LYMPHATIC FILARIASIS AT GOMOA OKYEREKO, AN IRRIGATION PROJECT COMMUNITY IN SOUTHERN GHANA: INFECTION, CLINICAL DISEASE AND VECTORS A Thesis Presented to The Board of Graduate Studies University of Ghana, Legon Ghana In Part Fulfilment of the Requirements for the Degree of Master of Philosophy (M. Phil.) Zoology By MAWULI DZODZOMENYO B. Sc. (Hons.), Legon. Department of Zoology Faculty of Science University of Ghana Legon. SEPTEMBER, 1996 University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Page DECLARATION............................................................................................................................. i DEDICATION..................................................................................................................................... ii ACKNOWLEDGEMENT........................................................................................................... iii LIST OF TABLES ............................................................................ v LIST OF FIGURES . . . . . . . . . . . vii SUMMARY . . . . . . . . . . . . . • viii CHAPTER 1 1.1 GENERAL INTRODUCTION . . . .................. . . 1 1.1.1 Prevalence and Distribution of Lymphatic Filariasis . . . 1 1.1.2 History and Classification of the Parasite 1 1.1.3 Vectors and Transmission . . . 2 1.1.4 Clinical Disease ........................ . . . 2 1.1.5 Social Impact of Lymphatic Filariasis Disease 3 1.1.6 Water Resources and Parasitic D is e a s e s .......... 4 1.1.7 Lymphatic Filariasis and Water Resources Development . . 5 CHAPTER 2 2.1 DESCRIPTION AND DEMOGRAPHIC CHARACTERISTICS OF THE STUDY VILLAGE . . . . . . . . . . 7 2.1.1 Location and Climatic F e a tu re s .................. . . . . . . . 7 2.1.2 Demographic Characteristics.................................. . . . . . 8 2.2 MATERIALS AND METHODS . . . . . 9 2.2.1 Village identification, community mobilization and ethical considerations................................................................. . . . 9 2.2.2 Census ............................. . . . . . . . 9 2.2.3 Data Entry and Analysis . . . . . . . . . . . . 11 2.3 Results . . . . . . . . . 12 2.4 DISCUSSION ................ 14 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 3.1 MICROFILARAEMIA AND CLINICAL MANIFESTATIONS IN LYMPHATIC FILARIASIS DUE TO Wuchereria b a n c ro fti .............................. 17 3.1.1 The Life Cycle of Wuchereria bancrofti . . 17 3.1.2 Clinical manifestations ................. ....................................... 18 3.1.2.1 Acute attacks .................. . . . . 19 3.1.2.2 Chronic D ise a se . . 19 3.1.3 Mechanism of pathology . . . 21 3.1.4 Diagnosis ................................ . . 25 3.1.4.1 Giemsa-stained thick smear . . 26 3.1.4.2 Counting Chamber Technique (CCT) ............................................................ 26 3.1.4.3 Membrane (Nuclepore) T echn ique .................................................................. 27 3.1.4.4 Knott’s Concentration Method . . 27 3.1.4.5 Immunodiagnosis ............................................................................. 28 3.1.4.6 Lymphatic imaging ....................................................................... 28 3.1.4.7 DNA-detection assays . . . . 28 3.2 MATERIALS AND METHODS 3.2.1 Study design ................. . 3 0 3.2.2 Blood Collection for CCT: . . . . 30 3.2.3 Using the CCT: ....................... . . . 31 3.2.4 Thick Blood Smear Preparation for Microfilariae Identification . 31 3.2.5 Giemsa-staining . . 32 3.2.6 Clinical Examination . . . . . 3 3 3.2.7 Data management and analysis . . . . . . . . . 33 3.3 RESULTS . . ............... . . . . 35 3.3.1 Distribution of Selected Persons by Age and S e x .................. . . 35 3.3.2 Microfilariae identification ............ . . . . . . . . 37 3.3.3 Microfilaraemia Prevalence . . . . . . . . . 37 3.3.4 Microfilarial In ten s itie s ..................... . . . . . . . . 40 3.3.5 Clinical Manifestations . . . . . . . . . . . . 42 3.3.5.1 Hydrocoele . . . . . . . . . , . . . . 42 3.3.5.2 Limb Elephantiasis . . 43 3.4 DISCUSSION . . . . . . 4 4 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 4.1 PERIODICITY STUDY OF LYMPHATIC FILARIASIS PARASITE . . 50 4.1.1 In troduction ........................................................................................................ 50 4.1.2 Mechanisms of P e rio d ic ity ............................................................................. 51 4.1.3 Implications of microfilarial Periodicity for Diagnosis .................... 53 4.2 Materials and M ethods........................................................................................ 54 4.2.1 Periodicity Data A na ly sis .................................................................................... 55 4.3 R e s u l t s .................................................................. . . . . 60 4.4 D ISCUSSION ....................................................................................... . . 64 CHAPTER 5 ................................................................................................................................... 66 5.1 ENTOMOLOGICAL STUDY OF THE VECTORS OF LYMPHATIC F ILAR IA SIS .............................................................. . . . . . . 66 5.1.1 Introduction . . . . . . . . . . . 66 5.1.2 Vector Abundance and Vectorial C apacity 68 5.1.3 Man-biting Rate and Vectorial C ap ac ity ................................. . 69 5.1.4 Vectors in Africa . . . . . 71 5.1.5 Vectors in Ghana . . . . . . 73 5.2 Materials and Methods . ............ . . . . 74 5.2.1 Adult mosquito collections . . 74 5.2.1.1 Pyrethrum Spray Catches (PSC) . . . . . 74 5.2.2.2 Human Landing Catches . 75 5.2.3 Identification and Dissection of Adult M osqu itoes ................................... 76 5.2.4 Definitions 76 5.3 RESULTS . . . . . 77 5.3.1 Mosquito abundance, infection and infectivity rates .............. 77 5.3.2 Worm Load in the vectors ................................. . . . 80 5.3.3 Intensity of Transmission ........................ . . . . 81 5.4 DISCUSSION ........................................................... . . . 94 CHAPTER 6 6.1 GENERAL DISCUSSION, RECOMMENDATIONS AND CONCLUSIONS 102 6.1.1 General D iscussion................................................................. . . . 102 6.1.2 Recommendations . . . . . 1 0 3 6.1.3 Conclusion . . . . . . . 105 Appendix 1 . . . . . . 106 REFERENCES 109 University of Ghana http://ugspace.ug.edu.gh I do hereby declare that except for other people’s investigations which have been duly acknowledged, this work is the result of my own original research, and that this thesis, either in whole or in part, has not been presented elsewhere for another degree. DECLARATION Mawuli Dzodzomenyo (STUDENT) Prof. W .Z Coker (SUPERVISOR) Dr. S.K Dunyo (SUPERVISOR; Mr. M. Appawu (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh DEDICATION I dedicate this work to my parents, SETH and GRACE. University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I wish to express my sincere gratitude to the Director of Noguchi Memorial Institute for Medical Research (NMIMR) of University of Ghana, Prof. F.K Nkrumah and all his staff especially those of the Epidemiology and Parasitology Units for making available to me their excellent laboratory facilities to carry out this work. I am particularly grateful to the members of the general laboratories of these two units for their companionship and tolerance during my stay with them. I am particularly indebted to my supervisors, Professor. W .Z Coker of the University of Ghana, Dr. S.K. Dunyo and Mr. M. Appawu both of NMIMR for their guidance, useful criticisms and valuable suggestions which steered this work to its successful end. I sincerely wish to express my heartfelt thanks to Dr. Dunyo for his good planning, care and encouragement throughout the period of the work and also for introducing me to the computer. I am also most grateful to Dr. Dunyo, Mr. Collins Ahorlu, Mr. S.K Dadzie and Mr. Opare (driver), all of NMIMR for missing their nights and helping during night blood collections at the study village and particularly to Mr. Dadzie for his immense contribution in the entomological aspects of this study. Many thanks go to Miss Cindy Amoabeng and Tina Aryeh for their various helps. University of Ghana http://ugspace.ug.edu.gh I wish to express my sincerely thanks to the Chief and people of Gomoa Okyereko, for their understanding and cooperation and to the Barclays Bank (Gh) Limited for providing financial assistance through the University of Ghana Graduate Fellowship scheme for my graduate study and also for partly funding this thesis. Finally, I wish to extend my warmest thanks to my brothers and sisters, Samuel, Regina, Hilda and Theodore, my colleagues and friends for their concern which did not only help me to endure the strenuous demands of graduate school but also contributed to the successful completion of this thesis. I wish to mention in particular Mr and Mrs Donya who helped me with their personal computer at a time when I urgently needed it and most especially, Miss Rose Tsekpo of University of Cape Coast for her understanding, encouragement and support throughout the study. iv University of Ghana http://ugspace.ug.edu.gh 12 36 38 40 42 43 61 62 63 83 84 85 86 LIST OF TABLES Population of inhabitants of Okyereko Distribution of study population by age and sex Microfilaraemia prevalence by age and sex Geometric mean intensity of microfilaraemia by age and sex at Okyereko Prevalence of hydrocoele with respect to age group Prevalence of elephantiasis with respect to age group Two-hourly microfilarial counts in the eight study individuals Observed and t theoretical microfilarial ratios in the eight study individuals Trigonometric analysis of the observed microfilarial ratios by the method of Aikat and Das (1977) Numbers of indoor resting and man biting mosquitoes collected in Okyereko (April 1995 to January 1996) Infection and infectivity rates of indoor resting mosquitoes in Okyereko Hourly distribution of mosquitoes caught biting human baits in Okyereko Infection and infectivity rates of mosquitoes caught on human baits in Okyereko v University of Ghana http://ugspace.ug.edu.gh Filarial larvae recovered in indoor resting mosquitoes collected in Okyereko Filarial larvae in mosquitoes caught biting human baits in Okyereko Frequency distribution of infective filarial larvae in night biting and indoor resting mosquitoes in Okyereko Estimated number of yearly infective bites of mosquitoes collected off human baits in Okyereko University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES 1. Map of the study area, Okyereko and its environs 1U 2. Population pyramid, Okyereko - 1995 Census 13 3. Prevalence of microfilaraemia in the different age groups 39 4. Geometric mean intensity in the different age groups 41 5. Observed and theoretical microfilarial ratios-Periodicity curve 59 6. Relative abundance of the different spieces of mosquitoes collected 91 7. Rainfall pattern for Okyereko area (Jan. 1995 to Jan. 1996) 92 8. The different species of mosquitoes collected in each hour 93 vii University of Ghana http://ugspace.ug.edu.gh SUMMARY A parasitological, clinical, periodicity and entomological study of lymphatic filariasis was earned out at Gomoa Okyereko, an irrigation project community in Southern Ghana to provide baseline information on the infection and its transmission in the community. Out of a total of 636 inhabitants, fifty percent of the residents were selected from the compiled demographic data using computer generated random numbers to constitute the study population. Quantitative examination of night blood sample (100/xl) between 21:00 and 01:00 hours from 296 (94.6%) selected persons using the counting chambers techniques (CCT) revealed high microfilarial prevalences and microfilarial geometric mean intensity (GMI) in the study group. Overall microfilarial prevalence of 26.4% was observed, and prevalence generally increased with age and was higher in females (27.6%) than in males (25%), though this was not statistically significant. Large variability was observed in individual mean intensities and an overall GMI among infected individuals was 819mf/ml of blood (1114.3mf/ml in males and 645.7mf/ml in females). There was however no statistical significance in GMI between these two sexes. Hydrocoele was the most common clinical manifestation of lymphatic filariasis and 9.3% of the males examination had hydrocoeles of grades 1-IV The prevalence of hydrocoele increased with age and the highest prevalence of 33.3 % was noted in 40-49 age group. Four female aged 13-55 had limb