SCHOOL OF PUBLIC HEALTH, COLLEGE OF HEALTH SCIENCES, UNIVERSITY OF GHANA PREVALENCE OF HEPATITIS B VIRUS CO-INFECTION AMONG HIV-SEROPOSITIVE PERSONS ATTENDING ANTIRETROVIRAL CLINICS IN THE EASTERN REGION OF GHANA BY GIDEON KYE-DUODU (10358920) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF MPHIL APPLIED EPIDEMIOLOGY AND DISEASE CONTROL DEGREE JULY 2012 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I do hereby declare that except for duly acknowledged citations and ideas, this thesis is an original work produced by me from a study personally undertaken under the supervision of Dr. Priscillia Nortey. This work has never on any previous occasion been submitted in part or whole to any institution or board for award of any degree. CANDIDATE: _________________________________ Gideon Kye-Duodu (20.02.2013) SUPERVISOR: _________________________________ Dr. Priscillia Nortey (20.02.2013) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION To my dearest one, Aba Kaale Kye-Duodu and my family, especially Kofi „Mungo Park‟, who were of tremendous support throughout the course of my study. It is also dedicated to all Persons Living with HIV and AIDS (PLHIV) in the Eastern Region of Ghana who made this study possible. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENTS First of all, I thank God for the opportunity to enrol in the MPhil Applied Epidemiology and Disease Control Programme, which made it possible for me to conduct this research and also providing wisdom and strength to complete it. I am grateful indeed to my academic supervisor, Dr. Priscillia Nortey of the Department of Epidemiology and Disease Control, School of Public Health and my field supervisor, Dr. S.B. Ofori of the Regional Hospital in Koforidua and Coordinator for the Eastern Regional AIDS/STI Control Programme for their suggestions, encouragements, constructive criticisms and corrections. I wish also to appreciate the support of my mentors and colleagues, especially Dr. Sherry Johnson, Joyce Der-Saayeng, Dr. Carl Stephen Osei and Dr. Joseph Opare, all graduates of Ghana Field Epidemiology and Laboratory Training (GFELTP) and Ms. Beatrice Tchuidjang Nganso of the African Regional Postgraduate Programme in Insect Science (ARPPIS), University of Ghana. I am thankful to all heads of institutions, laboratory personnel and HIV counsellors of hospitals and Antiretroviral clinics (ART) who gave permission to conduct the study in their facilities and also assisted me with the work. I am also grateful to the staff of the National Public Health Reference Laboratory (NPHRL), especially Mrs. Esther Aryee, Mr. Rowland Adukpo and Rexford Adade, as well as Mr. I.K. Boamah, Bernard Squire and Debbie of the Virology laboratory of the University of Ghana Medical School (UGMS) for assisting me with the laboratory investigations. University of Ghana http://ugspace.ug.edu.gh iv Many thanks go to the GFELTP for the academic, logistic and financial support. I am highly indebted to Prof. E. Afari (Coordinator, GFELTP), Dr. Fred Wurapa (Former Director, GFELTP), Dr. Kofi Mensah Nyarko (Director, GFELTP), Dr. Ohuanbunwo Chima (Former Resident Advisor, GFELTP), Dr. S.O. Sackey (Faculty Lecturer, GFELTP), and Dr. Nathaniel Yebuah (Adjunct Lecturer on “One Health”) for the lasting impact they have made on my fledgling academic and career path in Field Epidemiology. God richly bless you all. University of Ghana http://ugspace.ug.edu.gh v ABSTRACT Background: Hepatitis B virus and HIV infections are endemic in sub-Saharan Africa where 22.9 million individuals are estimated to be infected with HIV and nearly 50 million people are chronic HBV carriers. Due to similar routes of transmission, co-infections with the two viruses are common. Hepatitis B being severely infectious and found in HIV-positive patients, could pose more serious health problems. In Ghana, the prevalence of HBV infection is estimated at 15% of the adult population and the estimated HIV prevalence among antenatal clients in 2010 ranged from 0.4% in Krachi and Adibo, two rural sites to 7.4% in Agomanya, an urban site in Ghana. The guidelines for antiretroviral therapy (ART) in Ghana were reviewed in September 2011 to include for the first time the management of hepatitis B virus (HBV) co-infection with HIV. Clearly, the guidelines reveal a deficit of information to guide policy but understanding the extent of these dual epidemics is critical to the optimization of treatment. We determined the prevalence and risk factors of HBV co-infection in Persons Living with HIV (PLHIV) and assessed the knowledge of ART staff regarding the co-infection in the Eastern region. Method: A cross sectional study was conducted in five ART clinics to obtain data from a probability proportionate to size random sample of PLHIV in the Eastern region from March to June 2012 and a self-administered questionnaire to assess knowledge of health care workers was done. Descriptive and logistic regression models were used for analysis. Results: Of 320 PLHIV recruited for study, 28 were positive for HBsAg giving an overall prevalence of 8.8% and site specific prevalence ranged from 13.6% in Asesewa to 0% in Atua. More females 19 (67.9%) were found to be HBsAg positive and participants ages ranged from 