University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES PHENOTYPIC AND GENOTYPIC ANALYSIS OF ONCHOCERCA VOLVULUS RESPONSE TO IVERMECTIN TREATMENT JUDITH ESINAM YABANI DEPARTMENT OF MEDICAL MICROBIOLOGY SCHOOL OF BIOMEDICAL AND ALLIED HEALTH SCIENCES JULY, 2017 1 University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES PHENOTYPIC AND GENOTYPIC ANALYSIS OF ONCHOCERCA VOLVULUS RESPONSE TO IVERMECTIN TREATMENT BY YABANI, JUDITH ESINAM (10507341) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN MEDICAL MICROBIOLOGY DEPARTMENT OF MEDICAL MICROBIOLOGY SCHOOL OF BIOMEDICAL AND ALLIED HEALTH SCIENCES JULY, 2017 2 University of Ghana http://ugspace.ug.edu.gh DECLARATION i University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to the Almighty God, my parents and siblings. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT This work would not have been possible without the grace of God; therefore my utmost thanks go to Him for his strength, knowledge and protection throughout my three years of academic work. I am very grateful to my supervisors, Dr. Simon K. Attah and Rev. Prof. Patrick F. Ayeh-Kumi for their immense supervision and guidance for the successful completion of this research. I wish to express my heartfelt gratitude to Dr. Mike Osei-Atweneboana of the Water Research Institute (WRI), the Council for Scientific and Industrial Research Institute (CSIR) for his guidance, unflinching support, and for making this project a reality by providing the necessary logistics from his laboratory and research grant for the entire work. I am thankful to the entire staff of the Biomedical and Public Health Research Unit of the Water Research Institute, CSIR for their assistance and encouragements in making this research a success. My unreserved appreciations go to the lecturers, technicians and secretaries of the Medical Microbiology Department, School of Biomedical and Allied Health Sciences for the support they provided in the successful completion of this work. Lastly, my gratitude and sincere thanks go to my parents, Mr. and Mrs. E.K.G Yabani and my siblings for their unceasing encouragement, spiritual and financial support. iii University of Ghana http://ugspace.ug.edu.gh ABSTRACT Ivermectin remains the only potent drug for the control and mass treatment of onchocerciasis. Nonetheless, recent studies indicate sub-optimal responses and genetic changes in some populations of the adult Onchocerca volvulus. Hence, more studies are required to determine whether resistance is developing. This requires analysis of phenotypic and genotypic responses of O. volvulus to ivermectin treatment to determine the association between worm phenotype and genotype after treatment. Forty archived O. volvulus female worms were obtained from patients hailing from three Ghanaian endemic communities: Asubende, Kyingakrom and Agborlekame. These communities were known to harbour both good and poor ivermectin response groups. The female worms were retrieved from nodules removed from patients who previously had undergone 16-17 rounds of (150 µg/kg) ivermectin treatment with one year interval between treatments. The samples were obtained 3 months after the last round of treatment. Genetic analysis of the beta-tubulin gene of the adult worms and their pooled microfilariae (MF) on a 538 bp DNA fragment of the beta-tubulin gene as well as restriction was done to determine mutations in the beta-tubulin gene. It was detected that 70% (28/40) of the worms harboured greater than 70% of normal stretched MF in their genital tracts; these were considered as poor responders to ivermectin treatment. On the other hand, 30% (12/40) of the worms that harboured less than 10% of the stretched MF in their genital tracts were considered as good responders to ivermectin treatment. Molecular analysis indicated single nucleotide polymorphism (SNP) for both adult worm and their corresponding MF with the heterozygote mutant adult worms showing strong association with the MF. In conclusion, it is found in this study that some of the worms have responded poorly or sub-optimally to ivermectin treatment. This development stems from iv University of Ghana http://ugspace.ug.edu.gh mutations in the beta-tubulin gene of the adult female worms that have been passed on to their progenies. v University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS ATP Adenosine Triphosphate APOC African Programme for Onchocerciasis Control CDC Centers for Disease Control and Prevention CDTi Community Directed Treatment with Ivermectin DALY Disability-Adjusted Life Years DEC Diethylcarbamazine DNA Deoxyribonucleic acid dNTP Deoxynucleotide Triphosphate GDP Guanosine Di-Phosphate GTP Guanosine Tri-Phosphate L1s First stage Larvae of O. volvulus L3s Third stage Larvae of O. volvulus MDA Mass Drug Administration MF Microfilariae ML Macrocyclic lactone OCP Onchocerciasis Control Programme OEPA Onchocerciasis Elimination Programme for the Americas OSD Onchocerciasis Skin Disease PCR Polymerase Chain Reaction Pgp Permeability glycoprotein PM Peritrophic matrix RFLP Restriction Fragment Length Polymorphism vi University of Ghana http://ugspace.ug.edu.gh RPM Revolution per minute SNP Single Nucleotide Polymorphism SOR Sub-Optimal Response UV Ultra-violet WHO World Health Organization vii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT CONTENT PAGES DECLARATION i DEDICATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABBREVIATION v TABLE OF CONTENT vi CHAPTER ONE 1.0 INTRODUCTION 1.1 General Introduction 1 1.2 Problem Statement 3 1.3 Justification 3 1.4 Aim 4 1.5 Specific Objectives 4 CHAPTER TWO 2.0 LITERATURE REVIEW 5 2.1 The Parasite 5 2.2 Morphology of Onchocerca volvulus 5 2.2.2 Microfilariae 6 2.3 Life cycle of Onchocerca volvulus 7 viii University of Ghana http://ugspace.ug.edu.gh 2.4 The disease 8 2.5 Diagnosis 9 2.6 Socioeconomic consequences 10 2.7 Treatment 10 2.7.2 Suramin 11 2.7.3 Ivermectin 11 2.7.3.1 Pharmacodynamics and mode of action of ivermectin 12 2.7.3.2 Contraindications, adverse effects and precautions 12 2.7.4 Doxycycline 13 2.8.2 African Programme for Onchocerciasis Control (APOC) 14 2.8.3 Onchocerciasis Elimination Programme for the Americas (OEPA) 14 2.9 Onchocerciasis in Ghana 15 2.10 Onchocerca volvulus response to ivermectin treatment 16 2.11 Genes associated with ivermectin resistance in humans and helminthes 18 2.12 Tubulin gene and its function in drugs 18 2.13 Genetic responses of Onchocerca volvulus to ivermectin treatment 20 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study design 22 3.2 Study area and population 22 3.3 Inclusion criteria 23 3.4 Ethical issues 23 ix 538bp p University of Ghana http://ugspace.ug.edu.gh 3.6 Categorization of Onchocerca volvulus based on response to ivermectin treatment 24 3.7 Embryogramme 24 3.8 Extraction of the deoxyribonucleic acid (DNA) from beta-tubulin gene 25 3.9 Amplification 26 3.10 Agarose gel electrophoresis 26 3.11 Restriction Fragment Length Polymorphism (RFLP) 26 3.12 Statistical analysis 27 CHAPTER FOUR 4.0 RESULTS 4.1 Embryogramme 28 4.2 Amplification of adult female worm beta-tubulin gene 29 4.3 PCR-RFLP for adult female worms 30 4.4 Amplification of microfilariae beta-tubulin gene 31 CHAPTER FIVE 5.0 DISCUSSION 5.1 Embryogramme 34 5.2 Genetic changes in beta-tubulin and SOR 34 5.3 Genetic changes in beta-tubulin of adult worms and microfilariae 35 5.4 Conclusion 36 5.5 Recommendations 36 x University of Ghana http://ugspace.ug.edu.gh REFERENCES 37 APPENDICES 47 APPENDIX 1: Embryogramme result 47 APPENDIX 2: Certificate of approval 48 xi University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES FIGURES PAGES Figure 2.1: Adult males and females entwine 6 Figure 2.2: Microfilaria of Onchocerca volvulus 7 Figure 2.3: Life cycle of O. volvulus 8 Figure 2.4: Ribbon diagram of the tubulin dimer 9 Figure.3.1: A map of the study sites 23 Figure 4.1: Stretched normal microfilariae from a poor ivermectin responding subject 29 Figure 4.2: Abnormal microfilariae from a good ivermectin responding subject 29 Figure. 