VDR EM PACIENTES COM A FORMA INDETERMINADA E CARDÍACA DA DOENÇA DE CHAGAS”. Atualmente a DC é uma doença tropical negligenciada em todo o mundo e endêmica em 21 países latino-americanos, com mais de 25 milhões de pessoas em área de risco de transmissão. Afeta de 8 a 10 milhões de pessoas, sendo que no Brasil estima-se que existam 3 milhões de infectados, com 6 mil mortes por ano. Em sua fase crônica, tem a forma cardíaca como a manifestações mais importante da doença, tanto por sua frequência quanto por sua gravidade, com impacto social e financeiro, já que afetam os indivíduos na fase mais produtiva da vida (entre 30 e 50 anos), com altos índices de morbimortalidade. Além dos distúrbios no sistema de condução cardíaco, pode evoluir para manifestações mais graves como cardiomegalia, falência cardíaca e morte súbita. A inexistência de drogas eficazes pa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The text presented is part of the studies on Chagas disease (CD), its clinical forms and correlation with morbidities associated with obesity and aging and made for the title of Masters in Tropical Diseases. It is divided into two chapters, in which the first text comprises a literature review on Chagas disease, vitamin D3 and catatelicidin LL-37; and in the second chapter an article with descriptive data from the study population entitled "CARDIOVASCULAR RISK FACTORS ASSOCIATED WITH CHRONIC CHAGAS DISEASE" and an article entitled "VITAMIN D3, CATELICIDINE LL-37 AND VDR GENE POLYMORPHOSMS IN PATIENTS WITH THE UNDETERMINED AND HEART CHAGAS DISEASE FORM". Currently, CD is a neglected tropical disease worldwide and endemic in 21 Latin American countries, with more than 25 million people at risk of transmission. It affects 8 to 10 million people, and in Brazil it is estimated that there are 3 million infected, with 6 thousand deaths a year. In its chronic phase, it has the cardiac form as the most important manifestations of the disease, as much by its frequency as by its gravity, with social and financial impact, since they affect the individuals in the most productive phase of the life (between 30 and 50 years), with high morbidity and mortality rates. In addition to disturbances in the cardiac conduction system, it can progress to more serious manifestations such as cardiomegaly, heart failure and sudden death. The lack of effective drugs to treat the disease in its chronic ph... (Complete abstract click electronic access below) / Mestre

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Laboratório Multidisciplinar de Pesquisa em Doença de Chagas, 2008. / Submitted by Larissa Ferreira dos Angelos (ferreirangelos@gmail.com) on 2009-09-28T18:12:51Z No. of bitstreams: 1 2008_PerlaFdeAraujo.pdf: 1286808 bytes, checksum: 53fc99e52ecaefa2dd3e2e8055632df8 (MD5) / Approved for entry into archive by Gomes Neide(nagomes2005@gmail.com) on 2011-01-04T19:09:11Z (GMT) No. of bitstreams: 1 2008_PerlaFdeAraujo.pdf: 1286808 bytes, checksum: 53fc99e52ecaefa2dd3e2e8055632df8 (MD5) / Made available in DSpace on 2011-01-04T19:09:11Z (GMT). No. of bitstreams: 1 2008_PerlaFdeAraujo.pdf: 1286808 bytes, checksum: 53fc99e52ecaefa2dd3e2e8055632df8 (MD5) Previous issue date: 2008-07 / O presente trabalho é o primeiro relato de integração de seqüências provenientes de um protozoário para o genoma de células germinativas humanas. Neste estudo, nós documentamos a integração de minicírculos de kDNA de Trypanosoma cruzi no genoma de células germinativas e a transferência vertical dessas mutações para os descendentes. Foram analisadas amostras de esperma de 34 voluntários. Desses, 22 doadores foram agrupados em cinco famílias, cujos progenitores (F0) eram portadores da doença de Chagas. Doze amostras de esperma foram obtidas de indivíduos isolados. Foi possível observar a integração de kDNA de T. cruzi no genoma de todos os 17 chagásicos estudados. A transferência vertical apenas das seqüências de kDNA para as progênies F1 e F2 pôde ser observada em 6 descendentes de chagásicos. As análises das regiões do genoma humano que flanqueavam as seqüências de kDNA mostraram a integração de minicírculos de kDNA preferencialmente em elementos retrotransponíveis do tipo LINE-1, em 72,5% das seqüências quimeras. O encontro do mesmo locus em indivíduos chagásicos e seus descendentes é mais uma evidência mostrando a herança das mutações. Verificamos, também, que as seqüências de minicírculos de kDNA ligaram-se a outros elementos transponíveis (4%), tais como Alu, ERV e MER, e, também, em alguns genes (9%). Nos demais clones (14%), não foi possível determinar os loci de integração do kDNA no genoma devido á ausência de informação em bancos de dados. Os achados deste estudo sugerem que as mutações decorrentes da inserção do kDNA poderiam causar alterações genotípicas e fenotípicas implicadas