4 to 76 years with a mean age of 40.82 (± 12.7) years. There were statistically significant University of Ghana http://ugspace.ug.edu.gh vi relationships between HBV infection and increasing age (p=0.004), partner with history of HBV infection (p=0.010) and history of HBV infection in the past (p=0.020). ART staff obtained 84.2% (SD± 20.53; 95% CI: 89-98.1) and 53.1% (SD± 35.06; 95% CI: 13.0-88.9)) in the “general knowledge” and “management practice” indexes respectively. Conclusion: Prevalence of HBV-HIV co-infection was within worldwide estimates of 5-10%. Individuals with HBV positive partners or with history of HBsAg testing were more likely to have HBV infection. Knowledge of ART staff with regards to management practices of HBV- HIV co-infection and HBV vaccination coverage among PLHIV were found to be low. Trainings should be done on a regular basis to address needs gaps and the inclusion of PLHIV in HBV vaccination programmes should also be considered. Keywords: HIV, HBV, co-infection, HBsAg risk factors, Ghana. University of Ghana http://ugspace.ug.edu.gh vii TABLE OF CONTENTS DECLARATION ........................................................................................................................................... i DEDICATION .............................................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................................... iii ABSTRACT .................................................................................................................................................. v TABLE OF CONTENTS ............................................................................................................................ vii LIST OF ABBREVIATIONS ..................................................................................................................... xii CHAPTER ONE ......................................................................................................................................... 1 INTRODUCTION ....................................................................................................................................... 1 1.1 Background ............................................................................................................................................. 1 1.2 Problem Statement .................................................................................................................................. 3 1.3 Justification ............................................................................................................................................. 4 1.4 Main Objective ........................................................................................................................................ 5 1.4.1 Specific Objectives ................................................................................................................... 5 CHAPTER TWO ........................................................................................................................................ 6 LITERATURE REVIEW .......................................................................................................................... 6 2.1 Epidemiology of HBV-HIV co-infection ............................................................................................... 6 2.2 Prevalence of HBV-HIV co-infection in sub-Saharan Africa ................................................................. 7 2.3 The status of HIV epidemic in Ghana ................................................................................................... 10 2.4 The status of Hepatitis B in Ghana ....................................................................................................... 10 2.5 The natural history of HBV disease in HIV co-infected patients ......................................................... 11 2.6 Impact of HIV on Hepatitis B ............................................................................................................... 11 2.7 Impact of Hepatitis B on HIV ............................................................................................................... 12 2.8 Serological Markers of HBV Infection ................................................................................................. 13 2.9 Knowledge of health care workers (HCWs) regarding HBV-HIV co-infection ................................... 14 CHAPTER THREE .................................................................................................................................. 16 METHODS ................................................................................................................................................ 16 3.1 Study Design ......................................................................................................................................... 16 University of Ghana http://ugspace.ug.edu.gh viii 3.2 Study Setting ......................................................................................................................................... 16 3.3 Study Population ................................................................................................................................... 