HOST GENETICS AND EXPOSURE IN SUSCEPTIBILITY TO SOIL-TRANSMITTED HELMINTH INFECTIONS AMONG HOUSEHOLDS IN IGBO-ORA, NIGERIA BY Y AR R OLUFUNKE ABIODUN, OLUWATLOBIAB B. Sc (ILORIN), M.Sc. (IBAYDA N) MATRIC NO: 8I57T27 S A thesis in the Department of ZEooloRgy submitted to the Faculty of Science in partial fulfilmenItV of the requirements for the degree of N U DOCTOR OF PHILOSOPHY AN of the D UNIVERSITY OF IBADAN A IB MARCH 2019 i ABSTRACT Soil-Transmitted Helminth (STH) infections remain one of the major health challenges in developing countries. Susceptibility factors such as host genetics and exposure play significant roles in determining human helminth infection. However information is scarce on the quantitative patterns of spatial and genetic determinants of susceptibility, which is required for the refinement of the ongoing control activities in Nigerian communities. This study was carried out to determine the relative contributions of host genetic factors and the presence of parasite in soil toY the prevalence and intensity of STH infection among households in Igbo-OraA, NiRgeria. Igbo-Ora town was mapped into low, medium and high density areasR using GIS-based household cross-sectional design. A pre-tested structured queIstBionnaire was used to obtain demographic, risk factor and pedigree information fLrom 508 participants from 239 randomly selected households. Stool samples were collected from the participants to assess the prevalence and intensity of STH inIfectioYns using the Kato-Katz method. Also, 236 surface soil samples were collectedS arou Tnd the selected houses to determine the presence of parasite in soil. VariancRe component analysis was used to assess the 2 2impact of genetic relatedness, that is, heritability (h ), common shared household (C ) V 2and presence of parasite in the soEil (E ) on STH prevalence and intensity using Sequential Oligogenic LinkaIge Analysis Routine. Data were analysed using descriptive statistics and CNhi-square at α 0.05 The prevalence of hUookworm was highest in high density areas (24.9%), while Ascaris lumAbricNoides was spatially clustered in the medium density areas of Igbo-Ora. Risk faDctors such as living in crowded room and mud houses were significantly assoAciated with STH infection. The prevalence of STH infections was 18.5% B(hookworm), 16.7% (A. lumbricoides), 3.0% (Strongyloides stercoralis) and 0.8% I (Trichuris trichiura). Prevalence was similar in male (28.2%) and female (28.1%) participants. Majority of STH infection (81.1%) were of low intensity, while 6.6% cases were moderate and 12.3% were heavy intensity in Ascaris and hookworm infections. The prevalence of parasite ova in soil was 24.1% (Ascaris sp), 3.6% (hookworm), 0.7% (S. stercoralis), 1.5% (Taenia sp) and 0.7% (Schistosoma haematobium). Seven degrees of family genetic relationships resulting in 639 relative pairs from 296 individuals in 91 families were established. Heritability of Ascaris, and ii hookworm within the population was 22.7% and 1.1% and respectively. The common 2 shared household had a greater impact (C = 0.047, p = 0.023) on the prevalence of 2 Ascaris infection, whereas genetic relatedness had a significantly higher effect (h = 0.25, p=0.023) on the intensity of Ascaris infection. The presence of parasite in the 2 soil had greater and significant effect on both the prevalence and intensity (E =0.472, p=0.034) of hookworm infection. Host genetics is an important susceptibility factor in the intensity of Ascaris infection, while exposure factors are more important in hookworm infection. These suggesYt the use of individual approach in soil-transmitted helminth infection control. AR Keywords: Soil-transmitted helminth, Parasite prevalence, HoRst genetic factor, Common shared household B Word count: 475 LI TY RS I VEI N N U A D IB A iii CERTIFICATION I certify that this thesis was written by OLUFUNKE ABIODUN OLUWATOBA, in the Department of Zoology, University of Ibadan, Ibadan. Y …………………………… AR SUPERVISOR R Dr. Roseangela I. Nwuba IB B. Sc (Jos), M.Sc., Ph.D (IbYadan ) L Cellular Parasitology PIroTgramme, Cell Biology Rand SGenetics Unit, DepaErtment of Zoology IVUniversity of Ibadan N N U A AD IB iv DEDICATION I dedicate this work to my loving children; Gloria, Sharon, Beauty and Joshua. You are God‟s blessings in my life. AR Y BR Y LI SI T R IV E UN N AD A IB v ACKNOWLEDGEMENTS I like to register my particular thanks to the four village heads that were my entry point to their community and the households that participated in this study. Beyond the twist of fate is the divine hand that brought me again under the expertise of my supervisor, Dr. Roseangela. I. Nwuba who, together with Professor Mark Nwagwu, graciously launched me into the dynamic field of Cellular Parasitology over a decade ago as their Master‟s degree student. Standing out among so many other „beyond the call of duty‟ assistance, I benefited from my supervisor. She was acutely sensRitiviYty to my financial needs; she magnanimously met my financial needs in the name of giving me „pocket money‟ when she visited me on the field to see that I get itA right at that most crucial stage of my research work. For this, I owe her un-enBdingR gratitude. The positive atmosphere provided by the academic staLff Iof the Department of Zoology and their high taste for excellence in research i nspired me to swim against the negative current of unexpected academic rigouYr, mental and emotional strain, deflating field experiences and untold hoursI TS of laboratory analysis that were unavoidable in achieving my research obRjectives. I greatly appreciate their kind input, corrections and helpful direction. Their frank contributions and valuable insight to my pre-field presentation provided me Ewith a good head-start that tremendously helped not to „endanger‟ the genetic oIr mVolecular dimension to this research. I appreciate the effortsU of mNy foreign research consultants and collaborators, including Peter Kochunov, m y SOLAR instructor from University of Texas, USA, Emily Adams, RicAk MNaziel, and the CARTA. It is difficult to gloss over their strategic impartsD on me at different stages of the research. Most importantly, CARTA provided me Awith a fellowship that substantially sustained my research work in many ways-B(financial, supervisory, international exposure and networking with renowned experts I in the specialized area of my research). Lastly, worthy of mention is the encouragement I received from my senior colleagues in the Department of Medical Microbiology and Parasitology, College of Medicine, University of Ibadan. They include Professor Rasheed Bakare; Late Professor Anthony Oni, the immediate past Head of the Department; Prof. A. O Kehinde, and other senior colleagues in the department: Dr (Mrs) Hannah O. Dada-Adegbola, Dr (Mrs) Abiola Okesola, Dr. S.A. Fayemiwo, Dr O. B Makanjuola and Dr. Adeola vi Fowotade. It has been rewarding working as an academic staff together for many years. I must give due appreciation to my husband and children for the home support during this period. To God be the glory! AR Y LIB R ITY ER S V UN I N DA IB A vii TABLE OF CONTENT Title page i Abstract ii Dedication iv Certification v Acknowledgement vi RY Table of Content A viii List of Tables B R xiii List of Figures L I xvi List of Appendix Y xviii CHAPTER ONE SI T INTRODUCTION R 1.1 Background to the study VE 1 1.2 Rationale N I 4 1.3 Objective N U 6 1.4 Hypothesis 7 CHAPTDERA TWO LITAERATURE REVIEW IB2.1 The burden and Epidemiology of STH 8 2.2 Spatial distribution of Soil-transmitted helminths 10 2.3 Pathogenesis of Soil-transmitted helminths infections 13 2.3.1 Ascaris lumbricoides: the roundworm 13 2.3.2 Ancylostoma duodenale & Necator americanus: the hookworm 15 viii 2.3.3 Trichuris trichiura: the whipworm 19 2.3.4 Strongyloides stercoralis: the threadworm 21 2.4 Environmental risk factors predisposing to STH infection 23 2.4.1 Temperature, climate and season 23 2.4.2 Availability of clean water 24 2.4.3 Soil types 26 RY 2.5 Human behavior and socioeconomic factors A 27 2.5.1 Hygiene practices B R 27 2.5.2 Occupation, housing system and family size L I 29 2.6 Household clustering of infection Y 30 2.7 Host Genetic factors predisposing to SSTHI i Tnfections 30 2.8 Morbidity control through deworRming programmes 35 2.9.1 New way forward: TargeVt druEgs or vaccine 39 CHAPTER THREE NI MATERIALS AND MUETHODS 3.1 ResearchN area 43 3.2 HouAse mapping 43 3.3 D Questionaires for collection of demographic, behavioural and A observational data. 46 IB3.4 Household survey for pedigree information 46 3.5. Soil analysis 47 3.6 Stool sample collection 47 3.6.1 Laboratory analysis 48 Wet preparation 48 ix Kato-Katz concentration method 49 3.7 Data analysis 49 CHAPTER FOUR RESULTS 4.1 Socio-demographic factors of the participants 52R Y 4.2 Prevalence of Soil-transmitted helminthes infection in the A Sampled population B R 53 4.2.1 Overall prevalence of multiple helminthes infection Lin IIgbo-Ora 53 4.3 Intensity of STH infection in the sampled popuYlation 53 4.4 Associated risk factors of STH infectSion wI Tithin the infected population R 61 4.4.1 Multivariate logistic regrVessiEon of risk factors of STH infection 61 4.5 STH infection withNin hIouseholds in Igbo-Ora 61 4.5.1 Households i nUfection within each community 64 4.6 The prevNalence of specific STH parasite within the sampled DcomAmunities in Igbo-Ora 64 4.6.A1 Risk factors for hookworm infections 65 IB4.7 Prevalence and risk factors of infection in each of the community sampled 70 4.7.1 Pako Community 70 4.7.2 Isale-Oba community 75 Socio-demographic, risk factors and prevalence of STH x Infection in Isale-Oba, Igbo-Ora 75 4.7.3 Igbole Community 79 Socio-demographic, risk factors and prevalence of STH infection Infection in Igbole community 79 4.7.4 Saganun Community 83 Socio-demographic, risk factors and prevalence of STH RY infection in Saganun community A 83 4.8 Result of Soil Analysis B R 88 4.8.1 Human STH infection versus soil parasite within a hLousIehold 88 4.9 Spatial distribution of Soil-transmitted helmintYh infection 92 4.10 Assemblage of Pedigree Structure TS I 97 4.11 Heritability of STH in Igbo-Ora R 102 4.12 The effect of host geneticV facEtors, common shared household and the presence ofN soilI parasite in the environment on the prevalence o f SUTH 106 4.13 The effeNct of host genetic factors and common shared D houAsehold on Intensity of STH 106 4.13A.1 Effect on Ascaris lumbricoides intensity 106 IB4.13.2 Effect on hookworm intensity 107 4.14 Favoured model of the variance component analysis of A. lumbricoides and hookworm infection 107 xi CHAPTER FIVE DISCUSSION 5.1 STH infection, demographic structure and associated risk factors of infection 113 5.1.1 STH infection and demographic structure 113 5.1.2 Risk factors associated with STH infection 11R4 Y 5.2 Spatial distribution of STH infection in Igbo-Ora A119 5.3 Genetic variance component analysis B R 121 5.3.1 Genetic effect of relatedness on the prevalence and LI Intensity of STH Y 121 5.3.2 Effect of genetic relatedness on the prSevaIle Tnce and intensity of Ascaris lumbricoides and hookwRorm infection 122 5.4 Summary of the findingsV E 125 5.5 Conclusion and recNommIendation 126 Reference U 127 AppendiNx I: Ethical approval Certificate 151 DAppAendix II: Questionnaire 152 AAppendix III: Informed Consent form 156 IB Appendix IV: Pedigree information Collection Sheet 157 xii LIST OF TABLES Table 4.1: Demographic characteristics and STH infection among the study participant in Igbo-Ora 55 Table 4.2: The overall prevalence of multiple helminth infection in the study population. 58 Table 4.3: Intensity of STH infection in the four GIS mapped community RY in Igbo-Ora A 60 Table 4.4: Bivariate analysis of risk factors associated with Soil-transRmitted helminths infections within the infected populatioLn oIf BIgbo-Ora, Nigeria 62 Table 4.5: Multivariate logistic regression analysIisT of Yadjusted risk factor of STH infection in the study popSulation 63 Table 4.6: Distribution of STH infecEtionR per household in the four Communities IV 67 Table 4.7: Association of cNommunity density areas and specific STH Parasite i n UIgbo-ora using Pearson chi-square 68 Table 4.8: FAactoNrs associated with hookworm infections by multivariate D logistic regression analysis in the study population of Igbo-Ora 69 BTabAle 4.9: Risk factors and prevalence of soil-transmitted helminthes I infection in Pako community Igbo-Ora 72 Table 4.10: Prevalence and intensity of STH infection within Pako community 73 Table 4.11: Risk factors and prevalence of STH infection within the participants of Isale-Oba, Igbo-Ora 76 xiii Table 4.12: Prevalence and Intensity of STH infection within Isale-Oba community 77 Table 4.13: Risk factors and prevalence of STH infection within the participants of Igbole, Igbo-Ora 80 Table 4.14: Prevalence and Intensity of STH infection within Igbole Community Y81 Table 4.15: Risk factors and prevalence of STH infection within the R participants of Saganun, Igbo-Ora A 84 Table 4.16: Prevalence and Intensity of STH infection in Saganun coRmmunity 85 Table 4.17: Multiple helminthes infection in Saganun commLuniItyB Igbo-Ora 87 Table 4.18: The Prevalence of Soil –Transmitted helminth s in the environmental soil samples around parTticipYants households in the different communities S I 89 Table 4.19: The distribution of STH in thRe soil sample of household with different no of participanEts 90 Table 4.20: Summary tableN of IM Voran index value for the spatial distribution of AscarisU and hookworm in Igbo-Ora 93 Table 4.21. TheN pair wise Relationships among the 296 related members of A the Igbo-Ora pedigree 101 Table 4D.22: Heritability of STH intensity in relation to genetic relatedness B A using SOLAR 8.1.1.0 version 108 I Table 4.23: The variance component analysis of the effect of host genetic factors (relatedness), common shared household and presence of soil parasite in the environment on the prevalence of A. lumbricoides and hookworm in Igbo-Ora 109 Table 4.24: Effect of host genetic factors (relatedness) and common shared households and soil parasite in the environment on Ascaris xiv intensity 110 Table 4.25: Effect of host genetic factors (relatedness), common shared households and contaminated soil on Hookworm intensity 111 Table 4.26: Variance component analysis for the Ascaris and hookworm infections showing the favoured models after adjusting for significant covariates 112 RY RA LI B TY RS I VE UN I N AD A IB xv LlST OF FIGURES Fig. 2.1: Distribution of STH survey data for Nigeria and average district-level prevalence 12 Fig. 3.1: A GIS map showing the sampling grid of Igbo-Ora according to house clustering of the community 45 Fig.4.1: Micrograph of ova of ascaris and hookworm and photograph RY of adult worm of STH parasite seen in participant stool A 56 Fig.4.2: The prevalence of each STH parasite within the study BR population. L I 57 Fig. 4.3: The intensity of A. lumbricoides and hookwoYrm infection In relation with age group S I T 59 Fig. 4.4: The prevalence of specific STHR parasite with each of the four community sampleVd inE Igbo-Ora 66 Fig.4.5: Intensity of STH iNnfecItion in Pako community 74 Fig.4.6: Intensity of SUTH infection within Isale –Oba community 78 Fig. 4.7: IntAensiNty of STH infection within Igbole community 82 Fig. 4.8D: Intensity of STH infection within Saganun community 86 BFig.A 4.9: Picture of Ascaris and Taenia ova seen in soil samples using I Zinc sulphate floatation method 91 Fig 4.10: Spatial distribution of Ascaris and hookworm in Isale Oba. 94 Fig 4.11: Spatial distribution of Ascaris and hookworm in Igbole. 95 Fig 4.12: Spatial distribution of Ascaris and hookworm in Sagannu. 96 Fig. 4.13a: Showing an example of a nuclear family 98 xvi nd Fig. 4.13b: An example of a family tree with 2 generation and polygamy. 99 rd Fig. 4.13c: An example of a family tree with 3 generations 100 Fig. 4.14a: Presence of STH within an extended family with polygamous Marriage. 103 Fig. 4.14b: Presence of STH within an extended family 104 Fig. 4.14c: Presence of STH within a nuclear family Y105 R RA LI B ITY ER S IV N U N AD A IB xvii LIST OF APPENDIX Appendix I: Ethical approval Certificate 151 Appendix II: Questionnaire 152 Appendix III: Informed Consent 156 Appendix IV: Pedigree information Collection Sheet 157 Y RA R B Y LI T RS I VE NI N U DAA IB xviii CHAPTER ONE INTRODUCTION 1.1 Background to the study Soil Transmitted Helminth (STH) remains one of the major health challenges in the developing world. The important helminth includes Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis and the hookworms which are AncylosYtoma duodenale and Necator americanus. These parasites belong to the class ofR parasitic nematode worms causing human intestinal infections. Man acquires theseA parasites by coming in contact with the eggs or larvae that thrive in the worRld‟s tropical and subtropical countries that are warm and moist. The adult worm Bcan survive for many years in the human body. (Bethony et al., 2006). World H eLaltIh Organisation (WHO) declared that over a billion people are infected with at least one species of STH (WHO 2012), thereby accounting for up to I40T% Yof the global morbidity from infectious diseases. The global burden of inSfections with STH was estimated at 3.4 million disability adjusted life years (RDALYs) by Global Disease Burden (GBD, 2015). E The greatest number of infecItVions due to soil transmitted helminth occurs in the tropical and subtropical reNgions of Asia, especially China, India and Southeast Asia, as well as sub-SahaUran Africa (WHO, 2012). Several studies have shown that significant helminth infection prevalence occurs within the population of people from Southeast AAsia, NSouth America and Sub-Sahara Africa, and having multiple helminth infectioDn is a common phenomenon (Lili et al., 2000; Guinard et al., 2000, Tchuente TchAuente et al., 2003, Raso et al., 2004). Soil transmitted helminth remains a major Bcause of morbidity, and sometimes, mortality in developing tropical countries. In I children according to WHO (2012), they are important cause of physical and intellectual growth retardation, likewise causing malnutrition, stunting, cognitive and educational deficit. STHs remain largely neglected by the international and medical community, in spite of their economic, educational and public health importance, because the poor people of the world are the most affected. Bethony et al., (2006) reported that it is difficult to quantify the effect of infection due to STH, especially as it affects education and 1 economy, because it has insidious clinical presentation and causes chronic ill health. The unique effect of STH infection on school performance, attendance and future economic productivity have also been highlighted by other studies (Bleakley, 2003, Miguel and Kremer 2003). Multiple infections with STHs are commonly found in children, hence the need to diagnose each specie in order to estimate the prevalence and intensity of the infection. In determining the best control strategy for STHs, diagnosis thus becomes useful. A resolution to control STH infection was passed by the World Health AssembYly in 2001; countries which are highly affected were implored to participate inR the said control through wider spread use of anthelminthic drug for school-ageAd children in under developed countries. R In Nigeria, despite the WHO resolution of large-scale dewormiIngB programme, there is no record of such a mass deworming programme. The su rvLey carried out in 2011 by the Global Atlas for Helminth Infection was to develYop the distribution map of STH infection prevalence in all the six geopolitical zIoTnes in Nigeria. Several small-scale deworming programmes that were done werSe as a result of one research programme or the other. In many communities inR Nigeria, there have been records of high prevalence of STH infection, such Eas 71.5% prevalence seen in Benue State, North Central Nigeria (Jombo et al., V2007), 54.9% prevalence recorded in Ikenne, Ogun State (Ekpo et al. 2008); mNeanIwhile, Kirwan et al., (2009) recorded 50% prevalence in Ile-Ife, Western NUigeria, while Odu et al., (2011) in Port Harcourt Southern Nigeria, recordeNd 30.7% prevalence among preschool and school-aged children. In 2011, a totAal of 5.7 million (13.8%) school-aged children were predicted to be infectedD with STHs in Nigeria. OluAwole et al (2015) in their nation-wide estimates of STH infection in Nigeria, using IBBayesian geostatistical model, reported that hookworm, A. lumbricoides, and T. trichiura infections are endemic in 482 (86.8%), 305 (55.0%), and 55 (9.9%) out of 555 locations in Nigeria, respectively. Hookworm and A. lumbricoides infection co- exist in 16 out of 36 states, while the three species are co-endemic in 12 states. Overall, STHs are endemic in 20 of the 36 states of Nigeria, including the Federal Capital Territory of Abuja. They reported a prevalence range of 1.7% to 51.7% for hookworm in endemic locations, and prevalence range of 1.6% to 77.8% for A. lumbricoides, and 1.0% to 25.5% for T. trichiura. For hookworm, a model-based 2 prediction of 0.7% to 51.0% prevalence range was reported, while it reported a range of 0.1% to 82.6% for A. lumbricoides, and 0.0% to 18.5% prevalence for T. trichiura. Their models therefore suggested that dense vegetation and land surface temperature during the day are necessary in order to discover the spatial distribution of STH infection in Nigeria. Adewale et al., (2017) reported a prevalence of 34.8% among primary school pupils in Lagos. A recent systematic review and meta-analysis of the prevalence and distribution of STH infection among Nigeria children revealed an overall pooled prevalence estimate of 54.8% STH infection (Solomon 2018). Y The idea of school based deworming programme has been lately debated Ras to its effectiveness in control of STH infection. Anderson et al., (2013) cRonclAuded in their study that there is every possibility that school-age deworming will be of little benefit to the children. Also, that the treatment coverage and the frLequIenBcy of treatment must be increased within this age-group before the desired ben efits can be reflected in the larger community. Regular delivery of anthelminTtic dYrugs is the mainstay for global soil-transmitted helminth control. Deworming caImpaigns are often targeted to school- aged children, who are at high risk of soil-trSansmitted-helminth-associated morbidity. However, findings from modelling studiRes suggest that deworming campaigns should be expanded community-wide forE effective control of soil-transmitted helminth transmission. Results of this Vmeta-analysis suggest that expanding deworming programmes community-wideI is likely to reduce the prevalence of soil-transmitted helminths in the high-risNk group of school-aged children, which could lead to improved morbiNdity o Uutcomes (Clarke et al., 2017). EpidemiologAical studies revealed that in addition to exposure and household determiDnant, genetic factors account for an important proportion of variations in worm loadA (William-Blangero et al. 1999, 2012, Cuenco et al 2009, Quinell et al. 2010). IBAnd based on these, several facts have suggested that there is an important genetic influence defining the susceptibility to helminths infection. Holland et al. (2009) reported that helminths worm load in infected communities are over-dispersed with the majority of infection found within a few individuals. Reports from other parts of the world have shown that these infections aggregate in families, likewise host genetic factor (relatedness) and domestic environmental factors have been shown to have significant involvement on susceptibility and infection 3 intensity, but the facts about the aggregation in families and the involvement of host genetic factors in infection susceptibility are yet to be explored in this part of the world, that is, Nigeria. This study aims to investigate the impact of parasite contaminated soil and host genetic relatedness on the prevalence and intensity of Soil- Transmitted Helminth (STH) infection among household in Igbo-Ora, Oyo State, Nigeria. 1.2 Rationale Y Environmental and socio-economic factors with human behaviour inRfluences exposure to infection, while a range of known immunological, phyAsiological or nutritional and unknown factors may affect susceptibility. This has bReen reported that in spite of increasing knowledge of environmental and househIolBd risk factors (Pullan et al., 2008, Olsen et al., 2001, Traub et al., 2004, Raso eLt al., 2006) and protective immune responses (Quinnell et al., 2004, Hoffmann et al., 2002) in helminth infection, few studies have addressed their relIatTive Ycontributions to predisposition (Holland, 2009). S In the recent years, school-based dewormRing programme have experienced increase in financial and technical support for Eschool-based deworming, and quite a number of countries in sub-Saharan AfricIa Vimplement this nationwide control. These however do not signal the end of epideNmiological research, and detailed data on patterns and risk factors for infection aUre still required for the refinement of ongoing control activities. Nevertheless, onNly few epidemiological population-based studies of STH are available in Africa (PAullan et al., 2010). Those few studies that exist typically report on age-related Dchanges in infection prevalence and intensity, and demonstrate consistent incrAease with age, peaking in adults (Behnke et al., 2000, Palmer and Bundy, 1995, IBUdonsi 1984a, b, Chandiwana et al., 1989, Asaolu et al., 1992) pronounced aggregation of high infection intensity within individuals who are at high risk (Behnke et al., 2000, Palmer et al., 1995), and villages (Asaolu et al., 1992). Thus, Behnke et al.