elephantiasis, two were grade I and the other two grade III. University of Ghana http://ugspace.ug.edu.gh Microfilarial periodic pattern of Wuchereria bancrofti was determined in eight microfilaraemic persons. From each individual, a 100/xl finger prick blood was collected every two hours throughout one complete 24hours cycle. The peak hour (k) was calculated as 01:03 (i.e 3 minutes after 1 a.m) and the periodicity index (D) was 122.6, confirming the nocturnal periodicity of microfilariae of this parasite in Ghana. Entomological studies using pyrethrum spray collections and landing catches revealed that the species of man-biting mosquitoes in the community include Anopheles gambiae s .l , 4^. funestus, A. pharoensis, Culex quinquefasciatus and Mansonia species. Their relative abundance showed seasonal variation influenced by the amount of rainfall and the availability of favourable breeding places such as the irrigation canals. In the rainy season, the most abundant species was A. gambiae s .l while in the dry season, the most abundant species was A. funestus. During the period that the dam was opened, the most abundant species was A. gambiae s .l . Most of the mosquitoes were biting late in the night coinciding with the peak of microfilarial abundance in the peripheral blood. Overall infectivity rates of 7.8%, 3.9% and 9.1% were recorded for A. gambiae s .l , A. funestus, A. pharoensis respectively. A. pharoensis had a very high infectivity rate though very small numbers were collected. Other species collected in small numbers were Culex quinquefasciatus and Mansonia species and their role in the transmission of the disease was minimal. It was calculated that inhabitants in the area are at risk of getting 309.5 infective bites per man per year from A. gambiae s.l while both A. funestus and A. pharoensis may give 24.1 infective bites each. The main vector responsible for the transmission of lymphatic filariasis in the community therefore is A. gambiae s .l while A. funestus and A. pharoensis play minor roles. University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.1 GENERAL INTRODUCTION 1.1.1 Prevalence and Distribution of Lymphatic Filariasis Lymphatic filariasis is caused by infection with a group of slender elongated nematode worms which are called filarial worms. The disease in its various forms remains a public health problem of considerable magnitude in many tropical countries (WHO, 1992). According to WHO expert committee on filariasis, it is estimated that over 3 billion persons live in countries where the disease is endemic and some 757 million live in areas where transmission is known to occur (WHO, 1992). Its prevalence is increasing worldwide, largely because of rapid urbanisation in many endemic areas (Ottesen, 1995). 1.1.2 History and Classification of the Parasite The microfilariae of the parasite Wuchereria bancrofti were first found by Wucherer in the urine of a patient suffering from chyluria in Bahia, Brazil in 1866 (Kettle, 1990). However, Nelson (1978) reported that the parasites were observed earlier on by Demarquay in 1863. Since then interest in filariasis grew worldwide and many workers such as Lewis in India, Manson in China and Bancroft in Australia detected microfilariae in the blood of patients in subsequent years. Three main filaria worms cause the disease in man namely, Wuchereria bancrofti, Brugia malayi, and Brugia timori. The group of filaria worms that cause lymphatic filariasis belong to the superfamily Filarioidea, the order Spirurida and the class Nematoda. Of the estimated 90 million infections worldwide, Wuchereria bancrofti is responsible for over 80 million cases and is the only known aetiologic agent in the African Region where about 25.6 million people are 1 University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.1 GENERAL INTRODUCTION 1.1.1 Prevalence and Distribution of Lymphatic Filariasis Lymphatic filariasis is caused by infection with a group of slender elongated nematode worms which are called filarial worms. The disease in its various forms remains a public health problem of considerable magnitude in many tropical countries (WHO, 1992). According to WHO expert committee on filariasis, it is estimated that over 3 billion persons live in countries where the disease is endemic and some 757 million live in areas where transmission is known to occur (WHO, 1992). Its prevalence is increasing worldwide, largely because of rapid urbanisation in many endemic areas (Ottesen, 1995). 1.1.2 History and Classification of the Parasite The microfilariae of the parasite Wuchereria bancrofti were first found by Wucherer in the urine of a patient suffering from chyluria in Bahia, Brazil in 1866 (Kettle, 1990). However, Nelson (1978) reported that the parasites were observed earlier on by Demarquay in 1863. Since then interest in filariasis grew worldwide and many workers such as Lewis in India, Manson in China and Bancroft in Australia detected microfilariae in the blood of patients in subsequent years. Three main filaria worms cause the disease in man namely, Wuchereria bancrofti, Brugia malccyi, and Brugia timori. The group of filaria worms that cause lymphatic filariasis belong to the superfamily Filarioidea, the order Spirurida and the class Nematoda. Of the estimated 90 million infections worldwide, Wuchereria bancrofti is responsible for over 80 million cases and is the only known aetiologic agent in the African Region where about 25.6 million people are 1 University of Ghana http://ugspace.ug.edu.gh affected (WHO, 1984). The adult worms are found in the lymph nodes and lymphatic channels of humans. Male worms are about 40mm in length and 0 .1mm in width and are less than half the size of the females which measure 80-100 by 0.24-0.30mm. Both are creamy white, cylindrical, blunt tapering, threadlike nematodes with smooth cuticular surfaces and unarmed mouths. 1.1.3 Vectors and Transmission The vectors responsible for the transmission of lymphatic filariasis vary in the different geographical regions of the world. Mosquitoes belonging to three genera. Anopheles, Culex and Aedes are known to be involved in transmission of the disease worldwide (WHO, 1987). In Africa, the mosquitoes, Anopheles gambiae sensu latum and Anopheles funestus are the principal vectors except along the coast of the Indian Ocean (ie in Kenya and Tanzania) where Culex quinquefasciatus is known to be involved in transmission (WHO, 1987). Transmission occurs when the mosquito in the process of feeding on the blood of the host deposits the infective larvae on the skin in a small fluid and successful penetration is known to depend on the relative humidity of the external environment (Lindsay et al, 1984). 1.1.4 Clinical Disease Lymphatic filariasis produces a spectrum of infection and disease outcomes such that communities in endemic areas differ in the proportion of people who are microfilaraemic, in the mean densities of microfilariae that are detected and in the prevalence of clinical symptoms that suggest a difference in the intensity of transmission. The adult worms live in the lymphatic 2 University of Ghana http://ugspace.ug.edu.gh system and obstruct the flow of the lymph, causing local inflammation of the lymphatic vessels (lymphangitis), swelling of the lymphatic glands (lymphadenitis) and oedema of the affected area, frequently the legs (Maegraith, 1980). Chronic obstruction of the lymphatic system leads to permanent oedema and the development of fibrous tissue resulting in permanent elephantiasis. Elephantiasis particularly affects the legs either below the knees or the whole limb, the arms, breasts, labia and scrotum. In males hydrocoele is a common complication. Obstructed abdominal lymphatic vessels may rupture leading to the excretion of lymph in the urine, a condition known as chyluria. 1.1.5 Social Impact of Lymphatic Filariasis Disease Lymphatic filariasis persists as a major cause of clinical morbidity and thus a significant impediment to socioeconomic development in much of Asia, Africa and the Western pacific, as well as in certain regions of the Americas. The social impact of filariasis vary not only from place to place but also within the same communities and in general, the degree of stigma seems to be associated with the severity and visibility of the disease (Evans et al, 1993). Like many helminthic infections, filariasis is not considered to be a major cause of mortality, but filarial morbidity is devastating and is a crippling affliction that causes social and economic hardships at both the individual and community levels (Wamae, 1994). Hunter (1992) reported that the stigma attached to swollen limbs for instance, discourages young men from marry ing girls from areas that are known for filariasis in North East Ghana. In the Philippines, Lu et al (1988) indicated that cases of hydrocoele were considered serious only when they reach the size of a sack but generally , people with hydrocoele were the subject of considerable teasing. Kessel, University of Ghana http://ugspace.ug.edu.gh cited by (Evans et al, 1993) reported that in Polynesia, at least in the 1950s, people suffering from filariasis hid or retired to the background because they were the laughing stock of the community. The distribution and transmission of the disease are closely related to socioeconomic and behavioural factors in the endemic populations (Muhodwa, 1983). For example, rural-urban migration and urbanization interact to facilitate the spread of W. bancrofti (Mak, 1986). This is believed to occur largely as a result of inadequate waste disposal and sanitation facilities providing suitable breeding sites for the vectors. The crowding of people living in urban areas in developing countries is also known to aid transmission. Other forms of migration in endemic regions such as occupational migration may also aid transmission and Laurence (1989) linked the historical spread of lymphatic filariasis throughout the world with large scale migrations of infected peoples. 1.1.6 Water Resources and Parasitic Diseases Even though water resources development projects are essential for a wide range of human activities notably agriculture and energy production, it has long been recognized that inadvertent, unintended and adverse side effects of these projects can seriously threaten human health (Hunter, et al, 1993). Notable among the adverse side effects of such water development projects is the increase in certain parasitic diseases among the populations living nearby. In Ghana for instance, the Kpong Dam (which is about 25km below the Akosombo Dam), immediately after completion became infested with submerged and floating aquatic plants such 4 University of Ghana http://ugspace.ug.edu.gh as Ceratophyllum and Pistia which served as suitable habitats for intermediate host snails of schistosomiasis (Odei, 1977). Similar events were reported in Egypt (Dawood, 1951) and in the Selingue dam in Mali (Traore, 1989). The Barekese reservoir, the main source of water supply in the Ashanti Region of Ghana was invaded by Mansonia and other mosquitoes soon after its completion in the early 1960s (Hunter et al, 1993). 1.1.7 Lymphatic Filariasis and Water Resources Development The effect of water resources development projects on lymphatic filariasis transmission has received little attention. The incidence and prevalence of lymphatic filariasis is increased around small dams and the severe form of the disease may occur frequently because of the intensity of transmission (Hunter, 1992). In a reconnaissance survey of lymphatic filariasis in North East Ghana, Hunter (1992) reported that all communities reporting of filariasis as a public health problem were former beneficiaries of foreign assistance agricultural development projects such as small village dams which were constructed from 1958-1962. Notable among these dams are the Vea and Tono irrigation projects. The key understanding to this is that the irrigation canals provided additional habitats which are suitable for mosquito breeding leading to an increase in mosquito populations and therefore high intensity of mosquito-man contact which are necessary conditions for the transmission of lymphatic filariasis. 