17 3.4 Variables ............................................................................................................................................... 18 3.5 Sampling Methods ................................................................................................................................ 18 3.5.1 Sample Size Calculation ................................................................................................................ 18 3.5.2 Study Sites: .................................................................................................................................... 19 3.5.3 Persons Living with HIV and AIDS (PLHIV): .............................................................................. 20 3.5.4 Health care workers in ART clinics: .............................................................................................. 20 3.6 Inclusion criteria: .................................................................................................................................. 21 3.7 Exclusion criteria: ................................................................................................................................. 21 3.8 Specimen and Data Collection Techniques and Tools .......................................................................... 22 3.9 HBV (HBsAg ELISA) Laboratory Procedure ...................................................................................... 22 3.9.1 Principle of the Foresight HBsAg ELISA kit assay ....................................................................... 24 3.9.2 Validation and Quality Control of ELISA Testing ........................................................................ 25 3.10 Data Quality Control Measures ........................................................................................................... 27 3.10.1 Pretesting of questionnaires ......................................................................................................... 27 3.11 Laboratory Quality Control Measures ................................................................................................ 27 3.11.1 Running of control samples ......................................................................................................... 27 3.11.2 Replication of Assay .................................................................................................................... 28 3.11.4 Supervision of laboratory testing ................................................................................................. 28 3.12 Data Processing and Analysis ............................................................................................................. 28 3.13 Ethical Considerations ........................................................................................................................ 29 CHAPTER FOUR ..................................................................................................................................... 30 RESULTS .................................................................................................................................................. 30 4.1 General Overview ................................................................................................................................. 30 4.2 Socio-Demographic Characteristics of Study Participants (PLHIV=320) ............................................ 30 4.3 Clinical Characteristics of Study Participants ....................................................................................... 33 4.4 Laboratory Analysis .............................................................................................................................. 34 University of Ghana http://ugspace.ug.edu.gh ix 4.4.1 HBV Status (assessed as HBsAg ELISA) of Study Participants ....................................................... 34 4.4 Distribution of HBsAg Positive Study Participants in ART Sites, Eastern region ............................... 36 4.5 Hepatitis B Serologic “Markers” in HBV Positive Study Participants ................................................. 36 4.6 Characteristics of ART Staff (HCW) in Study Sites ............................................................................. 37 4.6.1 General Knowledge of Hepatitis B and HBV-HIV co-infection ....................................................... 38 4.8 Demographic and HBV Risk Factor Analysis among Study Participants ............................................ 41 CHAPTER FIVE ...................................................................................................................................... 43 DISCUSSION ............................................................................................................................................ 43 5.1 Introduction ........................................................................................................................................... 43 5.2 Characteristics of Study Participants .................................................................................................... 43 5.3 Prevalence of HBV-HIV Co-infection among Study Participants ........................................................ 