4.3: Image of agarose gels showing adult DNA bands of beta- tubulin gene amplicons 30 Figure.4.4: Agarose gel showing restriction bands for adult female worms 31 Figure. 4.5: Image of agarose gels showing microfilariae DNA bands of beta-tubulin gene amplicons. 32 Figure. 4.6: Image of agarose gel showing restriction bands for microfilariae 33 xii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES TABLES PAGES Table 4.1: Distribution of good and poor response female worms from subjects in three communities 28 xiii University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 General Introduction Onchocerciasis or “river blindness’’ is an infectious disease caused by the parasitic filarial nematode Onchocerca volvulus. This parasite exists in two main forms, the adult (male or female) worms and the skin-dwelling embryonic larvae called microfilariae (MF). The disease is a global burden and geographically, it is distributed in Africa, South and Central America and Yemen (MMWR, 2013). It is estimated that 37 million people are infected globally with the parasite out of which 99% live in Africa. Over 90 million people are at risk of acquiring the infection in 29 Sub-Sahara African countries. Farmers, hunters, fishermen and people who reside close to Simulium breeding sites are the risk population. Studies have shown that adult males are highly infected than females and children (CDC, 2013). The third stage infective larvae (L3s) of the parasite are transmitted into the skin of the human host when an infected Simulium fly bites to ingest blood. The L3s penetrate the bite wound and develop into adult males and females in the subcutaneous tissue where they mate and live up to about fifteen years (or an average of twelve years). The gravid female worm produces about 1,000 MF daily. These MF which can live for two years are responsible for most of the morbidity associated with the disease (CDC, 2013). Among infection related diseases that result in blindness, onchocerciasis is rated as the second leading cause of this disability after trachoma. It is also estimated that onchocercal skin disease (OSD) which causes troublesome itching accounts for 60% of healthy life-years lost annually due to the disability and mortality associated with the disease (Remme et al., 2002). Other effects of O. volvulus infection are epilepsy due to heavy infection, social ostracism, 1 University of Ghana http://ugspace.ug.edu.gh troublesome itching associated with disfiguring skin lesions which makes it difficult for one to work, study and interact socially, reduced life expectancy rate among infected individuals with high MF load, high mortality rate among the blind and reduced socioeconomic status of affected communities as a result of low productivity and abandonment of fertile river valleys (Prost 1986; Vlassof et al., 2000; Murdoch et al., 2002; Little et al., 2004; Pion et al., 2009). Other negative impacts associated with the infected individuals are increased health expenditure and diminishing income generating capacity (Pion et al., 2002). Ocular onchocerciasis is diagnosed through an ophthalmological examination of the visual function of the eye, presence of MF in the intraocular eye and other pathological changes related to the infection. Microscopy is used to detect the MF from skin biopsies, whiles the adult worms are detected after surgical removal of nodules from the body followed by their digestion with collagenase to expose the adult worms or by histological processing of the nodules (WHO, 1995). According to Zimmerman et al. (1994), DNA probes through polymerase chain reaction (PCR) have been developed to determine the presence of the Onchocerca parasite in routine skin snips. In order to control the disease, some strategies were developed and implemented. These include the establishment of the Onchocerciasis Control Programme (OCP), African Programme for Onchocerciasis Control (APOC) and the Onchocerciasis Elimination Programme for the Americas (OEPA). These programmes were tasked to control the disease by reducing its burden to a level where it will no more be of public health importance. According to the previous studies, ivermectin is the only safe drug recommended for the treatment and control of the O. volvulus infection and the only drug proven to be safe for the treatment of the disease (WHO, 2017). 2 University of Ghana http://ugspace.ug.edu.gh 1.2 Problem Statement There are reports of sub-optimal response (SOR) of O. volvulus to ivermectin treatment and this has been associated with mutations in the beta-tubulin gene of some female worms. Reports indicate that there is SOR in some endemic communities in Ghana (Awadzi et al., 2004; Osei-Atweneboana et al., 2007; Basáñez et al., 2008; Pion et al., 2013). It is not clear if the worms from the study sites have mutations in the beta-tubulin gene. Confirmation is required for the findings from these studies and the current study was carried out to do that. The apparent emergence of resistance of O. volvulus worms in some endemic communities to ivermectin may be attributed to the prolonged reliance of ivermectin for mass drug administration (Taylor et al., 2009). This situation is critical to the control of O. volvulus and poses a significant threat to the fight against onchocerciasis and a high probability of disruption of the success of the onchocerciasis control programme (OCP) and the African programme for onchocerciasis control (APOC). It is therefore imperative to ascertain if there is any emerging resistance in the endemic communities where the samples were collected from for this study. 1.3 Justification Ivermectin is the main drug being used for onchocerciasis control. Therefore, the development of resistance in the MF will throw the control of onchocerciasis out of gear. Early detection of resistance in the progenies of the adult worm will enable the deployment and integration of new management tools in the fight against the disease. 3 University of Ghana http://ugspace.ug.edu.gh 1.4 Aim The aim of this study was to analyze the phenotypic and genotypic responses of O. volvulus to ivermectin treatment. 