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Diagnosis of Chagas Disease by Detection of Trypanosoma Cruzi

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Diagnosis of Chagas Disease by Detection of Trypanosoma Cruzi Diagnosis of Chagas Disease by Detection of Trypanosoma cruzi DNA Fragments from Filtered Urine Samples by Ana G. Madrigal A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science. Baltimore, Maryland April 2019 Abstract Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life threatening disease endemic to Latin America that has re-emerged as a global public health issue due to globalization. While this disease has been known of for hundreds of years, detection methods are still not straight forward; especially for the chronic phase of the disease. There is no golden standard for diagnosis and current detection methods rely on indirect tests such as serology; which has proven to be a suboptimal approach due to immunological and geographical variability. Here, we report on a direct form of noninvasive diagnosis, focused on the detection of T. cruzi-derived cell-free DNA in the urine of Chagas Disease patients. For this study, samples were collected, stored, and processed using a filtration/extraction method. DNA was analyzed through conventional and nested PCR protocols, with three different primer sets targeting T. cruzi k-DNA or Sat-DNA repeat sections. The resulting positive PCR samples were confirmed through sequencing and compared against serological results. We found that T. cruzi DNA is indeed present in the urine of Chagas disease patients. While further studies with a larger population are needed for improved primer development, the detection of T. cruzi DNA in urine has the potential to provide a novel non-invasive diagnostic method for Chagas disease that can be employed to improve chronic phase detection, treatment-monitoring, early detection in congenital disease cases and the management of immunosuppressed patient. Primary reader: Dr. Alan L. Scott Secondary reader: Dr. Clive J. Shiff ii Acknowledgments First, I would like to thank both of my advisers for the opportunity to work in this project. Thank you Dr. Shiff and Dr. Scott for all the encouragement and advise through all steps of this project. I would also like to thank Miryam Romano for her mentoring and training, and Luz Toribio for all the brainstorming sessions; it was fun sharing a lab together. I would also like to thank Dr. Gilman for all the resources provided. Especially thanks to Yagahira Castro and Rachel Marcus who provided much help in sample collection. I would also like to thank Dr. Zavala for allowing us use to some of his lab equipment. Also, many thanks to Dr. Monica Mugnier for her insights and guidance in understanding sequencing results and parameters. I would also like to thank Anne Jedlicka for her aid in running the tape station and instruction on how to analyze the results. Secondly, thanks to Camilla Strother and the Casadevall lab members for being great lunch buddies. Thanks to my family for all their constant support throughout this process. Finally, thanks to my cats Miles and Kitty for all the stress relief and also for being special editors and having me re-write this thesis at least three times more. iii Table of Contents I. INTRODUCTION ........................................................................................................................... 1 BACKGROUND ............................................................................................................................................ 4 The Parasite ........................................................................................................................................... 4 Morphology & Lifecycle .................................................................................................................................. 4 Phylogenetic Relationship ................................................................................................................................ 7 Genome .............................................................................................................................................................. 8 Population Genetic Diversity ......................................................................................................................... 10 Epidemiology ....................................................................................................................................... 11 Epidemiology of Reduviid bug – ................................................................................................................... 11 Prevalence in Latin America– ....................................................................................................................... 11 Prevalence worldwide–................................................................................................................................... 13 Transmission in the U.S.A–............................................................................................................................ 14 Animal reservoirs in the U.S.A. – .................................................................................................................. 15 Economic impact in the U.S.A.– .................................................................................................................... 15 Clinical Manifestations ....................................................................................................................... 16 Acute – ............................................................................................................................................................. 16 Chronic – ......................................................................................................................................................... 16 Immune response to T. cruzi ............................................................................................................... 18 Acute – ............................................................................................................................................................. 18 Chronic – ......................................................................................................................................................... 19 Diagnosis .............................................................................................................................................. 20 Acute infection – ............................................................................................................................................. 20 Congenital – .................................................................................................................................................... 20 Chronic ............................................................................................................................................................ 20 Nucleic Acid-based detection of T. cruzi ....................................................................................................... 24 Treatment ............................................................................................................................................. 26 II. STUDY AIMS ............................................................................................................................. 28 RATIONALE. ............................................................................................................................................. 28 III. METHODS ................................................................................................................................ 29 PRELIMINARY STUDIES ............................................................................................................................ 29 Study Population. ................................................................................................................................. 29 Frozen Urine Extractions. ................................................................................................................... 29 DC COHORT SAMPLE ............................................................................................................................... 30 Study Population. ................................................................................................................................. 30 Sample Filtration. ................................................................................................................................ 31 DNA Extraction. .................................................................................................................................. 31 PCR Controls. ...................................................................................................................................... 32 Primer building. ................................................................................................................................... 33 PCR. ..................................................................................................................................................... 33 Analysis of PCR Products by Agarose Gel Electrophoresis. .............................................................. 36 DNA Band Extraction and Sequencing. ............................................................................................. 36 Bioinformatic analysis. ........................................................................................................................ 36
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