5 University of Ghana http://ugspace.ug.edu.gh Little has been done on lymphatic filariasis in Ghana. However, in recent years, studies were carried out in the North (Gyapong et al. 1993) and along the coast of Ghana (Dunyo et al. in press). This study was therefore carried out at Gomoa Okyereko, a village situated very near to an irrigation project, the Okyereko Irrigation Project (OIP), which involved the construction of an earth dam across a small tributary of the River Ayensu. Since the advent of the dam however, it has been realized that there is an increase in mosquito populations (Hunter et al, 1993), and cases of clinical manifestations of lymphatic filariasis have been observed in the community (Ahorlu et al, personal communication 1995). This study therefore aims at providing baseline information on the prevalence and intensity of lymphatic filariasis in this irrigated community by determining the prevalence of microfilaraemia, clinical manifestations and also entomological observations. It also aims at establishing the periodicity pattern of the responsible filarial parasite, in order to document the appropriate time to collect blood sample for optimum microfilariae detection in Ghana. 6 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 2.1 DESCRIPTION AND DEMOGRAPHIC CHARACTERISTICS OF THE STUDY VILLAGE 2.1.1 Location and Climatic Features Gomoa Okyereko is a village located between latitudes 5°N 5 ’/2°N in the Central Region of Ghana. It is located about one and half kilometres off the Accra to Winneba road and is forty nine (49) kilometres from Accra. This village is in the dry equatorial climatic region of Ghana. It has two rainfall maxima and the average yearly rainfall varies between seventy-four and eighty nine centimetres. This region is the driest in Ghana (Dickson et al, 1977). The main rainy season lasts from May to July with a minor season of light showers between September and October. Daily temperatures average 26°C and many of the small streams dry up during the dry season. The vegetation is coastal savanna interspersed with very few trees. On the western margin of the village is the river Ayensu which is the main source of water supply to the village. This river normally has a large volume of water in the rainy season leading to periodic flooding but reduces in volume in the dry season. The people are mainly peasant farmers and to permit sustained agricultural activities during the dry season, the installation of irrigation facilities was necessary. The construction of an irrigation facility was therefore started in 1973 and completed in 1974. This involved the construction of an earth dam across a small tributary of the river Ayensu. The project thus envisages an increase in the production of rice and vegetables during the dry season in the Winneba area. It was also intended to minimize the drift of the youth to the urban areas by allocating plots to farmers where it is expected that the income would go a long way to raise the living standards of the people. The Okyereko settlement itself is situated 7 University of Ghana http://ugspace.ug.edu.gh between the dam and the river Ayensu and most of the land surrounding the village is the irrigable land which is mainly used for the cultivation of cereals, mainly rice and maize and vegetables. 2.1.2 Demographic Characteristics The houses at Okyereko are grouped together into one cluster and the general extended family system prevails with most households headed by men. In order to provide information on the population size of the village in relation to sex and age distribution in order to explain any variations in disease prevalence should the need arise, a population census was conducted. The census was also aimed at explaining any observed changes that might have occurred in the population and its effects on the population growth. 8 University of Ghana http://ugspace.ug.edu.gh 2.2 MATERIALS AND METHODS 2.2.1 Village identification, community mobilization and ethical considerations Prior to the start of the study, a map of the study area was collected from the Irrigation Development Authority of the Ministry of Agriculture. This was followed by a visit to the community to check on the existence of the village. After this has been confirmed, a series of meetings were held with the chiefs, elders and political leaders of the community to explain the essence of the study and seek their consent. A day was selected for a forum with all the residents of the community to describe and explain the procedures involved and answer questions to clarify any doubts and suspicions on the study. Their consent and participation was sought and the consent and participation of children was obtained from their parents. 2.2.2 Census After approval for the study was given by the elders and responsible authorities of the Ministry of Health, the Irrigation Development Authority and the District Health Administration, all the houses in the village were numbered sequentially by the investigator with the assistance of five selected youth of the community nominated by the chief and elders. These youth were given prior training in simple demographic data collection such as house numbering and house to house registration of residents. A census was then conducted to register the usual residents of the community who are 1 year and above using the form in Appendix 1. During the census, all persons living in each house were registered by name, age, sex, and the duration of stay in the community. A map of the village was made depicting its location with reference to other landmarks such as water bodies, the irrigation dam and the streams flowing into it. F ig .l. 9 University of Ghana http://ugspace.ug.edu.gh 10 University of Ghana http://ugspace.ug.edu.gh 2.2.3 Data Entry and Analysis The data were entered into the computer using dbase program and age and sex compositions were analysed using SPSS. The individuals were divided into seven groups aged 1-9, 10-19, 20-29, 30-39, 40-49, 50-59 and >60. Data on age and sex compositions were used to construct a population pyramid. 11 University of Ghana http://ugspace.ug.edu.gh 2.3 Results Table 1 shows the results of the census conducted in the study village. Overall, there were 636 inhabitants living in 99 houses. This was made up of 298 males (46.9%) and 338 females (53.1%). The age and sex distribution of the population is shown in a population pyramid in Figure 2. The most populous age group is the youngest age group (ie 1-9). The younger ages (0-19) form about 50% of the total population. Therefore the population pyramid is broadest at the base, with 31.1% in the (1-9) age group. This reduced to 18.9% in the (10-19) age group. The reduction continues with the (20-29) age group contributing 12.7%. The (30-39) group formed 11.3%, while (40-49) group forms 8.6%. The (50-59) year group are 6.1% while those above 60 years form 11.3% of the population. Table 1. Population of inhabitants of Okyereko Males Females Males and Females Age-group (Years) No. % of total No. % of total No. % of total 1-9 109 17.1 89 13.9 198 31.1 10-19 68 10.7 52 8.2 120 18.9 20-29 35 5.5 45 7.1 80 12.7 30-39 26 4.1 46 7.2 72 11.3 40-49 29 4.6 26 4.1 55 8.6 50-59 13 2.1 26 4.1 39 6.1 £60 18 2.8 54 8.5 72 11.3 Total 298 46.9 338 53.1 636 100.0 12 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 2.4 DISCUSSION One important demographic fact about a population is its rate of growth. The population trend at Okyereko follows the "normal African population pyramid structure", which is broad at the base and narrowing at the apex with its consequent inbuilt dynamic growth. This follows also the age structure of Ghana’s population in contrast to the population pyramid for an average developed country which is shaped more like a cylinder, reflecting that the distribution of the population is more even for all age groups. The implication is that countries with low population growth rates have a high percentage of their total population in the economically active age group (15-64). Looking at the trend of population growth in the various age groups, we realize that the population reduces as the age increases. This is normally the case in most developing countries putting a strain on the little resources available to them due to increasing rate of growth of the population. This concept is referred to as inbuilt dynamism (ie a situation where a large population base also grows to reproduce another large base, with consequent population problems due to constancy of available resources. The rate at which a population is changing affects not only its size and numerical increase but also its composition. This is important in determining the provision of resources to match the population growth. In some populations, the number of males and females tends to be nearly equal and from the demographic viewpoint, a situation of near equality assures that each sex has an adequate supply of mates so that the population can replenish its losses from mortality. However, in most populations, the males outnumber females at the younger ages and females outnumbering males at the older ages. 14 University of Ghana http://ugspace.ug.edu.gh An unique feature of the population of Okyereko is that at the base of the pyramid (ie 1-9 age group), males outnumber the females. It is, however, known that more male babies die between the ages of 0-5, making the female population within this group more often than not more than the males. This is because it is known that a disproportionately high percentage of male foetuses are miscarried or stillborn, so that a population in a poor nutritional state or in poor health may be expected to have a lower sex ratio (which is the number of males per a hundred or thousand females) at birth, other things being equal. Also in most populations, from the first instant of life onward, death rates for males are higher than those of females. This seeems to be due to constitutional factors and this is because males are more susceptible to most diseases and have higher death rates at every age. Also, within the reproductive age group (ie normally 15-44), it is expected that more females than males die resulting in higher male population than females. However, in the pyramid of Okyereko,the female population is higher than that of males. This point can be explained as due to migration of the male population. Most of the males in the economically active group move out of the community in search of greener pastures and most of the males in this age group at Okyereko are said to be involved in cocoa and other cash crop farming in some other parts of the Central Region and the Western Region. This observation in itself defeats the main purpose of the irrigation project which is aimed at allocating plots of land to the youth to indulge in agricultural activities in order to reduce the rural-urban drift. Ghana’s population, like any other country's is influenced by births, deaths and migration and the present high level of the population growth is the result of persistent high birth rates and declining mortality rates over the years (Benneh, 1990). Until recently, fertility has been 15 University of Ghana http://ugspace.ug.edu.gh persistently high while mortality has consistently declined in the past three decades resulting in a population that is growing rapidly (Ghana Statistical Service, 1993). The fertility level of a country is the principal determinant of its rate of growth and rural women have a higher fertility rate of 6.4 compared to 4 in urban women. The women at Okyereko having a high fertility rate will apparently give birth to a lot of children resulting in a high population growth. For a variety of social and economic reasons, large families are attractive to many Ghanaians. The demographic data have therefore shown that at Okyereko there is the likelihood of a high rate of population growth due to the high fertility rate of the women, the broad base of the population pyramid with high numbers of infants. 