Epidemiology, Chemotherapy of Onchocerciasis and Development of Onchocerca-specific Polymerase Chain Reaction Based on a Pglycoprotein Gene
Epidemiology, Chemotherapy of Onchocerciasis and Development of Onchocerca-specific Polymerase Chain Reaction Based on a Pglycoprotein Gene.
ABSTRACT
Onchocerciasis is a disease caused by a filarial nematode Onchocerca volvulus, which is transmitted to man through the bite of infected black flies of the Simulium damnosum complex.
In Achi community (South Eastern Nigeria) – ivermectin treatments started since 1990 on a yearly basis under the auspices of a WHO/UNDP/WORLD BANK ONCHO. PROJECT.
During this study in Achi and Amansea, the participants medical history, onchocercomata and skin snips were obtained between January, 2009 to December, 2012 from the studied groups.
The studied population of 240 subjects was randomly selected thus: Achi (57), Amansea (52) and UNTH (131) (health seeking patients at Oncho. Project, University of Nigeria Teaching Hospital).
The studied groups were out patients of the WHO/UNDP/World Bank Oncho Project in the various villages. The patients were grouped into age, sex, occupation, residence, dosage levels, skin microfilariae and degrees of onchodermatitis.
The uterine contents of the female worms after ivermectin treatment of patients were qualitatively and quantitatively assessed using embryogram techniques.
TABLE OF CONTENTS
Title Page – – – – – – – – – – i
Certification Page – – – – – – – – – v
Dedication – – – – – – – – – – vi
Acknowledgements – – – – – – – – – vii
Table of Contents- – – – – – – – – – ix
List of Tables – – – – – – – – – – xii
List of Figures- – – – – – – – – – xiii
Abstract – – – – – – – – – – xvi
List of Abbreviations – – – – – – – – xviii
CHAPTER ONE. INTRODUCTION AND LITERATURE REVIEW – – 1
1.1 Introduction – – – – – – – – – 1
1.2 Justification of the Study – – – – – – – 6
1.3 Objectives of the Study – – – – – – – 8
1.4 Literature Review – – – – – – – – 9
1.4.1 The biology of the parasite,Onchocerca volvulus – – – – 9
1.4.2 Reproductive potential of O. volvulus- – – – – – 13
1.4.3 Vector biology – – – – – – – – 14
1.4.4 Vector ecology – – – – – – – 17
1.4.5 Onchocerciasis: Disease manifestation- – – – – – 21
1.4.5.1 Basic symptoms of onchocerciasis – – – – – – 21
1.4.5.2 Skin manifestations – – – – – – – 23
1.4.5.3 Ocular manifestations – – – – – – – – 24
1.4.5.4 Other manifestations – – – – – – – – 26
1.4.6 Geographical distribution and epidemiology – – – – – 27
1.4.6.1 Geographical distribution – – – – – – – 27
1.4.6.2 Epidemiology of onchocerciasis – – – – – – 31
1.4.7 Economic and public health importance – – – – – 33
1.4.8 Control of onchocerciasis – – – – – – – 37
1.4.8.1 History of early control methods – – – – – – 37
1.4.8.2 Antibiotic therapy – – – – – – – – 41
1.4.8.3 Treatment with ivermectin – – – – – – – 42
1.4.9 Possibility of resistance – – – – – – – 52
1.4.9.1 Molecular nature of resistance – – – – – – 55
CHAPTER TWO: MATERIALS AND METHODS – – – – 66
2.1 Description of the Study Area- – – – – – – 66
2.2 Collection of Study Materials- – – – – – – 71
2.3 Embryograms – – – – – – – – – 72
2.4 Molecular Biology Work; Search for MDR Genes – – – – 75
2.4.1 DNA extraction – – – – – – – – 75
2.4.2 PCRs – – – – – – – – – 76
2.4.3 Cloning and sequencing of the O.volvulus MDR genes – – – 76
2.4.4 Computer analysis – – – – – – – – 77
2.4.5 Designing of O. volvulus mdr primers (P11 – P12) – – – – 78
2.5 Protein Analysis: Search for the P-glycoprotein in O.volvulus – – 78
2.5.1 Preparation of protein antigens (OvAg/OvP) – – – – – 78
2.5.2 Separation of O.volvulus worms antigens (SDS-PAGE) – – – 79
2.5.3 Staining of the SDS-GELS to visualize the separated proteins – – 82
2.5.4 Western blotting and immunoblotting- – – – – – 83
2.6 Statistical Analysis – – – — – – – 86
2.7 Classification of Worms Samples used for the Study- – – – 86
CHAPTER THREE: RESULTS – – – – – – – 96
3.1. Prevalence of Onchocerciasis in three Different Communities- – – 96
3.1.1 Clinical manifestations of onchocerciasis – – – – – 96
3.1.2 Variation in content of nodules in Achi and Amansea – – – 100
3.1.3 Influence of occupation on the prevalence of onchocerciasis – – 102
3.1.4 Microfilaridemia level of ivermectin treated patients (possibility of resistance) 104
3.2 Embryonic Status of O. volvulus Female worms – – – – 107
3.3 Molecular Biology Results – – – – – – – 110
3.3.1 Isolation of the O. volvulus PGP genes- – – – – – 110
3.3.2 The O. volvulus PGP sequences – – – – – – 117
3.4 Protein Analysis and Immunoblotting – – – – – 130
3.4.1 SDS-PAGE Analysis – – – – – – – – 130
3.4.2 Western blotting and Immunoblotting- – – – – – 133
CHAPTER FOUR: DISCUSSION – – – – – 137
4.1 Field Study – – – – – – – – – 137
4.2 Molecular Biology Work – – – – – – – 145
4.3 Conclusion – – – – – – – – – 150
4.4 Future Studies- – – – – – – – – 151
REFERENCES – – – – – – 153
APPENDIX – – – – – – – – – 200
INTRODUCTION
Onchocerciasis is the disease resulting from infection with the filarial nematode Onchocerca volvulus, which is transmitted to man through the bite of the infected black flies belonging to the genus, Simulium (Family: Simuliidae) (WHO, 1991, Okuliez, 2008).
The global distribution of onchocerciasis includes endemic areas in tropical Africa, where 99% of the total infected persons are found, Central and South America (WHO, 1987).
It is estimated that 85.5 million people are at risk and that 17.7 million people are infected with O. Volvulus (WHO, 1995). Onchocerciasis is one of the leading causes of loss of sight in the world, responsible for about 270,000 blind and 500 000 partly sighted people (WHO, 1995).
The current global burden of the disease showed that an estimated 123 million persons were at risk of contracting the disease and 17 – 18 million were infected (WHO, 1999; Hoerauf et al., 2003; Boatin and Richrads, 2006).
According to IJPD (2009), more than 30 million Nigerians in 32 states and the Federal Capital Territory are estimated to be at risk for onchocerciasis.
About 360,000 people are projected to be blind (Abiose et al., 1993). The disease is present in all the states including the Federal Capital territory with the exception of Lagos, Rivers, and Akwa–Ibom states where the infections are sporadic. The vectors breed along fast-flowing rivers where infection intensity and morbidity are highest.
Onchocerciasis is also known colloquially as river blindness (WHO, 1985). The manifestations of onchocerciasis are predominantly dermal, lymphatic and ocular in character (Mackenzie et al., 1995),
But, several other features of uncertain association, etiology or pathogenesis have also been described, including low body weight, general debility, diffuse musculoskeletal pain and, in Africa, epilepsy and hyposexual dwarfism (WHO, 1987a; Nwoke, 1992; Kipp et al., 1994; Shu and Okonkwo, 1998).
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CSN Team.
