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Evaluation of Next-Generation Sequencing Technology in Determining Infectious Causes of Fever

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Evaluation of Next-Generation Sequencing Technology in Determining Infectious Causes of Fever ResearchOnline@JCU This file is part of the following reference: Susilawati, Tri Nugraha (2016) Evaluation of next- generation sequencing technology in determining infectious causes of fever. PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/46187/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://researchonline.jcu.edu.au/46187/ Evaluation of Next-Generation Sequencing Technology in Determining Infectious Causes of Fever Thesis submitted by Tri Nugraha Susilawati Bachelor of Medicine, Medical Doctor, Master of Medicine (Infection and Immunity) For the Degree of Doctor of Philosophy in the College of Medicine & Dentistry James Cook University January, 2016 ii Statement of Access I, the undersigned, the author of this thesis, understand that James Cook University Library, by microfilm or by other means, allows access to users in other approved libraries. All users consulting this thesis will have to sign the following statement: ‘In consulting this thesis, I agree not to copy or closely paraphrase it in whole or part without the consent of the author; and to make proper written acknowledgement for any assistance, which I have obtained from it.’ Beyond this I do not wish to place any restriction on access to this thesis. Signed: Date: 7 January 2016 iii Statement of Sources Declaration I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published, unpublished or oral work of others has been acknowledged in the text and a list of references is given. Every reasonable effort has been made to gain permission and acknowledge the owners of copyright material. I would be pleased to hear from any copyright owner who has been omitted or incorrectly acknowledged. Signed: Date: 7 January 2016 iv Statement of Contributions of Others Nature of Contribution Co-contributors assistance Intellectual The supervisory team provided Professor John McBride, MBBS, support assistance within the remit of their DTM&H, FRACP, FRCPA, PhD roles as described in the policy College of Medicine & Dentistry, The Role of the Advisory Panel in James Cook University, Cairns, Providing Regular Guidance and Australia Support to Research Student. Professor Alex Loukas, PhD Editorial assistance was provided Centre for Biodiscovery and to the candidate by both the Molecular Development of supervisory team and professional Therapeutics, Australian Institute of editors. Proofreading was Tropical Health and Medicine, James restricted to correcting the Cook University, Cairns, Australia presentation of the text to conform with standard usage and conventions and the provision of Aaron R. Jex, BSc, PhD advice in the matters of structure, Faculty of Veterinary and conventions of grammar and Agricultural Sciences, The University syntax, use of clear language, of Melbourne, Australia logical connections between phrases, clauses, sentences, Cinzia Cantacessi, PhD paragraphs and sections, voice and tone and repetition. Department of Veterinary Medicine, Cambridge Veterinary School, University of Cambridge, UK Elite Editing 213 Greenhill Road, Eastwood, South Australia 5063 Financial Project costs James Cook University support Stipend Far North Queensland Hospital Supplementary academic support Foundation (thesis editing and conference Australia Awards Scholarships presentation) v Acknowledgements Study Participants I would like to express my sincere appreciation to the participants of this study. The majority of participating patients were cooperative and happy to contribute. Contact with the study participants, especially with the patients, provided rewarding and unforgettable personal experiences. PhD Supervisors It was Professor John McBride who inspired me to keep up my spirit and persistence throughout this project. As my principal supervisor, he has guided me very well and was always available when I needed his help. He encouraged me to learn many things and try every opportunity. I thank him for his patience and careful supervision throughout the years of my candidature. My appreciation is also extended to Professor Alex Loukas, whose supervision has been always energetic, enthusiastic and thought provoking. I thank him for providing in-kind support and access to his laboratory. The biggest help in the molecular side of my project came from Dr Aaron Jex and Dr Cinzia Cantacessi, whose expertise has encouraged me to learn and learn more about the new fields in my life: molecular technique and genomic data analysis. Lastly, I