‟s (2000) submission provides evidence for condition responsible for low or high intensity infection. Investigations on spatial and genetic determinants of infection are few within African communities. A cohort study by Saathoff et al., (2005), which involves primary 4 school children of South Africa, demonstrated that within a small area, there is pronounced spatial clustering of infection. Thus, the infection was reported to have been strongly influenced by several environmental factors (Saathoff et al., 2005). In the same vein, many studies outside Africa have reported that both family environment and host genetics play significant role in hookworm infection intensity (Breitling et al., 2008, Quinnell et al., 2010, Pullan et al., 2009). A study on the genetic epidemiology investigation of hookworm in Zimbabwe, do not consider the effects of shared family household (William-Blangero et al., 1997). Also, a sYtudy done by Pullan et al. (2010) in a rural community in Uganda suggested that Rexposure related factors play a greater role in hookworm and that host genetic faActors is not a major determinant of infection. Till date, no study has investigatedR the role of host genetic relatedness in STH infection in Nigeria. LIB The significant role of human genetics in determinYing human helminth infection intensity has been reported, with heritability oIf Tup to 44% (Brooker et al., 2006, Breitling et al., 2008). Significant heritabSility in heavy or light infection is an indication that host genetics is an important factor for predisposition. However, Quinnell et al., (2010) confirm the EheritRability of initial and reinfection intensity has heritable phenotypes, but the exVtent to which host genetics affect predisposition was not examined. Brooker et aIl., (2006) in their study advocated for a scientific perspective that will integNrate the use of spatial analysis with statistical methods that will measure the eff ecUt of environmental factors, genetic factor and household factor separately, for aN better understanding of the determinants of infection. Two linkage scans study Aidentified a Quantitative Trait Loci (QTL) on chromosome 13q33.3 which has beeDn found to be associated with susceptibility to A. lumbricoides. This region harbAours the gene Tumor Necrotic Factor Superfamily 13B (TNFSF13B gene) and it IBhas been suggested to play an important role in antibody response to Ascaris. Also, information about the involvement of the gene in STH infection susceptibility is lacking in our environment. Therefore, this study sets out to conduct a population– based genetic epidemiological study of Soil-transmitted helminth infection in Igbo- Ora southwest Nigeria. This approach employed, firstly, negative binomial spatial modeling which investigated the spatial variation in the intensity of infection, taking into consideration the individual and household covariate. 5 Secondly, genetic variance component analysis was done using SOLAR a Unix software computer programme version 8.1.1.0. This is a genetic approach that takes into account genetic relationship among the selected individual households and between the different households as an aggregate of the entire communities and determine the relative contributions of host genetics (relatedness), domestic environmental factors, and other factors to the variation of infection intensity in the different density areas of Igbo-Ora, in southwestern Nigeria. Y 1.3 Objectives of the study AR This study seeks to determine the effect of parasite contaminated soRil around homes, host genetic relatedness and common shared household on Bthe prevalence and intensity of Soil-Transmitted Helminth (STHL) I infection among household/communities in Igbo-Ora, Oyo State, NYiger ia. Therefore, the specific objectives are: IT 1. to determine the prevalence, inStensity, variability and associated risk factors of STH infection wiRthin the different communities in Igbo-Ora, Nigeria; E 2. to determine the effeVct of house clustering density on prevalence of STH infection and spNatiaIl distribution of STH Infection in Igbo-Ora; 3. to determinUe the presence of STH in the domestic environment (soil) and its effect on human susceptibility; intensity of infection. 4. tAo dNetermine the effect of genetic relatedness on heritability of STH Dinfection within households in the community; and A5. to evaluate the contribution of host genetic relatedness, parasite B contaminated soil in the domestic environment, common shared I household, to the prevalence and intensity of STH infection. 1.4 Hypothesis: Hypothesis 1. Prevalence and intensity of STH infection within and between households Ho: There is no relationship between the prevalence and intensity of STH within and between households in Igbo-Ora. 6 2. Familial aggregation of STH infection within households and domestic environment. Ho: There is no significant correlation between familial aggregation of STH infections within household and domestic environment. 3. Estimated contribution of addictive genetic (relatedness) effect and domestic environment. Ho: The estimated contribution of addictive genetic effect and domestic environment on the intensity of STH infection is zero. AR Y BR LI TY R S I IV E N N U A AD IB CHAPTER TWO LITERATURE REVIEW 2.1. The burden and Epidemiology of STH 7 Soil-Transmitted helminth infections have been reported by researchers from different parts of the world to cause significantly large burden on the poor nations of the world, especially on people living in rural or undeveloped urban settings. (Hotez 2007, 2008, Knopp et al., 2008). Though Bethony et al., (2006) reported estimates of death from Soil-transmitted helminth infection to vary widely from 12,000 to 135,000. Murray and Lopez (1996) submitted that the infections have been noted to cause more disability than death. Death and disability, therefore, are the main dimensions for estimating the burden resulting from STH infection with the worldwide burdeYn of disease being typically assessed by disability-adjusted life years (DALYs). RHotez et al., (2006), however, was more specific putting the burden of the DALYAs around 39 million annually. According to Brooker (2010), the majority of DARLYs were lost in Southeast Asia (47%) and sub-Saharan Africa (23%). IB For Nigeria, the burden of STH infection estimates provi deLd by Bundy et al., (2000) was high in all class of people: preschool childrTen, Yschool-aged children and adult population. Although in Bundy‟s report, the Ihighest prevalence was recorded in school-aged children, intensity of infection wSas however observed to be high among adults. Bundy and Brooker global and loRcal estimates of STH burden reported above, underscore the importance of theE disease with a potential worldwide scale of infection. More researchers, acVcording to Bundy et al. (2000), have been giving recognition to the significNanceI of helminthic infections, which Bundy noted to have dramatically increas edU with the upsurge of HIV infections. The factors that drive STH infection globalNly are similar from the submission of several authors in different parts of the worldA where researches on STH have been carried out. These causal pathways include Denvironmental factors; Human behaviour; housing and human habitation pattAern and rural-urban migration (Ulukanligil et al., 2000). For instance in Nigeria, Bthe endemic level of STH infection have mainly been blamed on unhygienic I environmental situation, improper disposal of wastes, gross environmental pollution with agrochemical and industrial waste, domestic waste, plus steady contamination of surface and underground water (Oyerinde et al., 1980, Fagbenro-Beyioku and Oyerinde 1987. Asaolu et al., 1997, Jombo et al 2007, Egah and Akosu 2007, Ekpo et al. 2008). All these researchers found those factors listed above as contributing to environmental decay and ecosystem degradation, which are suitable conditions for high transmission and sustenance of many human diseases, especially parasitic 8 diseases. Soil transmitted helminth infection are therefore diseases of the low or poor socio-economic conditions of human existence, particularly in area with poor sanitation whether rural communities or urban setting. Humans become infected with STHs in two ways, firstly by the penetration of the infective third stage larvae of hookworms and S. stercoralis into the skin, and secondly by the ingestion of embryonated eggs in food or drink which has been feacally contaminated, as in the case of A. lumbricoides and T. trichiura, after which the larvae develop to adult worms and can survive in the human gastrointestinal Ytract for years. School age children have been identified as the most at risk of Rinfection with STHs (Colley et al., 2001; Dada-Adegbola et al., 2005, GirumR 200A5) and early childhood infections have been reported to contribute significantly to debilitation (Colley et al., 2001). These critical submissions have ledL reIsBearchers to prioritize school age children as target for epidemiological survey of STH and anti-helminthic treatment. Figure 2.1 shows the distribution of STTH Yin Nigeria according to Global Atlas for Helminth Infection (GAHI) 2011 surveIy. According to the map, the prevalence ofR <1%S recorded in few areas of Nigeria may be due to several unreported reasons, while the prevalence of >1-10% are reported in virtually all the six geopoliticaVl zoEnes of Nigeria, but more in the northeast and southeastern part of the nationI. The prevalence of >10-20 and >20-50 are reported mainly in the southwesteNrn part of the nation, this may be due to the weather condition which is fUavourable to the survival of the ova of the parasite in the environment or Navailability of adequate facility for diagnosis or the availability of published daAta. SevAeralD studies after the GAHI 2011 reports showed that the prevalence of STH Bwithin the nation remains as it was reported by GAHI. For instance, Babatunde et al., I (2013) reported a prevalence of 41.9% in Kwara State, while Dimejesi et al., (2014) in their study of STH among pregnant women attending tertiary health facility for their antenatal clinic in Enugu, reported a prevalence of 32.4%. Likewise, Ibrahim et al., (2015) reported a prevalence of 28.8% in a household study survey in a semi- urban community in the southwestern part of the nation. Aniwada et al., (2016) reported a prevalence of 25.6% prevalence among primary school pupils in Enugu. Adewale et al., (2017) reported a prevalence of 34.8% among primary school pupils 9 in Lagos. A recent systematic review and meta-analysis of the prevalence and distribution of STH infection among Nigerian children revealed an overall pooled prevalence estimate of 54.8% STH infection (Solomon 2018). Oluwole et al., (2018) reported 34.6% prevalence for STH infection among primary school pupils in Ogun State. 2.2 Spatial distribution of Soil-transmitted helminth In order to effectively control STH infection, there is a need for accurate descripYtion and understanding of the geographical distribution of infection. TheR use of geographical information systems, remote sensing and spatial statistiAcs, in recent years, has greatly enhanced the ability to map the distribution of STRHs (Brooker and Michael 2000, Brooker et al., 2006). Many epidemiological sItuBdies have been done by examining the stool sample of the inhabitant. These rLesults do not indicate the extent to which the residents are at the risk of parasitic infection but demonstrate the point prevalence of infection within the commuIniTty sYampled (Uga et al., 1997). The initial difficulty presented by the traditionalS cartography was one of the reasons for lack of information about the spatial pattRerns of infection. These difficulties have been dramatically resolved by the advent of the easy to use systems that will capture data, store it and analyse it, this incluEde global positioning systems and geographical information systems (Hay et alI.,V 2000). Anderson and May (1U991N) reported marked heterogeneity in the pattern of parasite distribution within in dividuals, households and communities. The use of geographical informationA systNems has now provided a novel understanding of ecology of infection. This haDs led to the development of low cost ways to identify target populations for treaAtment (Brooker and Micheal 2000, Raso et al., 2005, 2006). Saathoff et al., (2005) Bin their GIS-based studies in a cohort of South African primary school children I demonstrated that within a smaller area, there is a pronounced spatial clustering of infection, which according to the authors was strongly influenced by several environmental factors. Studies done by Brooker et al., (2006) in Brazil and that of Pullan et al., (2008, 2010) in a rural community in Uganda reported households clustering of helminth infections. A random distribution was reported for hookworm infection by Raso et al., (2006) in a study done in a rural community of Cote dˈIvoire. 10 There is paucity of information on the spatial distribution of STH infection in Nigerian community. Y AR LIB R ITY RS IV E U N AN AD IB Fig. 2.1. Distribution of STH survey data for Nigeria and average district-level prevalence. 11 Source: Global Atlas for helminth Infection (GAHI 2012) 2.3 Pathogenesis of Soil-transmitted Helminth infections In STH infections, the occurrence of the disease is directly correlated with the number of worms harboured in the host (Raso et al., 2004). Pullan et al., (2010) further clarified that the host worm load is what is described as infection intensity and which actually are the primary determinant of the transmission dynamics of soil-transmitted helminth infection and not the number of hosts infected. Thus, intensity of infectiYon is the major indices used to describe STH infections, such that in the laRboratory, quantitative egg counts, which is an indirect estimate of worm burden Abecomes the focus of analysis. R B 2.3.1 Ascaris lumbricoides: The roundworm LI Ascaris is usually reported as the most common helYmin thic infection in developing nations (WHO 2012). Studies have demonstraIteTd that in the majority of infected individuals, the adult worm presents minor oSr no symptoms, but in the case of heavy worm burden, intestinal obstruction, Rnausea, weight loss, and protein-energy malnutrition in children are commonE deleterious symptoms (Bethony et al., 2006). In addition to symptoms relating toV the intestinal phase of ascariasis, patients may also experience pulmonary symptoIms when the worms migrate through the lungs. During this phase, patients may Ndevelop a low-grade fever, cough, eosinophilia, and/or pneumonia. An asth mUatic reaction to the presence of the worms, which is allergic in nature, may alsoN occur. According to the estimate provided by Bethony et al (2007), about 807-1A221 million populations were infected with A. lumbricoides which is the most cDommon roundworm infection. However, a more recent WHO estimates repoArted that over 1billion people are infected with roundworm (WHO, 2012), IBdemonstrating the global increase in prevalence of roundworm infection and its continued global significance as a public health risk. Infection occurs when the host ingests eggs found in stool-contaminated soil. In the duodenum, larvae are released and enter the circulation via the enteric mucosa. Once in the capillaries (venous, arterial or lymphatic), it reaches the liver via the portal vein and then the lungs within the first week. In the lungs, they damage the alveolar membrane and mature in the alveolus. Eventually, the larvae are expectorated and 12 swallowed reentering the gastrointestinal tract. In the lumen of the small intestine, the larvae mature to adult worms in about 20 days. When the adult female and male worms are present, they copulate, and the female can produce approximately 200,000 eggs per day. They are later eliminated in the faeces to the soil. In the appropriate conditions of a moist, shady, and warm environment the eggs mature to infective form in two to eight weeks and remain viable for up to 17 months. They can be ingested and can restart the infective cycle (Sharma et al., 2017) Patients infected with ascariasis can be asymptomatic, only showing long-Yterm manifestations of growth retardation and malnutrition. If symptoms areR present, abdominal pain, bloating, nausea, vomiting, anorexia, intermittent diaArrhea are the most common manifestation. If the number of larvae passing thrRough the lung is significant, pneumonitis and eosinophilia (also known as LLoeIffBler syndrome) can be seen; symptoms include wheezing, dyspnea, cough, hemoptysis, and fever. In superinfection, adult worms can migrate to tubuTlar Ystructures like the biliary and pancreatic system causing cholecystitis, choIlangitis, pancreatitis, small bowel obstruction, volvulus, appendicitis, and intusSsusception. Children are more susceptible to complications than adults. Jung et al.R, (2004) reported the case of acute interstitial nephritis in association with A. lumEbricoides infection of a 48-year-old patient in Germany. Another case of a paItiVent from the Ecuadorean Amazon region with limited access to drinking water, Nshe presented to the emergency room with abdominal pain and a mass in the ab dUomen. After surgery, a mass with a perforation due to Ascaris was discoveredN. After discharge and due to lack of follow up and self-care, she became infeActed again, this time making her prognosis more complex and her clinical presentation more difficult (Molina et al., 2018). Another case of a 68-year-old JapaAnesDe man was presented with Ascaris lumbricoides discharge from his mouth. BThe infection was suspected to have occurred while the patient was in the Philippines. I This A. lumbricoides migration occurred because a proton pump inhibitor was used and Billroth I resection had earlier been performed on the man, which reduced gastric acid secretion and increased gastric pH to 6.8; this made it easy for A. lumbricoides to migrate past the stomach to the mouth. The number of imported foods, of infected migrants and refugees, and of overseas travels is increasing, and these factors may lead to an increase in A. lumbricoides infection even in countries with a typically low 13 incidence of such infections. Clinicians should bear in mind that parasitic infections may occur in non-endemic areas (Kobayashi and Tsuyuzaki 2018). The prevalence of A. lumbricoides differs in many communities in Nigeria: Chijioke et al. (2011) reported a prevalence of 19.1% in their study done in Enugu, in the eastern part of Nigeria. Okeke and Ubachukwu (2015) reported the prevalence of 76.9% among schoolchildren in Ebonyi which represent the same eastern part of Nigeria. The reason for the differences observed may be as a result of many factors. Kirwan et al. (2009) in their study in Ile-Ife, Osun State, Southwest Nigeria repoYrted A. lumbricoides (12.2%) has the dominant STH infection. Age, father's ocRcupation and dog ownership were identified as the significant risk factorsR in Athe minimal adequate model for A. lumbricoides. The odds of being infected with A. lumbricoides increased as the children got older. Children aged 12-17 monItBhs and 18-25 months were 8.8 and 12.4 times, respectively, more likely to harb oLur Ascaris than those aged 7-11 months. The odds of harbouring Ascaris for chYildren whose families owned a dog were 3.5 times that of children whose familiIesT did not own a dog. Children whose fathers were businessmen were 0.4 times lesSs likely to be infected with Ascaris than those whose fathers were farmers. The fRindings from their study suggested that many of the young children, who are at aE critical stage of development, are infected with Ascaris and that the prevalenceI oVf infection with this parasite increases with age. Reports from other parts oNf the world showed similar prevalence as that of Nigeria: Galgamuwa et al. (20U18) reported a prevalence of 38.4% for A. lumbricoides in their study among childre n in a tea plantation community in Sri Lanka. A prevalence of 60% and 72A% Nwas recorded for Ascaris and hookworm infection respectively in a cross-seDctional survey done among school children in poor neighborhoods of Port ElizAabeth, South Africa (Muller et al., 2016). IB2.3.2 Ancylostoma duodenale & Necator americanus: The hookworm Hookworm infection in developing countries remains one of the most important parasitic diseases of humans. Data published by Chan (1997) recorded that about 576- 740 million people were infected worldwide with either of the two hookworm species resulting to as many as 22million disability-adjusted life-year lost every year. A more recent WHO estimates stated that 740 million people are infected with hookworm (WHO, 2012). Chan data though over a decade ago still fall within the margin of the 14 WHO recent estimates, justifying the increasing focus of researchers on hookworm infection in particular. There are two known species of hookworm which are Necator americanus and Ancylostoma duodenale, and there are two primary differences between the two organisms. First, the geographic distribution varies slightly with each organism. About 72.5 million people are infected with A. duodenale with the majority in Asia. About 400 million people are infected with N. americanus all over the world with one million living in the United States. A. duodenale which is also known as old world hookworm was seen in India, Southeast Asia, southern Europe, JapanY and North Africa. A. duodenale causes a form of anaemia among the infected coRal miners in Belgium and Great Britain and also in tunnel construction workers Ain Germany, Italy and Switzerland. Meanwhile, Necator americanus, commonlyR known as New world or American hookworm was believed to have been brouIgBht in to the Americas during the slave trade, it‟s found in the Caribbean, Centra l Land South America. It has been found in China, India and Africa. Y Secondly and more importantly for identificatioInT purposes, the adult worms of each have minor morphologic differences. The egSg and larva stages, however, are basically indistinguishable. Adult hookworm livesR in the jejunum with the anterior end attached to the mucosa. Morphologically, thEey have a sharply bent back head and a large buccal capsule, male have an eIxpVanded caudal bursa. Thirdly, pathogenically, iNnfection as a result of Ancylostoma duodenale is more serious and the adul t Uworm is however larger than N. americanus. A. duodenale has two pairs of teeNth on the ventral wall of its buccal cavity while N. americanus has a pair of cuttiAng plate on the dorsal wall, a concave tooth on the medial wall and a pair of triangDle-shaped lancets on the ventral wall of its buccal cavity. BAccAording to Bethony et al., (2006), the adult hookworm become haematophagous, I feeding on blood in the human small intestine, resulting into chronic loss of blood, leading to iron deficiency anemia, particularly among children and pregnant women. The phase of getting infected with human hookworm can be said to be in three stages: (i) invasion of human via skin penetration (ii) migration/movement of the larva within the host and (iii) The establishment within the intestine. i. Invasion of the human skin through penetration: Invasion begins with the penetration of human skin by the infective L3 larvae, although little 15 damage is done to the superficial layer of the skin there is usually a granulomatous reaction which is as a result of the host cellular immune response during blood vessel penetration resulted in an urticarial condition usually known as ground itch. ii. The migration phase is the time when the larvae move from the capillary beds into the lung then enters into the alveoli and migrate up to the bronchi and then to the throat where they are swallowed back into the intestine. When the intensity is high, that is, large numbers of worm are presentY, the migration of the worms produce severe bleeding, otherwise sRymptoms such as dry cough and sore throat may also be observed inA the infected person. A phenomenon known as a stage of dormancy hasR been reported in A. duodenale infection in areas where re-infection iBs continual. This is a situation wherein the filariform larvae invade t hLe skIeletal musculature and remain dormant to resume development at a later time. It is known that dormancy can be caused by pregnancy aYnd development resume at the onset of parturition. The larvaSe cIan T be seen in breast milk, hence transmission of infection to breast feeding babies. iii. The establishment of thEe paRrasites within the intestine: This is the most serious stage of hooVkworm infection. Once the parasite is established within its host, the Iyoung worm uses its teeth or buccal capsule to burrow into the mucosaN and feed voraciously on the host blood. The anticoagulant propertie s Uin the salivary gland enable the parasite to feed to satisfaction without the blood getting clotted. The blood loss as a result of A. dAuodNenale infection is ten times more than that of N. americanus. Iron Ddeficiency anaemia with occasional abdominal pain, loss of appetite and A craving to eat soil are some of the symptoms. In a situation of heavy worm IB load, other symptoms such as protein deficiency, delayed puberty, distended abdomen, dry skin and hair severe anaemia, stunted growth , mental dullness, cardiac failure and death, although rare, can be seen (Zeibig, 2013). The prevalence of hookworm infection differs from region to region. In Nigeria, Yaro et al., (2018) reported the prevalence of 55.8% in Akwa Ibom and Kano States for hookworm infections. While Delta, Oyo and Benue States had prevalence of 38.08, 35.80 and 35.40%, respectively and another 22 states in Nigeria had prevalence of hookworm 16 below 20.0%. Humphries et al., (2011) carried out cross-sectional pilot study of hookworm infection among 292 subjects from 62 households in Kintampo North, Ghana. The overall prevalence of hookworm infection was 45%, peaking in those 11–20 years old (58.5%). In children, risk factors for hookworm infection included coinfection with malaria and increased serum immunoglobulin G reactivity to hookworm secretory antigens. Risk factors for infection in adults included poor nutritional status, not using a latrine, not wearing shoes, and occupation (farmYing). The most serious consequence of hookworm infection is Ranaemia, secondary to loss of iron and protein of gut (Hotez et al., 2010A). It has been estimated that a single A. duodenale ingests about 150 µRL blood per day, while N. americanus sucks about 30 µL (Midzi et aIl.B, 2010). In a situation where the worm burden is significantly high in Lan individual, infection is normally severe with iron deficiency anaemia , particularly in people with inadequate iron reserves or intake.I ITn mYost developing countries, for instance, anaemia in pregnancy hSas been associated with worm infestation, especially hookworm (Hotez 2009). Prevalence and intensity of hookworm infection have been assoEciateRd with age and sex (Verhagan et al., 2013). Walana et al. (2014) reported considerable variation in the age-intensity profile of hookworImV infestation in their study in Kumasi, Ghana. Their study revealed Nthat infected individuals in age groups of <1, 1 to 9, 10 to 19, 20 to U29 and 30 to 39 years were in the proportion of 1.3%(2), 10.8%N(17 ), 16.5% (26), 27.2% (43) and 23.4% (37) respectively. Their oAbservation agrees with other existing findings that prevalence of Dhookworm is high among children (Rayan et al., 2010, Mbae et al., 2013, A Osazuwa et al., 2011). The high prevalence seen among individuals within IB the age of 10 to 39 years could be attributed to the fact that they are physically active and are more likely to be involved in activities such as farming which exposes them to the infection (Brooker et al., 2007, Abu- Madi et al., 2010). There are other reports that hookworm infection in the elderly could be relatively high (Lim et al., 2009). 