44 5.4 Risk Factors for HBV infection in Study Participants .......................................................................... 46 5.5 Assessment of Health Care Workers Knowledge of HBV-HIV Co-infection ...................................... 48 5.6 Limitations of Study ............................................................................................................................. 49 CHAPTER SIX ......................................................................................................................................... 51 CONCLUSIONS AND RECOMMENDATIONS .................................................................................. 51 6.1 Conclusions ........................................................................................................................................... 51 6.2 Recommendations ................................................................................................................................. 52 REFERENCES ........................................................................................................................................... 53 APPENDICES ........................................................................................................................................... 60 APPENDIX I – ART sites in the Eastern region of Ghana......................................................................... 60 APPENDIX II – Informed Consent Form (Adult) and Questionnaire 1 ..................................................... 61 APPENDIX III – Informed Assent Form (Child) and Questionnaire 2 ...................................................... 67 APPENDIX IV - Antiretroviral Clinic Staff Questionnaire 3 .................................................................... 73 APPENDIX V – Hepatitis B Serologic Markers Assay (ELISA) ............................................................... 76 APPENDIX VI-Approved Ethical Clearance ............................................................................................. 77 University of Ghana http://ugspace.ug.edu.gh x LIST OF TABLES Table 1: Surveys of Hepatitis B Virus surface Antigen (HBsAg) Prevalence in HIV-infected Persons in sub-Saharan Africa, 1996-2011 .................................................................................................................... 9 Table 2: Age and Sex Distribution of Study Participants, ART Clinics, Eastern Region (n=320) ............. 31 Table 3: Socio-Demographic Characteristics of Study Participants, Eastern Region, 2012 ....................... 32 Table 4: Clinical Characteristics of Study Participants and HBV Infection, Eastern Region, 2012 ........... 33 Table 5: Age Distribution of Study Participants and HBV Infection (HBsAg ELISA) ............................. 35 Table 6: HBV Infection of Study Participants by Location of ART Site, Eastern Region, 2012 (n=320) . 35 Table 7: HBV Serologic 'Markers' among HBV Positive Study Participants, Eastern Region, 2012 (n=28) .................................................................................................................................................................... 37 Table 8: HCW 'General Knowledge' Items Frequency Distribution, Eastern Region, 2012 (n=54) .......... 38 Table 9: Frequency Distribution of ART Staff Knowledge Items for HBV-HIV Co-infection Management Practices, Eastern Region, 2012 (n=54) ...................................................................................................... 40 Table 10: Multivariate Analysis of HBV Infection of Study Participants and Risk Factors, Eastern Region, 2012 (n=320) ................................................................................................................................. 42 University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES Figure 1: Map of Eastern Region of Ghana showing ART Study Sites ..................................................... 17 Figure 2: Microwell Plate showing HBV ELISA Results (Source: Virology Lab; UGMS) ...................... 24 Figure 3: HBV Prevalence in ART Study Sites, Eastern Region (n=320) .................................................. 36 Figure 4: HBV Vaccination Status of Study Participants (n=307) ............................................................. 41 University of Ghana http://ugspace.ug.edu.gh xii LIST OF ABBREVIATIONS ALT - Alanine Aminotransferase AIDS - Acquired Immune Deficiency Syndrome Anti-HBc - Antibodies to Hepatitis B core Antigen Anti-HBs - Antibodies to Hepatitis B surface Antigen ART - Antiretroviral Therapy ARV - Antiretroviral CDC - Centers for Disease Control and Prevention CHPS - Community-Based Health Planning and Service CI - Confidence Interval EDTA - Ethylene Diamine Tetra-Acetic Acid EID - Early Infant Diagnosis ELISA - Enzyme Linked Immunosorbent Assay ESLD - End-Stage Liver Disease GAC - Ghana AIDS Commission GHS - Ghana Health Service HBeAg - Hepatitis B e Antigen HBsAg - Hepatitis B surface Antigen HBV - Hepatitis B virus HCC - Hepatocellular carcinoma HCV - Hepatitis C virus HIV - Human Immunodeficiency Virus HAART - Highly Active Antiretroviral Therapy University of Ghana http://ugspace.ug.edu.gh xiii HSS - HIV Sentinel Surveillance IDU - Injection Drug Users MoH - Ministry of Health MSM - Men who have sex with men NACP - National AIDS/STI Control Programme NMIMR - Noguchi Memorial Institute for Medical Research OR - Odds Ratio PCR - Polymerase Chain Reaction PPS - Probability Proportional to Size RS - Random Start SD - Standard Deviation SI - Sampling Interval UGMS - University of Ghana Medical School UNAIDS - Joint United Nations Programme on HIV and AIDS University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION 1.1 Background Globally, hepatitis B virus (HBV) and HIV infections present major public health challenges. They can both result in chronic disease, cancer and death and neither of them can be eradicated with the use of current therapies (Kourtis et al., 2012). Due to similar routes of transmission, co-infections with HBV and HIV are relatively common and pose increased risk for life threatening complications for people living with both infections (Ward, 2011). HBV prevalence has been established to be higher in HIV-infected individuals than in the general population (WHO, 2004; Burnett et al., 2005; Thomson and Main, 2008) and in some regions of the world, one of every three persons living with HIV (PLHIV) has HBV or hepatitis C virus markers, or both (Fabris et al., 2008). Sub-Saharan Africa has only 12% of the global population and yet it was reported in 2010 that about 68% (approximately 22.5 million) of all people living with HIV reside in the region as well as an estimated 50 million chronic HBV carriers (UNAIDS, 2010; Modi and Feld, 2007). In Ghana, the HIV epidemic is generalized with year on year prevalence rates consistently above 1% (HSS, 2011) and the prevalence of HBV infection is estimated to be 15% of the adult population (NACP, 2010). University of Ghana http://ugspace.ug.edu.gh 2 Due to the current use of highly active antiretroviral therapy (HAART), people living with HIV (PLHIV) are living longer and AIDS-related deaths are dramatically declining, especially in countries with longer treatment experiences (Palella et al., 2006). Consequently, liver diseases associated with HBV, have emerged as a major cause of morbidity and mortality in PLHIV (Mocroft et al., 2002; Konopnicki et al., 2005; Lewden et al., 2008). Although most individuals with only chronic hepatitis B do not develop hepatic complications; about 25 to 30% develop serious HBV-related disease during their lifetime, including liver failure and hepatocellular carcinoma (HCC) (Soriano et al., 2005). The presence of HIV infection negatively impacts the natural history of hepatitis B leading to increased rates of persistent infection, loss of protective antibody against HBV, increased liver-related mortality, and increased risk of liver cancer at lower CD4 counts among others (Thio et al.,2002; Hoffman et al., 2009). Again, HAART regimen including nevirapine, efavirenz and ritonavir used to treat HIV are toxic to the liver, which may already be damaged from hepatitis B infection (Sulkowski et al., 2002). Accurately diagnosing and assessing the state of HBV infection in the HIV-infected individual is critical because therapeutic management is dependent on the correct diagnosis and staging (Thio, 2009). Worldwide, guidelines for antiretroviral therapy (ART) including that of the Ghana Health Service therefore recommend the screening of HIV-infected individuals for HBsAg (NACP, 2011). Hepatitis B surface Antigen (HBsAg) is one of the first serological markers to appear during the course of HBV University of Ghana http://ugspace.ug.edu.gh 3 infection and regarded as a key marker for screening and laboratory diagnosis of HBV infection (Scheiblauer, 2010). It is easy to assume generally that health care workers (HCW) by virtue of their profession and proximity to the health facility should have adequate knowledge about diseases and other health conditions (Samuel et al, 2009), however current studies of provider knowledge, have reported the lack of awareness about chronic viral hepatitis B. Subjects of deficient knowledge include many factors among which are proper methods of testing and interpretation of test results, knowledge of hepatitis B serologic „markers‟ and proper follow up management of chronic infection (Anonymous, 2010). 1.2 Problem Statement There have been a number of studies that have examined the epidemiology of both HIV and HBV in various countries in sub-Saharan Africa, where both epidemics have hit so hard, but relatively few have studied HIV and HBV co-infection, and the effect that the increased prevalence of HIV may have on the epidemiology of HBV (Burnett et al., 2005). Dual infections may be even more problematic in regions of the world with a high or intermediate prevalence of HBV infection. HBV infection is highly endemic in Ghana, whereas the HIV epidemic in the country is generalized (WHO definition for a generalized epidemic is when the prevalence is 1% or greater in the general population) with some sites having rates above 5%. University of Ghana http://ugspace.ug.edu.gh 4 HAART has been introduced into Ghana with a subsequent reduction in the morbidity and mortality in PLHIV. The ART guidelines estimates that liver disease from HBV co- infection will become the most common non-AIDS related cause of death among people living with HIV. Considering the global impact of the co-infection, the guidelines for antiretroviral therapy in Ghana was reviewed and published in September 2011 to include for the first time the management of hepatitis B virus co-infection with HIV despite the clear deficit of information in Ghana to accurately establish the prevalence of hepatitis B in the HIV population. 