16 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 3.1 MICROFILARAEMIA AND CLINICAL MANIFESTATIONS IN LYMPHATIC FILARIASIS DUE TO Wuchereria bancrofti 3.1.1 The Life Cycle of Wuchereria bancrofti The life cycle of W. bancrofti was first worked out by Manson in Culex quinquefasciatus in China in 1878 (Manson-Bahr et al, 1987). It involves both the human and the insect vector as definitive and intermediate hosts respectively. The female worm is viviparous liberating embryos into the lymphatic system in large numbers. The embryos or microfilariae are enclosed in a membrane and are therefore said to be sheathed. They ultimately escape from the lymphatics and appear in the peripheral blood. The microfilariae of W. bancrofti are long and slender measuring 260ptm. They are taken up by the mosquito vector during feeding. Within an hour of entering the mosquito’s stomach, they shed their sheaths and penetrate the midgut epithelium. They may however be damaged at this stage by the bucopharyngeal armature of the mosquito. Bryan et al, (1988) confirmed the damage of microfilariae by mosquito foregut but emphasized that the size of the mosquito does not seem to influence the amount of damage inflicted on the microfilariae. The damage explains the differing infection rates of the vectors responsible for transmission (Manson-Bahr et al, 1987). At the end of an infective feed, the embryos collect at the anterior end of the stomach and then enter the anterior cylindrical portion of the midgut. Forward transportation from here is effected by reversed peristalsis until they are distributed over the whole cylinder. The proboscis of the mosquito exerts positive chemotaxis upon microfilariae therefore mosquitoes can abstract more 17 University of Ghana http://ugspace.ug.edu.gh embryos than would be present in a similar quantity of circulating blood. The mosquitoes normally abstract 1mm3 of blood at each feed and in so doing concentrates the embryos about tenfold (Manson-Bahr et al, 1987). McGreevy et al (1982) demonstrated a "concentrating effect" of 8.6-12 fold of W. bancrofti in Cx. quinquefasciatus, An. gambiae, and Aedes in Tanzania. The parasites then enter the thorax where they lie between the fibrillar flight muscle of the thorax and the pharyngeal muscle of the head of the mosquito. Within 2 days they develop into thicker, shorter, "sausage" forms which undergo two moults before developing into elongated infective larvae measuring about 1.5 X 0.02mm. Mature third stage larvae leave the thoracic musculature and enter the haemocoele in which they move around actively and accumulate in the head. The complete life cycle in mosquito takes 10-14 days at high temperature and in moisture but is retarded to 6 weeks by cold (Manson-Bahr et al, 1987). They escape during the next feeding of the mosquito by entering the labium and rupturing the labella. They are deposited in a drop of haemolymph and enter the host through the puncture made by the feeding mosquito (McGreevy et al, 1974). The presence of the fluid prevents desiccation of the larvae on the surface of the skin (Lindsay et al, 1984). There is no asexual reproduction taking place between the infective larvae introduced and the adult stage. In man, the development of the worm takes at least seven to twelve months before becoming mature (Sasa, 1976). 3.1.2 Clinical manifestations Clinical symptoms of lymphatic filariasis range from none to severe clinical manifestations such as elephantiasis and hydrocoele. The clinical manifestations may depend on host factors and parasite strain (Garcia et al, 1993). In a community where filariasis is endemic, individuals may be divided into the following clinical categories: 18 University of Ghana http://ugspace.ug.edu.gh i) No microfilaraemia and no evidence of disease. ii) Microfilaraemia with no evidence of disease. iii) Acute filarial disease with or without microfilaraemia and iv) Chronic filarial disease mostly amicrofilaraemic with or without acute attacks. In most endemic communities, disease manifestation begins in late childhood or early adulthood, with acute attacks such as filarial fevers, adenolymphangitis and epididymo-orchitis. 3.1.2.1 Acute attacks Early manifestations of filariasis is frequent high fevers, lymphangitis and lymphadenitis. Filarial fever usually begins with a high fever and chills that last one to five days before spontaneously subsiding and patients with filarial fevers in many cases do not have microfilariae. (Garcia et al, 1993). The attacks are often precipitated by hard labour in the fields but may occur without any apparent cause (Partono, 1984). The patient may be incapacitated for a few days, but occasionally may remain ambulatory. Acute episodes interrupt normal activities, rendering people unable to work, often confining them to bed. The events precipitating acute attacks are not known with certainty. 3.1.2.2 Chronic Disease When the adult worms enter the lymphatic vessel, an inflammatory reaction occurs and there is an infiltration of plasma cells, eosinophils and macrophages in and around the affected vessel. Repeated inflammatory attacks produce hyperplasia of the endothelium resulting in an increase in hydrostatic pressure due to the damage to the lymph vessel and this in turn leads to an 19 University of Ghana http://ugspace.ug.edu.gh increase in vessel wall permeability. The chronic leakage of high protein-containing fluid into the surrounding tissues results in a hard or brawny oedema, with thickening and varicous changes in the skin and this is known as elephantiasis. The onset of elephantiasis of the leg is frequently preceded by attacks of lymphangitis and lymphadenitis involving the inguinal glands draining the leg (Jordan, 1955). Lymphadenitis and lymphangitis develop in the lower extremities more commonly than in the upper, and apart from the limbs, there can be breast involvement. Another chronic disease state frequently resulting from the infection with W. bancrofti is hydrocoele. The epitrochlear and femoral lymph nodes become firm, discrete and tend to remain enlarged. These conditions are preceded by severe pain in the scrotum (Garcia et al, 1993). The development of hydrocoele is usually characterized by an attack of epidymo-orchitis, epididymitis and funiculitis and these conditions are frequently associated with oedema of the scrotal skin and a small collection of inflammatory fluid in the tunica vaginalis (Jordan, 1955). There is swelling of the reflection of the peritoneal lining that surrounds each of the testicles and a clear hydrocoele fluid accumulates in this closed sac as a result of lymphatic blockage in draining lymphatics located in the retroperitoneal and sub diaphragmatic areas. Hydrocoele is the most common disease manifestation of W. bancrofti (WHO, 1992). Obstruction of the retroperitoneal lymphatics may cause the renal lymphatics to rupture into the urinary tract resulting in excretion of chyle in the urinary tract: a condition known as chyluria. Even though this condition is painless, large amounts of dietary lipids, proteins and possibly fat-soluble vitamins are excreted leading to weight loss. 20 University of Ghana http://ugspace.ug.edu.gh The asymptomatic microfilaraemic state is not benign as was previously thought. Two sets of recent observations have revealed that this being clinically "asymptomatic" does not imply freedom from morbidity. It was recognized that most of the microfilaraemic individuals have haematuria and/or proteinuria which reflects low-grade renal damage which does not appear to be reversible after treatment (Dreyer et al, 1992). Secondly, using lymphoscintigraphy to visualize by radioisotope tracer techniques, it was found that almost all the microfilaraemic individuals even though asymptomatic, had markedly abnormal patterns of lymph flow (Freedman et al, 1994). 3.1.3 Mechanism of pathology Experimental model systems have provided clear evidence that portion of the pathology resulting with bancroftian and brugian parasites is derived from the direct action of the parasites themselves or their released molecules on the lymphatic tissue, while the remainder results from the host’s immune response to these parasites (Ottesen, 1991). It has also recently been observed that there is the danger of secondary bacterial and/or fungal infection in patients with lymph stasis and this may also contribute in part to the pathology in infected individuals (WHO, 1994). Most of the pathology associated with bancroftian filariasis derives from damage to the lymphatics which ultimately leads to chronic lymphoedema, elephantiasis or chyluria. Normal lymph vessels are delicate endothelium-lined channels leading from an extensive network of 21 University of Ghana http://ugspace.ug.edu.gh peripheral lymph capillaries through a series of collecting vessels and intermediate lymph nodes to the large cisterna chyli and thoracic duct that empties into the vena cava. In infected individuals, adult worms reside within these lymphatic vessels, generally in the afferent approaches or cortical sinuses of the lymph nodes. The parasite first causes dilatation of the lymphatic and then hypertrophy of the vessel wall. There is endothelial and connective tissue proliferation with polypoid growth protruding into the lumen, but so long as the worm remains alive, it appears the vessel stays patent. This patency, however, does not ensure normal lymphatic function and lymph stasis may occur while the worm is still alive. This may result in early lymphoedematous changes in the affected limb, genitals, kidney or breasts. Dreyer et al (1994) using ultrasonography demonstrated that adult worms located in the lymphatic vessels show marked stability in their location and did not appear to be disturbed by several potential sources of stress such as light, temperature change and manipulation of the lymphatic vessel of any kind. In ultrsonography, adult worms demonstrate a peculiar form of movement called " filarial dance sign" Lymphatic pathology resulting from the presence of living worms include lymphatic proliferation, dilatation and oedema formation. Adult worms frequently invade the inguinal glands resulting in lymph stasis and this is the main factor in the formation of elephantiasis. There is the formation of extensive fibrosis in these nodes and this is an indication of lymphatic obstruction (Jordan, 1955). Obstructive obliterative reactions in the lymphatics results from dead parasites. When adult worms die, their death is associated with distinctive pathological changes. An area of necrosis develops around the dead parasite, followed by a granulomatous reaction 22 University of Ghana http://ugspace.ug.edu.gh with the formation of cells and eosinophils. Collagen deposition results and the parasite is either resorbed or becomes partially calcified. It is during these inflamatory reactions that lymphatic obstruction occurs. The manner in which an infected individual reacts immunologically to filarial antigen may determine whether or not lymphatic pathology will develop (WHO, 1992). The immune response to infection varies considerably, with antibody response the lowest in asymptomatic microfilaraemic patients and highest in amicrofilaraemic patients (Ottesen, 1982). In humans, IgG3 antibody response to filarial antigens are known to be made almost exclusively by patients who develop lymphatic pathology whereas IgG4 predominates in asymptomatic but microfilaraemic individuals (Hussain et al, 1987). Ottesen (1984) reported that in an endemic area, all exposed individuals mount cellular and humoral responses to the parasite and the measured levels of these responses vary among individuals but they tend to be similar and characteristic for those with similar clinical manifestation of infection and as such, they appear to be the determinants of clinical expression of filariasis. The different types of immune responses and thus pathological reactions are known to be genetic-based and are determined by differences in genetically restricted major histocompatibility complex-linked antigen processing in different individuals (Kwan-Lie et al, 1990). Tropical pulmonary eosinophilia (TPE) syndrome is the result of an immunological hyperresponsiveness on the part of the host to the parasite (WHO, 1984). Observations on the pathogenesis of TPE suggests that microfilariae released into the circulation from adult worms dwelling in the lymphatics are rapidly opsonized with antifilarial antibody and are then cleared in the pulmonary vasculature (Udwadia, 1975). 23 University of Ghana http://ugspace.ug.edu.gh According to WHO (1992), patients with TPE have markedly increased numbers of inflammatory cells infiltrating the lungs and the majority of these cells are eosinophils. TPE is mediated by IgG antibody and the asthmatic symptoms resulting from the allergic responses is mediated through specific IgE antibodies bound to mast cells (Manson-Bahr et al, 1987). The acute phase is caused by an antibody-dependent mechanism while the chronic phase which is marked by pulmonary fibrosis may result from a type II delayed hypersensitivity response caused by an increase in or inadequately suppressed pulmonary lymphocyte reaction. This immunological hypersensitivity is found only in certain hosts and is probably of genetic origin. Prenatal conditioning could influence subsequent immunological and thus, pathogenic responses to filarial infection (Ottesen, 1991). Recent studies in Haiti have shown that maternal microfilaraemia predisposes to microfilaraemia in offsprings, and this is very important in explaining the differences in clinical manifestations of lymphatic filariasis among residents in endemic areas and those who migrate to such areas (WHO, 1992). In many patients, there is the occurrence of intermittent episodes of lymphangitis or adenolymphangitis (ADL). This may be the only indication of filarial infection for many patients (Ottesen, 1982). The characteristic features of these episodes include the development of painful, tender lymph nodes with or without "retrograde" extension of the inflammation to involve the lymphatic tracks. This happens with a usual frequency of once or twice a year (but sometimes five or six times yearly.) 