1.5 Specific Objectives The specific objectives of the study were to determine:  the effect of ivermectin on the adult female worms and their progenies particularly, the intrauterine MF,  if the genetic changes in the beta-tubulin of the female O. volvulus is associated with SOR to ivermectin treatment, and  if these genetic changes in the beta-tubulin have been passed on to the progenies of the female worm. 4 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 The Parasite Onchocerca volvulus is a filarial parasitic worm that belongs to the Superfamily Filarioidea. It is an obligate parasite that causes the disease, onchocerciasis. It exists as adult (macrofilariae) or microfilariae (Udall, 2007). The macrofilariae are found in the subcutaneous tissues entangled in painless, firm and usually palpable nodules called onchocercomata. The MF is found mainly in the skin although they can be found in other body specimens such as blood, urine and sputum when their numbers are high in the body (CDC, 2013). 2.2 Morphology of Onchocerca volvulus 2.2.1 Macrofilariae The macrofilariae comprise both adult male and female worms that are normally intertwined in the nodule. Both sexes are whitish in colour which changes to brown with maturity. Each worm has a blunt head, and a tapering posterior end or tail. The females are larger in size than the males and measure 33 to 50 cm by 270 to 400 µm with narrow anterior ends. The reproductive system of the females consists of paired ovaries, oviducts, seminal receptacles and uteri. Male worms measure 19 to 42 mm by 130 to 210 µm. They have a reproductive system that consists of a straight tube which runs through them (CDC, 2013). 5 University of Ghana http://ugspace.ug.edu.gh Figure 2.1: Adult males and females entwine; Source: Schulz-Key, 1988. 2.2.2 Microfilariae The MF measure 220 to 360 µm by 5 to 9 µm and is unsheathed. They have a blunt anterior portion and a tapering posterior end as shown in figure 2.2 below. 6 University of Ghana http://ugspace.ug.edu.gh Figure 2.2: Microfilaria of Onchocerca volvulus (arrow); Source: Original 2.3 Life cycle of Onchocerca volvulus The parasite has an indirect life cycle in which the blackfly (vector) serves as the intermediate host and humans serve as the definite host. An individual becomes infected when the L3s of the parasite are inoculated into the skin of humans after the blackfly takes a blood meal. The L3s, after two years, develop into adult worms which permanently reside in the subcutaneous tissues and live for almost 15 years. The adult male and female then mate and produce several MF that move to the skin, eyes and other organs of the body. The female worms reside in the nodules and produce the MF for approximately 9 years. The MF has a life span of two years (CDC, 2013). The transmission cycle continues when the female blackfly ingests the MF in the course of taking another blood meal. The blackfly ingests several MF during a single bite most of which are engulfed and digested in the peritrophic matrix (PM) secreted by the fly midgut. Only a few 7 University of Ghana http://ugspace.ug.edu.gh MF penetrate the PM into the stomach wall and further invade the epithelial layer of the stomach into the abdominal and thoracic cavities (CDC, 2013). The MF develops into the sausage-like first stage larvae (L1s) and later develops into L3s which measures up to 650 µm in length. The L3s then migrate to the blackfly’s proboscis to infect another human when the fly ingests a blood meal. Figure 2.3: Life cycle of O. volvulus (Accessed from: http://www.cdc.gov) 2.4 The disease 2.4.1 Clinical presentations The MF are the primary cause for the morbidity accompanied with the disease. When the MF die, they cause intense inflammatory reactions in the skin leading to skin rashes, lesions, and 8 University of Ghana http://ugspace.ug.edu.gh intense itching (pruritis). Inflammatory lesions or onchodermatitis appear as small, round red bumps on the skin. Prolonged scratching of the affected parts destroys the elastic fibres of the skin and causes the skin to appear thin and wrinkled, a condition referred to as like a lizard skin. Skin depigmentation in the affected area can be found in later stages of the infection and it is known as leopard skin. In other conditions, the skin may become thickened and it is referred to as elephant skin. Another uncommon complication associated with onchocerciasis is lymphadenitis which sometimes leads to hanging groins and elephantiasis of the genitals as well as epilepsy and hyposexual dwarfism. In persons with worm burden, the MF migrates to the eyes, die and release an endosymbiotc bacterium known as Wolbachia. The death of this bacterium causes the release of toxins which causes inflammatory responses that lead to the beginning of impaired vision and finally blindness (WHO, 1995; Brattig, 2004; Higazi et al., 2005). Some of the effects of O. volvulus infection are severe itching, blindness, epilepsy due to heavy infection, high mortality rate in the blind which leads to a short life expectancy rate and abandonment of fertile river valleys (Prost, 1986 ; Pion et al., 2002; Little et al., 2004). 2.5 Diagnosis There are different methods of diagnosing onchocerciasis, but the gold standard is the skin snipping. This method involves the use of a sclerocorneal punch, scapel or blade to obtain skin biopsy from the iliac crests, scapular and calves. The skin snips are placed in a 96-well plate containing about 100 µl of physiological saline (pH 7.0) and incubated to allow the MF to emerge from the skin snip over a 24 hour period. The MF is enumerated with the aid of an inverted microscope, or an ordinary light microscope. Skin snip microscopy is not a sensitive 9 University of Ghana http://ugspace.ug.edu.gh diagnostic tool in areas with low infection prevalence as a result of either low transmission rate or successful ivermectin treatment programmes. Nodule examination can be done by palpation of affected skin areas but this technique cannot be used to determine infectivity since some nodules are located in deep muscles, with the most active and very small nodules usually very difficult to palpate (Thylefors, 1978; Duke, 1993). Recent biochemical methods such as skin snip PCR, enzyme linked immunosorbent assays (ELISA), enzyme immunosorbent assay (EIAs) and antigen detection have been extensively used to develop more sensitive and reliable means of infection diagnosis (Ngomou, 1994; Alley, 2001). There are other methods such as: ultrasonography, histology and collagenase digestion of nodule, slit lamp microscopy for detecting MF and Mazzotti test (WHO, 1995). 2.6 Socioeconomic consequences Among people with onchocercal skin disease (OSD), males are faced with challenges related to their ability to achieve economic prospects and their sexual performances whiles females are challenged with the probability of getting married due to their disfigured physical appearances as a result of OSD (Vlassoff et al., 2000). Also, people with OSD have low income (Oladepo et al., 1997) and they suffer stigmatization which affects them in all facets of their lives (Mbanefo et al., 2010). 