2.3.3 Trichuris trichiura: The whipworm 17 Trichuris trichiura, also known as the human whipworm, is a roundworm that causes trichuriasis in humans. It is referred to as the whipworm because it looks like a whip with wide handles at the posterior end. The whipworm has a narrow anterior esophagus and a thick posterior anus. The worms are usually pink and attach to the host via the slender anterior end. The size of these worms varies from 3 to 5 cm. The female is usually larger than the male (Truscott et al., 2015). The female worm can lay anywhere from 2000 to 10,000 eggs per day. The eggs are deposited in soil from human faeces. After 14 to 21 days, the eggs mature and enter an infective stagYe. If humans ingest the embryonated eggs, the eggs start to hatch in the humRan small intestine and utilise the intestinal microflora and nutrients to multiply anAd grow. The majority of larvae move to the ceacum, penetrate the mucosaR and mature to adulthood. Infections involving a high-worm burden will typicaIlBly involve distal parts of the large intestine (Truscott et al., 2015) L Trichuriasis is 1 of 3 well-documented soil-transmittYed helminthiasis infections; the other 2 are ascariasis and hookworm infection. IITt is considered a neglected tropical disease by the World Health Organisation (SWHO) and Centers for Disease Control and Prevention (CDC). ER The egg of the whipworm is ItVhe infective stage, and favourable conditions for its maturation are warm and Nhumid climate. This is why most of the disease burden is seen in tropical climates, specifically in Asia and less often, in Africa and South America. It is also f ouUnd in rural parts of the southeast United States. It is estimated that worldwideN there are between 450 million to 1 billion active cases with most diagnoAsed in children. It is thought that there is partial protective immunity that devAelopDs with age (Ranjan et al., 2015, William-Blangero et al., 2008). IBWhipworm infection was estimated to affect about 1 billion people. Severe consequences, such as rectal prolapse and dysentery are associated with high worm load, with obvious and immediate influence on the health of infected individual (Stephenson et al 2000, Bethony et al., 2006). In contrast to the above data, a global prevalence of 795 million people was previously estimated by the World Health Organisation (WHO, 2012). The most common cause of trichuriasis is ingestion of infected eggs that are found in soil. This is often due to poor sanitary conditions, including open defecation and using human faeces as fertilizer. Some recent studies 18 show that people with certain chromosome traits may be predisposed or have increased susceptibility to acquiring trichuriasis (Brooker et al., 2015). A human host consumes infected eggs, typically while eating food. Once the embryonated eggs are ingested, the larvae hatch in the small intestine. From there, they migrate to the large intestine, where the anterior ends lodge within the mucosa. This leads to cell destruction and activation of the host immune system, recruiting eosinophils, lymphocytes and plasma cells. This causes the tyYpical symptoms of rectal bleeding and abdominal pain. Most infections are of lightR intensity and therefore are asymptomatic. Characteristic symptoms such as weightA loss nausea, bloody stools, pain in the lower abdomen, anemia and rectal prolapRse can be seen in chronic infection as a result of Trichuris trichiura. Adult Bworms can be seen externally, embedded in the rectal mucosa in the case of reLctalI prolapse. Patients will typically reside in or have visited areas that are endemic to the whipworm. The patient will usually complain of abdominal pain, painful Ypassage of stools, abdominal discomfort, and mucus discharge. Rectal prolaIpTS se is known to occur in a heavy infestation. Children may develop aneRmia, growth retardation, and even impaired cognitive development. The latter 2 are thought to be due to iron deficiency and poor nutrition secondary to worm bVurdeEn and are not a direct cause of the infestation (Bansal et al., 2018, Shear et aIl., 2018). N Anemia results whe n Uthe worms penetrate the intestinal wall, also some blood loss may be as a resNult of the worms ingesting host blood. Secondary bacterial infections are commonA feature of heavy infections, occasioned by the penetration of the mucosal lining bDy the worms, providing entry for pathogenic bacteria. The infection is usually assoAciated with malnutrition and numerous studies have documented this in most of IBthe developing worlds. Wilson et al. (1999) noted that children even with low worm loads manifest stunted growth. One of its most insidious effects reported in whipworm infection include Vitamin A deficiency and its debilitating effects on cognitive development (Stephenson et al 2000, Bethony et al.,2006) and deficiency in verbal fluency in children (Ezeamama, 2005). Ezeamama further cited the study conducted among Filipino children who were 7-18 years of age and that were carried out by Nokes et al. (1992) among Jamaica children 9-12 years of age as two evidential research reports that confirm the devastating influence of Trichuris 19 trichiura on verbal fluency in children. These deficits in childhood growth and development occasioned by whipworm infection have long-term implication. 2.3.4 Strongyloides stercoralis: The threadworm Strongylodiasis is best known in the developed world for the severe consequences of the hyperinfection syndromes linked to immunosuppression caused by diseases like lymphomas, leukemias, or the use of corticosteroids (Mejia and Nutman 2012). YThis is a small worm and in resource poor countries are associated with malnutriRtion. The most important presenting complaint of S. stercoralis infection reportedA is diarrhea, which in most cases is chronic. The infection may not show any syRmptom in normal immunocompetent individuals, but may be severe or fatal iIn Bindividuals who are immunocompromised, especially in HIV and advanced tubLerculosis patients, (Purtilo et al., 1974; Dada-Adegbola et al., 2010). Y According to Krolewiecki et al. (2013), the IpTrincipal features of Strongyloides stercoralis are: multiplication within Rthe Shost which resulted in autoinfection, morbidity which may be acute or chEronic, potential fatality, the main diagnostic stage is the larvae and the theraupeutiVc goal is cure. In immunocompetent host, the parasite usually infects the mucosa of Ithe small intestine and persists as an asymptomatic but often chronic infection. MNan gets infected with S. stercoralis through contact with the infective larvae in theU soil rather than the larvae in contaminated water. Human cases of strongyloidiaNsis are currently estimated at about 100 million to 200 million worldwide. AAn estimated case of 21 million infections occur in Asia, while about 900,000D cases were reported in the former USSR, and 8.6 million cases in Africa, whiAle 4 million in tropical America, 400,000 in North America, and about 100,000 IBcases in the Pacific islands. The free-living forms thrive best in warm, moist climates where sanitation is substandard. A reportedly high incidence of infection, among residents of mental institutions in the United States have been blamed on the prevalence of faeces with infective larvae or larvae capable of rapidly becoming infective combined with poor sanitation or personal hygiene. An 18% rate of infection was identified in a study among the mentally ill patients in New York City. The disease can be considered zoonotic because dogs and cats serve as reservoirs of infection for human infection (Bogitsh et al., 2013). 20 Epidemiologic studies looking into the distribution of S. stercoralis in communities have shown prevalence peaks in adolescents, remaining stable in adults, with a similar distribution as hookworms. Some studies have shown no gender difference and others have found it more prevalent in males, possibly representing differential exposure (Becker et al., 2012, Steinmann et al., 2007, Krolewjecki et al., 2011). Findings of higher burden in the adult population challenge the current policies of focusing interventions (and also drug donations) in school-age and preschool-age children. WHO‟s guidelines offer a clear stepwise approach to the community based treatYment of STH through anthelminthic therapy, with the 20% and 50% thresholds of cRombined prevalence for any of the major STH triggering the use of preventive cAhemotherapy interventions once or twice a year, respectively (WHO, 2006). WhiRle this strategy is in use around the world and delivering measurable benefits, thIeBre is room for further study of this strategy in order to provide scientific su pLport to the expansion or modification of this approach. Among these unsolveYd areas is the definition of an appropriate prevalence threshold that should tIriTgger community treatments for S. stercoralis, considering the particularities of the life cycle and treatment goals. The search for new diagnostic tools for S. stercoSralis should not hamper the development of strategies for the implementation ofR control programs. The use of the available, albeit moderately sensitive, direct Ediagnostic tools in sentinel sites should allow predicting a good enough pictuIrVe of the distribution of S. stercoralis that could justify a therapeutic interventionN. More evidence and data are needed, however, to define such prevalence thre sUholds. Krolewjecki et al. (2013), in their write up, brought out the following keNy point about S. stercoralis: (i) Direct parasitological techniques for the diagnosAis of S. stercoralis infections have suboptimal sensitivity, affecting adequatDe prevalence measurements, burden of disease estimations, and clinical trials desiAgn. (ii) The incorporation of S. stercoralis in the preventive chemotherapy IBstrategy in place of other STH requires special adjustment in the drug regimens. Ivermectin is the drug of choice; albendazole and mebendazole have no significant activity in single drug regimens. (iii) The life cycle of S. stercoralis, which replicates within the human host, makes cure rather than lowering the worm burden, the appropriate theraupeutic goal. (iv) Although multiplex molecular-based diagnostics and optimal treatment regimens for S. stercoralis and STH burden, the appropriate therapeutic goal, Infections, should be pursued as a pressing need, their development 21 should not delay the planning and implementation of strategies to control strongyloidiasis and STH with the existing tools. 2.4 Environmental risk factors predisposing to STH infection Several studies (such as Asaolu et al., 2002, Ugbomoiko et al., 2008, Ilechukwu et al., 2010, Mara et al., 2010) carried out in Nigeria and around the whole world have reported the association of STH infections with environmental risk factors, sucYh as lack of clean water, climate and season . The presence of refuse heaps, guRtters and sewage unit in and around human dwellings, which pollute the Aenvironment contribute to the thriving condition for soil-transmitted helminth (NRwosu and Anya, 1980). IB 2.4.1. Temperature, climate and season L Soil-transmitted helminth infections are widely distribYuted throughout the tropics and subtropics, and climate is an important detIerTminant of transmission of these infections. Adequate warmth and moistureS are key features for each of the soil- transmitted helminth. Ascaris lumbricoidRes and Trichuris trichiura eggs survive drier climate better than the hookwoVrm Elarvae. At low humidity (atmospheric saturation less than 80%) human ascarisI ova do not embryonate (Brooker and Michael 2000). The viability of hookwormN eggs and the survival of the larvae depends on whether egg-containing hum anU faeces are deposited in an environment where the ambient temperatures are high enough and the soil conditions appropriate for larval developmenAt (HNotez, 2008). Chandler (1929) reported that the ambient temperatures of betwDeen 20 °C and 30 °C are optimal for the development of the larval in the soil, but Athat larval viability still continues even when temperatures rise into the low 40s. It IBhas been discovered through the use of satellite mapping and remote sensing, that the environmental stages of hookworm have higher heat limits than other soil-transmitted helminth, such as Ascaris lumbricoides and Trichuris trichiura. This may be responsible for the observation that hookworm is endemic throughout most of Mali and southern Chad, in contrast to ascariasis and trichuriasis. On the other hand, hookworms exhibit a lower tolerance for cold temperatures than do either Ascaris or Trichuris, which explains why hookworms are seldom found at high altitudes. In Africa, the geographic range of temperatures and rainfall that make conditions 22 suitable for hookworm larvae in the soil are similar to those conditions required for the Anopheles mosquito vector that transmits malaria. Thus, there is a high degree of geographic overlap and co-endemicity between hookworm and malaria in sub- Saharan Africa. Mabasso et al. (2003) discovered that summer temperatures in KwaZulu-Natal, South Africa, play a primary role in the transmission of hookworm infection, with cold temperatures serving as an effective means of regulating its inland and southward distribution. Bogitsh et al., (2013) reported that Ascaris and whipworm (Trichuris) are found in areas with dense shade, warm climate, heavy rainfall and Ypoor sanitary conditions that are conducive to soil pollution. Children are moreR heavily infected than adults because they come into close physical contact with cAontaminated soil than adults. Stojčević et al. (2010), in their study on contaminRation of soil and sand with parasite elements as a risk factor for human healthB in public parks and playgrounds in Pula, Croatia, showed a higher contaminatioLn raIte in December than in June. This correlates that the increase in the number of p ositive samples, as well as the increase in the number of helminth eggs founId in YDecember to be attributed to the very suitable environmental and climate conditioTns, with moderate temperature and sufficient soil humidity. S ER 2.4.2 Availability of clean waIteVr: The availability of clean wNater for domestic purposes is a key factor in reducing the rates of diarrhoea, Uascariasis, schistosomiasis, and trachoma. The presence of sanitation facilities were also reported to decrease diarrhoea morbidity and mortality and the seveArityN of hookworm infection (Esrey et al., 1991). ConAtrarDily, Belyhun et al., (2010) carried out a population-based study among Bmothers and their infant in a rural area of Southern Ethiopia and found out that the I availability of piped water in the house compound was independently associated with increased risk of infant helminth infection, and the reason for this was suspected to arise from the poor quality of piped water. However, Bartram and Caincross (2010), in their article, suggested a five point summary about the importance of Hygiene, sanitation and water, which are: (1) that massive disease burden as a result of deficient hygiene, sanitation, and water supply can be largely preventable (2) that the costs of the interventions has a total benefits greater than the health benefits alone (3) 23 that the ambition of international policy on drinking water and sanitation is inadequate; likewise, hygiene, sanitation, and water supply should be a development priority. (4) that hygiene, sanitation, and water supply continue to have health implications in the developed world. (5) that active involvement of health professionals in hygiene, sanitation, and water supply should be accelerated to consolidate progress for health. Strunz et al., (2014) conducted a review analysis on the association of imprYoved Water, Sanitation and Hygiene (WASH) and the STH reduction; Water-relatRed access and practices were generally associated with lower odds of STH infectioAn. The meta- analyses examine the association of piped water access and use ofR treated water on STH infection. Using treated water (filtered or boiled) was aBssociated with lower likelihood of having any STH infection (k=3, OR 0.46, L95I% CI 0.36–0.60). The quality of evidence for the analysis was low, as all thrYee s tudies used for the analyses were observational. The use of piped water was InTot associated with STH infection in general (k=5, OR 0.93, 95% CI 0.28–3.11)S. The quality of evidence for the pooled 2estimate was very low due to high heterogeneity (I =98.6%, 95% CI 98%–99%, Qp- value, 0.01) among the studies. TheE useR of piped water was associated with reduced likelihood of A. lumbricoides infection (k=4, OR 0.40, 95% CI 0.39–0.41) and T. trichiura infection (k=3, OR 0I.5V7, 95% CI 0.45–0.72). Evidence quality for these two meta-analyses was low, baNsed on four studies and three studies respectively. They did not find a sufficie ntU number of studies to conduct a similar meta-analysis for hookworm infecNtion, although Nasr et al., (2013) found a significantly lower adjusted odds of infeAction (OR 0.59, 95% CI 0.34–0.91) for Malaysian children with access to piped wDater. Cundill et al. (2011) and Kounnavon et al. (2011) found no statistically signAificant associations between piped water access and hookworm infection. IB In one study, examining storage of water, Quintero et al., (2012) found a significantly higher adjusted odds of T. trichiura infection for Venezuelan children and adults collecting water in „„inappropriate‟‟ receptacles (OR 1.12, 95% CI 1.09–1.15) [69]. Belyhun et al., (2010) found a beneficial association of using an outside water pipe compared to an indoor tap for infection with any STH among Ethiopian infants (OR 0.21, 95% CI 0.09–0.51). Matthys et al., (2007) found that having a private well significantly increased the odds of hookworm infection for farming households in 24 western Cote d‟Ivoire (OR2.32, 95% CI 1.24–4.05). No evidence was found of an association between public or private water source and S. stercoralis infection (Hall et al., 1994). Having „„inadequate water supply‟‟ in schools was strongly associated with increased infection with any STH among school children living on Pacific islands (OR 4.93, 95% CI 2.24–10.88) (Hughes et al., 2004). 2.4.3 Soil types: Y The Ascaris eggs best survive with increases depth and in less permeable cRlay soils, (Crompton, 2001) and it prevents egg dispersal by water. Hookworm eAggs hatch in soil and give rise to first-stage larvae, which molt to infective Rlarval stage that penetrates the skin of man. Soil type is an important factor in IthBat the developmental stages of soil transmitted helminth, this includes the ova tLhat develops to the larval stage in the environment that is the soil. Clay soils Yhave low permeability because their interstitial pores are small and therefore lIarTval development is better in sandy soils rather than the clay soil. Hookworm mSobility is greater and better in sandy soils than in clay soils. According to MabRasso et al 2003, the coastal areas in the developing world exhibit particularlEy high hookworm endemicity because they have sandy soil, and they are at lowV altitudes (and therefore high temperatures). Studies have shown that the occurrenIce of high hookworm prevalence at low altitudes was significantly associated Nwith well-drained sandy soil types. Low hookworm prevalence at highe r Ualtitude was associated with poorly drained weakly developed soil types. AlsoN, Mabasso et al. (2004) in another study found out that the particle size distribuAtion of sand fractions, organic matter and clay content in the soil influence the survDival of hookworm larvae and hence the parasite‟s transmission. Sandy soils, dueA to their geological characteristics, being formed by sand particles with diameters IBranging from 0.02 to 2 mm, and with the ability to retain water between the spaces of soil particles, represent an important source of human infection by parasites. Helminth larval stages are notably aquatic, and a high humidity of the soil is essential for their survival. The development of eggs into embryos and in the viable larva stages are strongly influenced by the amount of sun to which the infected soil is exposed, the rate of evaporation, in addition to the rain pattern. Frequent rain throughout the year in places with sandy soil which are not directly exposed to sun and are protected from 25 intense evaporation provides ideal conditions for rapid parasite development (Rocha et al., 2011). 2.5 Human behaviour and socioeconomic factors: Human behaviour and socio-economic factors are important predisposing factors to Soil-transmitted helminth infection (Girum 2005; Rukmanee, 2008). Several studies have highlighted hygienic practices such as hand washing habit and dirty fingernails, shoe wearing habit, proper sewage disposal and level of education as importantY and major contributory factors for STH infections (Ulukanligil 2001; FatireRgun and Oluwatoba 2008). Conditions that promote environmental decay incluAde not well planned housing and human habitation patterns. This is becauseR urbanisation in developing countries usually results from unplanned, uLncoInBtrolled and constant migration of people from rural areas to the urban center s in search of employment opportunities (Fashuyi 1983, Ozumba et al 2002). FuYrthermore, the combined factors of ecosystem degradation with socio-cultural anId Tagricultural practices of the people create conditions favourable for high transmSission and sustenance of many human diseases, especially parasitic diseases (HRolland et al., 1989, Kaliappan et al., 2013). 2.5.1 Hygiene practices VE Hygiene practices such asN hanId washing habit before eating and after the use of the toilet, dirty finger naiUls, and shoe wearing habit has been reported by various studies to be positively relat ed to STH infections (Rukmanee 2008; Fatiregun and Oluwatoba 2008; GirumA 20N05). Mara et al. (2010) reported that out of human excreta, faeces are the mosDt dangerous to health. According to their submission, one gram of fresh faeces fromA an infected person can contain around 106 viral pathogens, 106–108 bacterial Bpathogens, 104 protozoan cysts or oocysts, and 10–104 helminth eggs. I Bartram and Carincross (2010) compared intervention studies of hand washing with soap carried out in child-care centers in the United States and Australia with some developing countries and found similar reductions in diarrhoea of roughly 50%. Their observation further underscores the role of personal hygiene in the spread of STH infection. Three randomised controlled trials, two carried out in China and one in the Peruvian Amazon, found strong benefits for interventions that focused on promoting hygiene in schools (Xu et al ., 2001, Bieri et al., 2013, Gyorkos et al., 2013). Xu et 26 al., (2001) assessed a randomized intervention that promoted handwashing with soap, both before eating and after defecation among 657 school children in three schools. All the infected children were treated at baseline. At the 1-year follow-up, A. lumbricoides prevalence for children in the experimental group had declined by 35.7% (pre-intervention prevalence, 68.3%; post-intervention cumulative infection rate, 43.9%) compared with an increase in the control group of 78% (pre-intervention, 41.4%; post-intervention, 73.7%); this was a statistically significant difference (p, 0.01). The study‟s primary limitation was that schools were the unit of randomizaYtion, with two primary schools becoming controls and the third receiving the inteRrvention. With so few clusters, it is highly possible that confounding factoArs were not comparable between the control and experimental groups. Bieri et aRl. (2013) reported on a single-blind, unmatched, cluster-randomised interventionI Btrial involving 1,718 children (aged 9–10) in 38 schools over the course of on e Lschool year. Schools were randomly assigned to a health-education package, which included an entertainment- education cartoon video, or to a control packaIgTe, wYhich only displayed a health-education poster. All participants were trSeated with albendazole at baseline. At follow-up at the end of the school yeRar, knowledge about STH was significantly higher in the intervention group, aEnd almost twice as many intervention children (63.3% versus 33.4%, p, 0.01)V reported washing their hands after defecating. The incidence of STH infection (prIedominantly T. trichiura and A. lumbricoides) was also significantly improved in tNhe experimental. U 2.5.2 OccupAatioNn, housing system and family size The sprDead, distribution and sustenance of various helminth infections may also be faciAlitated by the living conditions of the people in crowded or unhealthy situations IB(Udonsi and Amabibi, 1992). On many occasions, occupation has been used to determine the socioeconomic status of individuals and low-socio-economic condition is a risk factor for helminth infection (Hotez, 2008). Housing system in terms of the structure and materials used for building, likewise the availability of toilet system in the house is another major determinant in STH infection within household. Holland et al., (1988) in their study in Panama found 27 houses made from wood and bamboo associate with significantly higher rates of Soil transmitted helminth infection than concrete houses. Strunz et al., (2014) discovered that sanitation access (availability or use of latrines) was associated with lower likelihood of infection with any STH (k=8, OR 0.66, 95% CI 0.57–0.76), T. trichiura (k=7, OR 0.61, 95% CI 0.50– 0.74), and A. lumbricoides (k=6, OR 0.62, 95% CI 0.44–0.88). The quality of evidence for these meta-analyses was low due to the observational nature of included studies. They did not Yfind evidence that sanitation access was associated with hookworm infection (Rk=6, OR 0.80, 95% CI 0.61–1.06), which had very low evidence quality due toA imprecision. They found limited evidence that use of shared or private saRnitation facilities influenced odds of STH infection. Worrell et al., (2013) IfBound in Kenya that participants using toilets located outside of their househol d Lpremises had significantly increased odds of infection with any STH. In conYtrast, another study found that sharing latrines with neighbouring households, cIomTpared with private latrine use, was associated with significantly lower odds oSf hookworm infection (Matthys et al., 2007). R Another longitudinal study by GenEser et al. (2006) in urban Brazil found that the major risk factors for diarrhoeaI iVn the first three years of life were low socioeconomic status, poor sanitation coNnditions, presence of intestinal parasites, and absence of prenatal examinatio n.U The study concluded that diarrhoeal disease rates could be substantially decNreased by interventions designed to improve the sanitary and general living condAitions of households. Adekunle et al. (1986) in their study of family influencDe on incidence of intestinal parasites among Nigerian children found ascaris prevAalence and high worm burden to be higher among children from large family size IBand the order in which a child is born in large family also affects the likelihood of becoming infected. 