would like to thank my former supervisor, Dr Jason Mulvenna, who had already made a major contribution in preparation of my proposal and literature review. Funding Bodies The study was made possible by an annual grant from the Far North Queensland Hospital Foundation and from the College of Medicine and Dentistry, James Cook University. I also thank AusAID for the Australia Award Scholarship that covered my tuition fees, living expenses and other necessities. A top-up scholarship from College of Medicine and Dentistry, James Cook University provided an additional stipend during my PhD. vi Cairns Hospital Staff I should highlight the outstanding contributions of the staff from the Clinical Research Unit: Sue Richmond, Sue Dixon, Donna Kreuter and Debra Horold. They provided great help in enrolment of participants and blood collection. The study was conducted with the permission and assistance of Dr Richard Stone (Director of the Emergency Department), Dr Peter Boyd (Director of the Medicine Department), Dr Drew Wenck (Director of the Intensive Care Unit), Mark Porton (Director of the Pathology Department), Peter Hiatt and Deborah Moffat (staff of the Pathology Department). I thank them for their cooperation in helping me obtain the samples I needed. I also thank the staff of the Medical Record Department of Cairns Hospital with their assistance in providing more than a thousand medical records that I requested. Colleagues and Mentors Over the last four and half years, I have received wonderful advice and encouragement from a number of people. They include Intansari Nurjannah, Mercy Rampengan, Lalu Adi Gunawan, Mangalasiri Jayathunge, Sri Warsini, Yvonne Hodder, Lyn Kerr, Dr Cindy Woods, Dr Gregory Maes, A/Prof Malcolm McDonald and Prof Jane Mills. I learnt a great deal from the wonderful mentors I had at the Queensland Tropical Health Alliance laboratory, including Ivana Ferreira, Darren Pickering, Dr Mark Pearson, Dr Severine Navarro, Dr Annette Dougall, Dr Javier Sotillo-Gallego and Dr Paul Giacomin. Thanks so much to all of you who helped me to survive PhD life. Family I sincerely thank my husband, Dr Atik Susianto, for his understanding, continuous support and encouragement during this seemingly never-ending journey. And, to our children, Fatyanaura Arleya, Elena Zedya Renata and Kinza Altair Rashad, thank you so much for giving me a rich life experience as a PhD student with three young children and for keeping my spirits high by simply looking at me with your sparkling eyes and wonderful smiles. vii Abstract Undifferentiated fever (UDF) is a common complaint in clinical practice, but its aetiology is not always determined due to non-specific symptoms and laboratory findings. While fever of unknown origin (FUO) is a common medical term for fever without obvious cause, this condition is distinguished from acute undifferentiated fever (AUF) in terms of duration, progression of illness and underlying causes. In FUO, fever must exist for more than 3 weeks and can persist for a very long period unless the underlying cause is found and eliminated. In contrast, AUF is more limited in duration and many episodes spontaneously resolve, presumably due to self-limiting infectious diseases. The problem of determining the infectious causes of fever has received considerable attention, particularly in tropical countries. Previous studies in South and Southeast Asia reported high prevalence of infection as the main cause of AUF. This prompted the hypotheses that infection-related AUFs are common in the tropical region of Far North Queensland, Australia, and that a significant proportion of AUFs in this region are undiagnosed. Diagnosing infectious causes of fever is a challenge for clinicians. With hundreds of possible agents and a limited number of specific tests that can be performed, it is very likely that doctors will miss the true cause of fever. Moreover, current diagnostic approaches rely on prior knowledge of the pathogens being sought, thus precluding the detection of unsought or novel pathogens. Thus, the prevalence of undiagnosed undifferentiated fever (UUDF) indicates either that clinicians are failing to order appropriate tests, that current diagnostic methods are inadequate, or that there are causes of fever that are yet to be discovered. This diagnostic challenge necessitates good clinical skills, knowledge of the pattern of signs and symptoms associated with particular infections and a broad diagnostic tool for determining infectious causes of fever. Since there is a wide range of pathogens, the diagnostic tool should be able to distinguish one pathogen from another as well as identify multiple pathogens simultaneously.
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