2.7 Treatment 2.7.1 Diethylcarbamazine The World Health Organization (WHO) introduced diethylcarbamazine (DEC), a microfilaricide for the treatment of the disease after the Onchocerciasis Control Programme 10 University of Ghana http://ugspace.ug.edu.gh (OCP) was launched in West Africa with the aim of eliminating the disease. However, it was realized that, treatment of the disease with this drug had intrinsic toxic side effects and could not be used for mass treatment ( Francis et al., 1985; Greene et al.,1985 ; Awadzi and Gilles, 1992). 2.7.2 Suramin Suramin is a macrofilaricide that was later introduced for the treatment of onchocerciasis but it also has little effect on the MF. It is not safe (Thylefors and Rolland, 1979) so, it is recommended to be administered to patients under the supervision of an experienced physician. 2.7.3 Ivermectin Ivermectin, a semisynthetic drug with a broad spectrum antiparasitic activity, belongs to the avermectin family. It was isolated from the fermentation products of Streptomyces avermitilis. It contains a mixture of 5-O-dimethyl-22, 23-dihydroavermectin A1a and 10% 5-O-dimethyl- 25-de (1-methylpropyl)-22, 23-dihydro-25-(1-methylethyl) avermectin A1a, with molecular weights of 875.10 and 861.07 respectively (Merck & Co, 2007). This is a potent drug for veterinary animals, which was introduced in 1987 for the treatment of human onchocerciasis. It was soon added as an adjunct to vector control after it was donated free of charge by its manufacturer (Merck & Co, 2007) to the OCP for as long as needed. Ivermectin is not a macrofilaricde. Studies have indicated that administering it annually is very safe, prevents onchocerciasis and onchodermatitis, and has an enormous impact on reducing the transmission of the disease (Greene et al., 1985; Remme, 2004). 11 University of Ghana http://ugspace.ug.edu.gh Ivermectin is widely used against both endoparasites and ectoparasites. In human medicine, ivermectin is used in dermatology against arthropods such as human head and body lice (Strycharz et al., 2008) as well as scabies (Dourmishev et al., 2005; Khan & Yasmin, 2007). An analysis has shown that oral ivermectin can be considered as an alternate method of treating severely crustered scabies lesions in immunocompromised patients compared to topical treatment which is expensive and less effective (Fawcett, 2003). A recent study has shown the success of ivermectin as a parenteral veterinary formulation in treating disseminated strongyloidiasis (Marty et al., 2005). Additionally, it has been shown that topical ivermectin is of great importance in treating worm larvae infestation (myiasis) (Dourmishev et al., 2005) and cutaneous larval migrans (Caumes et al., 1993; Bouchaud et al., 2000). Also, ivermectin is used in treating other filarial infections (Bockarie et al., 1998; Boussinesq et al., 1998 ; TCC, 2013). 2.7.3.1 Pharmacodynamics and mode of action of ivermectin The avermectin and milbemycin exhibit two basic effects on nematodes; the inability to pump food into the pharynx and paralysis. Ivermectin has high affinity for and binds with glutamate gated chloride ion channels (GluCl) found in the cells and muscles of nematodes. When ivermectin binds with GluCl, it irreversibly activates GluCl receptors which depolarize the pharyngeal muscle leading to a high level of internal chloride ions. The presence of GluCl on the pharyngeal muscle prevents the pharynx from pumping food to the worm and starves it to death (Pemberton, 2001). Secondly, ivermectin inhibits neuromuscular transmission and paralyzes the worm’s body wall muscle (Wolstenholme and Rogers, 2005). 12 University of Ghana http://ugspace.ug.edu.gh 2.7.3.2 Contraindications, adverse effects and precautions Itching, ocular irritation reactions, headache, arthralgia, myalgia, lymphadenopathy, fever and oedema can occur. In patients with co-infection of Loa loa and O. volvulus, severe reactions such as encephalopathy occur if the MF of Loa loa exceeds 30,000 MF/ ml, and in case of more than 8,000 MF/ml functional impairment can occur. Thus, the drug is to be administered with caution especially in communities where Loa loa with O. volvulus is co-endemic. Pregnant women and children below 15 kg body weight are excluded in ivermectin treatment since safety is not guaranteed (WHO, 2013). 2.7.4 Doxycycline One of the several drugs currently under consideration is doxycycline. Reports indicate that doxycycline has a sterilizing effect on the macrofilariae and clears all developmental stages as well as the Wolbachia and had no adverse effect on treated subjects (Turner et al., 2010). 2.8 Control of Onchocerciasis 2.8.1 Onchocerciasis Control Programme (OCP) The Onchocerciasis Control Programme (OCP) was launched in West Africa in 1974 as a collaborative initiative of four United Nations (UN) agencies namely: WHO, United Nations Development Programme (UNDP), the World Bank and the Food and Agriculture Organization of the United Nations (FAO). The OCP employed the strategy of spraying the breeding sites of the blackfly vector in fast flowing rivers and streams to kill its larval stages with environmentally friendly insecticides over a period of three decades. The onset of 13 University of Ghana http://ugspace.ug.edu.gh ivermectin for the treatment of human populations changed the principle of control from the sole reliance on larviciding to a combination of both vector control and parasite control. The OCP successfully controlled the disease as a public health burden in its area of operation. Other achievements of the OCP include the freeing of 18 million children who have at the risk of blindness, the prevention of 600,000 people from becoming blind and reclamation of 250, 2 000 km of abandoned (Harlem, 2002; Hopkins, 2005). 2.8.2 African Programme for Onchocerciasis Control (APOC) The African Programme for Onchocerciasis Control (APOC) was launched in 1995 as a sole antiparasitic agency with the main aim of consolidating and extending the OCP gains to 19 other endemic African countries which did not benefit from vector control by the OCP. The APOC adapted the strategy of community directed treatment with ivermectin (CDTi) and was successful in the various communities that it operated within (Seketeli et al., 2002). After a successful significant reduction of infection was observed in communities that had taken the medication for more than 15 years (Borsboom et al., 2003), it was predicted that, the disease will not be a public health problem for more than 20 years if the treatment is suspended (Plaisier et al., 1995). It was deduced that, if CDTi is used to cover 70% of endemic areas, and 80% of these areas are able to maintain annual treatment at 65% coverage for a period of less than 15 years, 26 million DALYs would be prevented over a period of 25 years. 