2.6 Household clustering of infection According to Anderson and May (1991), intestinal parasites are neither evenly nor randomly distributed among hosts, but tends to be aggregated in a few infected individuals in communities where the prevalence of infection is high. There are 28 evidences of household clustering of infected individuals in most of the diseases as a result of helminth infection due to ascaris, trichuris, and strongyloides. This clustering can persist through time, as reported by Hotez et al. (2008) in their study of familial predisposition to heavy infection with Ascaris lumbricoides and Trichuris trichiura in Mexico. According to Ellis et al. (2007) individuals with high intensity of STH infection have been found to aggregate in families. Similarly, significant clustering both at a household and familial level was demonstrated in population endemic for A. lumbricoides in Nepal (William-Blangero et al., 1999) and susceptibility tYo T. trichiura infection has also been shown to be common in families (Chan et aRl., 1994). In Nigeria, where prevalence as high as 50% has been recorded amoAng preschool children, the household aggregation of STH infection remains uRnexplored, most studies were carried out among school aged and preschool Bchildren. The study conducted by Asaolu et al. (1992) among all age groups inL foIur different villages in Oyo State, recorded the significance of householdY si ze as an important factor influencing egg output. Hence, there is a need fIoTr this present study to look into the STH infections within and between householSds in southwestern Nigeria. R 2.7 Host Genetic factors predisposEing to STH infections Helminth parasites are regardIedV as master manipulators of host immune response (Williams-Blangero et al.N, 2012 Bethony et al., 2006; Quinnell et al., 2010). On helminth-host immu niUty relationship, Bentwich et al. (1995, 1999) have reported that the ability to mNount an effective immune response to other infections, particularly HIV and tubAerculosis is gravely impaired by helminth infections. Research reports on STH geDnome indicate Interleukin-10 as the most abundantly produced regulatory cytoAkine in soil-transmitted helminth infection (Jackson et al., 2004; Bethony et al., IB2006). The survival success of STH is attributed to their secretomes which interact with host tissues and maintain existence (Bethony et al., 2006). Two key observations and implications emerge from these experts‟ findings above: The significance of host immune response to STH infection, implying a possible connection between individual genetic component and STH infection and secondly, the worm genetic effects. This points out the relative importance of both host genetic factors and worm genetic factors on disease distribution. 29 Several facts suggest that there is an important genetic influence defining the susceptibility to helminth infections. It is typical that worm loads are over-dispersed in the infected communities with 20% of the individuals harboring 80% of the parasites (Holland, 2009). Individual predisposition to get infected by heavy or light worm loads is maintained in treatment-reinfection studies (Hlaing et al., 1987), and aggregates in families (Chan et al., 1994). Epidemiological studies revealed than in addition to exposure and household determinants, genetic factors account for an important proportion of the variation in worm loads (William-Blangero et al., 1Y999, 2012, Cuenco et al., 2009, Quinnell et al., 2010). R Genetic factors have been also found to influence the levels of protectiAve antibodies against helminths, as shown for the IgG levels to larval and aIdBult w Rorm antigens in humans infected by W. bancrofti (Cuenco et al., 2009). TheL heritability of circulating antibody levels against helminths (particularly IgG1 aYnd I gG2) ranges between 70 and 80% depending upon time and isotype (GasbarreT et al.,1993), and gene expression analyses have found that many genes differentiIally expressed between resistant and susceptible animals are indeed implicated inS antibody synthesis (Araujo et al., 2009). A. lumbricoides is very allergenic and, aRs explained earlier, induces in the host a type- 2 skewed immune profile with sVeveEral features that resemble the allergic response to non-parasitic allergens, includIing the synthesis of specific IgE (Fitzsimmons et al., 2014). The role of this isoNtype in the resistance to Ascaris is controversial, but some studies have shown thUat worm loads and the proportion of re-infected children after treatment is signNificantly lower in individuals with the highest levels of anti-Ascaris IgE antibodAies (Hagel et al., 2008). In endemic populations, most of the infected individuDals produce specific IgE to Ascaris without developing allergic symptoms, sugAgesting that the regulation of the antibody response to helminths is polygenic, and IBgenetic loci influencing the strength and specificity of the IgE response to helminths do not necessarily confer susceptibility to allergic asthma or IgE sensitisation. Researchers have largely been investigating diseases, including STH infection, from population-based approach, particularly with an important objective of finding out how the differences observed in susceptibility to many infectious diseases are greatly influenced by host genetic factors (Ellis et al., 2007, Quinnel et al., 2010, William- Blangero et al., 2012, Kaliappan et al., 2013). According to William-Blangero (2012), 30 the host genetic factors that are found within a specified population is important in determining patterns of infection rather than host population structure. The identification of specific host genes responsible for susceptibility to infection or the phenotypic response to infection is most important in determining more effective intervention for assessment of the impact and control of STH in a population. The high density genetic markers that were used in identification of specific genes were specifically used to perform quantitative trait locus (QTL) which was used to track segments of DNA that are passed identical-by-descent across generations in ordYer to co-localize them with phenotypic variations. R William-Blangero (2002) reported a strong evidence for two distinct locAi influencing susceptibility to Ascaris infection. The strongest signal was found neRar the q terminus (13q32-q34) of chromosome 13, the observed logarithm of oddI (BLOD) score represent strong evidence that a gene in the area of chromosome L13 harbours an important quantitative trait locus influencing human host AscYaris burden. The second clear evidence for quantitative trait locus influencing sIuTsceptibility to Ascaris infection was on chromosome 1. On this chromosome, theS LOD score exhibits a sharp peak at 1p32 with maximum value 3.01, genome-widRe p-value 0.033. The peak is significant and provides clear evidence for a secondE locus influencing the Ascaris worm burden trait. A further research in Nepal IinVvolving a genome scan of the Jirel tribe pedigree provided strong evidence Nfor two distinct quantitative trait loci (QTL) influencing the susceptibility to Asc arUis infection. The genes are on chromosomes 1 and 13 and there was an evidenceN for a third locus influencing Ascaris burden on chromosome 8. There are evidencAes that the latter quantitative trait locus was significantly less than the former Dtwo loci (Williams-Blangero et al. 2002). A subsequent genome scanning of the AJirel pedigree now in a higher number of host individuals, identified additional six IBand three chromosomal regions with evidence for QTLs influencing the intensity of and susceptibility to Ascaris infection and within the QTLs multiple immune-related genes were also identified. (Williams-Blangero et al., 2008). The genome wide linkage analyses study done by William-Blangero et al (2008) in Eastern Nepal among the Jirel population, provide evidence for two quantitative trait loci (QTL) influencing susceptibility to whipworm infection, one located on chromosomes 9 (LOD score 3.35; genome wide P= .0138) and the other located on 31 chromosome 18 (LOD score 3.29; genome wide P=.0159). The heritability (or familiality) of the whipworm egg count in this sample of individuals in the Jirel 2 -17 population is highly significant (h = 0.38+ 0.06; P =6.5 x 10 ). On chromosomes 9 is found the PDCD1LG1 which is also known as B7H1 genes, this gene are known to be involved in the stimulation of T cell response and have a particularly strong effect on the production of interleukin(IL) -10 (Dong et al., 1999). Levels of IL-10 among other cytokines in humans, have been shown to be factors predictive of the susceptibility to whipworm infection (Jackson et al., 2004). Y Nevertheless and considering the overlap in biological pathways implicateRd in the immune responses to helminths and allergens, it has been hypothResiseAd that some genetic variants influencing the IgE response to helminths may also predispose to develop IgE sensitisation with non-parasitic allergens (MuLlleIr Bet al., 2007) or even predispose to allergic diseases (Hopkin 2009, Fumagalli et al., 2010). Although the empirical evidence is limited, it can be noted that (Yi) the major histocompatibility complex (MHC) participate in determining susceIpTtibility to helminthic infections (De Angelis et al., 2012) (ii) some genetic varianSts are associated to both, susceptibility to helminthic infections and allergic sensitRisation, as shown for the genes encoding for interleukin 13 (IL13), the signalV traEnsducer and activator of transcription 6 (STAT6), and chitotriosidase; and (iii) Iother genetic loci regulate the antibody response to helminths without predispoNsing to allergy. Studies in humans anUd other mammals support the role of genetic factors in the predisposition toN Ascaris (William-Blangero et al., 1999, Nejsum et al., 2009) but few genes have Abeen identified so far (Mohler et al., 2007, William-Blangero et al., 2002, 200A8, PDeisong et al., 2004,Acevedo et al., 2009). The enhanced resistance to parasitic Bworms through genetic variation has been observed in Th2 immune signaling genes I and some also contribute to allergic susceptibility. Peisong et al. (2004) found an association between a common genetic variant of the 3′-UTR regulatory elements of STAT6 and Ascaris egg counts in China (Peisong et al., 2004). In addition, a cross- population comparison between haplotypes in China and United Kingdom revealed a negative correlation between worm burden and expected risk of asthma (Mohler et al., 2007). The 5q31 locus is another example of a common locus for the susceptibility to 32 helminthic infection and allergic diseases. It contains genetic variants in the IL13 gene associated with the worm burden of Ascaris in a Chinese population. Genes encoding for human chitinases have been also identified as a potential link between ancestral responses to invertebrates and the susceptibility to allergic phenotypes. In humans, chitinases promote Th2 responses. The acidic mammalian chitinase (AMCase) is induced in epithelial cells and macrophages by an IL-13- mediated pathway and is expressed at high quantities in human asthma (Zhu et al., 2004). Genetic polymorphisms in chitinase genes have been associated with asYthma (Bierbaum et al., 2005), and asthmatic children exhibited increased chitinasRe activity and increased YKL-40 levels in BALF (Goldman et al., 2012). GeneRtic vAariants in the gene encoding chitotriosidase (CHIT1) have not only been associated with the response to filarial infection but also with asthma (Sinha et IaBl., 2014, Kim et al., 2013). LY Experiments using different strains of mice aTnd rats demonstrated the MHC- restriction in the recognition of excretory-secIretory products of Ascaris and the genetic control of the antibody responsRe to Sits antigens (Kennedy 1989, Kennedy et al., 1990), however, the relationshipE between human MHC alleles and the specificity of the antibody response to A. luVmbricoides is unknown. Linkage studies identified a quantitative trait locus (QTL) aIccounting for the variability in Ascaris egg counts and total IgE levels in chromoNsome 13q33-34 (William-Blangero et al., 2002). A second linkage-based genom eU scan, including 1258 members of a single pedigree identified three potential QNTLs influencing susceptibility to A. lumbricoides with genome-wide significanceA, localised on chromosomes 8, 11, and 13 (William-Blangero et al 2008). Of AthesDe regions, the 13q33 locus is of great interest because it contains the gene BTNFSF13B encoding for the cytokine B cell activating factor (BAFF). They studied I the role of common variants in the 13q33 locus on the IgE and IgG levels against Ascaris and the putative resistance marker ABA-1, and identified a region of 125 kb harbouring two polymorphisms significantly associated with antibody levels against Ascaris (Acevedo et al., 2009). The effect of this variant was observed in both non- asthmatic and asthmatics, suggesting that participates in pathways implicated in antibody synthesis under physiological conditions and is not directly associated with allergic sensitisation and/or asthma. At this point, it is unclear if the association in 33 LIG4 is functionally related with this gene or resulted of the linkage disequilibrium with other variants. Furthermore, the polymorphism rs10508198 (3980G>C) in the TNFSF13B gene was associated to the specific IgG levels to Ascaris. The carriers of the wild-type genotype GG have the highest levels of specific IgG to Ascaris in both non-asthmatics and asthmatics, suggesting that TNFSF13B may regulate the strength of the antibody levels against Ascaris. There was no association between markers in the 13q33 locus and the IgE levels to House Dust Mites (HDM) or the presence of asthma (Acevedo et al., 2009). Y In Nigeria, the data about the involvement of this human genetics in STH inRfection is lacking, hence there is need for the information about the heritabilitRy ofA STH among the Nigerian population. LIB 2.8 Morbidity control through deworming programYme In the 1980s, several oral drugs developed aInTd commonly used in veterinarian practices were found to be highly effectiveS against human worm pathogens. These included praziquantel and albendazole (RHotez et al, 2004). This development, for the first time, allowed the systemVaticE examination of the impact of these parasitic infections on more subtle aspIects of morbidity, such as childhood growth stunting (Adams et al, 1994, McGNarvey et al 1996, Olds et al 1999), delayed intellectual development (Noke sU et al, 1992, Kimura et al, 1992), cognitive impairment (McGarvey et aNl, 1996), decreased function work capacity and anemia (Ezeamama et al, 2005, FriAedman et al, 2005). ThrAoughD research, it became clear that a significant amount of morbidity induced by Bworms was based on a complex interaction between the host and the specific parasite. I In children, for example, the growth stunting effect of parasites was most marked in early childhood or during the adolescent growth spurt; and it depended (to a large extent) on the baseline nutritional status of the population, i.e., populations which already had moderate to severe malnutrition, suffering disproportionately from the same degree of parasitic infection (Olds et al, 1996). Fortunately, growth stunting could often be reversed with curative chemotherapy, but it was critical to keep the child free of rapid reinfection (Olds et al, 1996). Cognitive impairment from parasitic 34 infections was also most pronounced in the youngest children and could often be reversed rapidly, following parasitological cure. Anemia was perhaps the most complex of the worm-induced morbidities. As one might expect, the impact of worms on anemia was greatest in populations who were already iron deficient and among women and growing children of both sexes. With infections, such as hookworm, anemia was principally driven by blood loss (Hotez et al, 2004). As a result, the development of anemia could be arrested with parasitological cure, but improvements in blood counts required iron supplementYation (Hall, 2007). AR It was also recognised that growing children had long-term dBetriRmental effects of chronic parasitism that could be reversed if regularly “dLewoIrmed” (Cooper et al, 1995). The international health communities, specif ically the World Health Organisation (WHO), began to look into the feasibilitYy of deworming populations on a scale similar to childhood immunizations and IdTistribution of oral rehydration fluid (WHO 2002a, 2002b, 2006). The evolvingS plan was to use a combination of anti-helminthic drugs to treat several parasitic infections, at the same time it was important to determine the most cost-effectiveE waRy to deliver treatment, particularly to school- aged children. Such an approaIcVh also had to be both safe and effective. The WHO commissioned a double-blNind placebo-controlled trial in four locations (two in Africa, two in Asia) using theU identical protocol to make this determination (Cioli et al, 1995, Olds et al 1999). Th e study showed that the two most important drugs, albendazole and praziquanteNl, could be administered together and that the side effect profile was so low that Athey could be administered by school teachers and lay community health worAkersD. This study and several additional ones were used to show that mass Bdeworming could have a positive impact on growth stunting, anemia, and cognitive I performance. As a result, the concept of deworming a significant percentage of the at- risk children of the world was brought to the international agenda in early 2000 (WHO 2002a 2002b). Miguel and Kremer (2004) analyse the impact of deworming on children‟s health and education. They evaluated International Christelijk Steunfonds Africa‟s Primary School Deworming Project, which targeted children in 75 primary schools in the Busia region of Kenya with anti-STH and anti-schistosome treatment. Their 35 evaluation results were inspiring: school-based deworming treatment kick-started a virtuous cycle of lower worm loads, improved growth, and fewer school absences among children. The Kenyan government and donors, impressed with their findings, seized the opportunity to incorporate school-based deworming into the new national school health policy. The government used a nationwide fecal survey and mapping exercise to identify high-burden areas and guide the design of the intervention. In 2009, school-based deworming went live at scale in Kenya. Encouraging results from the Busia trial provided a strong basis for national scalYe-up. Researchers‟ analysis showed that one year after the intervention, childRren who received deworming children treatment were 44 percent less likely to havAe a moderate or severe worm infection. As a result, they were significantly talRler, less likely to report being sick, and less likely to miss school than they woIuBld have been without treatment (Miguel and Kremer, 2004). By late 2014, four hLigh-burden provinces saw dramatic reductions in infection, with STH prevalenceY falling to just 6 percent (Kenya Ministry of Education, Science and TechnologIy,T 2014 Report). These results have come at a remarkably low cost. In Kenya, itS costs about US$0.56 per treated student per year. An analysis of Kenya‟s prograRmme conducted for Millions Saved yielded a cost-effectiveness ratio of US$47V.20E per DALY averted. Kenya‟s efforts illustrateN thaIt delivering deworming drugs on a national scale necessitates a sophistUicated programme that coordinates across schools, government ministries, and dono rs. It also shows that pairing decentralised delivery with measures to promote natioNnal accountability can work. And that public health intervention can impact morAe than health, reinforcing the need to look at synergistic gains in eduAcatioDn, productivity, and growth. In Kenya, deworming‟s wide-ranging benefits Bchanged the lives of millions of children for the better. But the programme is I vulnerable given its reliance on donors, and the sustainability of the effort may be in question as Kenya faces funding battles for other disease priorities. The use of anthelminthic drugs on a large scale for school-aged children in less developed countries was at the center of the 2010 resolution of the World Health Assembly. This led to the WHO 2010 resolution on achieving a 75% preventive chemotherapy coverage for pre-school and school-aged children by the end of that year yielded at the global level only 31% with 38% in India (WHO 2012). The reason 36 for the decision was based on the facts that school-based studies have the tendency to improve compliance and further improve infrastructure. It has been reported that as few as ten roundworm can affect the growth of school-age children, and that moderate whipworm infections can cause retardation and anemia. In case of more severe infection, children infected with Trichuris dysentery syndrome show “catch-up growth” after treatment (Drake 2000). Several studies in Nigeria have demonstrated the efficacy, acceptability and cost-effectiveness of school-based control of Soil- Transmitted infections (Nworgu et al., 1998, Ogbe et al., 2002, Kirwan et al., 20Y09). Studies conducted among preschool aged children (Kirwan et al., 2009) aRnd other adult groups (Egwunyenga and Ataikiru 2005, Rukmanee 2008, Pullan Aet al., 2010) show they harbour significant level of parasites that will allow the coRntinual existence of the STH infection in the entire population; this will leadB to reinfection after treatment. High rate of re-infection in endemic region dLespIite several rounds of school-based deworming implies, therefore, that reduc tion of morbidity due to decrease worm burden is the purpose of deIwTormYing and not the reduction of prevalence. The severity, extent, and lonSg-term consequences of morbidity are reduced following the WHO recommendRation of regular administration of single-dose anthelminthic drugs. Amongst adulEts, prevalence and intensity of infection remains high (Kirwan et al., 2009, RukmVanee 2008, Pullan et al., 2010). Meanwhile, Ellis et al. (2007) in their study suggesIted that targeting “infected households” is an effective, practical and rapid methodN of identifying and treating helminth-infected adults in the community. U According tAo KNatherine Parks (2017), Deworm the World has helped treat over 196 million Dchildren in Kenya, India, Ethiopia, Vietnam and Nigeria as at 2016, with some prelAiminary support introduced in Pakistan. Deworm the World‟s two biggest IBprogrammes are in Kenya and India. In Kenya, the Deworm the World Initiative has helped implement the government of Kenya‟s national deworming programme in schools since 2012. The collaborative project treated 6.41 million children in 16,708 schools in 2016. The programme served 83 percent of children in at-risk areas. Deworm the World‟s average cost of treating a child in Kenya is $0.71. India is the country with the most worm infections; 220 million children are at-risk of infection by STH. In India, the Deworm the World Initiative supported the government with the development and launch of National Deworming Day. National Deworming Day 37 targets all children between one and 19 years old and is implemented in schools. In 2017, the government of India treated over 260 million children. Deworm the World‟s average cost of treating a child in India is $0.34. In 2016, 69 percent of at-risk children were treated for STH, a 6 percent increase from 2015. Coverage in African countries was 65 percent in 2016 (Katherine Parks, 2017). 