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Diagnosis and Epidemiology of Zoonotic Nontuberculous Mycobacteria Among Dromedary Camels and Household Members in Samburu County, Kenya

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Diagnosis and Epidemiology of Zoonotic Nontuberculous Mycobacteria Among Dromedary Camels and Household Members in Samburu County, Kenya DIAGNOSIS AND EPIDEMIOLOGY OF ZOONOTIC NONTUBERCULOUS MYCOBACTERIA AMONG DROMEDARY CAMELS AND HOUSEHOLD MEMBERS IN SAMBURU COUNTY, KENYA LUCAS LUVAI AZAALE ASAAVA (M. Sc.) I84/29435/2014 A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY (IMMUNOLOGY) IN THE SCHOOL OF PURE AND APPLIED SCIENCES OF KENYATTA UNIVERSITY SEPTEMBER, 2020 ii DECLARATION iii DEDICATION To my parents, family, relatives, and friends. iv ACKNOWLEDGEMENTS I sincerely acknowledge the mentorship, guidance and support from my supervisors Professor Michael M. Gicheru, Kenyatta University and, Dr. Willie A. Githui, Head, Tuberculosis Research laboratory, CRDR, KEMRI. My gratitude goes to the both of them for their guidance in shaping this study through their technical input from conception, fieldwork, laboratory analysis and preparation of this thesis. I wish to thank Kenyatta University for the tutorial fellowship and am greatly indebted to NACOSTI and NRF for the PhD research grant received without which this study would not have been possible. Special thanks to Dr. Willie Githui for accepting to host me at the TB Research Laboratory, CRDR, KEMRI and for material support without which this study would have been impossible. My sincere gratitude goes to Ernest Juma and Ruth Moraa, for guiding me through TB laboratory diagnostic techniques. Special thanks to Kenneth Waititu and Rose Kahurai of Institute of Primate Research- National Museums of Kenya, pathology laboratory for guiding me through histopathology. Special thanks to my field assistants Benedict Lesimir, Adan Halakhe, Leon Lesikoyo as well as the Samburu County veterinary team who were very resourceful. Special thanks to Moses Mwangi and Edwin Mwangi for guiding me through study design, data management and statistical analysis of my data. Special thanks to the Chairperson and staff of Zoological Sciences department for their support and for facilitating the smooth progress of this study. The Samburu pastoralists’ cooperation was key to the success of this work and my greatest gratitude goes to them. My sincere gratitude to my family and friends. Finally, thanks be to the Almighty God for blessing me with the endurance, discipline, meticulousness, thoroughness and inquisitiveness required throughout this work. v TABLE OF CONTENTS DECLARATION ....................................................................................................... ii DEDICATION ......................................................................................................... iii ACKNOWLEDGEMENTS ..................................................................................... iv TABLE OF CONTENTS .......................................................................................... v LIST OF TABLES .................................................................................................... ix LIST OF FIGURES .................................................................................................. xi DEFINITION OF OPERATIONAL TERMS ...................................................... xv ABSTRACT ............................................................................................................ xvi CHAPTER ONE: INTRODUCTION ...................................................................... 1 1.1 Background information ........................................................................................ 1 1.2 Statement of the problem ..................................................................................... 10 1.3 Justification of the study ...................................................................................... 12 1.4 Research questions .............................................................................................. 13 1.5 Hypotheses .......................................................................................................... 14 1.6 Objectives ............................................................................................................ 14 1.6.1 Broad objective ................................................................................................. 14 1.6.2 Specific objectives ............................................................................................ 14 1.7 Significance of study ........................................................................................... 15 CHAPTER TWO: LITERATURE REVIEW ...................................................... 16 2.1 Mycobacteria ....................................................................................................... 16 2.1.1 Zoonotic Mycobacteria .................................................................................... 20 2.1.2 Tuberculosis in camels ..................................................................................... 23 2.1.3 Epidemiology of zoonotic Mycobacteria ......................................................... 24 2.2 Pathophysiology and immunopathogenesis of Mycobacterial diseases .............. 27 2.3 Clinical signs of tuberculosis and other clinically important mycobacterioses .. 32 2.4 Molecular epidemiology of Mycobacteria of zoonotic importance .................... 34 2.5 Risk factors associated with zoonotic mycobacterial infection ........................... 37 2.6 Diagnosis of Mycobacteria infections ................................................................. 39 2.6.1 Tuberculin test .................................................................................................. 40 2.6.2 Culture .............................................................................................................. 42 2.6.3 Polymerase chain reaction ................................................................................ 43 2.6.4 Genotyping ....................................................................................................... 44 vi 2.6.5 Serology ............................................................................................................ 45 2.6.6 Human TB diagnosis in Kenya ......................................................................... 47 2.7 Control and treatment of mycobacterial diseases ................................................ 47 2.7.1 Treatment in man .............................................................................................. 49 2.7.2 Control of TB in livestock ................................................................................ 50 2.8 Drug resistance in Mycobacteria of zoonotic importance ................................... 51 2.9 Future perspectives and knowledge gaps addressed by the current study ........... 54 CHAPTER THREE: MATERIALS AND METHODS ....................................... 56 3.1 Study area ............................................................................................................ 56 3.2 Study design ........................................................................................................ 58 3.3 Inclusion criteria ................................................................................................. 60 3.4 Exclusion criteria ................................................................................................. 61 3.5 Sample size determination ................................................................................... 61 3.5.1 Manyatta camels sample size ........................................................................... 61 3.5.2 Slaughter house camel sample size .................................................................. 62 3.5.3 Household sample size ..................................................................................... 62 3.6 Definition of people presumed to have tuberculosis ........................................... 63 3.7 Data collection ..................................................................................................... 63 3.7.1 Tuberculin skin testing and collection of camel milk samples ......................... 63 3.7.2 Investigation of the pathology of Mycobacteria in slaughtered camels ........... 66 3.7.3 Human sputum samples .................................................................................... 68 3.7.4 Determination of risk factors ............................................................................ 69 3.8Clinical and laboratory methods ........................................................................... 70 3.8.1 Mycobacteriology ............................................................................................. 70 3.8.2 Histopathology ................................................................................................. 75 3.8.3 Immunochromatography to differentiate between MTBC and NTM .............. 76 3.8.4 GeneXpertMTB/RIF®diagnosis ....................................................................... 77 3.8.5 Mycobateria speciation using GenoType®Mycobacterium CM line probe assay ................................................................................................................. 77 3.8.6 Identification of unknown Mycobacteria using partial DNA sequencing of 16SrRNA gene .................................................................................................. 79 3.8.7 Drug sensitivity testing (DST) using resistant ratio (RR) method on LJ solid media ........................................................................................................ 81 3.9 Data handling and analysis .................................................................................
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