2.8.3 Onchocerciasis Elimination Programme for the Americas (OEPA) The Onchocerciasis Elimination Control Programme for the Americas (OEPA) was launched with the intension of eradicating onchocerciasis in the Americas after several years of control 14 University of Ghana http://ugspace.ug.edu.gh using biannual treatment regimen. The transmission of the disease was interrupted in four of the six endemic countries in the Latin America by the end of 2012. The American programme used bi-annually ivermectin Mass Drug Administration (MDA) coupled with health education and mobilization of affected communities in endemic foci in the six affected countries (Blanks et al., 1998; Sauerbrey, 2008). The endemic communities targeted for MDA were classified into three categories namely; hyperendemic, mesoendemic and hypoendemic. After the MDA which was carried out from 1993 to 2012, the OEPA was found to be successful in interrupting transmission in 11 out of 13 foci (CDC, 2013). Transmission was found to have been interrupted in Colombia and Ecuador in 2007 and 2009 respectively and in Mexico and Guatemala in 2011. Colombia, Ecuador and Guatemala were declared free of onchocerciasis in 2013, 2014 and 2015 respectively. Effort to eliminate the disease is now focused on the Yanomami of Brazil and Venezuela (WHO, 2017). 2.9 Onchocerciasis in Ghana A study report revealed that onchocerciasis is endemic in nine out of the ten regions of Ghana; the exception being the Greater Accra region (Taylor et al., 2009). It was realized that, there are 3,204 endemic communities from 66 districts in the 9 regions. Of these 3,204 communities, 247 are hyperendemic areas and have been described as a special intervention zone. The findings from the entomological and epidemiological surveillance activities carried out by the APOC indicated the necessity to improve monitoring for fly infectivity levels and infection in humans. Surveys done in 2005 and 2006 showed high infectivity rates of parous flies from the Pru river basin sites and in 2006 within the White Volta, Kulpawn, Anum and Pra river basins (Taylor et al., 2009). In co-endemic communities with lymphatic filariasis and onchocerciasis, 15 University of Ghana http://ugspace.ug.edu.gh ivermectin treatment were introduced in 2001 and by 2005, 61 endemic communities were covered. 2.10 Onchocerca volvulus response to ivermectin treatment 2.10.1 Phenotypic responses A longitudinal follow-up study was carried out by Klager et al. (1996) to assess O. volvulus nodules from 77 patients in Sierra Leone in a randomized controlled trial. The patients received either 4 annual or 10 monthly doses of ivermectin for more than 6 years. Nine (9) months after the last ivermectin treatments, excised worms from these patients were examined for changes in morphology, viability and reproducibility. The findings indicated a high reduction in the proportion of live male worms in the nodules, and 90% reduction in reproduction in female worms. Turner et al. (2010), in a double blinded randomized field trial study, administered 200 mg/day of doxycycline only and doxycycline in combination with ivermectin (150 µg/kg) to 150 Onchocerca infected patients. Twenty two (22) individuals co-infected with Loa loa and O. volvulus were given the same treatments. The effects of the medications were determined at 4, 12 and 21 months after treatment. It was realized that at the end of the 12 months, doxycycline and ivermectin treated individuals had lower microfilaridermia compared with doxycycline or ivermectin treated individuals only. In order to determine emerging ivermectin resistance in O. volvulus, Osei-Atweneboana and colleagues (2011) conducted a follow up study on 268 microfilaridermia subjects in 9 Ghanaian communities who were previously administered with 10 to 19 annual doses of ivermectin treatment and an ivermectin naïve community. Skin snips were done 364 days after 16 University of Ghana http://ugspace.ug.edu.gh the initial ivermectin treatment. Skin snips and nodules were taken 90 days after the second ivermectin treatment, to determine the morphological age and MF contents. It was detected that 90% of the worms were classified as middle aged or older. Additionally, the study results also indicated SOR in some adult female worms (Osei-Atweneboana et al., 2011). Katabarwa et al. (2013a) assessed the transmission of onchocerciasis and its effect in three health districts in the Northern part of Cameroon, where mass ivermectin treatment had been going on (on annual basis) for 12 to 17 years. In these two year studies, skin snips were assessed for MF, nodules were palpated and MF in the eye was detected using a slit lamp microscopy and vector flies were dissected for the determination of larva rates. The findings revealed that adult subjects had Mf rates of 4.8%, nodule rates of 13.6% and 5.5% of patients had intra ocular MF. The study also revealed significant evidence of ongoing transmission in one of the health districts with 2.6% prevalence rates for children below 10 years of age. An onchocerciasis transmission study was carried out in West Cameroon where annual mass ivermectin treatment has been going on for 12 to 17 years by Katabarwa and colleagues (2013b). There was a follow up in 2005, 2006 and 2011 to assess MF load in skin snips and palpation examination for nodules. The Simulium vector species were dissected to determine larval infection rate from 2011 to 2012. The study results revealed a reduction in MF prevalence in adults from 68.7% to 11.5%; similarly, nodule prevalence dropped from 65.9% to 12.1%. It was also detected that only 3 out of the 11 districts studied were close to interrupting the transmission. There is evidence that transmission is still on going in two of the three fly infested sites where the flies were collected. In view of this, Katabarwa et al. (2013b) recommended that annual mass drug administration with ivermectin should proceed after 15 years, since discontinuing the treatment will lead to the increase in transmission rate. 17 University of Ghana http://ugspace.ug.edu.gh 2.11 Genes associated with ivermectin resistance in humans and helminthes The high rates at which anthelmintics are used in humans and animals are likely to result in selection for resistance. There is strong evidence that resistance is occurring in ivermectin treatment. Ivermectin resistance in veterinary parasites has been genetically associated with permeability glycoproteins (P-gp) and the beta-tubulin gene (Taylor et al., 2009). There is evidence that, ivermectin resistance has occurred in the P-gp gene of the veterinary parasites, Haemonchus contortus (Xu et al., 1998) and Caenorhabditis elegans (James & Davey, 2009). Recent studies have associated ivermectin resistance with the beta-tubulin gene of O. volvulus (Eng & Prichard 2005; Eng et al., 2006; Bourguinat et al., 2007; Osei-Atweneboana et al., 2007; Tawiah-Gyan, 2013). 