2.9 New way forward: Target drugs or vaccine Y The long-term use of anthelminthic drugs could promote drug resistaAnceR, thereby lowering the effectiveness against STH (Bethony et al 2006, WilliaRm-Blangero et al 2008, Kirwan et al 2009). According to Bundy et al 1987 and othBer numerous studies, individuals are readily re-infected after receiving anthelminthicI treatment, and it is an indication that acquired host immunity to infection is lYittle ( LBundy et al 1987, Bradley and Jackson 2004, Narain et al 2004). Helminth cTo-infections may also diminish the efficacy of future vaccines. It is generally acScepIted that vaccines for STHs would be the most ideal control agents as they wRould prevent re-infection, which anthelmintic drugs do not. However, there are no licensed vaccines (Noon and Aroian 2017). Currently, chemotherapy is deliVvereEd via mass drug administration (MDA), stirring grave concern for the strong Iselection of drug resistance, especially with limited efficacious anthelmintic Ndrugs (Geerts and Gryseels, 2001). Immunity to STH infections does not deUvelop upon clearance, and represents a vast issue for humans who are rapidlyN re-infected after chemotherapy (Bethony et al. 2006). Thus, vaccines would go a lAong way towards STH elimination. An AimpDortant distinction between STH infections in humans and livestock, ruminants Bin particular, is that in the latter, protective immunity often develops with age, but in I the former, this is not the case. So, although the duration of vaccine-induced protection in livestock may only need to last long enough to protect younger animals, for humans this protection would need to last throughout their lifetime. Thus, developing vaccines for humans will likely be a much greater challenge than for livestock due to the high likelihood that repeated immunisations will be needed throughout life. 38 Bartsch et al. (2016) developed a target product profile (TPP) for a recombinant subunit vaccine against human hookworms and evaluated the vaccines economic and epidemiologic impact on hookworm infection in endemic Brazil. A modelled human hookworm vaccine administered in a single dose to infants (78% coverage rate) with an efficacy of 80% at preventing L3 maturation and providing at least 10 years of protection with a single booster at 15 years of age (78% compliance), and costing $1 per dose, was highly cost-effective and economically dominant compared with no intervention or annual MDA (Bartsch et al. 2016). Thus, a human STH vaccine Ywith such a TPP should be ideal. R Many vaccine candidates for human parasite are undergoing clinical Atrials phases. Glutathione S-transferases (GSTs) play critical role in parasiteI-Bhost R interactions, and because it has demonstrated protective effect against some parasites, it has been targeted for pharmaceutical and vaccine purposes. F orL instance, a GST from schistosome is currently a leading vaccine candidaYte for human schistosomiasis. Parasite like hookworm which are blood- feeIdTers need to maintain a cytotoxic- metabolic requirement balance and hence SGST is a potential heme regulator. For hookworm Na-GST-1 adjuvanted with ARlhydrogel has been shown to elicit protective immunity in two vaccine trials Vin aE permissive hamster model (Zhan et al 2010). A recombinant bivalent subunit vIaccine containing rNa-GST-1 expressed in P. pastoris and a catalytically inactivNe, but still immunogenic, rNa-APR-1 expressed in tobacco plants (used as an al teUrnative to P. pastoris since expression was low) and formulated on Alhydrogel iNs currently in Phase I clinical trials for hookworm infection (Hotez et al. 2016). CAlinical testing is also evaluating if including synthetic Toll-like receptor agonistsD glucopyranosyl lipid A (GLA) or CpG oligodeoxynucleotide will help achAieve acceptable immunogenicity (Hotez et al. 2016). Surprisingly, to the best of IBour knowledge, there are no published results on the level of protection provided by the bivalent vaccine. Nevertheless, this vaccine is admittedly not expected to prevent hookworms from establishing infection within the small intestine (Hotez et al. 2010; Hotez et al. 2016), but the evidence does suggest that it will reduce clinicopathological parameters below threshold levels. Thus, although there are still many unanswered questions for this vaccine, there is cautious optimism for significant impact. There are no published studies for recombinant subunit vaccines for any Trichuris sp, vaccination of mice with adult Trichuris muris Excretory Secretory 39 products resulted in almost complete protection against T. muris egg challenge, in association with a classical TH2 response (Dixon et al. 2010) The first study that tested recombinant subunit vaccines against Strongyloides spp. used a DNA vaccine approach against experimental S. stercoralis L3 infection in BALB/cJ mice (Kerepesi et al. 2005). Strongloides stercoralis deoxycholate-soluble L3 antigens tropomyosin (Ss-TMY-1), Na+-K+ ATPase (Ss-EAT-6) and galectin (Ss- LEC-5) were shown to be recognized by serum IgG isolated from human plasma obtained from exposed Haitian donors, and this serum was shown to passYively transfer protection to BALB/cJ mice (Kerepesi et al. 2005). Currently, theRre are no published results for recombinant subunit vaccines against A. lumRbricAoides, while several studies have been published for A. suum and B. schroedBeri. Given the recent common ancestries between these ascarid species and A. lLumIbricoides, such studies are highly relevant to this most prevalent human STH (No on and Aroian 2017). In order to effectively control the prevalence and incidYence of STH in Nigeria, there is a dire need for an improved understanding of theI bTiological determinant of differential susceptibility to Soil transmitted helminRth inSfection in Africa, particularly in Nigeria. The result of this study provides poEssibly the first step towards the identification of which factor, that is, human gVenetic factor or environmental factor, has greater influence on the differential sIusceptibility to Soil–transmitted helminth infection in Nigeria. N U DA N IB A 40 RY RA LI B CHAPTER THREEY MATERIALS ANDS MIE TTHOD 3.1 Research Area R The study was conducted in IgbVo-OEra, one of the most popular town in Ibarapa local government under Oyo State,I southwestern Nigeria. Igbo-Ora lies in the savannah area of the country, haNving numerous small streams as one of its prominent geographical feature s.U The town has three main rivers that pass through her; these are River Opeki, OfNiki and Ayin. Igbo-Ora is about 120km from Ibadan, the state capital; the majorityA of the people that make up the town are Yoruba. The climate consists of a coolerD rainy season (April – October) and warm dry season (November – March). TheA majority of the men of the town are farmers and hunters by occupation, and the IBwomen are peasant farmers and retail traders. Igbo-Ora town was divided into six local communities, and each community has a traditional leader referred to as „Baale‟ who are identified as Baba-aso of Igbole, Onisaganun of Saganun, Oludofin of Idofin, Olu of Ibeerekodo, Olu of Pako and the overall head, that is, the Kabiyesi is the Olu of Igbo-Ora. 3.2 House mapping 41 The town was geographically mapped, using Geographic Information System (GIS), into three density areas: Low density, medium density and high density areas, depending on the household clustering in each area. Seven clusters of house density was identified which comprise of 4 low, 2 medium and 1 high density areas. The GIS grid recorded a total of 88, 160 and 209 grids for the high, medium and low density areas respectively, out of which 30% of each was randomly selected for the study (Fig. 3.1). Each grid can have between 1-5 households, depending on the density of the area. All households within the randomly selected grid are eligible and were gYiven a code number and individuals in those households were allotted a RPersonal Identification Code (PID). The longitude and latitude of each householdA participating in the study was determined using hand-held GeoExplorer Global PoRsitioning System (GPS). A sufficient satellite reception was ensured by taking readBing at a resolution of about 2m away from the front door or as near as possib leL. InI all, an average of 10 readings of the co-ordinate was taken. Map waYs created using ArcGIS 10.3 (Environmental System Research Institute Inc., RIeTdland, CA, USA). S VE R I U N N AD A IB 42 Y AR LIB R TY SI Fig. 3.1. A GIS map showing theE samRpling grid of Igbo-Ora according to house clustering of the community. V NI U AN D IB A 43 3.3 Questionnaire for collection of demographic, behavioural and observational data Approval for the protocol was obtained from the Ethical Review Committee of the College of Medicine, University of Ibadan, Nigeria with approval number UI/EC/12/0267. (Appendix 1) Inclusion criteria: (i) The individual enrolled in this study must reside in the selected area Yover the last 2 years. R (ii) Consents of the members of the selected household were soAught, and in case of minors, the parents or guardians gave consent Bon tRheir behalf. Exclusion criteria: I (i) The individual attending school or working outsi deL the study area, (ii) Received anthelminthic treatment within theY last 1 year. All individuals in the study were intervSieweId T with a structured questionnaire. Demographic information for age, sex, Reducation, occupation, past history of worm infection and treatment, availabilityE of toilet facilities, shoe wearing habit with hand washing practices were obtaineVd with the questionnaire and physical observation. (Appendix II) I 3.4 Household surveyU forN pedigree information Information aboNut r elationships between all household members was provided by household heads during interview and this was further verified by asking close or related DneigAhbours. All adult subjects and parents or guardians of minors gave their writAten or verbal consent (Appendix III). The pedigree data collection adopted the IBformat designed by William-Blangero and Blangero (2006) and modified to suit the cultural ideology of the African race, especially the Yoruba norms about asking for information about relatedness. The collected information was recorded in a separate pedigree interview sheet for each household (Appendix IV). The family relationship was grouped as: First degree relative – parent offspring, second degree are siblings, third degree relative - grandparent/grandchild and fourth degree relative - cousins. Names of other first or second degree relatives living in other households within the study area were collected. 44 Individuals were assembled into a pedigree if they are related to or married to anyone in a pedigree. Pedigree was assembled and was virtualised using genetic pedigree software called PROGENY. The family information was coded into the pedigree file, while all other data were coded into the phenotype file in the SOLAR programme. 3.5 Soil analysis Two soil samples were taken from two different locations at 20m distance fromY the entrance of each household according to Stojčeviić et al. (2010). At each siteR, 500g of top soil was taken with a small shovel in the area inside a square of 25A x 25cm and 10cm in depth. This was put inside a labeled plastic bag and taBkenR to the laboratory (Stojčeviić et al. 2010). These samples were analysed in the labIoratory using saturated ZnSO4 solution in centrifugation floatation method as dLescribed by Cheesbrough (2000) and modified by Stojčeviić et al. 2010. 500gY of the soil or sand sample was stirred and100g was placed in a cup and stirred aIgTain with 100mL water and sieved to remove larger particles. The homogenised Ssolution was placed into sedimentation cups, filled with 500mL water and left oRvernight. After the supernatant was decanted, 20g of the sediment was re-suspendEed with 50mL water, placed in 2 centrifuge tubes and centrifuged at 1500 rpm foVr 5mins. Finally, the sediment was re-suspended in 15mL saturated ZnSO4 soNlutioIn and poured into centrifuge tubes which were filled to the brim and the coveUrslip was superimposed. The samples were centrifuged at 1500 rpm for 5mins, the c overslip was removed onto a microscopic slide and examined for the presenceA ofN parasites eggs under the microscope at x10 and x40 magnification. PicturesD were taken by Optika Microscope Italy a digital binocular microscope 35MAL). IB 3.6 Stool sample collection After the administration of the pre-tested questionnaire, a wide mouth screw-capped 100 mL container pre-labelled with the participant PID was given to all subjects for the collection of their stool sample the next day. Uneducated participants were asked to give a mark or signs by which they could recognise each Child‟s and ward‟s sample bottles. Their ability to recognise their names was counter-checked. Each 45 subject was instructed to scoop a thumb size stool sample using a provided scoop into the container. Parents and guardians were also instructed to monitor their children during the sample collection to ensure that they placed their stool samples into the right containers. All study subjects were asked to provide moderately large stool sample (at least 10g) so that both the wet preparation and Kato-Katz techniques could be performed (Katz et al., 1972). A single stool sample collection was done contrary to the original design, because it was difficult to convince the participants against their cultural belief of giving away their stool samples for any purpose whYether scientific research or not. R The presence of any parasite was determined by using normal salinRe weAt preparation methods. This was necessary to identify parasite when the loaBd is high and also to identify actively motile parasite such as Strongyloides stLercoIralis. Actively motile parasite such as Strongyloides stercoralis cannot be identified with Kato-katz concentration method adopted in this study and it canY be missed out once the parasite dies. Kato-katz thick smear technique was usedI aTs the concentration method and the procedure was carried out for the stool sampSle collected, this method is appropriate in case of low parasite load. For the intensiRty of infection, quantification of ova/eggs per gram of faeces (epg), the ova was Ecounted per gram of faeces. To make up for the impossibility of collecting two Vstool samples per participants, two slides were taken from each stool sample anNd weIre examined within 45 minutes of slide preparation. U 3.6.1 LaborAatorNy analysis Wet PrDeparation BTwoA drops of normal saline was placed on a clean slide, spatula was used to pick a I speck of faecal material. The faecal material was then mixed with the normal saline on the slide until it was dispersed, then a cover slip was gently placed on the slide with the smear in order to avoid bubble, the slide was placed on a microscope and viewed under x10 for general observation and x40 for confirmation. Kato-Katz concentration method 46 A small mound of faecal material was positioned on a glass tiles, a cover screen was placed on top of the faecal material and some faeces were sieved through the screen. A flat-sided spatula was used to scrape the upper surface of the screen and the sieved faeces accumulate on the spatula. A template with holes was placed on the center of a microscope slide, the faeces from the spatula was added to the hole until it was completely filled and excess faeces was removed from the edge of the hole. The template was removed carefully from the slide, leaving mounted on it only the cylinder shaped faeces which was then covered with the already pre-soYaked cellophane strip. AR Another clean slide was used to firmly press the faecal sample wiRth the cellophane strip; with mild pressure the faecal sample was evenly spreadI bBetween the slide and the strip. The slide was gently removed without detachi ngL the cellophane strip; the slides with faecal-smeared samples were placed on bench until water evaporated while glycerol in the strip cleared the faeces. FIorT allY, except hookworm eggs, slides were kept for at least one hour at ambient temperature prior to the examination under the microscope. The smears were systeRmatSically examined to report the number of each parasites species. These were Elater multiplied by 24 to give the number of the eggs per gram faeces and a quVantitative variable scoring (light infection/low worm burden, moderate infection/meIdium worm burden and heavy infection/massive worm burden), following the stanNdard procedure used by World Health Organisation (WHO, 2002) was created for Ueach helminth. N 3.7 Data anAalysis All AparDasitological and questionnaire data were double-entered into an SPSS 15.0 IBdatabase and STATA 12 (Stata Corp., TX USA) software and it was cross-checked. Bivariate analysis was calculated for all variables to determine what the strength of association between the exposure and the outcome variable might be. Odds ratio with 95% confidence interval and significance was at p <0.05. Multivariate logistic regression was done using selected variables in relation to the presence of STH infection. Chi-square test p< 0.05 was significant. 47 Prevalence of infection was expressed in percentage, and intensity was measured by egg count per gram (EPG). The parasites isolated from each member of a particular household was grouped and treated as a single unit for spatial and genetic analysis. Spatial analysis adopted a Bayesian geo-statistical approach which uses Moran index of Z-score at 2.3 and p < 0.05 level of significant, putting into consideration both spatial correlation and non-spatial clustering at a household-level which provided the spatial distribution of the infection in the community. Y Variance components analysis was done by using a Unix-based software prRogramme called Sequential Oligogenic linkage Analysis Routine (SOLAR). FamilyA relationship information was recorded according to the file format requirementRs of the SOLAR 8.1.1 software package. The advantage of using SOLAR is its Bability to incorporate covariate effects into the models to account for potential cLonIfounding factors and a Kullback-Leibler R-Squared value is estimated to indi cate the effect size of the covariates in the model. ITY The family information was coded into theS pedigree file, while all other data were coded into the phenotype file in the SOLRAR programme. Models were developed that 2 allowed the estimation of heritabilitEy (h ) that is the proportion of variance in STH prevalence/intensity, attributabIleV to genetic factors and the proportion of variance in 2prevalence/intensity, attribNutable to the effect of common shared household C . The software implement aU maximum likelihood method which provides a log-likelihood estimate for each mo del. 2 The environAmenNtal model adopts the use of common shared households C and the 2presencDe of parasite in the soil around household (e ) as an additional factor, while hosAt genetic factor also included the genetic relatedness. IBThe analysis of genetic factor was based on Genetic Variance Component Analysis (VCA) whereby the relative importance of genetic susceptibility, other domestic factors and (non- genetic) individual factors in determining infection intensity was assessed. Genetic variance component was used to define the contribution of genetics: the degree of relatedness and environmental influence on intensity of infection, which provided the overall estimate of the total cumulative effect of individual factors (the “addictive genetic” effect, or heritability) on overall variation. 48 RY BR A LI Y SI T VE R UN I AN AD IB CHAPTER FOUR RESULTS 4.1 Socio-demographic factors of the participants. 49 The study population of 673 individuals from 239 households was recruited from four representative communities in Igbo-Ora. The communities were Igbole and Pako, representing the low density area; Isale-Oba in the medium density area; and Saganun in the high density area. A total of 508 consented individuals provided proper stool samples and complete demographic and behavioural information. Two hundred and thirteen (42%) were males and two hundred and ninety five (58%) were females. The age ranged from seven months to eighty six years, with the mean age of 22.8 (95% Cl 21.1-24.6). Table 4.1 showed that 10.2% of the sample population were children Y<1-5 years of age, while 44.3%, 18.1%, 14.6% and 12.8% were aged 6-15, 16-2R9, 20-49 and ≥50 years respectively. A More than 50% of the study participants were students while 3.1B% oRf the participants were civil servants. The skilled workers were 5.9% which LincIluded professions such as hair dressers, fashion designers, mechanics, electric ians and transporters. The unskilled workers were the petty traders and farmers Ywhich accounted for 18.3% and the unemployed were 9.8% of the study populatioInT. Table 4.1 also showed the frequency oRf 68S.7%, 27.4%, 3.1% and 0.8% for single, married, widowed and separated marEital status of the participants. The STH infection prevalenceI aVmong the study participants showed that both male and female were equally inNfected with 28.2% prevalence in male and 28.1% in female participants. The highUest infection prevalence of 34.5% was seen among the children in age group ≤1-5 years, while the age group 6-15 and 16-29 years had similar prevalence Aof N28.9% and 28.3% respectively. The lowest but not significantly differenDt prevalence was seen among the age group ≥50 years. STHA infection in relation to occupation showed that participants who were civil IBservants had the lowest prevalence of STH infection with 18.7%, while the highest was seen among the skilled workers (33.3%), followed closely by the unskilled workers (30.1%). There was no significant association between the level of education of the participants and STH infection prevalence within the study population (Table 4.1). 4.2 Prevalence of STH infection in the sampled population 50 The overall prevalence of STH in the four communities sampled was 28.1%. This reflects the number of individuals infected with any one of the STH parasites. Cases of co-infection were counted once. The parasites identified were Ascaris lumbricoides, Trichuris trichiura, hookworm, and Strongyloides stercoralis (Fig. 4.1a, b, c &d). Figure 4.1c & d showed the adult worm passed out in faeces by one of the participants during the study. Hookworm infection was the predominant STH infection in the population sampled with 18.5%, followed by Ascaris lumbricoides with 16.7%, while Strongyloides stercoralis was 3.0% and Trichuris trichiuraY was 0.8% in the population from the four communities sampled. (Fig. 4.2) R RA 4.2.1 Overall prevalence of multiple helminth infection in IgIbBo-Ora The result in table 4.2 showed that multiple infections we reL observed in 36.4% of the infected population in double and triple helminth Yinfections. Forty nine (94.2%) participants had double infections while 3 parIticTipants (5.8%) had triple helminth infections. A. lumbricoides/hookworm was 8S9.8% of the double helminth infections, while A. lumbricoides /S. stercoralis, hoRokworm/S. stercoralis were 4.1% each and A. lumbricoides /T. trichiura was E2.0%. Triple helminth infections were A. lumbricoides/hookworm/S. stercVoralis with 66.7% prevalence, and A. lumbricoides /hookworm/T. trichiura wNas 33I.3% of the triple helminth infections (Table 4.2). U 4.3 IntensitAy ofN STH infection in the sampled population The maDjority of the STH infections were of light intensity (table 3). Intensity was classified according to the WHO (2002) standard. For A. lumbricoides; 1 -4,999 ova IBwer Ae light intensity, 5,000 – 49,999 ova were moderate intensity, while >50,000 ova were heavy intensity. For T. trichiura: 1-999 ova for light intensity, 1,000-9,999 ova for moderate and ≥ 10,000 ova for heavy intensity. For hookworm: 1-1,999 ova as light intensity, 2,000-3,999 for moderate and ≥ 4,000 ova for heavy intensity. Few cases of moderate and heavy intensity were seen in A. lumbricoides and hookworm infections. In this study, intensity of A. lumbricoides and hookworm increases with age and both have two peaks of infection at different age groups (Fig 4.3) 51 Y RA R B LI Y IT RS IV E U N DA N BAI Table 4.1. Demographic characteristics and STH infection among the study participants in Igbo-Ora (N=508) Demographic Number (%) STH Infection Pearson P- value factor Yes Chi-square Sex Male 213 (42%) 60 (28.2%) Female 295 (58%) 83 (28.1%) 0.0001 0.993 Age 52 ≤1-5 52 (10.2%) 18 (34.6%) 6- 15 225 (44.3%) 65 (28.9%) 16-29 92 (18.1%) 26 (28.3%) 30- 49 74 (14.6%) 19 (25.7%) ≥50 65 (12.8%) 15 (23.1%) 2.19 0.70 Occupation Student 319 (62.8%) 95 (29.8%) Civil Servant 16 (3.1%) 3 (18.7%) Skilled worker 30 (5.9%) 10 (33.3%) Unskilled worker 93 (18.3%) 28 (30.1%) Unemployed 50 (9.8%) 7 (14.0%) 6.64 0.16 Y R Level of Education Primary 216 (42.5%) 58 (26.8%) A Secondary 168 (33.1%) 56 (33.3%) R Tertiary 34 (6.7%) 8 (23.5%) No formal 90 (17.7%) 21 (23.3%) 3.80 B education Y LI 0.28 Marital status Single 349 (68.7%) 104 (29.8%) Married 139 (27.4%) 31 (22.3%) IT Divorced 16 (3.2%) 7 (43.7%) Widowed 4 (0.8%) 1 (2R5.0%S) 4.76 0.19 E NI V U AN BA D I 53 Y A AR B A. lumbricoides ova HoBokwoRrm ova LI SI TY R VE UN I C N D Fig. 4.1D. MiAcrograph of ova and photograph of adult worm of STH parasite seen in participants stool sample. BA : APicture of Kato-Katz microscope slide showing ova of A. lumbricoides. PIN 369 I B: Picture of Kato-Katz microscope slide showing ova of A. lumbricoides and hookworm. PIN 404 C & D: Picture of large adult worm passed out with faeces by one of the participants. PIN 262 54 20 18.5 Y 18 R 16.7 16 BR A 14 LI TY 12 RS I 10 E % prevalence 8 IV 6 U N 4 AN 3 2AD 0.8 IB0 Ascaris Hookworm Trichuris trichiura Strongyloides lumbricoides stercoralis Fig 4.2: The prevalence of each STH parasite within the study population. N=508 55 Table 4.2: Overall prevalence of multiple helminth infection in the study population. Multiple helminth infection No of infected individuals (%) Prevalence Double helminth 49 94.2%Y A. lumbricoides /hookworm 44 A 8R9.8% A. lumbricoides /S. stercoralis 2 R 4.1% A. lumbricoides /T. trichiura 1 L I B 2.0% Hookworm/ S. stercoralis 2 Y 4.1% Triple helminth 3 I T 5.8% Asc/Hk/Strongy R 2S 66.7% Asc/Hk/T. trichiura E 1 33.3% Total I V 52 36.4% UN *Asc = A. lumbrNicoi des Hk= hookworm Strongy= S. stercoralis AD A B I 56 0.35 0.3 0.25 0.2 0.15 0.1 0.05 AR Y 0 Age group 1 2 3 4 5 BR Age group: 1 =<1-5 years, 2 = 6- 15 years, 3 =16- 29 years, 4 = 3L0- 4I9 years, 5 = ≥50 years. Fig. 4.3: The intensity of A. lumbricoides and hookworm infection in relation with age group ITY S ER NI V U N DA IB A 57 Table 4.3: Intensity of STH infection in the four GIS mapped community of Igbo-Ora Infection Intensity Community Pako (n) Isale-Oba(n) Igbole(n) Saganun(n) Ascaris Light 16 10 23 17 Moderate 1 - 1 2 Heavy 2 4 1 9 Hookworm Light 16 8 15 39 Moderate 2 1 2 4 AR Y Heavy 1 3 2 2 Strongyloides Light 3 4 5 R 3 Moderate - - - B - Heavy - - L- I - Trichuris Light 2 - Y 1 - Moderate - - T - - Heavy - S- I - - R Ascaris lumbricoides: 1-4,999 ova Elight intensity, Moderate intensity 5,000-49,999 ova, and heavy intensity ≥50,000V ova. Hookworm: 1-1,999 ova iNs ligIht intensity, 2,000-3,999 ova is moderate intensity and ≥4,000 ova is heavy intensity. Trichuris trichiura: 1-U999 ova light, 1,000-9,999 ova is moderate and ≥10,000 ova is heavy intensity. DA N B AI 4.4 Associated risk factors of STH infection within the infected population 58 The results from table 4.4 showed the STH infection and associated risk factors of infection in the 143 infected participants. The people living in mud houses are 2 times more likely to be infected with STH infection than those living in concrete houses (OR 1.69 95% CI 1.14- 2.49 p< 0.05). Likewise, those living in crowded room with more than four people in a room and not having toilet facility are 1.65 and 1.55 likely to be infected with STH infection (OR 1.65; 95% CI 1.12 – 2.44 p=0.01 and OR 1.55; 95% CI 1.04 – 2.30 p=0.03) respectively. Other factors such as past history of worm infection, defecating in bushes around homes, hand-washing before eYating and after toilet, wearing of shoes and having dirty finger nails do not have siRgnificant association with STH infection in this study (p>0.05). A 4.4.1 Multivariate logistic regression of risk factors of STH inBfectRion The multivariate regression model of the associated risk LfacItors for STH infection adjusted for the following variables: age, sex, occupYation , hand washing after using the toilet, type of toilet facility, house type, living in crowded room, house density area and having access to toilet facilities (Table I4.TS 5). The result showed that living in mud houses and overcrowded rooms wRith more than four individuals in a room are important risk factors of STH infectEion (OR 1.80; 95% CI 1.15 -2.81 p =0.01 and OR 1.65 95% CI 0.99 – 2.74 p= 0.05V respectively). NI 4.5 STH infection w iUthin households in Igbo-Ora STH infectiAon wNas found in 95 households out of the 239 households sampled. The numberD of participants ranged from 1 to 14 individuals per household. In 150 houAseholds, only one person from each household participated in the study. Overall, B39.7% of the households were infected with STH infection. Eighty-nine (89) I households (35.7%) had two or more participants per household and STH infection was seen in 51(57.3%) out of the 89 households. Twenty-eight (28) out of 51 households had more than 50% of the household