24 University of Ghana http://ugspace.ug.edu.gh Recently, bacterial and fungal infections have been identified to be responsible for part of the pathology due to W. bancrofti. Recent evidence both from astute clinical observations and from immunohistological and bacteriological studies of tissues from lymphoedematous limbs of affected patients, has suggested that bacterial or fungal superinfections of limbs with compromised lymphatic function play the primary role in triggering most episodes of adenolymphangitis which, themselves, actually cause or exercebate the elephantiasis changes in affected patients (Addiss et al, 1994). 3.1.4 Diagnosis Definitive diagnosis of lymphatic filariasis requires direct demonstration of the microfilariae since the adults worms are extremely difficult to detect. There are several methods available for diagnosis but the choice of method depends on its availability and the requirements of the particular study. The traditional or conventional methods of detecting microfilariae in the blood of infected persons involve the use of giemsa-stained smears, the counting chamber technique (CCT), the membrane filtration technique and the Knott’s concentration method. However, diagnostic methods based on detection of microfilariae are often of low sensitivity because microfilariae can be present in very low numbers or sequestered in inaccessible sites, or they can be absent as in prepatent cases. Blod sampling may also be inconvenient particularly in those geographical regions where they exhibit nocturnal periodicity (Wamae, 1994). Alternatively, more recent techniques involving immunodiagnostic tools, lymphoscintigraphy and ultrasonography have been devised, even though they are not routinely employed for diagnosis. 25 University of Ghana http://ugspace.ug.edu.gh 3.1.4.1 Giemsa-stained thick smear: This method involves the collection of blood by finger prick unto a clean slide by using a sterile lancet, drying the blood and dehaemoglobinizing in clean tap water. The sample is dried again and fixed in methanol after which it is stained in Giemsa solution. The method is used to identify and quantitatively determine microfilarie. It is the most widely used method for field surveys. It is also a reliable method for qualitative and quantitative microfilarial studies and the processing of the blood slides need not be done on the spot. The method also provides permanent records. However, it is argued that some of the parasites could be lost during processing and staining of the blood films (Denham et al, 1971). 3.1.4.2 Counting Chamber Technique (CCT): This method involves the use of a counting cell which holds 100 cubic millimetres of liquid lmillimetre deep over an area of 50 X 20 millimetres. The base is divided into 1 millimetre squares. A cover glass traps liquid to the correct depth and a known volume of capillary blood (eg. 100/xl, 60/xl) washed into 3% acetic acid is poured into the chamber. By observing the liquid through a low magnification microscope, objects contained in each cubic millimetre can be identified and counted. The method allows fast and accurate counts of microfilariae to be made and it has been shown by (Denham et al, 1971) to be 30-40% more sensitive than the conventional stained blood smear. It also has a great advantage over other techniques due to the fact that positive results are demonstrable at the site of examination making it an excellent visual aid for education in the communities. However, this method cannot be used to distinguish species and should only be used in areas where the parasite species has previously been determined (Wamae, 1994). 26 University of Ghana http://ugspace.ug.edu.gh 3.1.4.3 Membrane (Nuclepore) Technique: This method involves the use of a nuclepore membrane filter with a pore size of 3-5/xm into a filter holder and adding already collected anticoagulated venous blood by the use of a syringe and plunger. The blood is expressed through the filter to filter out microfilariae which are collected. The filter is then stained with Giemsa and examined under the microscope. This technique is sensitive and allows large volumes of blood to be examined and therefore enhances the detection of low level microfilaraemia (Wamae, 1994). McMahon et al (1979) working in Tanzania, compared this method with the CCT and the conventional thick smear and concluded that the use of the membrane filtration detected a proportion of infected individuals who would have been reported negative by the other methods. Membrane filters are however to be used in areas where low microfilaraemia is expected. The use of this method also involves venipuncture which is opposed by many communities. 3.1.4.4 Knott’s Concentration Method: In this method, 1ml of blood is diluted in 9ml of distilled water and 1ml of formalin and centrifuged in a tube. After leaving the tube for some hours, the supernatant is poured off and the sediment which contains the microfilariae is examined. This method is however impractical where venipuncture is unacceptable (Wamae, 1994), and examination of the precipitate may be difficult since it may have a jelly-like consistency (WHO, 1994). 27 University of Ghana http://ugspace.ug.edu.gh 3 .1.4.5 Immunodiagnosis: Immunodiagnostic procedures have become available for the diagnosis of filariasis for sometime now. Filarial nematodes are known to release antigenic products and elicit strong humoral antibody responses in human hosts (Ottesen, 1982). Both antigen and antibody detection assays have been developed as tools for the diagnosis of filarial infection. The importance of these assays lies in their ability to detect circulating antigens in both microfilaraemic infections (regardless of the time of day the blood is collected) and amicrofilaraemic cryptic infections. The antibody detction assays however have their drawbacks. Thus antibody responses appear to be persistent even after clinically defined cure has been achieved and they are therefore unable to discriminate between past and present infection (Lammie et al 1988). Also,the magnitude of the responses bear no relationship to parasite burden (Ottesen, 1984). More promising are recently developed assays for detection of filarial specific circulating antigens. Thus the Og4C3 antigen detection ELISA, which has recently become commercially available (Turner et al, 1993), appears to have a sensitivity and specificity close to 100%. 3.1.4.6 Lymphatic imaging: Recently, lymphoscintigraphy using radiolabelled colloid, albumin or dextran injected intradermally or subcutaneously and traced by a gamma-counter is being used to demonstrate the presence of lymphatic abnormalities in asymptomatic microfilaraemic individuals with no evidence of oedema. Their lymphatics are markedly dilated, with collateral channels. With this diagnostic method, it has now become possible to make a clear and precise analysis of the lymphatic system function in patients at risk (WHO, 1994). 28 University of Ghana http://ugspace.ug.edu.gh 3.1.4.7 DNA-detection assays: DNA-based technology can now be used for diagnosis of filarial infection both in humans and in the mosquito vectors by polymerase chain reaction (PCR-based) assays. Species-specific DNA probes developed so far detect DNA sequences that are highly repeated in the filarial genome and they are theoretically sensitive enough to detect DNA from a single filarial larva (WHO, 1992). 3.1.4.8 Ultrasonography: By using the method of ultrasonography, adult worms can be visualized in the lymphatics. Amaral et al (1994) used this method to describe adult worms performing " filaria dance" in the lymphatics. The visualization of adult worms by ultrasound is now available as a potential tool for direct assessment of the efficacy of adulticidal drugs. 29 University of Ghana http://ugspace.ug.edu.gh 3.2 MATERIALS AND METHODS 3.2.1 Study design Every individual registered during the census was assigned a unique project number (PN). The PN of an individual indicated the village coded by two letters, followed by the house number, and a serial number within the community. For example, a man living in a house with house number OK045 who was registered as the 100th person in the community will have his PN as OK045100. This method is unique and has the added advantage of locating or tracing the person should the need arise. Fifty percent of the residents were selected from the compiled demographic data to constitute the study population. A simple record form which contained information on personal history, clinical examination and blood sampling time with the volume of blood collected was designed for each study individual. Refer to appendix 2. 3.2.2 Blood Collection for CCT: Before each blood collection day, sampling tubes which are small 3ml test tubes were thoroughly cleaned with their lids and dried. Each was filled with 900/xl of 3% acetic acid and stored in a rack. A central point was selected in the community where blood samples were collected. Blood collection was carried out between 21:00 and 01:00hrs. The pulp of the finger was cleaned with mediswab and the surface of the skin allowed to dry by allowing the excess alcohol to evaporate. A sterile lancet was used to make a perpendicular cut on the finger. The blood was allowed to flow freely and collected into a 100/d heparinized capillary tube and with a rubber bulb, the blood was transferred into the sampling tubes and mixed gently. It is stored well in the rack for examination at a later date. The tube was labelled with the date of blood examination and the PN of the individual. The volume of blood collected and the time of 30 University of Ghana http://ugspace.ug.edu.gh collection were recorded on the record form. Occasionally if it became difficult to get the desired amount, the estimated volume taken was recorded and taken into consideration in the analysis. 3.2.3 Using the CCT: For the examination and counting of the specimen, the fluid was transferred from the sampling tube to the counting chamber by pouring gently. A pasteur pipette was used to spread the fluid out to fill the whole well of the counting chamber, care taken not to damage any microfilariae. Any air bubble in the specimen was removed with a needle. The counting chamber with the specimen was placed under the microscope and left to stand for approximately 3-5 minutes, to enable the microfilariae settle at the bottom of the chamber. The specimens were then examined under the microscope with a magnification of 40X. The CCT enables speedy and accurate counts of microfilariae to be made. 3.2.4 Thick Blood Smear Preparation for Microfilariae Identification Glass slides were cleaned in a watery solution of detergent and immersed in 96% alcohol. They were then dried and polished with a grease-free cloth, then stored in a slide box and taken to the field. To collect blood samples, the pulp of the finger was cleaned thoroughly with a mediswab, the excess alcohol allowed to evaporate and sterile lancet used to make a perpendicular cut on the finger which was held firmly between the collector’s thumb and index finger. Care was taken not to squeeze the finger too much to dilute the blood sample with tissue fluid. The blood was allowed to flow and drawn into the 100/xl heparinized capillary tube. Care 31 University of Ghana http://ugspace.ug.edu.gh taken to avoid air bubbles in the tube which may result in underestimation of blood volume. The blood was expelled onto the cleaned microscope slide which has been already labelled with the individual’s PN and date, using a rubber bulb. A rectangular thin smear of uniform thickness of blood was made and allowed to dry at room temperature in a horizontal position in a dust-free environment. Dry specimens were then sent to the laboratory for staining and examination later on. The blood samples for thick smear were collected at the same time blood was collected for CCT. 3.2.5 Giemsa-staining Before staining, the smears were dehaemoglobinized in clean tap water for approximately 1-2 minutes, by immersing the slide with the dried blood in a vertical position in a staining jar in which tap water has been placed. The blood cells lyse and the pigment was seen falling into the water. This was done only for few minutes to avoid the likelihood of microfilarae falling off. The slides were removed and allowed to dry in the open air and fixed in methanol for approximately lminute and allowed to dry again. The fixed smears were then stained in a diluted Giemsa solution (one part of Giemsa stock solution in 14 parts of buffered distilled water of pH 7.2) in a staining jar for half an hour. The slides were then rinsed briefly in clean water to remove excess stain and dried in upright position. They were then examined under the microscope with magnification of 40-100X under oil immersion. 