2.12 Tubulin gene and its function in drugs 2.12.1 Beta-tubulin gene and its role in ivermectin resistance Tubulin is a member of the protein superfamily of the globular protein. There are six families of tubulins in this superfamily known as alpha, beta, gamma, delta, zeta and epsilon. Gamma (γ) plays crucial role in the nucleation and polar orientation of microtubules. The delta (δ) and epsilon (ε) tubulins function in the mitotic spindle during mitosis found in centrioles. Zeta is found in kinetoplastid protozoa. The alpha (α) and beta (β) tubulins polymerize into dynamic microtubules. Both are slightly acidic and have an isoelectric point between 5.2 and 5.8 with a molecular weight of about 50,000 Daltons. Microtubules are components of cytoskeleton and are made of basic structural unit of alpha-beta tubulin heterodimer. Microtubules can grow as long as 50 micrometer and are very dynamic in eukaryotic cells and in some bacteria (Pilhofer 18 University of Ghana http://ugspace.ug.edu.gh et al., 2011). They play very crucial functions in structural support, cytokinesis, motility and transport of cellular structures around cells. In the Guanosine triphosphate (GTP) bound state, the α- and β-tubulin dimers bind to GTP and gather at the positive ends of microtubules (Heald & Nogales, 2002). The β-tubulin subunit is exposed on the plus end while the α-tubulin subunit is exposed at the minus end of the microtubule. After the incorporation of the dimer in the microtubule, the GTP bound state of the β-tubulin subunit hydrolyses into Guanosine diphosphate (GDP) through interdimer contacts along the microtubule protofilaments (Heald & Nogales, 2002). The stability of the dimer in the microtubule is influenced by the binding of the β-tubulin to either GTP or GDP. In order for the dimers to assemble in the microtubule, they bind to GTP and to disassemble, they bind to GDP. Thus, the GTP cycle is a requisite for the dynamic instability of the microtubule and is therefore a target for anticancer drugs in humans (Giannakakou & Zhou, 2005). - GTP α-TB GDP β-TB + Figure 2.4: Ribbon diagram of the tubulin dimer; Source: Heald & Nogales, 2002. 19 University of Ghana http://ugspace.ug.edu.gh The figure above illustrates the front view of the microtubule which shows the alpha-tubulin (α-TB) with bound GTP (top) and beta-tubulin (β-TB) containing GDP (bottom). The arrow indicates the direction of the protofilaments and microtubule axis. The alpha-tubulin is exposed to the minus (-) end whiles the beta-tubulin is exposed to the plus (+) end. The beta-tubulin monomer consists of the drug binding or intermediate domain where ivermectin binds, a nucleotide binding or N-terminal domain and a mitochondrion associated or C-terminal domain. Polymerization of the alpha-beta monomer is prevented from forming microtubules when ivermectin binds to the drug binding domain. This prevents the structural conformation of the drug binding domain to ivermectin. Furthermore, the occurrence of microtubules is prevented, thereby inhibiting the normal functioning of the cell, tissues and organs. On the other hand, mutations in the drug binding domain will alter that domain by inhibiting ivermectin from binding to it and conferring resistance to O. volvulus. 2.13 Genetic responses of Onchocerca volvulus to ivermectin treatment In a study by Huges et al. (2012) to determine whether there is emerging ivermectin resistance in some category of gravid female worms obtained from subjects in hyperendemic communities in Cameroon, the beta-tubulin gene of the worms were assessed for single nucleotide polymorphisms. Samples were collected prior to initial ivermectin treatment and after three years post ivermectin treatments. The study revealed four SNPs in the beta-tubulin gene of the worm. The SNP which occurred at 1545 (A/G) indicated an immense increase in the frequency of female worms after ivermectin treatments. A twenty one (21) month epidemiological investigation was carried out in ten (10) Ghanaian endemic communities by Osei-Atweneboana et al. (2012) to analyze the response of the beta- 20 University of Ghana http://ugspace.ug.edu.gh tubulin gene of O. volvulus to ivermectin by analyzing worm DNA for association between genotype and parasitological phenotypic response. Embryogramme analysis indicated poor response worms from subjects responding poorly to ivermectin treatment had a significantly higher reproductive activity than good response worms from subjects responding well to ivermectin treatment. An analysis of the phenotypic and genotypic response of O. volvulus to ivermectin treatment showed that the genotype (1183GG/1188CC/1308TT/1545GG) has been selected and showed strong association with the resistance phenotype. 21 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study design The study was a longitudinal one. It was geared towards analyzing the phenotypic as well as genotypic changes in the worms from ivermectin treated subjects. Female worms were excised from the treated subjects from June 2004 to October 2006. Samples taken were preserved in absolute ethanol and stored in a fridge at a temperature of -4°C from June 2004 to October 2006. Forty (40) female worms were analyzed at the Molecular Laboratory of the Council for Scientific and Industrial Research (CSIR) in Accra. 3.2 Study area and population The worms that were archived were collected from Asubende in the Pru district, Kyingakrom in the Tain district and Agborlekame in the Bole district (the Pru and Tain districts are located in the Brong Ahafo region and the Bole district is also located in the Northern region). The Pru o o o o district lies between latitudes 7 50 N and 8 22′ N and longitudes 0 30′ W and 1 26′ W with a land area of 2,195 sq. km. The primary occupations of these people are mainly farming and ° ° fishing. The Bole district lies within 9 2′ N and 8° 45′ N, and longitudes 2 29′ W and 1° 47′ W with a land surface area of 4,800 sq. km. The main occupation of these people is farming. The ° ° ° ° Tain district is located within latitudes 7 5′ N and 8 45′ N and longitudes 2 52′ W and 0 28′ E. It has a land surface area of 4,125 sq. km. The Tain River Basin and parts of the Black Volta River Basin are located within the Tain district. The primary occupation of the inhabitants is farming. 22 University of Ghana http://ugspace.ug.edu.gh Fig.3.1: A map of the study sites (represented by symbol ); Source: Map Tech Systems 3.3 Inclusion criteria The worms were obtained from subjects whose ages were between 18 and 65 years. These were people who had previously undergone at least 16 rounds of (150 µg/kg) ivermectin treatment and consented written to partake in the study. 3.4 Ethical issues Approval for the study was sought from the Ethical and Protocol Review Committee of the College of Health Sciences, University of Ghana prior to the commencement of the study. Please, find attached the certificate of approval from the committee (appendix 1). 