participants infected with STH within each household in the sample population. Forty-four (44) (that is, 29.3%) households out of the 150 households with one participant had STH infection. Table 4.4: Bivariate analysis of risk factors associated with Soil-transmitted helminth infections within the infected population of Igbo-Ora, Nigeria. N=143 59 Risk factors Prevalence of STH Odds 95% Confidence P-value (%) ratio Interval Toilet access Yes 54 (23.4%) 1.0 (ref) No 89 (32.1%) 1.55 1.04 -2.30 0.03 Toilet type Water closet 26 (22.8%) 1.0 (ref) Pit latrine 27 (23.3%) 1.02 0.55 – 1.89 0.93 Bush around 90 (32.7%) 1.62 0.97 - 2.68 0.061 home Past history of worm Y Yes 93 (29.2%) 1.0 (ref.) R No 50 (26.5%) 0.87 0.58 - 1.31 A0.51 Treated for worm in the past Yes 28 (22.6%) 1.0 (ref.) R No 115 (30.1%) 1.46 0.91 L- 2.I35B 0.11 Living in overcrowded room ≤4 64 (23.4%) 1.0 (ref.) >4 79 (33.6%) 1.65 ITY1.12 – 2.44 0.011 House type Concrete 68 (23.5%) 1.0 (ref) Mud 75 (34.2%) R1S.69 1.14 – 2.49 0.008 Hand washing before eating Yes 138 (28.9I%V) E 1.0 (ref.) No 8 (27.4%) 0.88 0.54 – 1.46 0.57 Hand washing after toileting Yes U106 N (27.0%) 1.0 (ref) No 37 (32.2%) 1.19 0.82 - 2.01 0.27 wear dirty finger nNails YesA 54 (29.8%) 1.0 (ref) No 89 (27.2%) 0.87 0.59 -1.31 0.53 wear shoDe regularly A Yes 70 (28.9%) 1.0 (ref.) B No 73 (27.4%) 0.99 0.63 -1.37 0.71 I Community density Low density area 66 (46.1%) 1.0 (ref.) Medium density area 25 (17.5%) 0.69 0.41 - 1.19 0.41 High density area 52 (36.4 %) 0.93 0.61 – 1.44 0.61 Table 4.5: Multivariate logistic regression analysis of adjusted risk factor of STH infection in the study population 60 Variables Odds Ratio P>z [95% Conf. interval) Age group (years) <1 -5 1.00 6--15 0.48 0.07 0.21 -1.06 16 -29 0.65 0.37 0.25 – 1.69 30 – 49 0.41 0.13 0.13 – 1.30 ≥50 0.42 0.15 0.13 – 1.37 Sex Male 1.00 Female 0.93 0.74 0.60 – 1.43 Y Occupation Student 1.00 R Civil servant 0.73 0.67 0.16 – 3.2A2 Skilled worker 1.21 0.75 0.39R – 3.76 Unskilled worker 0.96 0.93 0.37 – 2.47 Unemployed 0.22 0.01 LI B0.08 – 0.63 Wash hand after using the toilet Yes 1.00 No 0.71 0Y.24 0.40 – 1.26 Toilet type T Water closet 1.00 SI Pit latrine 0.75 0.39 0.39 – 1.45 Bush around the house 2.80 R 0.35 0.33 – 23.88 House density areas Low density 1.E00 Medium density IV0.66 0.29 0.31 – 1.42 High density N 1.03 0.92 0.63 – 1.67 Living in overcrowded room ≤ 4 U 1.00 >4 N 1.65 0.05 0.99 – 2.74 House type A Concrete 1.00 Mud D 1.80 0.01 1.15 – 2.81 HavAe access to toilet facility BYes 1.00 I No 0.48 0.50 0.06 – 4.01 4.5.1 Households infection within each community 61 Table 4.6 showed the distribution of infected household per community. A total of 45, 52, 66, and 76 households were sampled in Saganun, Igbole, Pako, and Isale-Oba respectively. The highest number of STH infected household was found in Saganun, with 23 (51.1%) out of 45 households infected. Meanwhile, in Igbole, 22 (42.3%) out of 52 households were infected, Pako had 25 (37.9%) out of 66 participating households infected with STH, while 25 (32.9%) out of 76 households in Isale-Oba had STH infection. RY 4.6 The prevalence of specific STH parasite within the sampled comAmunities in Igbo-Ora. R The prevalence of each soil-transmitted helminth infection in IeaBch of the community sampled showed that the triad of A. lumbricoides, hookw oLrm and S. stercoralis was seen in all the four communities. T. trichiura was seeYn only in the two communities representing the low density communities, that IisT, Igbole and Pako, with 0.8% and 2.2% prevalence respectively. A. lumbricoidSes had the prevalence of 18.5, 21.5, 13.0 and 15.5% in Igbole, Pako, Isale-ObaR and Saganun respectively. Meanwhile for hookworm, the prevalence of 13.8, E21.3, 11.1 and 24.9% was seen respectively. The prevalence of S. stercoralis wIaVs almost the same in three out four communities in Igbo-Ora. Figure 4.4 showNed the flow chart of STH infection within each of the four communities with the Uerror bar at 5% value. The associationN be tween the community density and the prevalence of each STH parasite wasA analyzed, and it was found that there is a significant association with hookwoDrm infection (p<0.01) while other STH parasite, that is A. lumbricoides T. tricAhiura and S. stercoralis do not have significant association with community IBdensity (Table 4.7). 4.6.1 Risk factors for hookworm infections 62 The significant association of hookworm infection with high density area, as shown in table 4.7 informed the decision to further investigate other associated risk factors for hookworm infection. Table 4.8 showed the bivariate and multivariate logistic regression analysis of hookworm infection with associated risk factors of STH infections. In the bivariate analysis, people who lived in high density area, in crowded rooms, mud houses and had no access to toilet facilities, had higher odds of acquiring hookworm infections OR 2.39 95% CI 1.23 -4.63; p< 0.01, OR 2.20 95% CI 1.39 – 3.49; p<0.001, OR 1.87 95% CI 1.19 – 2.94; p<0.01, and OR 1.64 95% CI 1.Y03 – 2.59; p < 0.04 respectively. Wearing of shoe was not significantly associatedR with an increased risk of hookworm infection in this study. In the multivariateA analysis the variables tested and included in the model were; toilet access, oveRrcrowding, house type, shoe wearing, density and toilet type. LI B TY RS I IV E UN AN D IB A 63 30 25 20 Ascaris 15 RhoYokwormA StrongyloidesTrichuri 10 LIB R 5 Y 0 IT Igbole Pako IsaSle-Oba SaganunR Fig 4.4: The prevalence of specific SETH parasite within each of the four communities sampled in Igbo-Ora. V . NI U AN D B AI Table 4.6: Distribution of STH infection per household in the four communities 64 Density Area Community Infected households Not infected households Total No (%) No (%) No (%) Low density Igbole 22 (42.3) 30 (57.7) 52 (100) Pako 25 (37.9) 41 (62.1) 66 (100) Medium density Isale-oba 25 (32.9) 51 (67.1) 76 (100) High density Saganun 23 (51.1) 22 (48.9) 45 (100) 95 (39.7) 144 (60.3) 239 (100) Y R BR A LI SI TY R VE NI U AN D IB A Table 4.7: Association of community density areas and specific STH parasite in Igbo- Ora using Pearson chi-square. 65 STH parasite Community density areas Total Pearson p-value 2 chi Low Medium High Ascaris lumbricoides 43 14 28 85 2.63 0.27 (19.6%) (13.0%) (15.5%) (16.7%) Hookworm 37 12 45 94 9.14 0.01 (17.0%) (11.1%) (24.9%) (18.5%) Strongyloides stercoralis 8 4 3 15 1.65 0.44 (3.6%) (3.7%) (1.7%) (3.0%) Trichuris trichiura 3 0 0 3 3.9 Y 0.14 (1.4%) (0.6%) R A LIB R ITY ER S IV UN AN AD IB Table 4.8: Factors associated with hookworm infections by multivariate logistic regression analysis in the study population of Igbo-Ora. 66 Variables Unadjusted OR (95% Sig. Adjusted OR (95% CI) Sig. CI) Density Low (Reference) 1.0 1.0 Medium 1.99 (0.93 - 4.24) 0.07 0.91 (0.39 - 2.13) High 2.39 (1.23 - 4.63) 0.01 2.12 ( 1.23 - 3.65) 0.01 Shoewear Yes (Reference) 1.0 1.0 No 1.16 (0.74 - 1.81) 0.52 0.62 (0.41 - 1.0Y9) 0.12 Living in crowded room R ≤4 (Ref.) 1.0 1.0A >4 2.20 (1.39 - 3.49) 0.001 2R.19 (0.54 - 3.25) 0.01 Have access to toilet facility B Yes (Ref.) 1.0 I 1.0 No 1.64 (1.03 - 2.59) Y0.04 L 11.6 (0.62 - 21.8) 0.1 House type Concrete (Ref.) 1.0 IT 1.0 Mud 1.87 (1.19 - 2.94) S 0.01 1.67 (1.02 - 2.74) 0.04 Toilet type R Water closet (Ref.) 1.0 E 1.0 Pit latrine 1.99 I(0V.93 - 4.24) 0.07 1.39 (0.62 - 3.08) 0.41 Bush around the house N2.39 (1.23 - 4.63) 0.01 1.30 (0.09 - 18.7) 0.86 U AN AD IB 4.7 Prevalence and risk factors of infections in each of the community sampled 4.7.1 Pako Community 67 Pako community, according to the GIS mapping, was in the low density area of Igbo- Ora. The total population of participants from this community was 89. Forty three (48.3%) participants were males while forty six (51.3%) were females. The prevalence of infection was almost same for both sexes: 37.2% for male and 37.0% for female. Children between the ages of 11-15 years accounted for 71.9% of the participants in this community and had the highest prevalence of STH infection (42.2%). This was followed by a prevalence of 12.1% for the 6-10 years age group. Only 5 adults participated in the study; the reason for this was because the paYrents refused to take part in the study though allowed their children and Rwards to participate. Some parents collected the stool bottle only to return it emptyA without any sample. It was later discovered they held a meeting and agreed that oRnly their children should participate in the study. The reason for this decision wasB best known to them (Table 4.9). LI The prevalence of STH infection was 42.9% amoTng tYhose that had no toilet facilities in their home; while 28.6% prevalence was oIbserved in those using water closet systems; and 34.6% prevalence in pit latrineS users. However, the highest prevalence of 42.9% was seen in the category thRat defecated in bushes around their homes. Participants with more than four (4)E people in a room had 41.5% prevalence for STH infection, while 25.0% prevaleIncVe was seen among those living in mud houses (Table 4.9). N All the participants Ureported hand washing practices, but when asked about the frequency of thNeir hand washing practices, only 65 (73.0%) reported they washed their hands Aregularly and 24 (27.0%) reported they washed their hands occasionally, either bDefore eating or after going to the toilet. Some 39.3% of the participants repoArted that they wear shoes while in the house but not into their rooms while 60.7% IBwore their shoes occasionally. The result from table 10 showed the prevalence of STH infection to be 35.4% among those that washed their hand regularly, while 31.9% prevalence of STH was seen in those that practised hand-washing after toilet the use of the toilet and 42.6% prevalence in those that wore shoe occasionally. The prevalence of STH infection was 38.2% among those with dirty finger nails and 38.9% in those with dirty house surroundings (Table 4.9). 68 Result in table 4.10 showed that the majority of the infections was light intensity in the community. The prevalence of A. lumbricoides and hookworm infection was the same, 21.3%; likewise similar intensity of infection was also seen for the two parasites. However, for A. lumbricoides, heavy worm load was observed in 10.5% of the infected individuals. The overall prevalence of STH infection in Pako community was 37.1%. Multiple helminth infection prevalence was 27.3% in the infected population; double helminth infection was 66.7% of the multiple infections. A. lumbricoides/ hookwormY, A. lumbricoides/T. trichiura, and A. lumbricoides/S. stercoralis were double Rhelminth infection with 11.1% prevalence each, while the prevalence foRr triAple helminth infection with A. lumbricoides/hookworm/T. trichiura was also 11.1%. Figure 4.5 showed the bar chart presentation of STH infection intensityL in IPBako community. Y IT RS E NI V U N DA IB A Table 4.9: Risk factors and prevalence of soil-transmitted helminth infection in Pako community, Igbo-Ora. 69 Risk factors Intestinal helminth 2 Yes No Total x P-value No (%) No (%) No (%) Sex Male 16 (37.2%) 27 (62.8%) 43 (100%) Female 17 (37.0%) 29 (63.0%) 46 (100%) 1.0 0.58 Age group <1 – 5 0 (0%) 1 (100%) 1 (100%) 6 – 10 4 (12.1%) 15 (78.9%) 19 (100%) 11 – 15 27 (42.2%) 37 (57.8%) 64 (100%) 16 – 25 1 (100%) 0 (0%) 1 (100%) 26 – 40 0 (0%) 1 (100%) 1 (100%) 41 – 50 1 (100%) 0 (0%) 1 (100%) 51 – 70 0 (0%) 2 (100%) 2 (100%) Y >70 0 (0%) 0 (0%) 0 (0%) 8.56 R 0.20 House clustering ≤ 4 living in a room 6 (25.0%) 18 (75.0%) 24 (100%) A > living in a room 27 (41.5%) 38 (58.5%) 65 (100%) R 2.05 0.11 House type Concrete 19 (31.7%) 41 (68.3%) 60 (L100I%B) Mud 14 (48.3%) 15 (51.7%) 2 9 (100%) 2.31 0.10 Toilet Access Yes 15 (31.9%) 32 (69.1%) T Y 47 (100%) No 18 (42.9%) 24 (57.1%I) 42 (100%) 1.14 0.20 Toilet type Water closet 6 (28.6%) 1R5 (71S.4%) 21 (100%) Pit latrine 9 (34.6%) 17 (65.4%) 26 (100%) Bush around house 18 (42.9%) E 24 (57.1%) 42 (100%) 1.32 0.50 Hand washing habit Before eating V Regularly 23 (N35.4%I) 42 (64.5%) 65 (100%) Sometimes 10 (41.7%) 14 (58.2%) 24 (100%) 0.30 0.38 After the use of toilet Yes N U 23 (35.9%) 41 (64.1%) 64 (100%) No 10 (40.0%) 15 (60.0%) 25 (100%) 0.12 0.45 Shoe wearingA habit DRegularly 10 (28.6%) 25 (71.4%) 35 (100%) AOccasionally 23 (42.6%) 31 (57.4%) 54 (100%) 1.79 0.13 Dirty finger nails B Yes 12 (35.3%) 22 (64.7%) 34 (100%) I No 21 (38.2%) 34 (61.9%) 55 (100%) 0.08 0.48 House surroundings Neat 12 (34.2%) 23 (65.8%) 35 (100%) Dirty 21 (38.9%) 33 (61.1%) 54 (100%) 0.19 0.42 Table 4.10: Prevalence and intensity of STH infection within Pako community Helminth Prevalence Intensity 70 Light Moderate Heavy A. lumbricoides 19 (21.3%) 16 (84.2%) 1 (5.3%) 2 (10.5%) Hookworm 19 (21.3%) 16 (84.2%) 2 (10.5%) 1 (5.3%) T. trichiura 2 (4.6%) 2 (100%) 0 (0%) 0 (0%) S. stercoralis 3 (2.2%) 3 (100%) 0 (0%) 0 (0%) RY BR A LI ITY RS VE UN I AN BA D I 71 18 16 14 12 10 Light infection 8 Moderate infection 6 HeavyR infecYtion4 2 RA0 Ascaris Hookworm Trichuris Strongyloides B lumbricoides trichiura stercor alLis I Y Fig 4.5: Intensity of STH infection within Pako cIoTmmunity. RS E NI V U N DA IB A 4.7.2 Isale-Oba community 72 Socio-demographic, risk factors and prevalence of STH infection in Isale–Oba, Igbo-Ora One hundred and eight individuals, 48 male (44.4%) and 60 female (55.6%), participated in the study. The prevalence of STH was highest in female (26.7%) than male (18.8%) at p-value 0.23. The highest prevalence of infection was observed in the 11-15 years age group with 32.1% prevalence. A total of 21.3% of the people who lived in concrete houses were positive for STH infection, while 27.3% of those infected lived in mud houses. The prevalence of STH was 25.0% for the 28 peYople (25.9%) that had toilet facilities and 22.5% for the remaining 80 participantsR (74.1%) that had no toilet facilities. None of the participants reported living wiAth more than four people in a room in this community. The result from table 4.1R1 showed that 88 participants with 23.9% STH prevalence regularly washed thIeiBr hand before eating, while the 20 participant that reported occasional hand was hLing before eating had 20% prevalence for STH. Sixty-eight (68) participantsT repYorted washing their hands after the use of toilet, while 40 participants reporteId not practicing hand washing after toilet. The prevalence of STH infection amoSng those that wore shoe regularly, had no dirty finger nails, and had neat house sRurroundings were 25.5%, 23.5% and 25.4% respectively (Table 4.11). E The prevalence of STH infectIioVn in this community was 23.1% while 18.2% of the infection was double hNelminth infection: A. lumbricoides/hookworm, and A. lumbricoides/S. sterc oUralis. T. trichiura was not seen in this community (Table 4.12). Figure 4.6 sAhowNed the intensity of STH infection within Isale –Oba community of Igbo-OrDa. Each of the STH infection was of light intensity in this community, althAough heavy worm intensity was observed in A. lumbricoides (28.6%) and Bhookworm (20.0%) infections. I Table 4.11: Risk factors and prevalence of STH infection within the participants of Isale-Oba, Igbo-Ora. 73 Risk factors Intestinal helminth 2 Yes No Total x P-value No (%) No (%) No (%) Sex Male 9 (18.8%) 39 (81.2%) 48 (100%) Female 16 (26.7%) 44 (73.3%) 60 (100%) 0.94 0.23 Age group <1 – 5 0 (0%) 2 (100%) 2(100%) 6 – 10 2 (8.7 %) 21 (91.3%) 23 (100%) 11 – 15 9 (32.1%) 19 (67.9%) 28 (100%) 16 – 25 14 (28.0%) 36 (72.0%) 50 (100%) 26 – 40 0 (0%) 1 (100%) 1 (100%) Y 41 – 50 0 (0%) 3 (100%) 3 (100%) 51 – 70 0 (0%) 1 (100%) 1 (100%) R >70 0 (0%) 0 (0%) 0 (0%) 6.7R4 A 0.35 House type Concrete 16 (21.3%) 59 (78.7%) 75 (100%) Mud 9 (27.3%) 24 (72.7%) 33 (100%L) I B 0.45 0.33 Toilet Access Yes 7 (25.0%) 21 (75.0%) 28 (100%) No 18 (22.5%) 62 (77.5%) 80 Y(100%) 0.07 0.49 Toilet type IT Water closet 5 (29.4%) 12 (70.6%) S 17 (100%) Pit latrine 1 (10.0%) 9 (90.0%) 10 (100%) Bush around house 19 (23.5%) 62 (76.5%R) 81 (100%) 1.35 0.51 Hand washing habit Before eating E Regularly 21(23.9%) I 6V7 (76.1%) 88 (100%) Sometimes 4 (20.0%) N 16(80.0%) 20(100%) 0.14 0.48 After the use of toilet Yes 14 (20.6%) 54 (79.4%) 68 (100%) No N 11 (2 7 U.5%) 29 (62.5%) 40 (100%) 0.68 0.28 Shoe wearing habit Regularly 12 (25.5%) 35 (74.5%) 47 (100%) OccasionDally A 13 (21.3%) 48 (78.7%) 61 (100%) 0.27 0.39 DirtAy finger nails BYes 6(22.2%) 21 (77.8%) 27 (100%) I No 19(23.5%) 62 (76.5%) 81 (100%) 0.017 0.56 House surroundings Neat 17 (25.4%) 50 (74.6%) 67 (100%) Dirty 8 (19.5%) 33 (80.5%) 41 (100%) 0.49 0.32 Table 4.12: Prevalence and Intensity of STH infection within Isale-Oba community 74 Helminth Prevalence Intensity Light Moderate Heavy A. lumbricoides 14 (13.0%) 10 (71.4%) 0 ( 0%) 4 (28.6%) Hookworm 12 (11.1%) 8 (66.7%) 1 (8.3%) 3 (25.0%) T. trichiura 0 (0%) 0 ( 0%) 0 ( 0%) 0 ( 0%Y) S. stercoralis 4 (3.7%) 4 (100%) 0 (0%) 0R ( 0%) RA LI B TY RS I VE 16 NI 14 U12 N 10 DA Heavy infection8 Moderate infection B6AI Light infection4 2 0 Ascaris Hookworm Trichuris Strongyloides lumbricoides trichiura stercoralis Fig 4.6: Intensity of STH infection within Isale-Oba community. 75 RY A BR Y LI IT ER S V 4.7.3 Igbole Community NI Socio-demographic , Urisk factors and prevalence of STH infection in Igbole community, IgNbo-Ora. One HundreAd and thirty individuals, 40.8% male and 59.2% female, participated in this comDmunity. The prevalence of STH infection was highest with 28.3% among malAe participants. The age group distribution cut across all classes of age and the IBhighest prevalence was recorded among age group 6-10 years (55.6%); followed by age group 41-50 years (42.9%), and age group <1-5 years with 35.3% prevalence (Table 4.13) The prevalence of STH infection was seen to be highest among those living with >4 people in a room (26.9%). Those living in mud houses had 27.8% prevalence of STH infection, while 28.6% prevalence of STH infection was observed in those living in houses with no toilet facility. Defecating in bush around the house accounted for 76 23.5% STH prevalence of infection. A total of 25.4% STH prevalence was recorded 2 in those that reported regular hand washing before eating (x =0.001), while 35.7% prevalence was seen in those that did not practice hand washing after toileting. Those 2 that wore shoe occasionally had 25.0% prevalence for STH infection x =0.01 p=0.54 (Table 4.13). The prevalence of STH infection in this community was 24.5%. A. lumbricoides was the predominant helminth in this community with 18.5% prevalence of infection; this was followed by hookworm infection with 13.8% prevalence; S. stercoralis with 3.8% prevalence and lastly T. trichiura 0Y.8% prevalence. Multiple helminth infection was responsible for 42.4% of the iRnfection, while single infection with any of the helminth was 57.6%. A. luAmbricoides / hookworm was the only double helminth infection seen, and this paRttern was seen in 13 participants, while triple helminth infection was A. lumbriBcoides/ hookworm/S. stercoralis and was seen in only one participant. The re suLlt fIrom table 4.14 showed that STH infection intensity was 92.0% and 78.9% light infection for A. lumbricoides and hookworm infection respectively, while modeYrate and heavy infection was observed in hookworm infection with 10.5% eaIcTh. No moderate or heavy infection intensity was seen in T. trichiura and S. RstercSoralis. Figure 4.7 showed the intensity of STH infection within Igbole commuEnity of Igbo-Ora. Table 4.13: Risk factors and prevaIlenVce of STH infection within the participants of Igbole, Igbo-Ora. N Risk factors U Intestinal helminth 2 Yes No Total x P-value N No (%) No (%) No (%) Sex MaleA 15 (28.3%) 38 (71.7%) 53 (100%) DFemale 18 (23.4%) 59(76.6%) 77 (100%) 0.40 0.33 AgeA group B <1 – 5 6 (35.3%) 11 (64.7%) 17 (100%) I 6 – 10 5 (55.6%) 4 (44.4%) 9 (100%) 11 – 15 4 (26.7%) 11(73.3%) 15 (100%) 16 – 25 4 (28.6%) 10(71.4%) 14 (100%) 26 – 40 3 (13.0%) 20 (87.0%) 23 (100%) 41 – 50 6 (42.9%) 8 (57.1%) 14 (100%) 51 – 70 3 (13.0%) 20 (87.0%) 23 (100%) >70 2 (13.3%) 13 (86.7%) 15 (100%) 12.4 0.08 House clustering ≤ 4 living in a room 15 (23.8%) 48 (76.2%) 63 (100%) >4 living in a room 18 (26.9%) 49 (73.1%) 67 (100%) 0.16 0.42 House type Concrete 11 (21.6%) 40 (78.4%) 51 (100%) Mud 22 (27.8%) 57 (72.2%) 79(100%) 0.65 0.28 77 Toilet Access Yes 7 (17.9%) 21 (82.1%) 39 (100%) No 26 (28.6%) 65 (71.4%) 91 (100%) 1.63 0.15 Toilet type Water closet 7 (24.1%) 22 (75.9%) 29 (100%) Pit latrine 1 (9.1%) 10 (90.9%) 11 (100%) Bush around house 25 (27.8%) 65 (72.2%) 90 (100%) 1.84 0.40 Hand washing habit Before eating Regularly 31 (25.4%) 91 (74.6%) 122 (100%) Sometimes \ 2 (25.0%) 6 (75.0%) 8(100%) 0.001 0.6Y7 After toileting Yes 28 (24.1%) 88 (75.9%) 116 (100%) R No 5 (35.7%) 9 (64.3%) 14 (100%) 0.88A 0.26 Shoe wearing habit R Regularly 12 (17.1%) 52 (82.9%) 70 (100%) Occasionally 15 (25.0%) 45 (75.0%) 60 L(100I%B) 0.01 0.54 Dirty finger nails Yes 15 (30.0%) 35 (70.0%) 50 (100%) No 18 (22.5%) 62 (77.5%T) Y 80 (100%) 0.19 0.23 House surroundings Neat 28 (24.8%) 85S (75.I2%) 113 (100%) Dirty 5 (29.4%) 12 (70.6%) 17 (100%) 0.17 0.44 R Table 4.14: Prevalence and IntVensiEI ty of STH infection within Igbole community N Helminth UPrevalence Intensity N Light Moderate Heavy A A. lumbDricoides 25 (18.5%) 23 (92.0%) 1 (4.0%) 1 (4.0%) BHooAkworm 19 (13.8%) 15 (78.9%) 2 (10.5%) 2 (10.5%) I T. trichiura 1 (0.8%) 1 (100%) 0 (0%) 0 (0%) S. stercoralis 5 (3.8%) 5 (100%) 0 (0%) 0 (0%) 78 30 AR Y 25 20 IB R 15 L Heavy infectionY Moderate infection10 IT Light infection 5 RS0 Ascaris HookwormVETrichuris Strongyloideslumbricoides I trichiura stercoralis Figure 4.7: Intensity Uof SNTH infection within Igbole community N A BA D I 79 4.7.4 Saganun Community RY Socio-demographic, risk factor and prevalence of STH infection Ain Saganun community, Igbo-Ora. R One hundred and eighty one individuals participated Lin ItBhe study from this community. Sixty-nine (38.1%) were males while 112 (61.9%) were females. The male participants had 29.0% prevalence of STH infYection while 28.6% prevalence was observed in the females (p<0.004).The agIe Tdistribution table showed that the highest prevalence of STH infection was in tShe age group 51-70 (45.5%), followed by age group <1-5 (36.4%) and the lowRest prevalence was in age group 6-10 years (20.0%). Those living in mud hoEuses had 38.5% prevalence of STH infection (p<0.01); 42.2% STH prevalenIcVe was seen in those that did not have toilet facility in their homes (p<0.003); wNhile 43.1% STH prevalence (p-value 0.005) was seen in those that defecated inU the bush around the house (Table 4.15). The prevalenceN of S TH infection was 31.3% and 28.3% in those that reported hand washing beAfore eating and after the use of toilet respectively. For participants practiciDng occasional shoe wearing, 29.6% prevalence of STH infection was seen whiAle 27.9 % STH prevalence was seen in those with dirty finger nails and 15.6% IBprevalence (p <0.002) in those with dirty house surroundings. The prevalence of STH infection in this community was 28.7%. The predominant helminth infection was hookworm with 24.9% prevalence, followed by A. lumbricoides with 15.5% prevalence and S. stercoralis with 1.7% prevalence. However, T. trichiura was not seen in this community. The intensity of infection was highest in A. lumbricoides with 32.1% resulting from heavy worm load as shown in table 4.16. Multiple helminth infection was observed in 21 (40.4%) individuals and A. 80 lumbricoides/hookworm double helminth infection was 85.7% of the double infection, while triple helminth infection with A. lumbricoides/hookworm/S stercoralis was seen only once (Table 4.17). Figure 4.8 showed the intensity of STH infection within Saganun community of Igbo- Ora. Majority of hookworm and A. lumbricoides were of light intensity, although 32.1% of A. lumbricoides were of heavy intensity. Table 4.15: Risk factors and prevalence of STH infection within the participants of SagRanunY Igbo-Ora. A Risk factors Intestinal helminth 2 Yes No Total B R x P-value No (%) No (%) No (%) I Sex Male 20 (29.0%) 49 (71.0%) 69 (1 00L%) Female 32 (28.6%) 80 (71.4%) 1Y12 (100%) 0.004 0.54 Age group <1 – 5 12 (36.4%) 21 (63.6%) I T 33 (100%) 6 – 10 9 (20.0%) 36 (80.0%) 45 (100%) 11 – 15 5 (23.8%) 16 (76.2%) 21 (100%) 16-25 4 (25.0%) 12 (75.0%S) 16 (100%) 26 – 40 8 (27.6%) 21 (7R2.4%) 29 (100%) 41- 50 4 (30.8%) E 9 (69.2%) 13 (100%) 51 – 70 10 (45.5%) V 12 (54.5%) 22 (100%) >70 0 (0%) I 2 (100%) 2 (100%) 6.83 0.45 House clustering ≤4 living in a room 18 (23.1%) 60 (76.9%) 78 (100%) > 4 living in a room 34 (U33.0%N) 69 (67.0%) 103 (100%) 2.14 0.10 House type Concrete 22 (21.4%) 81 (78.4%) 103 (100%) Mud N 30 (38.5%) 48 (61.5%) 78 (100%) 6.34 0.01 Toilet Access Yes D A 25 (21.4%) 92 (78.6%) 117 (100%) No 27 (42.2%) 37 (57.8%) 64 (35.4%) 8.76 0.003 ToilAet type IBWater closet 8 (17.0%) 39 (83.0%) 47 (100%) Pit latrine 16 (23.2%) 53 (76.8%) 69 (100%) Bush around house 28 (43.1%) 37 (56.9%) 65 (100%) 10.72 0.005 Hand washing habit Before eating Regularly 42 (31.3%) 92 (68.7%) 134 (100%) Sometimes 10 (21.3%) 37 (78.7%) 47 (100%) 1.72 0.13 After toileting Yes 41(28.3%) 104 (71.7%) 145 (100%) No 5 (13.9%) 25 (86.1%) 36 (100%) 0.07 0.47 Shoe wearing habit 81 Regularly 23 (27.7%) 60 (72.3%) 83 (100%) Occasionally 29 (29.6%) 69 (70.4%) 98 (100%) 0.08 0.46 Dirty finger nails Yes 21 (30.0%) 49 (70.0%) 70 (100%) No 31 (27.9%) 80 (72.1%) 111 (100%) 0.09 0.45 House surroundings Neat 38 (37.2%) 64 (62.8%) 102 (100%) Dirty 10 (15.6%) 54 (84.4%) 64 (100%) 8.95 0.002 Table 4.16: Prevalence and Intensity of STH infection in Saganun community RY Helminth Prevalence Intensity A Light Mo LIB R derate Heavy A. lumbricoides 28 (15.5%) 17 (60.7%Y) 2 (7.1%) 9 (32.1%) Hookworm 45 (24.9%) 39 (I86T.7%) 4 (8.9%) 2 (4.4%) T. trichiura 0 (0%) S0 (0%) 0 (0%) 0 (0%) S. stercoralis 3 (1.7%) R 3 (100%) 0 (0%) 0 (0%) VE NI U N AD A IB 82 50 45 40 35 30 Heavy infection 25 Moderate infection 20 Light infectYion 15 AR10 5 R 0 Ascaris Hookworm Trichuris StrongyloidesIB lumbricoides trichiura stercor alLis Fig. 4.8: Intensity STH infection within SaganIunT coYmmunity. ER S V NI N U AD A IB 83 Table 4.17: Multiple helminth infection in Saganun community Igbo-Ora. Helminth infection No of individuals % prevalence A. lumbricoides/hookworm 18 85.7% Y Hookworm/S. stercoralis 2 9.5R% A. lumbricoides/hookworm/S stercoralis 1 R A 4.8% Total 22 I B 100% L ITY S R VE UN I AN AD IB 84 4.8: Result of Soil Analysis Two hundred and thirty six soil samples were obtained from the surroundings of 137 households. Many households were clustered together, some households were surrounded with graves and hence soil samples cannot be taken from such places. Twenty-five (25) (that is 10%) households had concrete floor surrounding among the participating households. Majority of the soil samples were sandy soil 173 (73.3%) from 91 households, while 36 soil samples from 27 households were mixture of sand