32 University of Ghana http://ugspace.ug.edu.gh 3.2.6 Clinical Examination A house was selected in the study village as a private examination room and all the selected persons were examined during the day by the clinician supervisor. All individuals aged 2r6 months were physically examined for evidence of acute and chronic signs and symptoms of bancroftian filariasis. Physical examination of males included the genitals, the legs and arms, and the lymph glands in the groin and axillae. Female examination was usually restricted to the legs and arms. The breast and genitals were to be included only when there was a history of their involvement. However, no such involvement was observed. Clinical manifestations were graded as described by Estambale et al (1994). Chronic manifestation of hydrocoele were graded into four stages. Grade I showed a swollen spermatic cord with either fluid accumulation somewhere along the cord, a lymphocoele above the testis or a testis larger than 6cm in longitudinal diameter without fluid accumulation. True hydrocoeles were graded as grades II- IV Grades II,III and IV were 6-10cm, ll-15cm and >15cm respectively, in longitudinal diameter with accumulation of fluid. Leg elephantiasis was graded as I when there is loss of contour and pitting oedema. Thickened skin with loss of elasticity and non-pitting oedema was graded as II, and grade III was evident elephantiasis with skin folds and papules. During the examination, males were asked if they had had a hydrocelectomy. 3.2.7 Data management and analysis The number of parasites counted for each microfilaraemic individual were multiplied by a conversion factor and expressed as microfilariae per millilitre of blood. The geometric mean intensity (GMI) of microfilarial intensity was calculated as [Elog (x+ l)/n ], where x is the 33 University of Ghana http://ugspace.ug.edu.gh number of microfilariae per millilitre of blood in microfilaraemic subjects examined and n is the number of microfilaraemic subjects examined. Data on microfilariae prevalence, GMI, and the results of clinical manifestations resulting from infection of the parasite were entered into the computer using the dBase programme and analysed with SPSS. Microfilaraemia prevalence in different groups of subjects (sex and age groups) were compared statistically using the chi- squared test and microfilarial GMI in the different groups of subjects were compared with Student’s t-test and ANOVA on log-transformed values. All P values of less than 0.05 were considered to indicate statistical significance. 34 University of Ghana http://ugspace.ug.edu.gh 3.3 RESULTS 3.3.1 Distribution of Selected Persons by Age and Sex Table 2 shows the distribution of persons who were selected to participate in the study by age and sex and the proportion of those examined. Overall, 312 persons were selected and out of these, 296 making 94.4% of the selected population were examined for microfilaraemia and clinical manifestations of bancroftian filariasis. In most of the age groups, more females participated than males. Participation in the older age groups (ie >50 years) was high and in two age groups, 50-59 and ^6 0 , all the selected persons reported for examination, making 100% participation in these two age groups. The age group 40-49 however was poorly represented as only 57.1% of those selected came for examination. In the age group 10-19, 54 persons reported for the study out of the 60 selected, giving a coverage of 90%. However, during examination, 14 additional volunteers were examined resulting in 68 persons being examined. 35 University of Ghana http://ugspace.ug.edu.gh Table 2. Distribution of study population by age and sex No. examined (% of selected) Age-group Total (Years) No. selected Males Females Total 1-9 99 54(54.5) 35(35.4) 89 (89.9) *10-19 68 42(70.0) 26(43.3) 68(113.3) 20-29 40 15(37.5) 23(57.5) 38 (95.0) 30-39 36 7(19.4) 23(63.9) 30 (83.3) 40-49 28 9(32.1) 7(25.0) 16 (57.1) 50-59 19 7(36.8) 12(63.2) 19(100.0) .>60 36 6(16.7) 30(83.3) 36(100.0) Total 312 140(44.9) 156(50.0) 296 (94.4) *: 14 additional volunteers participated in this age group. 36 University of Ghana http://ugspace.ug.edu.gh 3.3.2 Microfilariae identification Microfilariae of Wuchereria bancrofti were identified after staining with Giemsa. In all, 50 samples were prepared and of these, 7 were identified as containing varied numbers of microfilariae. Identification was based on morphological characters such as pointed tail free of nuclei and body curve sinous, among other criteria. 3.3.3 Microfilaraemia Prevalence Results of microfilaraemia prevalence are presented in Table 3 and Figure 3. There were a total of 78 individuals infected with W. bancrofti, giving an overall prevalence rate of 26.4% in the community. Generally, prevalence was slightly higher in females than in males. However, the difference in prevalence between males and females was not statistically significant in any age group (x2 : P > 0.05) and in total prevalence in the two sexes (x2: P > 0.05). Of the 78 microfilaraemic individuals, 43(27.6%) were females while 35(25%) were males. In both sexes generally, microfilaraemia prevalence increased with age in the first 3 age groups after which it decreased in the 30-39 age group. After this, prevalence increased again until the 50-59 age group and reduced in persons above 60years. The same trend of increasing prevalence with increase in age is noted in the total prevalence of each age group until it again reduced in the 30-39 year group with subsequent increase thereof. The highest prevalence of 66.7% was in males in 40-49 age group while the lowest of 5.7% occurred in females 1-9 years. The youngest microfilaraemic person is a 3year-old girl with microfilarial intensity of lOmf/ml of blood. 37 University of Ghana http://ugspace.ug.edu.gh able 3. Microfilaraemia prevalence by age and sex \ge-group No. No.with microfilariaeforevalence in %) Years) examined Males Females Total 1-9 89 6(11.1) 2(5.7) 8(9.3) 10-19 68 11(26.2) 4(15.4) 15(22.1) 20-29 38 5(33.3) 10(43.5) 15(39.5) 30-39 30 2(28.6) 7(30.4) 9(30.0) 40-49 16 6(66.7) 1(14.3) 7(43.8) 50-59 19 4(57.1) 6(50.0) 10(52.6) .>60 36 1(17.1) 13(43.3) 14(63.9) Total 296 35(25.0) 43(27.6) 78(26.4) 38 University of Ghana http://ugspace.ug.edu.gh P R E V A L E N C E (% ) FIG. 3 PREVALENCE OF -MICROFILARAEMIA IN THE D IF F E R EN T AGE GROUPS -“ -M ALES + F E MALES X t o t a l S T University of Ghana http://ugspace.ug.edu.gh 3.3.4 Microfilarial Intensities Microfilarial intensities varied from 10-19240mf/ml of blood among micrifilaria positive individuals. For all microfilaraemic individuals in the community, the geometric mean intensity (GMI) was 819mf/ml of blood. It was higher in infected males (1114.3mf/ml) than in infected females (645.7mf/ml). However, microfilarial GMIs did not differ significantly between males and females in any age groups (two-way ANOVA; P > 0.05) or in total GMI between the two sexes (students’ t-test; P > 0.05). In most age groups, the GMI was higher for males than for females. In males, except for the age groups 30-39 and 50-59, GMI increased with age. In females however, the variation of GMI with age does not follow a commendable order. Males in the age group, 20-29 had the highest GMI of 6194.4 compared to females in this age group who had 613.8. The lowest GMI of 218.8 ocurred in 40-49 age group (Table 4). The highest individual microfilarial intensity was 19270mf/ml. Table 4. Geometric mean intensity of microfilaraemia by age and sex at Okyereko Age-group (Years) Geometric Mean Intensity Males Females Total 1-9 1091.4 83.2 562.3 10-19 1422.3 995.4 1358.3 20-29 6194.4 613.8 1000.0 30-39 524.8 677.6 774.5 40-49 582.1 218.8 517.6 50-59 298.5 867.0 501.2 >60 724.4 744.7 1000.0 Total 1114.3 645.7 819.0 40 University of Ghana http://ugspace.ug.edu.gh GM I (m f/ m l in th ou sa nd s) GEOMETRIC MEAN INTENSIFY ' (GMI) OF MICROFILARAEMIA BY AGE AND SEX AT OKYEREKO 4-1 University of Ghana http://ugspace.ug.edu.gh 3.3.5 Clinical Manifestations 3.3.5.1 Hydrocoele Of the 140 males examined clinically for hydrocoele, 13 had hydrocoeles of grades I-IV giving a hydrocoele prevalence of 9.3 %. All hydrocoele cases were however seen in males aged £ 18 years. There were 8 true hydrocoeles (ie grades II-IV) giving the prevalence of true hydrocoeles as 5.7% while 5 of the hydrocoele cases were grade I (ie 3.6% prevalence rate)(Table 5). The prevalence of hydrocoele increased with age and the highest prevalence of 33.3% was noted in the 40-49 age group. This was followed by 28.6% prevalence in the 50-59 age group. The commonest grades of hydrocoele were I and II occuring in 80% of hydrocoele cases. The only grade IV hydrocoele was in a 19 year old boy. Three males with hydrocoele were microfilaraemic including the case of the grade IV hydrocoele who had microfilarial intensity of 1130 mf/ml. This gives a prevalence of microfilaraemic hydrocoele cases as 23.1% The other two aged 42 and 56 and both with grade I hydrocoeles had microfilarial intensities of 20 and 70 mf/ml respectively. Table 5. Prevalence of Hydrocoele in males with respect to age group. Age-group ; Years) Hydrocoele No. Examined No. with Grade I No. with Grades II-IV Total no. (Prevalence in %) 0-9 54 0 0 0 (0.0) 10-19 42 1 2 3 (7.1) 20-29 15 1 2 3 (20.0) 30-39 7 1 0 1 (14.3) 40-49 9 1 2 3 (33.3) 50-59 7 1 1 2 (28.6) ^.60 6 0 1 1 (16.7) Total 140 5 8 13 (9.3) 42 University of Ghana http://ugspace.ug.edu.gh 3.3.5.2 Limb Elephantiasis The results of limb elephantiasis prevalence are shown in Table 6. Of the 296 persons examined clinically, 4 had elephantiasis and all 4 were females aged 13 to 55. This gives an overall prevalence of limb elephantiasis as 1.4%. Two were grade I and the other two grade III. There was no grade II elephantiasis. As in the case of hydrocoele, the prevalence of elephantiasis increased with age. The highest prevalence of 12.5% was in the 40-49 years age group and this formed 50% of the cases who were all grade III. Generally, elephantiasis was predominant in the older age groups (ie 40 years and above) but there was a case of grade I elephantiasis in a 13 year-old girl. One of the elephantiasis females was microfilaraemic with intensity of 220mf/ml and elephantiasis of grade III. Table 6. Prevalence of elephantiasis in both sexes with respect to age group. Elephantiasis A.ge-group Tears) No. Examined Grade I Grade II Grade III Total Prevalence (%) 0-9 89 0 0 0 0 0 10-19 68 1 0 0 1 1.5 20-29 38 0 0 0 0 0 30-39 30 0 0 0 0 0 40-49 16 0 0 2 2 12.5 50-59 19 1 0 0 1 5.3 >.60 36 0 0 0 0 0 Total 296 2 0 2 4 1.4 43 University of Ghana http://ugspace.ug.edu.gh 3.4 DISCUSSION Information on the prevalence and distribution of any disease is necessary for determining its public health importance and the subsequent planning of any control programme. This is because the evaluation of any control programme must have a reference point if it is to be meaningful. According to WHO (1992), data on the prevalence of lymphatic filariasis in sub- Saharan Africa is very limited. As a result therefore, the true picture of the disease burden is not known. This has made it difficult to assess the public health importance of the disease in this region. This study has identified lymphatic filariasis as a major public health problem in the study community. Apart from earlier epidemiological surveys in the Kassena-Nankana District (Gyapong et al, 1994), a national filariasis survey (Gyapong et al, 1996) and reports of an epidemiological study along the coast of Ghana (Dunyo et al, 1996), a detailed information on the prevalence and distribution of the disease in an irrigation project community with conditions that favour the profuse breeding of the mosquito vectors involved in transmission have not been documented in Ghana. Earlier reports by Hunter (1992) identified small village dams in north east Ghana as potential transmission areas for lymphatic filariasis. In this report, Hunter pointed out that the closer a house is to the main canals in an irrigation community, the greater the frequency of filarial disease and the key to understanding this observation is the intensity of mosquitoes biting. 