23 University of Ghana http://ugspace.ug.edu.gh 3.5 Nodule digestion and Isolation of adult Onchocerca volvulus The nodules were preserved in liquid nitrogen and transported from the field to the CSIR. All unwanted tissues were removed from the nodules. The nodules were then placed in 50 ml tubes which contained 10 ml of 0.5% collagenase in sterile medium 199 solution. They were o digested at 37 C in a shaking waterbath for 10–24 hours. The worms were washed clean with normal saline. Using a dissecting microscope, live and dead worms at the time of nodulectomy were determined (Live worms were intact in morphology whiles dead worms were not). Intact female worms were stored in absolute ethanol before further work was done on them. 3.6 Categorization of Onchocerca volvulus based on response to ivermectin treatment Female worms were categorized as good or poor response worms. Good response female worms were characterized by the presence of more degenerate or abnormal MF found in the genital tracts obtained from subjects who responded well to ivermectin treatment for over ten years without repopulation whilst poor response worms were those characterized by the presence of more normal stretched MF obtained from subjects who responded poorly to the treatment. 3.7 Embryogramme Out of the forty (40) female worms assessed by the embryogramme technique, 18 were from Asubende, 10 were from Kyinakrom and 12 were from Agborlakeme. One (1) to two (2) cm of the anterior and posterior portions of each female worm was cut. The rest of the worm was homogenized in 1 ml of saline in sterile plastic mortar. The uterine contents of the worm were squeezed out with a plastic pestle. Aliquots of the homogenates 24 University of Ghana http://ugspace.ug.edu.gh were charged with a counting chamber. The viability of the intra-uterine MF was determined and enumerated under a light microscope. Intact stretched MF were considered as normal or alive whiles degenerated or beaded MF were considered as abnormal or dead at the time of nodulectomy. Using a calibrated pipette, 10µl of intra-uterine MF of each female worm was pipetted onto a ring slide. Using a 10X magnification of an inverted microscope, each sample was scanned for abnormal and normal MF. Using a 40X magnification to observe, five groups of pooled MF from each sample was pipetted into PCR tubes containing 2µl of distilled water. The MF were cautiously pipetted ensuring the absence of any other tissues. 3.8 Extraction of the deoxyribonucleic acid (DNA) from beta-tubulin gene The cut ends of the female worms that were collected were separately placed in labeled 1.5 ml eppendorf tubes and macerated with a sterile plastic pestle. DNA was extracted from female worms using a slightly modified protocol of chelex DNA extraction by Walsh et al. (1991). Eighty (80 µl) of 10% chelex solution and 1.5 µl of proteinase K was added to degrade o proteins. The samples were incubated at 60 C for an hour and mixed thoroughly by vortexing for 15 to 30 sec (to ensure thorough mixing) and centrifuged at 8,000 rpm for one (1) min. The o mixture was incubated at a temperature of 95 C for 45 mins. Vortexing was repeated for 30 sec and centrifuged at 10,000 rpm for 1 to 2 mins. The supernatant which contained the DNA was pipetted into labeled new 1.5 µl eppendorf tubes. For the pooled MF, 19.7µl of PCR direct and 0.3µl of proteinase K was added to degrade the proteins. The mixture was incubated at a temperature of 65°C overnight, 55°C for sixteen hours and deactivation at 85°C for 45 mins. 25 University of Ghana http://ugspace.ug.edu.gh 3.9 Amplification The region 538bp of the beta-tubulin gene was amplified with the set of primers: Ov IVM Tub- 1 1 1 F2 (5 -GAGATGGATAATATGGACTAG-3 ) and Ov IVM Tub-R2 (5 - 1 GATCCACCAAATTGCACCTG-3 ). The fragment 538 bp of the beta-tubulin was amplified in a PCR machine for each sample. The gene was amplified in a 15 µl master mix containing 7.5 µl of syber mix, 0.2 µl each of the forward and reverse primers, 2.1 µl of ddH2O, and 5 µl of DNA. The PCR mix was subjected to 45 amplification cycles, with each cycle consisting of ° o ° 30 secs denaturation at 94 C, 45 sec annealing at 54 C, and 1 min final extension at 72 C. ° o Holding was done at 22 C for the final PCR products before storing at -20 C. 3.10 Agarose gel electrophoresis Agarose gel (1.5%) was prepared and stained with 5 µl of 1X ethydium bromide. Seven (7 µl) of each PCR products was mixed with 3 µl of 1X DNA loading dye. A 50 base pair DNA ladder (New England Biolabs Inc.) was ran on all gels. Gel electrophoresis was done at 80 V and ran for an hour. After electrophoresis, the gel was visualized on a bench top Ultra-violet trans-illuminator. 3.11 Restriction Fragment Length Polymorphism (RFLP) The restriction enzyme Mph1103I was used to digest the PCR products. The reactions for the ° restriction were incubated at 37 C for 2 hrs and then analyzed on a 2% agarose gel, 1X TAE, 26 University of Ghana http://ugspace.ug.edu.gh electrophoresis at 100 V for 1 hr. The gels were stained with ethydium bromide (5 µl), and visualized with Ultra-violet trans-illuminator. 3.12 Statistical analysis The effect of ivermectin was based on the viability of the MF, that is whether dead or alive MF (binary variable). Similarly, the phenotypic outcome was classified as Normal MF and Abnormal MF. The chi-square test was used to determine significant differences in poor and good response worms. Significant level was set at α = 0.05. 27 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS 4.1 Embryogramme Poor response female worms were more compared to good response female worms as shown below (Table 4.1). P- Value= 0.01. Names of Number of Number of good Number of poor Total number Communities subjects response female response female of worms worms (%) Worms (%) Asubende 3 4(22%) 14(78%) 18 Kyingakrom 5 4(40%) 6(60%) 10 Agborlekame 8 4(33%) 8(67%) 12 Total 16 12(30%) 28(70%) 40 Table 4.1: Distribution of good and poor response female worms from subjects in three communities 28 University of Ghana http://ugspace.ug.edu.gh X20 Figure 4.1: Stretched normal microfilariae (arrow) from a poor ivermectin responding subject X20 Figure 4.2: Abnormal microfilariae (arrow) from a good ivermectin responding subject 4.2 Amplification of adult female worm beta-tubulin gene Only 14 DNA samples showed amplification on the 1.5% agarose gel as shown below (Fig. 4.3). The gel showed no amplification bands for negative template control (NTC) indicating that the various samples were not contaminated. 29 University of Ghana http://ugspace.ug.edu.gh Fig. 4.3: Image of agarose gels showing adult DNA bands of beta- tubulin gene amplicons Key: L: 50 bp DNA marker; S2, S4, S6, S7, S9, S10, S11, S13, S14, S16, S17, S18, and S19: DNA samples with amplified gel bands; NTC: Negative template control sample of gel; S1, S3, S5 and S8, S15, S20, S21, and S22: DNA samples with no positive amplification gel bands 4.3 PCR-RFLP for adult female worms Out of the fourteen (14) amplified samples from adult female worms which were restricted, only nine (9) gave successful bands. The result showed one (1) homozygote wild type and eight (8) heterozygote mutants as shown below (Fig. 4.4). 30 University of Ghana http://ugspace.ug.edu.gh Fig.4.4: Agarose gel showing restriction bands for adult female worms Homozygote wild type: S8; Heterozygote mutants: S2, S3, S4, S5, S6, S7, S9, S10, S12 S1, S11 and S13: Samples with no positive restriction gel bands. 