and clay soil (15.3%) and 27 soil samples from 19 households were clay soilR (11.Y4%). In all, parasites were seen in soil samples from 38 households (27.7%). A The prevalence of soil parasite in Igbo-Ora was 17.7% and the parasRite isolated are A. lumbricoides 33 (24.1%), hookworm 5 (3.6%), Taenia ova 2 (1.B5%) and Schistosoma haematobium 1(0.7%) and S. stercoralis 1 (0.7%) (Fig 4 .9L). ITable 4.18 showed the distribution of STH parasites in the soil within tYhe different four communities sampled in Igbo-Ora. Soil samples were collecteIdT from between 31 to 38 households in each community and the result showed 16 out of 36 households in Saganun have STH parasite in the surrounding soil, coRntriSbuting about 11.7% of the prevalence of soil parasite in Igbo-Ora communityE, while the least was found in Pako, contributing 4.4% of infection (6 out of 3V8 households). The distribution of parasites in the environmental soil samples waIs found to be significantly associated with the different community areas in Igbo-ONra. (Chi-square 7.89, p-value 0.048). The result of infec tiUon per household revealed that all households with 10-14 participants AhadN A. lumbricoides infection, while no hookworm was found in them. MeanwDhile, in households with 7-9 participants, 6 out of 8 had A. lumbricoides and no hAookworm infection (Table 4.19). IB4.8.1: Human STH infection versus soil parasite within a household. Human STH infections was found in 57 (41.7%) out of the 137 households from where soil were sampled. Human STH infection was significantly associated with the 2 presence of parasite in the environmental soil (ᵪ 5.74, p-value 0.017). Households with large number of participant were significantly associated with the presence of 2 parasite in the environment soil (ᵪ 33.73 p-value 0.0000). Table 4.18: The Prevalence of soil -transmitted helminth in the environmental 85 soil samples around participants households in the different communities. Parasite No of household per community area where soil Total Present samples were taken Igbole Pako Isale-oba Saganun Yes 8 6 8 16 38 (5.8%) (4.4%) (5.8%) (11.7%) (27.7%) No 24 32 23 20 99 (17.5%) (23.4%) (16.8%) (14.6%) (72.3%) Total 32 38 31 36 137 (23.5%) (27.7%) (22.6%) (26.3%) (100%Y) R Chi-square 7.89 p= 0A.048 R LI B TY I ER S NI V N U A B A D I 86 Table 4.19: The distribution of STH in the soil sample of household with different no of participants STH infection No of household participant 1-3 4-6 7-9 10-14 Total Chi- P-value square Ascaris Yes 14 9 6 4 33 No 88 11 2 0 101 35.37 0.45 Hookworm Yes 2 3 0 0 5 Y No 100 17 8 4 129 8.69 0R.06 BR A LI ITY RS IV E UN DA N IB A 87 A B Y RA R LI B ITY RS C IV E UN N Figure 4.9: AAscaris and Taenia spp. ova seen in soil samples using Zinc Sulphate floatatiDon method. A: AAn ova of Ascaris from soil sample IBB: Teania ova from soil sample C: Disrupted ova of Teania from soil sample 88 4.9: Spatial distribution of Soil-transmitted helminth Infection Soil-transmitted helminth infection was generally seen to be randomly distributed in Igbo-Ora community. The location specificity was then assessed; A. lumbricoides was seen to be clustered in the medium density area of the community, Isale –Oba (Fig 4.10). While the distribution of Ascaris and hookworm in all other communities within Igbo-Ora was randomly distributed. Using Moran‟s index to analyse the statistical significance of the distribution, the result showed the z-score of 2.3 with p- value =0.02 indicating a significant pattern of clustering for Ascaris infection wYithin that community. (Table 4.20). R BR A LI SI TY ER IV U N AN AD IB 89 Table 4.20: Summary table of Moran index value for the spatial distribution of Ascaris and hookworm in Igbo-Ora. Location Moran's Expected Variance z-score p-value Type Pattern Index Index Igbole -0.016884 -0.0417 0.00316 0.44092 0.65927 Ascaris Random Igbole 0.042867 -0.0417 0.0079 0.9511 0.34155 Hookworm Random Isale Oba 0.314964 -0.0435 0.02425 2.30176 0.02135 Ascaris Clustered Isale Oba 0.019422 -0.0435 0.09859 0.20033 0.84122 Hookworm Random Saganun -0.02481 -0.0303 0.00783 0.06206 0.95051 Ascaris YRandom Saganun 0.022089 -0.0303 0.0079 0.58933 0.55564 HooAkwRorm Random R LIB ITY S R VE NI N U A B A D I 90 RY RA IB A: Pattern of Ascaris distribution. L ITY ER S V UN I AN AD IB B: Pattern of Hookworm distribution. Fig 4.10 A and B: Spatial distribution of STH in Isale-Oba, Igbo-Ora. The z-score of 2.30176 for Ascaris falls within the significant level of a clustered distribution. The z- score of 0.200328118247 for hookworm show the pattern to be random. 91 AR Y LIB R Y A: Pattern of Ascaris distribution. IT ER S V NI U DA N BAI B: Pattern of Hookworm distribution. Fig 4.11 A and B: Spatial distribution of STH in Igbole, Igbo-Ora The z-score of Ascaris and hookworm in this community falls within the range of - 1.65 to 1.65 which is an indication of random distribution. . 92 RY RA IB A: Pattern of Ascaris distribution L SI TY ER NI V U AN AD IB B: Pattern of Hookworm distribution. Fig 4.12 A and B: Spatial distribution of STH in Saganun, Igbo-Ora The z-score of Ascaris and hookworm in this community falls within the range of - 1.65 to 1.65 which is an indication of random distribution. 4.10. Assemblage of Pedigree Structure 93 Complete questionnaire and parasitological data were available for 508 individuals (75.5% of the total participant). Pedigree information was available and confirmed for 296 (58.3%) individuals and they were assembled into 7 degrees of relationships with 2- 14 phenotypic individuals; the first degree are parent- offspring, second degree are siblings, third are grandparent – grandchildren, fourth are half siblings, while fifth are cousins, sixth are second cousins and the seventh are uncle/aunt. One hundred and twenty two individuals out of the 508 had no phenotype relatives in the study area, while 90 individuals phenotype cannot be confirmed. The large pedigree spannYed 3 generations of phenotype individuals. There were 639 informative relative pRairs; this include 181 first degree relatives, 141 second degree relatives and 147A third degree relatives, 24 fourth degree relative, 117 fifth degree relative, 27 sixRth degree relative and 2 seventh degree relatives (Table 4.21). The pedigree wBas virtualized using genetic pedigree software called PROGENY. Figure 4.13 aL, b Iand c was examples of pedigree structure in the population sampled. SI TY R VE UN I AN BA D I 94 Y RA R LI B ITYS Fig. 4.13a: Showing an example of a nRuclear family. Kinship relationship visualized using PROGENY; a genetic pedVigreEe software The family tree showing: NI st 1 = Parent – Child U nd 2 = SiblingA relNationship. DMale Female IB A 95 Y RA R B Y LI SI T VE R I U N nd Fig. 4.13b: An eNxample of a family tree with 2 generation and polygamy The famDily tAree of one of the participating household showing the following degree of relaAtionship: st IB1 = Parent - Child nd 2 = Sibling rd 3 = Grandparent - grandchild th 4 = Half- sibling 96 RY RA LI B TY RS I E rd Fig 4.13c: An example of a faImVily tree with 3 generations. The family tree of one of tNhe participating household showing the following pedigree structures: st U 1 = Parent-Child nd 2 = Sibling N rd 3 = GrDAandparent-grandchild th B5 =A Cousins I th 6 = Second cousins th 7 = Uncle-Niece Table 4.21. The pairwise relationships among the 296 related members of the Igbo- 97 Ora pedigree. Relationship Kinship coefficient Pairwise Relationship Number of pairs degree matrix First 0.5-0.5625 Parent –offspring 181 Second 0.25-0.375 Siblings 141 Third 0.125-0.219 Grandparent-grandchild RY147 Fourth 0.0625-0.117 Half-sibling RA 24 Fifth 0.031-0.055 CoLusinIsB 117 Sixth 0.0156-0.027 SYecond cousins 2 IT Seventh 0.0078-0.017 RS Uncles/Aunt 27 IV E UN N AD A IB 4.11: Heritability of STH in Igbo-Ora. 98 The presence of STH infection within the family unit was virtualized on PROGENY. Figure 4.14a, b and c showed the STH infection within 3 families out of the 91 families that participated in this study, the family are 66, 33 and 68 respectively. 2 Meanwhile, table 23 shows the heritability of A. lumbricoides, hookworm as h 22.7% and 1.1% respectively. In family 66, the presence of hookworm was coded as condition 3. Family 66 comprises of a grandfather and grandmother, their son and his two wives with their children. RY Family 33 comprises of three brothers, of the same parent, two out Aof which are married, each had a daughter that are married, one daughter had twRo children, a boy and a girl, and the other daughter had a son. IB Family 68 is a nuclear family that comprises of a father , mLother and their children; two boys and a girl. Y IT ER S V UN I AN BA D I 99 Y RA R LI B ITY ER S V I U N Fig 4.14a: PrNesence of STH within an extended family with polygamous marriage. A A• DAscaris was found in only one member of the family, while hookworm was B found in five family members. I • Hookworm was found in 5 members of the family. 100 RY BR A LI SI TY R Fig. 4.14b: Presence of STH wIiVthin E an extended family. The presence of STH withNin this family unit of 12 members. • Ascaris prese nUt in 5 out of 12 family members. • HeavAy ANscaris intensity was found in only one member of the family • DHookworm was found in 3 out of 12 family member IB A 101 Y RA R LI B ITY RS IV E Fig. 4.14c: Presence of STNH within a nuclear family. • Heavy Ascar isU intensity was found in only one sibling N AD A B I 102 2 2 4.12: The effect of host genetic factors (h ), common shared household (C ) and 2 presence of soil parasite in environment (e ) on the prevalence of STH. The result on table 4.23 showed that the host genetic factors and the presence of soil parasites in the home environment (soil around homes) had no contributory effect on the prevalence of A. lumbricoides while the common shared household was responsible for 4.7% of the variation observed in the prevalence of Ascaris infection. 2 For hookworm infection, both the host genetic factors (h ) and the presence ofY soil 2 parasite around home (e ) contribute greatly to the variation observeRd in the hookworm prevalence. The presence of soil parasite in the environmenAt contributed 47.2% to the prevalence of hookworm (Table 4.23). R LIB 4.13: The effect of host genetic factors and coYmm on shared household on intensity of STH: T Ascaris lumbricoides and hookworm infectioSn wIas the only STH infection taken into consideration, because of the low prevaRlence of T. trichiura and S. stercoralis, they were not considered for these analysEes. IV 4.13.1: Effect on AscaUris Nlumbricoides Intensity 2 Table 4.24 showNed t he effect of host genetic factors (heritability=h ), common shared 2 2household (Ahousehold=C ) and presence of soil parasite in the environment (e ) on AscarisD intensity among related individuals. The SOLAR trait taken into conAsideration was the egg count per gram (epg), this was analysed within the related IBfamily members within households and the effect of shared environment and the effect of the presence of parasite in the soil of the house environment. The result showed that relatedness have significant effect on the intensity of Ascaris infection within families, common shared household have significant effect on Ascaris intensity. Looking at the effect of common shared environment within family 2 and between families living together, a household model (C ) was developed and 149 pedigree was merged into 76 household group by SOLAR. The result showed that 103 host genetic factors (relatedness) contributed to the intensity of Ascaris (25.0%), while common shared environment was responsible for 21.3% of the intensity observed in the Ascaris infection. Meanwhile, the presence of parasite in the soil around homes contributed 9% to the intensity. 4.13.2: Effect on Hookworm Intensity The effect of host genetic factor (relatedness) and common shared environmeRnt oYn the intensity of hookworm within and between families was analysed. The Aselected trait was the hookworm ova count and the covariance were the related family members irrespective of where they live, and household effect within and betwReen families and common shared environment. The result showed that the iIntBensity of hookworm infection was not affected by any of the above mention eLd factors, p-value remain consistently greater than 0.05 (p value > 0.05) (Table 4Y.25). TSI 4.14: Favoured model of the variance Rcomponent analysis of A. lumbriocides and hookworm infection E The summary of the favouredI Vmodels of the variance component analysis for the Ascaris and hookworm inNfections after adjusting for significant covariates, revealed that household mod eUl was the favoured model for Ascaris prevalence while host genetic factor wNas the favoured model for the intensity. Meanwhile, in hookworm infection, thAe presence of soil parasite in the environment was consistently the favoureDd model for the prevalence and the intensity (Table 4.26). IB A 104 Table 4.22: Heritability of STH intensity in relation to genetic relatedness using Sequential Oligogenic Linkage Analysis Resource 8.1.1.0 version. 2 Trait model h C h i - s q u a r e p - v a l u e l o glikelihood Intensity of Ascaris Polygenic 0.227 3.3016 0.035 - 1 843.91Y9 Intensity of R Hookworm polygenic 0.011 0 . 0186 0.446 R - A 1 737.2252 IB L TY RS I VE UN I N AD A IB 105 Table 4.23: The variance component analysis of the effect of Host genetic factors (relatedness), common shared household and presence of soil parasite in the environment on the prevalence of A. lumbricoides and hookworm in Igbo-Ora. Infection Standardized parameter loglikelihood p-value Kullback- 2 estimate Leibler R Y Ascaris Genetic R 2 (relatedness) h =0.000 -126.157 R A0.500 0.007 Household 2 (common shared household) c = 0.047 -125.84I1 B 0.023 0.006 Environment 2 (soil parasite) e = 0.000 -12 6L.157 0.500 0.017 Hookworm Genetic Y 2 (relatedness) h =0S.401I T -131.381 0.065 0.007 Household 2 (common shared household) cR = 0.080 -131.172 0.308 0.017 Environment 2 (soil parasite) V E e = 0.472 -130.778 0.034 0.005 NI U DA N B AI 106 Table 4.24: Effect of host genetic factors (relatedness), common shared households and soil parasite in the environment on Ascaris intensity. Model Loglikelihood Chi p-value Std. Error Genetic related 2 member (famid) h -1839.048 0.250 4.010 0.023 0.132 Common shared households (hhid) Y 2 C A R -1838.103 0.2129 3.880 0R.090 0.122 Contaminated environment (soil around homes) IB 2 e L -2237.102 0.0921 1Y.544 0.146 0.090 IT RS VE UN I N DA B AI Table 4.25: Effect of host genetic factors (relatedness), common shared households and contaminated soil on Hookworm intensity. 107 Model Loglikelihood Chi p-value Std. Error related family member 2 h -1447.225 0.011 0.010 0.459 0.113 common shared households Y 2 C R -1447.197 0.000 0.011 0.459A 0.113 contaminated environment R 2 (soil hookworm) e B -1446.488 0.012 0.01 1L I 0.459 0.113 ITY RS IV E N N U DA A IB Table 4.26: Variance component analysis for the Ascaris and hookworm infections showing the favoured models after adjusting for significant covariates 108 Infection variable Favoured model Standardised parameter estimates Kullback-Leibler Likelihood 2 Genetic Household Environment R a Ascaris lumbricoides Household ( 0) 0.047 (0) 0.039 -125.841 b Ascaris lumbricoides Genetic 0.250 0.213 0.092 0.022 -2237.841 a Hookworm Environment 0.401 0.080 0.472 0.065 -131.381 b Hookworm Environment 0.011 ( 0) 0.012 0.010 -1446.488 A.lumbricoides/hookworm Polygenic 0.774 0 0.221 0.005 -77.335 RY A R LI B SI TY R VE NI N U DA BA I CHAPTER FIVE DISCUSSION 109 5.1: STH infection, demographic structure and associated risk factors of infection 5.1.1: STH infection and demographic structure Population-based studies of soil-transmitted helminth infection are remarkably few in sub-Saharan Africa (Pullan et al., 2010). Although prevalence and intensity of STH infection have been extensively studied in Nigeria, information on prevalence and intensity of STH infection within households and different household clusteYring density are rare. Likewise, the information on the influence of human geRnetics on susceptibility is scarce in this part of sub-Saharan Africa. The findings froAm this study showed that STH infection is an important public health problRem in Igbo-Ora, hookworm infection and A. lumbricoides are the predominaIntB STH parasite in the population sampled. L The prevalence of STH in this study showed 28.1%, Ywhich was lower than what was seen in the study carried out in Ile-Ife, about a IdeTcade ago by Kirwan et al., (2009), where the prevalence was 50% and A. lumbSricoides was the predominant helminth. Adewale et al (2017) in Lagos and ORluwole et al (2018) in Ogun reported STH prevalence of 34.8% and 34.6% amEong primary school pupils respectively, no recent study reported the prevalence oIfV STH among households in Nigeria. Kaliappan et al., (2013) in a research in a Ntribal area in South India reported the STH prevalence of 39% among all age grUoups and hookworm was the predominant helminthes. This was also the predominan t parasite in this study. N There wDas Ano significant difference in STH infection between male and female (p>A0.05), the possible reasons being that men and women have similar occupations Band behavioural patterns in this community. Studies have reported that infection rates I increases significantly with age, from childhood to school age, adolescent and further in older age (Asaolu et al., 1992, Kaliappan et al., 2013). In contrast, the result from this study showed Ascaris had two peaks, one at pre-school age group and a decline at young adulthood, and gradually increases at middle age group and a decline in old age. Hookworm infection had similar pattern, but the first peak of infection was at the school age group, Humphries et al (2011) reported a peak at age 11-20 years for hookworm infection in their household study in Kintampo North, Ghana. This may 110 explain the reason for the World Health Assembly resolution for mass preventive chemotherapy programme primarily targeting pre-school and school-age children (WHO 2001). Irrespective of the benefits of school-based de-worming programme, it is obvious that such an approach will miss potentially heavily infected groups outside of the target population (Walker et al., 2011). There was no significant association between the occupation of the participants and STH infection in this community; the majority of the participants are students, followed by unskilled workers, who are either farmers or petti traders. Also, the level of education of the participants waYs not significantly associated with STH infections; about 17.7% of the participanRts had no formal education. These demographic factors did not have significant eAffects on the prevalence of STH in the community sampled. R IB 5.1.2: Risk factors associated with STH infection L Several studies within and outside Africa have highYlighted several associated risk factors of STH infection, (Adekunle et al., 1986,I ATsaolu et al., 2002, Rukmanee et al., 2008, Pullan et al., 2010, Kaliappan Ret aSl., 2013) but only few have taken the advantage of the new technologyE of geographical mapping, using geographic information system to evaluate the possibilities of clustering of infection. This study highlighted the specific parasitIe Vdifferences in terms of prevalence and intensity in the different community densiNty areas; that is the low, medium and high density areas in Igbo-Ora. The highe stU prevalence of hookworm infection (24.9%) was observed in the GIS mapped high density area of the studied community. Meanwhile the highest prevalence fAor AN. lumbricoides was seen in the two communities, representing the low density area (21.3% and 18.5%). In the high density area, the houses were structured in sAuch Da close proximity that there were no spaces between the houses, the last room IBin a building led directly to the entrance of another house, and the majority of the houses were built with mud. This may explain the reason why living in houses built with mud was a significant risk factor for hookworm infection. In the two communities representing the low density areas, the majority of the house structures were built with concrete and there were spaces between each house structure which in many cases were used as vegetable gardens, where some of the vegetables consumed in those households were planted. Some of the houses, although 111 modern in structure, did not have toilet facilities. Ascaris lumbricoides had the highest prevalence in the low-density area, and this may be explained from the infection acquisition point, because many of the houses in the area were not congested, with plots of land around homes/houses used as vegetable gardens, as well for defecation. The ova passed out with faeces from the infected individual, within the household, will contaminate the vegetables planted within the garden, increasing the chance of acquisition of infection by ingestion of those faecally contaminated vegetables. STH parasites such as Ascaris lumbricoides, and Trichuris trichiura are acquired thrYough this means. This may explain the reason for the high prevalence of A. lumRbricoides observed in these two communities, representing the low density areas. ALikewise, no T. trichiura infection was found in medium and high density areas oRf this community, and this could be due to the geographical distribution, becauseB the low density area mimicked the urban setting in the house structure and arraLngeIment and studies have shown that T. trichiura are predominant in urban are as (Sorensen et al., 1994, Kaliappan et al., 2013). This finding is in agreeImTent Ywith previous studies (Albonico et al., 1997) which reported high prevalenceS of A. lumbricoides in urban settings and high prevalence of hookworm infection Rin rural settings. For S. stercoralis, almost the same prevalence was seen in the mEedium density and low density areas, while the lowest prevalence was from theV high density areas of the community. No previous study investigated clusteringI of different parasite species between different community densities in IgNbo-Ora. Similar GIS mapped study carried out by Ibikounle et al (2018) in the URepublic of Benin showed a countrywide predominance of hookworm in all th e 77 districts of Benin with the prevalence of 17.14%, while Ascaris andA TriNchuris were found in 48/77 with 5.35% prevalence and 37/77 with 1.15% pDrevalence respectively. Their findings confirmed that Ascaris and hookworm infeActions are endemic in all the departments of Benin and that the prevalence of STH IBin Benin varied steadily over localities. The study clearly highlighted the predominance of hookworm infections nationwide with several hotspots where the prevalence reached 50% and more (Ibikounle et al., 2018). In the bivariate analysis, there was no significant association between STH infections and the following risk factors: toilet type, past history of worm and treatment, hand wash before eating and after the use of toilet, regular shoe wearing, and wearing of dirty finger nails (p>0.05). A potential shortcoming for the observed differences may 112 be as a result of reporting bias from the respondents; although information on dirty finger nails was obtained as an observational report. Toilet access, living in crowded rooms with more than four individuals in a room and houses built with mud were significantly associated with STH infection in the multivariate analysis (p<0.05). Pullan et al (2010) also reported living in households with mud floor as a significant associated risk factor for hookworm in a study carried out in rural Uganda. In hookworm infection, studies have shown that skin contact is required for this infection (Hotez et al., 1990) and footwear usage is a protective factor (Bethony eYt al., 2002, Hotez et al., 2003). In this study, there was no association bAetwReen shoe wearing and hookworm infection, the reason may be as a result of report bias, because the information about shoe wearing was got from the participants‟ qRuestionnaire and human tends to change their behaviour/attitude when theyL arIe Bbeing observed for a reason. Similar report of no association was made by K aliappan et al. (2013) in a tribal Southern India population. The high prevaleTnceY of hookworm was found to be significantly associated with living in high deInsity area, where the houses were closely clustered together and the structuresS were mainly built with mud, with large number of people living together in closeR contact. Walker et al., (2011) discoveredV thatE individual predisposition is extremely weak once the clustering of household is Iaccounted for, which means that exposure to infectious roundworm eggs shared Nby household members are important determinants of household clustering . UThis finding is in agreement with Souza et al. (2007) where STH clusteringN was found to be associated with socio-economic status and overcrowdinAg. Because of the absence of toilet facilities in most of the houses, defecatiDon takes place in the bushes around the houses and many do not wear shoes, andA this may increase the risk of infection. In the bivariate and multivariate analysis IBof the risk factors; shoe wearing and toilet types were not significantly associated with STH, neither were they significant for hookworm infection. The observed parasite- specific differences in community density areas are possibly a reflection of variation in exposure and host factors. In this study, the bivariate analysis of risk factors with each specific STH parasites was analysed for A. lumbricoides and hookworm, but S. stercoralis and T. trichiura were not considered for analysis because their prevalence were low, that is 3.0% and 0.8% respectively. 