44 University of Ghana http://ugspace.ug.edu.gh The overall microfilaraemia prevalence of 26.4% reported in this study indicates a high level of endemic filariasis, according to the World Heath Organisation (OMS, 1988). The prevalence of microfilaraemia is similar to those reported in Kassena-Nakana (Gyapong et al, 1994), the endemic areas on the coast of Western Region of Ghana (Dunyo et al, 1996) and Indian Ocean coast of Kenya and Tanzania (Estambale et al, 1994, Wijers,1977, Meyrowitsch et al, 1995, and Simonsen et al, 1995). A lower prevalence rate of 9% had been reported in the Igwun Basin of Nigeria (Udonsi, 1988). The slightly higher microfilaraemia prevalence in females than males does not conform with the trend in most reports (Estambale et al, 1994, Gyapong et al, 1994, Simonsen et al, 1995). Several investigators have suggested that females, especially those of childbearing age, are more resistant to infection of same age group and this was supported by serological studies which showed a higher antibody positivity to adult worm antigen in females (Brabin, 1990). However, observations by Brunhes (cited by Wijers, 1977) concluded that in high transmission areas, the differences in microfilariae rates generally observed between males and females tends to disappear because of the higher microfilarial densities in these areas. Jordan (1960) stated also that in many parts of Africa, the night-biting A. gambiae s.l and A. funestus were the main vectors and it was unlikely that one sex would be more exposed than the other. Specific physiological as well as behavioural and occupational differences between the two sexes therefore could be a likely predisposing factor affecting parasite establishment in the two sexes. However, very little differences between male and female occupations and behaviour patterns were observed in the community. 45 University of Ghana http://ugspace.ug.edu.gh Microfilaraemia was generally rare in young children and prevalence generally increased with age and were high in the older age groups. If young children are equally exposed to moquito bites in the community, then the rare prevalence rate in them is surprising, especially when the pre-patent period of only 7 to 8 months is expected for the establishment of W. bancrofti (WHO, 1992). However, the rare occurrence of microfilaraemia in young children might be related to the finding in transmission studies that high numbers of infective bites over a prolonged period of time are necessary before establishment of a patent infection with microfilaraemia will occur (Southgate, 1992). Parasite induced tolerance is also said to appear to play a role for survival of filariae in their hosts (Ottesen, 1989) and long exposure period may be necessary for the parasites to induce a state of tolerance in the host before an infection can develop to patency. Steel et al (1994) in explaining the role of the immune system in the development of lymphatic pathology have shown that the microfilaraemic status of the mother during pregnancy influences the immune response of an individual to microfilarial antigens and thus children bom of microfilaraemic mothers are much less capable of responding to microfilarial antigens than those of noninfected mothers even 17 years after birth. The overall mean microfilarial intensity (GMI) was higher in males than in females. However, an immense variation was observed in individual microfilarial intensities. For most age groups, GMI was higher in males than in females. But this was not significant in any age group. The higher GMI in males than in females in most age groups is in accordance with reports by Bundy (1988) that males are more susceptible to parasitic infections than females. As most blood samples were obtained around the same time (ie between 21:00 and 02:00hrs), microfilarial 46 University of Ghana http://ugspace.ug.edu.gh periodicity cannot be assumed to be resposible for these variations. However, the abnormally very high GMI of the five males in the 20-29 age group can be explained that all of them reported for blood sampling late (four of them were sampled at 01:25, 01:35, 01:45and 02:00hours) and this period happens to coincide with the peak hour from the periodicity study when maximum numbers of the parasite appear in the peripheral blood. Adult worms are only accidentally recovered from humans, and whether microfilarial intensity reflects adult worm burden is unknown. Studies on circulating antigens from adult W. bancrofti by Lammie et al (1994) have shown a significant, although weak correlation between antigen concentrations and microfilarial intensities. Therefore what actually determines the variations in microfilarial intensities in infected individuals (whether adult worm burden, host regulation mechanisms or both) and the reasons for the great variability between individuals remain unclear (Meyrowitsch et al, 1995). The GMI of all microfilaraemic persons were comparable with what has been found in other parts of Ghana and Africa. Gyapong et al (1994) recorded a GMI of 794mf/ml of blood in Kassena-Nankana. Along the coast of Ghana, Dunyo et al (1996) working in six villages and three urban communities recorded an overall mean intensity of 321-1172mf/ml of blood in microfilarial positive villages. In the Kwale District, Kenya, Estambale et al (1994) reported a lower GMI of 223mf/ml of blood. In North-eastern Tanzania, Meyrowitsch et al (1995) reported a range of 331-1202mf/ml. 47 University of Ghana http://ugspace.ug.edu.gh Hydrocoele was the commonest chronic clinical manifestation of bancroftian filariasis in the community. A prevalence of 9.3 % was seen in males aged £ 18 years. The prevalence of limb elephantiasis was 1.4% of those examined. It is considered that hydrocoeles may form more easily than limb lymphoedema because of the smaller increase in lymphatic pressure involved (Harinath, cited by Brabin, 1990). A higher prevalence of hydrocoele was reported in Kassena- Nankana (Gyapong et al, 1994) and (Dunyo et al, 1996). In other parts of Africa, higher prevalences were reported (Meyrowitsch etal, 1995, Wijers, 1977, Estambale et al, 1994). The prevalence of hydrocoele generally increased with age and the highest prevalence was noted in males above 40 years. All persons with elephantiasis were females and the fact that this condition has severe personal and social consequences cannot be underemphasized. Generally, the prevalence of elephantiasis increased with age and it is predominant in the older age group (ie above 40 years). This may have a detrimental effect on the mobility, working capacity and social acceptance of the affected individuals (Evans et al, 1993). All the elephantiasis cases were seen only in females and similar higher prevalence in females than males was reported by Gyapong et al (1994) and Dunyo et al (1996) from other parts of Ghana. There was also a higher prevalence of microfilaraemia in females than males in the study community. However, with the very low numbers, this may be due to chance if not occupationaly-related. The study community is predominantly a farming community with majority of inhabitants being peasant fanners engaged actively in working on the rice farms on the irrigated plots with the two sexes being equally exposed to frequent lacerations or cuts at the lower extremities. Therefore, occupational differences could not be the likely factor. 48 University of Ghana http://ugspace.ug.edu.gh There is similarity in the prevalence of microfilariae in persons with the two clinical outcomes of bancroftian filarial infection in the community (ie hydrocoele and limb elephantiasis). Three males with hydrocoele were microfilaraemic, and one of the elephantiasis cases was also microfilaraemic. A similar pattern of low microfilaraemia in elephantiasis patients has been reported by Simonsen et al (1995) in three endemic communities in Tanzania. A correlation between the severity of lymphatic lesions and host response, either cellular or humoral to parasite antigens has been described by Ottesen (1992) and if host responses are enhanced during reproductive years as proposed by Brabin (1990), then clinical disease might be expected in these groups of persons. However, in the study, there seems to be very little evidence that this occurs and overall, elephantiasis is more prominent in the older age groups. 49 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 4.1 PERIODICITY STUDY OF LYMPHATIC FILARIASIS PARASITE 4.1.1 Introduction Infection of humans with Wuchereria bancrofti begins with a bite by an infected mosquito which introduces the parasite into the host. The adult worms live in the lymph channels and paired adult worms produce millions of larval forms called microfilariae that circulate in the bloodstream. The density of microfilariae present in the peripheral blood varies throughout a 24-hour cycle. Depending on the time of the day that the maximum numbers appear in the peripheral blood in a 24-hour cycle, the periodic cycle may be referred to as nocturnal or diurnal (McMahon et al, 1996). Also, variations in completeness (ie the difference between the numbers of parasites present at the peak and the low point of the cycle) results in the cycle being referred to as periodic or subperiodic. In nocturnally periodic W. bancrofti for example, this difference is almost 100%, whereas in Dirofilaria immitis in the dog, although a definite peak occurs during the evening, approximately 30% of this number is present throughout the whole cycle and this state is defined as subperiodic (WHO, 1967). The nocturnally periodic type thus shows a marked peak of microfilarial density in the peripheral blood during the night hours, very few if any, during the day. In the nocturnally subperiodic and diumally subperiodic types, microfilariae can be found in the peripheral blood at all hours but their density increases slightly during the night or day respectively. Manson first observed the phenomenon of periodicity in 1879 in Amoy (South China) where he found that the parasites were numerous in the blood of infected patients at night but became rare or absent from the blood during daytime. The periodicity occurs as the result of retention of large numbers of microfilariae in the lung 50 University of Ghana http://ugspace.ug.edu.gh capillaries during certain hours of the day and their release into the circulating blood during other hours (Sasa, 1976). Various hypotheses or theories have been proposed as to the host or environmental factors responsible for such a circadian rhythm. In most instances, the pattern of periodicity of a form of microfilaria appears to be synchronized with the biting habits or activity of the mosquito vectors in the same area, thus ensuring a high level of transmission of the infection (Hawking, 1967, Sasa, 1976). Periodicity of microfilariae however can be easily converted by a change in the rhythm of the day (Manson-Bahr et al, 1987). Thus the periodic cycle is stable under "natural" conditions in a given locality but depends upon the activity of the host in relation to the day to night cycle. The numbers of microfilariae are influenced by sleeping and respond to waking and bodily activity. By reversing the hours of sleeping and waking, the periodicity is disturbed for 3 days and then reversed to diurnal periodicity. The parasite thus has the ability to adjust its cycle, after a brief delay, to changes in the host’s activity cycle. This has been demonstrated many times for W. bancrofti and for other filariae in humans. 