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Infectious Etiologies of Febrile Illnesses in Cameroon

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Infectious Etiologies of Febrile Illnesses in Cameroon INFECTIOUS ETIOLOGIES OF FEBRILE ILLNESSES IN CAMEROON A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCES (TROPICAL MEDICINE) MAY 2017 By Kenji Obadia Mfuh DissertatiOn COmmittee: Vivek R. Nerurkar, Chairperson Axel T. Lehrer Diane W. TaylOr Richard Yanagihara Alan R. Katz, External Member ACKNOWLEDGEMENTS I thank GOd almighty for seeing me through this journey. I have experienced your guidance day- by-day. I wOuld like to express my sincere appreciatiOn to my advisOr Dr. Vivek R. Nerurkar, yOu have been a tremendous mentor for me. I would like to thank you for encouraging me, believing in me and for allowing me to grow as a researcher. Similar, prOfOund gratitude gOes tO my cOmmittee members and mentors, Dr. Axel T. Lehrer, Dr. Diane W. TaylOr, Dr. Alan R. Katz, Dr. Richard Yanagihara and Dr. ROse Leke fOr yOur brilliant cOmments and suggestiOns. I wOuld equally like to thank the Directors and Health Care WOrkers of the MarOua regiOnal hospital, CNPS hospital Maroua, Catholic health center Nkolbisson and PMI Nkwen, who suppOrted me in recruiting patients fOr my study. Words cannOt express hOw grateful I am tO the staff and students Of the department Of TrOpical Medicine, Medical Microbiology and PharmacOlogy, University Of Hawaii. It has been such a wonderful intellectual and cultural experience. MAHALO! A special thanks tO my family members, friends, the international Baptist fellowship/ministry and the CamerOOnian cOmmunity in Hawaii for their mOral suppOrt, which incented me to strive towards my gOals. My training was possible thanks to the financial suppOrt frOm the FOgarty International Center of the United States NatiOnal Institutes of Health (GID Training Grant – D43TW009074 FIC/NIH). My field research in Cameroon and laboratory work was supported by COBRE Pacific Center for Emerging InfectiOus Diseases Research – P30GM114737 NIGMS/NIH, NatiOnal Medical Research Institute, IMPM, CamerOOn, Contract: W81XWH-15-C-0101, USAMRAA, DoD and GID Training Grant – D43TW009074 FIC/NIH. ii ABSTRACT Background: DiagnOsis Of febrile illnesses in malaria-endemic countries focuses primarily on confirming Or ruling Out malaria, since malaria is considered a majOr health threat. But because of the high rate of malaria over-diagnosis, other febrile illnesses are often misdiagnosed or overlooked. Thus, there remains a large gap in Our understanding Of pathOgen prOfiling in mOst malaria-endemic countries. Because knowing the cause of fever is the first step in deciding appropriate treatment and improving disease outcomes, the overall objective of this dissertation research was tO identify infectiOus causes of febrile illnesses in Cameroon. Methods: We recruited 550 febrile patients seeking care at selected hOspitals in CamerOOn. BlOOd samples were cOllected frOm patients and used to conduct malaria tests by thick film micrOscOpy, rapid diagnOstic testing and PCR. Plasma samples were tested for antibodies against dengue, West Nile, chikungunya and respiratory viruses, Leptospira interrogans and Salmonella typhi, using RDT, micrOsphere immunoassay (MIA) and ELISA. Collected stoOl samples were cultured for the isolation of Salmonella and Shigella sp. RecOmbinant proteins of EbOla, Sudan, Marburg and Lassa viruses were expressed using DrOsophila S2 cells and used to develOp a MIA for viral hemOrrhagic fevers (VHF). MIA was pre-validated using human Or humanized monoclonal antibodies, and 408 previously collected plasma samples were screened fOr the presence Of IgG antibOdies fOr hemorrhagic fever viruses. Results: AlthOugh malaria was the main cause Of febrile illnesses in CamerOOn, the accuracy Of clinical diagnOsis Of malaria was pOOr. That is, 38% of the patients clinically diagnosed as having malaria had fever caused by other pathogens, including acute respiratOry tract infections, typhOid fever, amebiasis, toxOplasmOsis, shigellOsis, dengue fever, West Nile fever, and chikungunya virus infectiOn. We develOped and pre-validated a multiplex MIA fOr the diagnOsis of VHF, and identified samples (5/408) suspected to be positive for Ebola, Lassa and Marburg viruses. iii Conclusion: Collectively, Our data prOvide the first evidence Of several pathOgens causing febrile illnesses in CamerOOn and may prOvide the basis for develOping an algOrithm for the management Of febrile illnesses. This study alsO repOrts fOr the first time the develOpment Of a MIA fOr the diagnosis of VHF. iv TABLE OF CONTENTS Acknowledgements................................................................................................... ii Abstract...................................................................................................................... iii List of Tables……………………………………………………………………………….. viii List of Figures............................................................................................................. ix Abbreviations………………………………………………………………………………. x Chapter 1. Hidden Dengue Burden in the Shadow of Malaria in Africa…………. 1 Abstract……………………………………………………………………………………… 3 BackgrOund…………………………………………………………………………………. 4 MethOds……………………………………………………………………………………… 4 BeyOnd malaria: nOn-malaria febrile illnesses in the trOpics…………………………… 5 DiagnOstic challenges Of nOn-malaria febrile illnesses in the trOpics…………………. 5 The hidden burden Of dengue in Africa…………………………………………………… 7 The burden Of malaria in Africa……………………………………………………………. 11 Malaria Over-diagnosis……………………………………………………………………… 11 Is dengue and malaria cO-infection pOssible in Africa?............................................... 12 Clinical parameters and implicatiOns Of dengue and malaria cO-infection……………. 14 References.................................................................................................................... 21 Chapter 2. Dissertation Scope…………………………………………………………. 32 BackgrOund................................................................................................................. 33 Long-term gOal, Objective and hypOthesis.................................................................. 43 Specific aims............................................................................................................... 43 Specific aim 1………………………………………………………………………. 43 Specific aim 2………………………………………………………………………. 43 v Specific aim 3…………………………………………………………………………. 44 Significance.................................................................................................................... 44 InnOvatiOn ……………………………………………………………………………………… 46 References..................................................................................................................... 47 Chapter 3. Flipping the Switch to Accurately Diagnose Malaria: A Comparison of Clinical Diagnosis, Rapid Diagnostic Test, Thick-Film Microscopy and Polymerase Chain Reaction in the Diagnosis of Malaria in Cameroon………………………….. 48 Abstract......................................................................................................................... 50 IntrOductiOn.................................................................................................................... 51 Materials and MethOds.................................................................................................. 52 Results.......................................................................................................................... 55 DiscussiOn..................................................................................................................... 60 References.................................................................................................................... 64 Chapter 4. Infectious Causes of Febrile Illnesses in Cameroon…………………… 67 Abstract......................................................................................................................... 69 IntrOductiOn.................................................................................................................... 70 Materials and MethOds................................................................................................... 71 Results........................................................................................................................... 77 DiscussiOn...................................................................................................................... 82 References...................................................................................................................... 86 Chapter 5. Development of Microsphere Immunoassay Incoporating Recombinant Protein Antigens for Detection of Viral Hemorrhagic Fevers in Cameroon………. 89 Abstract........................................................................................................................... 91 IntrOductiOn...................................................................................................................... 92 Materials and Methods..................................................................................................... 94 vi Results............................................................................................................................. 98 DiscussiOn........................................................................................................................ 105 References......................................................................................................................
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