113 The majority of the infections were of light intensity for hookworm (ranges from 66.7% - 86.7%) and A. lumbricoides (ranges from 60.7% - 92.0%) in the four communities sampled within Igbo-Ora. Few individuals harboured heavy intensity of hookworm infection (4.4%-25.0%) and A. lumbricoides (4.0%-32.1%). This finding is similar to work done by Kirwan et al. (2009) where 85.7% of A. lumbricoides infection was light intensity, while 14.1% was moderate intensity. Pullan et al. (2010) reported a similar light intensity for hookworm in their population-based study in a rural community in Uganda. Ibikunle et al (2018) in Benin Republic reportedY that moderate to heavy hookworm and Ascaris infections was observed in severaRl districts of the country, but most of the detected infections had a light parasiteA load. Other studies have demonstrated that intensity of infection increases with Rage, with heavier worm burdens among children aged 5-15 years (O‟Lorcain 2000B; Kirwan et al., 2009; Pullan et al., 2010). In this study, intensity of A. lumLbricIoides and hookworm increases with age and its peak was found in adult. The reason for this may be as a result of repeated exposure to the parasite, and non-treYatment of the existing infection over a long period of time, which enhanced Sthe cIh Tance of increased population of the parasite within the host, since helminRth do not multiply within their host. Other studies have also demonstrated thatE females have heavier worm burden (Holland et al., 1989; Kightlinger 1998). V Multiple infections were NobseIrved in one-third (33.3%) of the infected population with double and trip leU helminth infections. Similar report was given in a study carried out by Dada-AdNegbola et al. (2005) in rural communities in Nigeria where 49.1% of the infectedA population had multiple helminth infection. The majority of the multiple infectioDns were A. lumbricoides and hookworm infections. This may be largely due to tAhe transmission of these two parasites that are associated with poor hygiene Bbehaviour and lack of adequate sanitation (Raso et al., 2004, Ellis et al., 2007). I A strong cluster of STH infection was observed in the community with the small number of household participating in the study, but had the highest number of participants, and the highest number of infected households was found in it, that is, the high density area of the community. STH infection has been found to be highly aggregated, with A. lumbricoides at household levels and T. trichiura in families (Ellis et al., 2007). This finding is in line with the submission of Adekunle et al. (1986) on the importance of family size in relation to STH infection in Ibadan. 114 Similarly, a strong clustering of STH was reported by Souza et al (2007) in a Brazilian study, where 48% of the infected individuals were from 5% of the households. The reason may be the fact that living in close proximity increases the chance of having similar exposure to infection. This may explain the reason for the observed significant association in those living in overcrowded rooms with more than 4 individuals in a room and STH infection. This fact about retention of STH infection within households may suggest the control measure adopted by WHO should be revisited. This becomes necessary because an infected adult living in the same hYouse with the treated school children may serve as source of infection for the treaRted child. This resolution is in agreement with Ellis et al.‟s (2007) suggestion Athat infected households should be targeted for treatment, in that this will provRide an effective, rapid and practical method of identifying and treating helmintIh-B infected adult in the community. L Considering the individual community and the risTk faYctors of STH infection, the risk factors were not significantly associated with ISTH infection in two communities representing the low density area and the mSedium density area. This may be partly due to the none participation of sizeableR number of the adult population in two of the communities, that is, Pako and IsaleE-oba, and partly due to the small sample size of the participants from this two aIreVas This is one of the major limitations of this study. In the high density area, wNhere the participants were evenly distributed among all age groups, the signific anUtly associated risk factors included house type, toilet access, defecating in bNushes around the house and dirty house surrounding. Some of the following riAsk factors have been found in other studies to be associated with STH infectioDn (Tchuem Tchuente et al., 2003, Raso et al., 2004). The reason why most of the Aknown associated risk factors, like shoe wearing and hand-washing, was not IBsignificant may be because the people lived in close community and they communicated; so they were informed about the study and a kind of attitudinal change was put up by the participants during the course of the study in the community, also a report bias may be playing an important role in the analysis. 5.2 Spatial distribution of STH infection in Igbo-Ora 115 In this study, the presence of STH parasite in the environment, that is, in human was used to develop the spatial distribution of STH infection in Igbo-Ora. The justification for this was in the positive correlation found in the prevalence of human STH infection and the presence of STH parasite in the soil around homes of the study participants. Pullan et al. (2010) reported that only few studies within African communities have addressed the spatial determinants of infections. In this study, Ascaris lumbricoides was the major parasite found in the soil followed by hookworm and only one S. stercoralis was found, other parasite foundR incYlude Taenia and S. haematobium ova. Similar result was recorded by Adekeye et al (2016) in their study done in Ibadan, wherein A. lumbricoides was the predomiAnant parasite, followed by hookworm and S. stercoralis and other parasites aside SRTH were isolated. The pattern of parasite prevalence was slightly differentL froImB what Ogbolu et al (2011) recorded, in their study in Ibadan; hookworm was the most frequently encountered parasite followed by S. stercoralis, and AY. lumbricoides was found to be fourth on the line. The reason for the highest preIvaTlence seen may be explained by the structure of the Ascaris ova, wherein the cutSicle is thick, it can withstand desiccation and it can survive better in the environmRent than any other human parasite. Also, the season wherein the samples were coEllected may contribute to the availability of one parasite specie than the other. IV The presence of soil parasNite has been said to be an avenue by which human infection is made possible, be caUuse the ova in the soil can be transferred directly to the mouth (Kobayasi, 1999N) or to the vegetables and from vegetable to mouth when eaten raw or poorly cookAed. This is practically opposite for hookworms (Ancylcostoma duodenale and NecDator americanus) because of the susceptibility and sensitivity of their free-livinAg larvae and thin-shelled eggs to desiccation and cold temperatures. This reduced IBtheir chance of being seen often in the soil samples. On the contrary, there are no free-living larval stages of Trichuris trichiura (whipworm) but rather thick-shelled eggs. This allowed for its survival over a long period of time even under harsh or unfavourable environmental conditions. The observed prevalence of 28.1% of human STH infection and 17.7% of STH parasite in the environmental soil is a pointer that there is a need to determine the distribution pattern, exposure factor, the source of infection and possibly the 116 environmental factor responsible for the presence of the infection. The geographic information system revealed that both A. lumbricoides and hookworm were randomly distributed in all the sampled areas except for Ascaris that was found to be clustered in medium density area of the community. The observed cluster of Ascaris in this region showed the possibility of a major environmental factor, predisposing individuals in this area to infection. The physical factor that was seen that could be responsible for the unique clustering of Ascaris in this location was the major market. The market, Towobowo by name was, on a normal day, a residential place, but eYvery household and space becomes a market stall every 5 days. This is being patroRnised by everybody in the community, and there is usually a large influx of peoplAe bringing in their farm produce for sales from neighbouring communities. BuyersR from other large cities come to take advantage of the cheap farm produce. Data Bfrom this community revealed that less than a quarter of the participants from thiLs loIcality reported to have access to toilet. Less than 16% had water closet toilYet ty pe, 10% had pit latrine and the remaining 74% of the participants living in thIisT place defecated in the bush around their homes. S Another important indicator is that therRe is no public toilet in this location and the large influx of people into thisV plaEce every five days increased the chance of the people making use of open spIaces around the dunghills to increase, thereby making the parasite to be readily aNvailable in the community soil, increasing the exposure of the resident to infec tiUon. Also, during the rain, the ova in the soil could be washed down to farm laNnd and plants are contaminated with the ova of Ascaris. According to the EuropAean Commission, the definition of „contaminated site‟: a site where there is a conDfirmed presence, caused by human activities, of hazardous substances to such a deAgree that they pose a significant risk to human health or the environment, taking IBinto account land use (Science Communication, 2013). Hence the presence of a large volume of excreta in this community, qualify it as being marked as a contaminated area, and therefore, the health of the resident of this area is at risk of STH infection. 5.3 Genetic variance component analysis. Many studies outside Africa have reported the significant role of host genetic factors in identifying STH intensity (Breitling et al., 2008, Quinnell et al., 2009, Pullan et al., 117 2009) and the only two known studies done in Africa by William-Blangero et al. (1997) in a rural Zimbabwe and Pullan et al. (2010) in a rural community in Uganda. These two studies suggested the possible role of human genetics in hookworm intensity. The only known association study on STH, in Nigeria, that suggested that genetic factors may influence susceptibility to helminthic infection, was an analysis of A. lumbricoides worm loads in Nigerian children, that suggested the involvement of major histocompatibility complex (MHC) in determining resistance to infection (Holland et al., 1992). The paucity of information on the role of human genetYic in infection intensity is one of the reasons for which this study was undertaken. R The genetic analyses was designed to explicitly evaluate the relative Aeffect of the genetic factors and environmental factors contributing to humanR susceptibility to Soil-Transmitted helminth infection in Igbo-Ora, representLingI thBe south western part of Nigeria. The genetic factors employed for this study wa s investigating the effect of genetic relatedness of the study participant on helTminYth infections. This made use of pedigree information. However, confounded variIable of household and environmental risk factors affect heritability, because relSated individuals are likely to be living together in the same place, resulting in Rhaving similar exposure risk factors, likewise unrelated individuals may have simEilar exposure with related individuals. Therefore, household and environmental rIisVk factors were taken into consideration for this study. N 5.3.1 Genetic effect oUf relatedness on the prevalence and Intensity of STH Heritability Ais foNrmally defined as the proportion of phenotypic variation that is as a result oDf variation in genetic values. According to Ekstrom (2009), heritability estiAmation is usually considered the first step in unravelling the genetic basis of a IBdisease or trait. The effect of genetic relatedness on the prevalence of various STH infections revealed heritability of 22.7% for Ascaris lumbricoides and a much higher heritability for other STH parasite. The heritability demonstrated in this study is in line with the research result in the Jirel population of the eastern Nepal that demonstrated that susceptibility to Ascaris is heritable, with between 30% and 50% of the variation being attributable to genetic factors. Acevedo et al. (2009) in their research reported that specific antibodies against Ascaris are associated with resistance and their result supported the 118 possibility of a direct causal relationship between 13q33 locus and Ascaris susceptibility. 5.3.2 Effect of genetic relatedness on the prevalence and intensity of Ascaris lumbricoides and Hookworm infection In determining patterns of infectious disease, according to William-Blangero et al., (2012), host population structure factors are generally not more important than wiYthin- population host genetic factors. The result from this study revealed Athat Rthe host genetic factors and the presence of parasite in the soil around homes have little or no effect on the prevalence of Ascaris lumbricoides infection wBithiRn the population 2 2sampled; h and e were 0.000 and the p- value 0.500. MeanLwhIile, the common shared 2household, C , was responsible for 4.7% of the prevale nce within households and there was a significant influence of the common sharYed household on the prevalence of Ascaris within related individuals (p=0.023).I TThis result is in agreement with the report of Ellis et al. (2007) in the study carriSed out in Poyang Lake in China, wherein the variance component analysis indicatRed common shared environment as the major risk factor and there is no addictive Egenetic effect for A. lumbricoides infection in the area; this is contrary to WilliamI-VBlangero et al., (1999). Shaw and Quinnell (2009N) reported that helminth infection susceptibility in human population varies a mUong ethnic group and that pedigree study is required in investigating thNe effect of genetic relatedness. The environmental factors that may mask the effAect on the phenotype is taken care of or eliminated in pedigree study. This is becauDse pedigree study allows for the analysis of a variant phenotype in unrelated indiAviduals living in the same household and related individuals living in different IBhouseholds. It is however observed, in this study, that shared common household has greater and significant effects on the prevalence of A. lumbricoides within the population sampled than the host genetic relatedness. This can be explained by the mode of acquisition of Ascaris infection which is by ingestion of faecally contaminated food or drinks. The population sampled are people living together and having close communal relationship. There, people within the same household can exchange food and drink items, hence the possibility of one infected individual transmitting the infection to the other people within the same household, as a result of 119 their communal living lifestyle. Another possibility is that they get their food or vegetable from the same source, especially those with vegetable garden in and around their homes; hence they have the same exposure to infection. On the contrary, for hookworm infection, host genetic and house environment have significant effect on the prevalence of hookworm infection in this study. Heritability 2 h , the proportion of variance explained by relatedness was 40.1%, while additional variance of 47.2% was explained by the presence of parasite in the soil around home 2 e . This result is the third hookworm heritability report to the best of my knowledYge in Africa. The first report was given by William-Blangero et al (1997) in ZimbaRbwe and the second report in the continent of Africa was in Uganda by Pullan et aAl. (2010) and this present study provide third heritability report of hookworm inRfection in Africa. This present result showed that the relative contribution of IhBost genetics, that is, relatedness to the prevalence of hookworm infection, was noLt significant (p= 0.065) as compared to the significant contribution of presence oYf contaminated soil in the home environment (p=0.034) sampled. Similar report IwTas given by William-Blangero et al (1997) wherein heritability of 37% was repoSrted among the Zimbabwean for Necator americanus. On the contrary, the heRritability report from Ugandan community reported heritability of 11.2% andE 17.8 percentage of variance from unidentified domestic effects. Thus, host geInVetic relatedness was not considered to be a significant determinant of infection inNtensity in that community. Various studies hav e Uindicated the importance of clean environment in relation to being disease free (Narain et al., 2000; Asaolu and Ofoezie, 2003). According to the biology of hookNworm infection, humans acquire the infection by the presence of the filariforDm laArvae L3, of hookworm in the soil and when this comes in contact with humAan skin, it penetrates and it is carried into the human circulation and, hence the IBlife cycle continues. This finding is in line with Brookers et al. (2007) wherein it was concluded that though the host genetic factors cannot be ruled out, the variation in exposure and parasite life cycle, are primarily responsible for the species-specific differences in the patterns of infection by environment. Quinnell et al. (2010), in their research in a Brazilian community, reported that predisposition to human hookworm infection is due to the combined factors of host genetics and consistent differences in exposure. The exposure being determined by the effects or factors of the environment and the households. 120 Forrester et al. (1990), in their epidemiological study of Ascaris lumbricoides and T. trichiura in individuals and families in Mexico, suggested that there is considerable diversity in A. lumbricoides susceptibility among subjects living under the same conditions and that infection intensity is typically overdispersed, with 10-20 % of the population harbouring most of the parasites. In this study, host genetic component accounts for 25% of the variation observed in the worm burden due to A. lumbricoides, while 21.3% of the intensity observed was accounted by the common shared environment and the presence of the parasite in the soil around homes accYount for 9.3% of the variation. These imply that in this study variance componentR analysis for A. lumbricoides intensity identified host genetic factor as the major risAk factor. Similar results were seen by William-Blangero et al. (1999) in the rResearch involving the genome scanning of individual human host from a Jirel poIpuBlation in East Nepal. In their study, about 30-50% of variation in worm burde nL was accounted for by the host genetic, while 3-13% of the variation in Ascaris lYumbricoides load was due to the effect of a shared environment. Further workI Tdown by William-Blangero et al. (2000) among the Jiri population revealeSd a strong evidence for two distinct quantitative trait loci (QTL), influencRing the susceptibility to Ascaris infection. Holland et al. (1992), in their stuEdy among children in Ile-Ife Nigeria, reported evidence for a role of the majoIrV histocompatibility complex in determining pattern of Ascaris infection. N Although several he riUtability studies have been done on hookworm infection, none of those studies toN the best of my knowledge have been able to look at the effect of a particular geAne on the prevalence or on the intensity of hookworm infection in Africa. The onDly heritability study done in Africa was in Uganda, wherein heritability of hooAkworm intensity was evaluated as 11.2 % while 17.8 percent variation was from IBunidentified domestic effect (Pullan et al. 2010). This study provides information on the genetic epidemiology of hookworm intensity wherein heritability of hookworm intensity was 1.1% and the variance explained by the common shared environment and the presence of parasite in the soil around homes was same as that of the host genetic relatedness that is 1.1%. This result implied that none of the factors has greater effect than the others on the intensity of hookworm infection in this community despite the differences in the effect on the prevalence. Several other studies done outside Africa recorded a greater variance effect of host genetic 121 relatedness e.g. Quinnell et al (2010) reported a heritability for hookworm intensity in the pretreatment as 17% and 25% after reinfection in a Brazillian community. The 2 shared household environment c accounted for 16.3% and 9.5% of residual variance in both the pretreatment and reinfection respectively. 5.4 Summary of the findings Hookworm is the predominant STH infection in the community sampled followed by Ascaris lumbricoides. Predisposing factors associated with intensity are living in Yhigh density areas, living in rooms crowded with more than four individuals, Rliving in houses built with mud. Ascaris lumbricoides infection was found to beA significantly clustered in GIS mapped medium density area of Igbo-Ora. SpatialR analysis showed that hookworm infection was randomly distributed in all theI GBIS mapped areas of Igbo-Ora. L Heritability of A. lumbricoides was 22.7%. Host Ygenetics and common shared 2 environment had significant effect on the intensiItyT of A. lumbricoides infection. (h = 2 0.250 p-value 0.023; c =0.213 p-value 0.01S). In hookworm infection, human genetic 2 relatedness accounted for 40.1% of the vRariation in the prevalence of infection (h = 0. 401 p-value 0.065) while the presencEe of parasite in the soil around homes contributed 2 significantly 47.2% to the prIevValence of hookworm infection (e = 0.472 p-value 2 2 20.034). All the factors h ,N c and e equally contributed, but not significantly, to the intensity of hookwormU infection in this study. N 5.5 ConAclusion and recommendation HumAan Dgenetic relatedness have significant effect on Ascaris infection intensity, IBwhile the presence of soil parasite plays important role in hookworm infection. The different factors responsible for human susceptibility displayed by Ascaris and hookworm in this study is an indication that specific approach should be put in place to reduce or curtail the prevalence and intensity of STH infection rather than the assumption that the environmental condition is the only factor responsible for exposure. The relative contributions of human gene can further be explored. Exposure related factors also played significant role as determinants of STH infection, especially in hookworm infection, in these communities. The use of statistical 122 method, that partitioned addictive effect of genetic factors from that of environmental factors and shared household, enabled a better understanding of the determinant of soil-transmitted helminth infection in this community. Hence, the variation observed in the pattern of the factors that contributed to the prevalence and intensity of the specific STH infection is an indicator that different prevention and control strategy should be employed towards the elimination of each of the STH infection, which has been marked by WHO as one of the Neglected Tropical Diseases. RY BR A Y LI IT ER S V NI References Abu-Madi, M.A., Be hUnke, J.M. and Doiphode, S.H. 2010. 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InSfection and Immunity vol. 78:4, 1552-1563 doi 10:1128/IAL00848-E09 V UN I N AD A IB 146 APPENDIX I: ETHICAL CERTIFICATE AR Y R LI B ITY ER S NI V N U AD A IB 147 APPENDIX II: QUESTIONNAIRE IMPACT OF HOST GENETICS AND ENVIRONMENTAL FACTORS RISK FACTORS ON SOIL-TRANSMITTED HELMINTH (STH) INFECTIONS AMONG HOUSEHOLDS IN IGBOORA, NIGERIA. QUESTIONNAIRE Y Household Number………… Personal ldentification R Number…………. A Demographic information. BR 1. Age……….. Weight (Kg)…………L… I Height (cm)………………. Y 2. Sex: Male Female T SI 3. Level of Education: (1) PrimRary School (2) SEecondary School I (3V) Tertiary Institution N (4) No formal Education 4. Marital SNtatu U s: (1) Single DA (2) Married (3) Widowed A (4) Separated IB 5. Present Occupation:…………………………………….. 6. Religion: (1) Christianity (2) Islam (3) Others…………………………………. 7. Ethnicity: (1) Yoruba 148 (2) Ibo (3) Hausa (4) Others specify……………………… Environmental/Household Description 8. How many of you live together in the house? (1) 1-3 (2) 4-6 (3) 7-9 Y (4) 10-12 AR(5) More than 12 BR 9. Type of house: (1) Concrete LI (2) Mud (3) Others specify…………IT……Y…. 10. Do you have access to a toilet? R YesS No 11. Where is the toilet locateVd? E(1) Within the compound I (2) Attached to the building N (3) The community toilet U 12. How maNny o f you use the toilet………………. 13. DTypeA of toilet: (1) Water closet A (2) Pit latrine IB (3) Bucket/Potty latrine (4) Nylon bags (5) Bush around the house Others specify………………………………….. Behavioural activities. 14. Do you wear shoes when at home? Yes No 149 15. Do you wear shoes when working or relaxing/playing outside the house? Yes No 16. How often do you wear shoe?(1) Regularly (2) Occasionally (3) Never Y (4) Only when going out for occasion R RA 17. How often do you wash your hands before eating? (1) BRegularly L I (2) Sometimes Y (3) Never 18. How often do you wash your hands aSfter Ito Tileting? (1) Regularly R (2) Sometimes E (3)Never 19. Do you eat uncooked vIegVetable? Yes No UN Past history of Nwor m infection 20. HaveA you ever had about or seen a worm before? Yes No AD B21. Have ever passed out worm either from mouth, nose or faeces? Yes I No 22. Have you been treated for worm in the last one year? Yes No Observational data 23. Is your interviewee wearing long nails? Yes No 150 24. Are the nails dirty? Whether long or short. Yes No 25. Is the house neat or dirty? Yes No 26. Is the surrounding of the house neat or dirty? Yes No RY RA LI B ITY S VE R UN I AN D IB A 151 Appendix III INFORMED CONSENT FORM Dear Family Head, My name is Olufunke Oluwatoba, a postgraduate student from Department of Zoology, University of Ibadan. I will like to find out about the health status of every member of your household, especially their state of health as regard worm infestation. I will need to ask your house member few questions about their health and will need to collect two stool samples on two different days, this will be processed in the laboratoryY to ascertain whether they have worm or not, the test will be at no cost to you. The resRult of the test will be made available to you and all member of your household for appropriate action to be taken if they have worm. A Your household will be given a number and every member of the househoRld as well and this will be written on the form to be filled during the interview with the hBousehold member. Likewise, a small proportion of soil around your house will be taken Ias sample to be tested in the laboratory; this is to be sure of where the parasite is in you r Lenvironment. Your household was chosen because it falls within they selYected area from our sampling method and you are free to refuse to take part in thiIs pTrogramme. You have the right to withdraw at any given time if you choose to. ThaSnk you. Consent: Now that the study has been wEell Rexplained to me and I fully understand the consent of the study process, I will be willing to allow every member of my household and myself to take part in the programImVe. N U Signature/ThumbNprint of household head/date Signature of Interviewer with date DA IB A Signature/Thumbprint of Witness with date 152 APPENDIX IV: PEDIGREE INFORMATION COLLECTION SHEET Date: Locality: Househo ld ID: Birth Father's Father's Father's Mother's Mother's PID Name Sex Date Birthplace Name Birthplace Clan Name Birthplace RY RA B L I ITY S R E I V N U N A D B A I 153