4.1.2 Mechanisms of Periodicity Two mechanisms for periodicity have been suggested. These include the alteration in oxygen tension of the blood and phototaxis on the part of the microfilariae. The periodicity of W. bancrofti may depend on changes in the difference in oxygen tension between venous and arterial blood by day and night (Manson-Bahr et al, 1987). During daytime, the microfilariae accumulate in the lungs where the oxygen tension is high and they manage to hold themselves in the pulmonary capillaries by some force which is increased by the rise in oxygen tension and 51 University of Ghana http://ugspace.ug.edu.gh decreased by its fall. This force seems to be switched on and off every 12 hours by an unknown mechanism inside the microfilariae (Manson-Bahr et al, 1987). It is presumed that microfilariae gather together in the capillaries and other vessels of the lung during their absence from the peripheral blood by the power of agglutination and thigmotaxis (which is the movement of the microfilarie in response to the stimulus of contact or touch to the lungs). By injection of an anticoagulant, heparin, to the drawn blood, a curious agglutinative phenomenon has been described. Intravenous injection of this subtance during daytime releases microfilariae into the peripheral blood for a short period. Hawking (1967) presents evidence that the migration of microfilariae is related to responses to circadial variations in host’s physiological factors such as oxygen tension and temperature. For example, in man, the venous-arterial difference in oxygen tension is lower by night (40 mmHg) than by day (55 mmHg) with the result that microfilariae of W. bancrofti pass through the lungs by night but accumulate there by day. This pattern can thus be upset by causing a patient to breathe oxygen at night. A different mechanism for periodicity has been suggested in which the microfilariae are said to possess a photosensitive substance containing a vitamin A-like carotenoid similar to visual pigments in flourescent granules in the epidermis which causes them to leave the peripheral circulation in daylight and collect in the lungs. Periodic microfilariae therefore possess numerous granules in contrast to subperiodic forms which have few or none, Masaya, cited by (Manson-Bahr et al, 1987). Studies by a number of workers in various regions of the world have revealed that there exists different forms even within the same species which differ in the pattern of microfilarial periodicity (Sasa, 1976). Thus differences in the nature of the periodic cycle may occur not 52 University of Ghana http://ugspace.ug.edu.gh only between species of filariae but between "varieties" within a single species. W. bancrofti and Brugia malayi both occur in two forms within humans. Throughout the major part of its geographic range, W. bancrofti is nocturnally periodic, but in the South Pacific region, a diurnally subperiodic form exists (Sasa et al, 1972). 4.1.3 Implications of microfilarial Periodicity for Diagnosis Diagnosis of bancroftian filariasis is generally dependent on the finding and identification of microfilariae in blood specimens (Wamae, 1994). To know the best time to take blood samples to detect microfilaraemia, there is the need to determine the periodicity of the parasite in the locality (WHO, 1987). Results of various surveys in many parts of the world have indicated that the different types of filarial parasites exhibit different periodic patterns in the diferent geographic regions. Results of surveys in Asia and the Pacific have indicated that at least five forms differing in type of microfilarial periodicity occur in the region. These include the nocturnally periodic W. bancrofti which is found widely in the tropical and subtropical zones, the diurnally subperiodic form in the South Pacific islands, the nocturnally subperiodic W. bancrofti recently discovered in West Thailand, the nocturnally periodic Brugia malayi widely distributed in South and East Asia and the nocturnally subperiodic B. malayi found in some rural areas in Malaysia, Indonesia and the Phillipines (Sasa et al, 1972). Ealier reports by many workers in these regions in which the periodicity index and peak hours were reported agreed with the above findings (Ramachandran et al, 1964, Rosen, 1955). 53 University of Ghana http://ugspace.ug.edu.gh In Ghana, early works by Muirhead-Thomson (1954) reported of a plateau of microfilarial density rather than a peak when working on three subjects in Weija near Accra. A somewhat similar report was documented earlier on in Liberia where although a somewhat peak actually occurred between midnight and 02:00hours, a high microfilarial density near maximum was obtained between 21:00 and 04:00hours (Poindexter, 1950). These reports, although representative of the phenomenon, do not conform to the current acceptable mathematical analysis of periodicity data. The nocturnal periodicity of W. bancrofti microfilariae using the statistical method was confirmed recently in Kenya and Tanzania (Gatika et al 1994, Simonsen et al, 1996), There is however generally very little information on microfilarial periodicity in Africa as a whole. Surveys on W. bancrofti have been conducted at night in Ghana without documentary evidence of its periodicity and peak hour. This study therefore aimed at establishing the periodic pattern of the parasite since accurate information on the pattern of periodicity will be a useful guideline with respect to the optimal sampling time. 4.2 Materials and Methods One month after blood samples were read and microfilaraemic individuals identified (see previous chapter), eight males with ages between 17 and 56 years were selected to participate in the periodicity study. The study participants were transported to Accra and accomodated at the Legon Hall of residence of the University of Ghana from the evening of the first study day, until the end of the following day. Each of the participants’ age was noted and individual numbers were assigned to them. 54 University of Ghana http://ugspace.ug.edu.gh Blood samples were collected from them every two hours starting from 18:00hours and ending at 16:00hours the next day. At each examination time, the pulp of the finger was cleaned thoroughly with mediswab, excess alcohol allowed to evaporate and a sterile lancet used to make a perpendicular cut on the finger which was held firmly between the examiner’s thumb and index finger. The blood was allowed to flow and drawn into a 100/xl heparinized capillary tube and expelled into the labelled test tube with the help of a rubber bulb. The tube was labelled with the date, time of collection and the individual number of the subject. During the daytime, subjects were made to perform simple excercises like walking around before blood was collected in order not to simulate night conditions. The blood samples were transferred from the sampling tubes into the counting chamber and the microfilariae for each individual counted for each hour. 4.2.1 Periodicity Data Analysis Analysis of microfilarial periodicity was until recently done by just recognizing the presence or absence of microfilariae in blood examinations carried out both by day or by night. Such simple and non-quantitative methods obviously harbour the risk of causing various misunderstandings of the real nature of the phenomenon. Since the individual microfilaria counts are known to be subject to considerable errors of variation, efforts were made to develop mathematical methods for analysis of microfilarial data based on statistically most reliable measures such as the mean and the standard deviation. The basic idea employed for the statistical analysis of the microfilarial periodicity is that it is a biological phenomenon which can be interpreted simply as a wave of the microfilarial density in the circulating blood in relation to the hour of day. The time length required for the wave for one phase, or the wave period, is 24hours and is common 55 University of Ghana http://ugspace.ug.edu.gh to all filarial forms. Therefore, the conventional terms "periodic", "subperiodic", and "nonperiodic" can be denoted by the relative amplitude of the wave and the terms "nocturnal" or "diurnal" can be defined more precisely by the time of day at which the peak density of microfilariae is encountered, ie peak hour (Sasa et al, 1972). Generally, a wave formula is determined by various factors such as the amplitude, the wavelength, the period, the phase and the wave profile. In the case of microfilarial periodicity, the factors involved are simple because the wavelength and speed are fixed to 24-hour rhythms. Therefore the only three factors necessary to determine the character of such a wave are: the relative amplitude (the grade of difference between the maximum and minimum densities), the phase (the hour of day the density becomes maximum and minimum), and the wave profile. In general, a wave profile such as the microfilarial periodicity can be expressed by the following simple formula: y = m + a(fx) (1) It was shown by Sasa (1976) that the microfilarial periodicity followed the simplest example of such a wave, namely the harmonic form (sine or cosine curve). The density of microfilariae y at the hour h can be expressed by the following simple formula: y = m + acosx ; (2) 56 University of Ghana http://ugspace.ug.edu.gh As 24 hours correspond to 360°, the hours 0 to 24 are multiplied by 15 to correspond to angles 0° to 360°. Therefore, when x = 15(h-k) y = m + acosl5(h-k) (3) where a is the amplitude, m is the mean density and k the peak hour of microfilarial density in the blood. By expanding (3) according to Aikat and Das (1977), we obtain y = m + a {cos(15h -15k)} = m + a {cosl5h cos 15k + sinl5hsinl5k} = m + acosl5hcosl5k + asinl5hsinl5k or y = m + bcosl5h + csinl5h (4) where b = acosl5k (5) and c = asinl5k (6) so that a2 = b2 + c2 (7) and tan 15k = c/b. (8) Thus the problem of estimating the parameters m, a and k of (3) is reduced to that of estimating the parameters of (4) and then application of relationships (7) and (8). The least square estimates of m, b and c of equation (4) are as follows: m = 1/n Ey (9) b= 2/n 'Ey cosl5h and (10) c= 2/n Ey sinl5h. (11) 57 University of Ghana http://ugspace.ug.edu.gh In estimating these parameters, the following procedures were followed. Microfilarial intensities at the hours of examination were recorded for each individual and the arithmetric mean microfilarial intensity calculated. Taking the mean intensity as the common denominator, microfilarial ratios were calculated for each blood sampling time as: Microfilarial ratio = microfilaria intensity _____________ X 100 mean density The mean of the microfilarial ratios for the eight study individuals was calculated for each hour and were farther expressed as a percentage of the maximum value. These represented the observed microfilarial ratios which were used to draw the observed microfilarial periodicity curve. Trigonometric analysis of the observed microfilarial ratios by the method of Aikat and Das (1977) making use of equations (7) and (8) and expressions (9), (10) and (11) enables the calulation of the m, b and c. These values were used to arrive at the harmonic wave equation for the microfilarial periodicity in the study area, which can be expressed as: y = 100.2 + 116.7cosl5h + 32.8sinl5h This represented the theoretical microfilarial equation which was used to calculate theoretical microfilarial ratios and the ratios were further expressed as a percentage of the m ax im um value. These were used to draw the theoretical microfilarial periodicity curve (Fig.5). 58 University of Ghana http://ugspace.ug.edu.gh su i| j y i-u il l % of m a x im um o I ro o o 03 o 03 o o o 2 0 01 o CO CO H S3 2!a M o S3H 1-3 O>r 1 o S3 o Ir-> >Ir- S3>-3 Ow T3 Pd 2oo a •-3►c OC S3< University of Ghana http://ugspace.ug.edu.gh 4.3 Results Microfilarial intensities at the hours of examination in the periodicity study are presented in Table 7. Overall, the 2-hourly microfilarial intensities in the individuals ranged from 0- 15740mf/ml of blood over the 24-hour period. Generally, except in a few samples, the mf intensities were low from the early hours of the morning (ie 06.00hrs) and increased gradually until m ax im um numbers from 22.00hours to 04.00hours. The individual mean microfilarial intensities ranged from 105.8 to 6150mf/ml. Table 8 shows the results of microfilarial ratios calculated as the ratio of the two-hourly count to the mean 24-hour intensity for each individual. Microfilarial ratios ranged from 0 to 303. The means of the ratios were calculated and the results expressed as observed mean microfilarial ratio. The observed mean microfilarial ratios increased from low values during the day to high values in the night until a peak of 217.5 occurs at 02.00hrs. The mean microfilarial ratios were further expressed as percentage of the maximum value (217.5 which is the observed peak hour value) and these percentages have been used to draw the observed microfilarial periodicity curve shown in Figure 3. It is obvious that the observed and theoretical periodicity curves follow each other closely, especially between 20.00 and lO.OOhours and the harmonic wave equation thus expresses rather accurately the relationship between microfilarial ratio and the time of day for the study area. Table 9 is the trigonometric analyses of the microfilarial periodicity by the method of Aikat and Das (1977). From this, the periodicity index (D) has been calculated as 122.6 and the peak hour (k) is 1.03 (ie 01:03hrs) 60 University of Ghana http://ugspace.ug.edu.gh Ta bl e 7. Tw o- ho ur ly m ic ro fil ar ia l in te ns iti es in the eig ht stu dy in di vi du al s. 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