4.4 Amplification of microfilariae beta-tubulin gene Of the fourteen (14) worms that amplified, six (6) had stretched normal MF (poor response) while the remaining eight (8) had abnormal MF (good response). No statistical difference (p>0.05) was found between these figures. Only fourteen (14) out of the 24 groups of the pooled MF samples from 6 poor response worms gave amplification bands on the 1.5% agarose gel. The gels showed no amplification bands for the negative template control (NTC) (Fig.4.5). 31 University of Ghana http://ugspace.ug.edu.gh Fig. 4.5: Image of agarose gels showing microfilariae DNA bands of beta-tubulin gene amplicons. Key: L: 50 bp DNA marker; S1, S3, S12, S14 and S17: DNA samples with no positive amplification gel bands; S2, S4-S11, S13, S15, S16, S18 and S19: DNA samples with amplified gel bands; NTC: Negative template control sample of gel; PTC: Positive template control sample of gel 4.5 PCR-RFLP for microfilariae Only eleven (11) groups of pooled MF restricted. The RFLP result showed sample 1 was a homozygote wild type whiles samples 2 to 11 were heterozygote mutants and sample 12 and 13 did not show any bands as shown below (Fig.4.6). 32 University of Ghana http://ugspace.ug.edu.gh Fig. 4.6: Image of agarose gel showing restriction bands for microfilariae Homozygote wild type: S1; Heterozygote mutant: S2, S3, S4, S5, S6, S7, S8, S9, S10, S11 Samples with no positive restriction gel bands: S12 and S13 33 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION 5.1 Embryogramme Majority of the worms belonged to the group of poor response worms from patients responding poorly to ivermectin treatment. This observation implies that some of the adult female worms population are not responding to the anti-fecundity effect of ivermectin treatment. This finding is similar to those found in poor response subjects in other studies (Awadzi et al., 2004; Awadzi et al., 2012). Studies show that poor response subjects to ivermectin treatment are subjects who are not responding well to the treatment and have worms undergoing active embryogenesis despite more than 10 annual standard doses of ivermectin treatment (Huges et al., 2014). Findings from this study show that the poor response female worms have stretched normal MF in their genital tract. Furthermore, this study implies ivermectin SOR has been selected for in O. volvulus which conforms to findings of Awadzi et al. (2004). Whiles these researchers found that some of the poor response worms were mostly characterized by abnormal MF; this study result shows that poor response worms were mostly characterized by stretched normal MF. Also, this recent study finding indicates that some populations of the female worms are responding to the anti-fecundity effects of ivermectin treatment which implies ivermectin kills the stretched MF. This finding is similar to those of Huges et al. (2014) who found abnormal MF in the genital tract of good response female worms from patients who received repeated doses of ivermectin treatment. 34 538bp p University of Ghana http://ugspace.ug.edu.gh 5.2 Genetic changes in beta-tubulin and SOR In general, all the poor response worms which amplified in this study had SNPs. This observation implies that there are genetic changes in the beta-tubulin of the poor response female worms. This finding is similar to those found in other studies (Eng & Prichard, 2005; Eng et al., 2006; Bourguinat et al., 2007). Studies show that poor response adult female worms from SOR patients have SNPs in their beta-tubulin gene (Huges et al., 2012; Osei- Atweneboana et al., 2012). Findings from this study showed the poor response worms have SNPs (Fig. 4.4) in their beta-tubulin gene. Also, results from this study shows that ivermectin SOR has been selected for in O. volvulus with SNPs in the heterozygote genotype which is in agreement with findings from Huges et al. (2012) and Osei-Atweneboana et al. (2012) studies. While these researchers found strong selection at 4 and 6 SNP positions, results from this study shows SNPs occurred at one position because the restriction enzyme digested the DNA at only one position. In addition, their findings indicated strong association of the genotypes at all four SNP positions with the resistance phenotypes. On the contrary, this study finding showed that poor response phenotype worms were strongly associated with genotypes at a single SNP position. There was association (p<0.05) between genetic changes in the beta-tubulin of heterozygote mutant female worms and poor response worms from patients who responded poorly to ivermectin treatment. The result from this study is consistent with those of Eng et al. (2006) which showed that ivermectin has been selected for and causes changes in the alleles of the beta-tubulin gene of O. volvulus and H. contortus. Likewise, this study finding agree with those of Bourguinat et al. (2007) who also found that ivermectin selects for beta-tubulin in heterozygotes female worms. 35 University of Ghana http://ugspace.ug.edu.gh 5.3 Genetic changes in beta-tubulin of adult worms and microfilariae Single nucleotide polymorphisms were found in poor response worms and their corresponding intra-uterine MF. One of the factors that affect genetic variation is mutation. These include the replacement of one DNA base pair for another, the deletion or insertion of one or more base pairs as well as changes in the chromosomes such as inversions and translocation. For genetic changes in the beta-tubulin of adult worms and their corresponding MF, findings from the present study indicate that adult female worms have passed on the mutations to their progenies. This study result clearly indicates genetic selection has occurred in O. volvulus. 5.4 Conclusion Despite several rounds of ivermectin treatment, MF still survived in the genital tracts of the adult female worms even after further treatment. Genetic changes in the beta-tubulin of the female O. volvulus are associated with SOR to ivermectin treatment. It was also detected that some adult worms have passed some mutations on to their progenies. 5.5 Recommendations 1. Molecular markers should be incorporated in monitoring resistance in subsequent onchocerciasis studies. 2. More work in this area should be done by way of increasing the sample size and extending the work to other areas of Ghana in order to confirm the findings from this work and previous work. 36 University of Ghana http://ugspace.ug.edu.gh 3. Patients who are responding poorly to ivermectin treatment should be treated with doxycycline to avoid the spread of the resistance gene in the population. REFERENCES Alley, W. 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The Journal of Infectious Diseases,169(3): 686–689. 46 University of Ghana http://ugspace.ug.edu.gh APPENDICES Appendix 1 Embryogramme result Number Number Microfilariae enumerated of of Adult Total Percentage Percentage Communities subjects worms normal abnormal Asubende 3 18 5,228,000 4 96 47 University of Ghana http://ugspace.ug.edu.gh Kyingakrom 5 10 748,015 60 40 Agborlakame 8 12 532,000 7 93 Total 16 40 6,508,015 8.53 91.47 48 University of Ghana http://ugspace.ug.edu.gh Appendix 2 Certificate of approval 49