na auto-imunidade descrita na doença de Chagas. De fato, a incorporação de DNA exógeno na célula humana nos mostra a complexidade das interações do parasito com o hospedeiro humano. A descrição de mutação, recombinação e deriva genética, alterando genótipo e fenótipo do hospedeiro, poderão sugerir a base da auto-imunidade que se associa à patogênese da doença de Chagas. / The present work shows by the first time the integration of Trypanosoma cruzi kDNA minicircle sequences into the human genome. This eukaryote-to-eukaryote lateral transfer of DNA is constantly seen in each patient harbouring the infections. The kDNA mutations are inherited by F1 and F2 progeny and undergo genetic drifts, recombination hitchhiking from primary to secondary chromosomes. We analysed 22 samples of sperm of volunteers from five families whose founders have the active infections. Independently, we further analysed haploid cells DNA from 12 individuals. The vertical transfer of kDNA minicircle sequences was detected in 17 family members. Among these there were 11 with active T. cruzi infections, showing nDNA and kDNA signatures. The remaining six F1 and F2 progenie had the kDNA signatures only. In each of these 17 individuals we showed sequences of minicircles of kDNA from T. cruzi integrated into germ line cells. The main hotspot for the kDNA integration was retrotransposons LINE-1 (72.5%). In 9% of cases the integreations entered coding regions of genes. Besides, kDNA minicircles sequences integrated nonautonomous transposable elements (4%) Alu, ERV and MER, Finally, in (14%), the kDNA mutations entered undetermined sites into the human genome. The presence of the kDNA mutations are in agreement with the findings of lateral transfer of kDNA in somatic cells of same individuals (Hecht, M.M, Thesis, University of Brasilia, 2008). Furthermore, the demonstration of kDNA mutations in haploid cells is brought here in the absence of active T. cruzi infections. Actually, the vertical inheritance could be appreciated in two marriages to which the marriages generated offsprings with kDNA mutations showing sequence alignments similar to the father. In fact, an exogenous DNA incorporation by the human cell let us to see more complex process of parasite-host`s interaction. The evolution of living creatures suggests that lateral transfer of exogenous DNA, mutations and hitchhiking, genetic drifts and social heterosis could be considered ongoin forces leading to genetic diversity and evolution.

BACKGROUND: Although Chagas disease is not an endemic health concern in the United States, it is prevalent with estimates of approximately 300,000 infected individuals. Of this, an estimated 20 – 30% will develop severe, life-threatening consequences. While what is known about Chagas disease is extensive, there is limited knowledge of this disease, especially among medical providers and clinicians in the United States. In order to bridge the gap between what is known about Chagas disease and those who lack this knowledge, adequate and effective education interventions must be developed and delivered. Education that is tailored for medical providers and clinicians most likely to encounter individuals at greastest risk for having Chagas disease is essential. Once this knowledge is gained, accurate evaluation, diagnosis, and treatment of indivuals with Chagas disease may be pursued, ultimately decreasing and preventing disease-associated morbidity and mortality. PROPOSAL: Clinicians from six departments (infectious disease, cardiology, internal medicine, family medicine, pediatrics, and obstretrics and gynecology) will be recruited from Boston Medical Center where they will be given an educational module about Chagas disease. Their knowledge of Chagas disease and how to clinically apply it will be assessed prior to the educational module, immediately following the educational module and 1-month and 6-months following the educational module. CONCLUSION: Chagas disease is a burden to health systems in many countries worldwide including the United States, and awareness of Chagas disease is lacking among medical personnel of multiple specialities in the United States. Educational interventions have provided knowledge of various diseases leading to protocol development, ultimately influencing clinical practice to a degree that reduces morbidity and mortality. The same is needed with respect to Chagas disease. The goal of this educational intervention is to provide a knowledge base through teaching and resources for clinicians to learn, understand, and review the steps needed to clinically evaluate, diagnose, and treat patients with Chagas disease. Using the reseach identified in this study as well as the proposed educational intervention, it is hoped that this disease burden can be alleviated.