1.3 Justification To the best of our knowledge, there were no documented studies examining HBV-HIV co-infection rates among PLHIV seeking care and treatment in ART clinics in primary care settings in rural and urban areas in Ghana at the time of the study. This study therefore provided baseline data to allow for an estimation of the extent of HBV-HIV co- infection problem in the Eastern region as well as serve as a reference for prevalence studies on the co-infection in Ghana. It also provided more information regarding differences in prevalence between various groups of patients including rural and urban populations, males and females, children and adults and will be useful for planning of preventive measures and development of hepatitis B vaccination programmes for HIV and AIDS patients in the future. University of Ghana http://ugspace.ug.edu.gh 5 Knowledge and level of awareness of ART staff with regards to the HBV-HIV co- infection was also assessed to help the Ghana Health Service to address needs gaps and training modules for health care workers. 1.4 Main Objective This study sought to assess the burden of HBV and HIV co-infection in clinics providing Antiretroviral Therapy (ART) services in the Eastern region of Ghana. 1.4.1 Specific Objectives 1. To determine the prevalence of HBV (assessed as HBsAg) among Persons Living with HIV and AIDS (PLHIV) in selected ART clinics in the Eastern Region. 2. To determine risk factors of HBV among PLHIV in the selected ART clinics. 3. To assess the knowledge of staff of ART clinics regarding HBV-HIV co-infection. University of Ghana http://ugspace.ug.edu.gh 6 CHAPTER TWO LITERATURE REVIEW 2.1 Epidemiology of HBV-HIV co-infection The Joint United Nations Programme on HIV and AIDS (UNAIDS) in their 2001 epidemic update described AIDS as “the most devastating disease humankind has ever faced”. At the end of 2010, the number of people globally living with HIV and AIDS was 34 million with 2.7 million new infections and 1.8 million AIDS-related deaths in that year alone (UNAIDS, 2011). Clearly, sub-Saharan Africa has been hit hardest by the HIV epidemic resulting in unprecedented impact in all aspects of human life. The World Health Organization (WHO) in 2012 reported that more than 2 billion people alive today have been infected with HBV at some time in their lives and that a third of the world‟s population are living in areas where there are high levels of infection. Arbuthnot and Kew in 2001 estimated that 387 million people (5% of the global population) were chronic carriers of HBV with sub-Saharan Africa estimated to have approximately 50 million of this number (Kotzee et al., 2006). Co-infections of HBV and HIV are common due to similar routes of transmission. The prevalence of HBV varies significantly among different HIV-infected populations with modes of transmission usually characterized by the geographical origin of infected patients. In areas of low HBV prevalence (less than 2% HBsAg-positive), such as North University of Ghana http://ugspace.ug.edu.gh 7 America and Western Europe, injecting drug use (IDU) and unprotected sex are the primary modes of transmission, and affect essentially the adult population. The result is a prevalence of co-infection of between 5% and 10%, 10 times higher than that of the general population. On the contrary, in Africa and Asia, where HBV endemicity is high (greater than 8% and often approaching 15% HBsAg-positive), most HBV infections occur within the 5 years of life through perinatal transmission, as in Asia or close contact within households and medical or cultural procedures, such as scarification and tattoos as in Africa (Adewole et al., 2009). In such countries with intermediate and high HBV endemicity, HIV-HBV co-infection rates are 10-20% (Diop-Ndiaye, 2008; Lee, 2008; Nyirenda, 2008). 2.2 Prevalence of HBV-HIV co-infection in sub-Saharan Africa Though Africa makes up 12% of the global population, 68% of persons living with HIV reside in sub-Saharan Africa (UNAIDS, 2011). UNAIDS‟s 2002 report clearly showed that southern African countries were experiencing the worst of the pandemic with adult prevalence rates ranging from 38.8% (Botswana) to 31% (Lesotho), whereas central and west African countries were still in the early stages of the HIV epidemic, with prevalence rates ranging from less than 1% (Senegal) to 12.9% in the Central African Republic. Though the basic epidemiology of HBV transmission in sub-Saharan Africa is poorly understood, current studies conducted in the region in the last three decades have clearly shown HBV infection to be highly endemic and a major public health problem in the region where the major route of HBV transmission is horizontal, that is, transmission University of Ghana http://ugspace.ug.edu.gh 8 unrelated to recognized sexual, perinatal, or parenteral exposure. A vast majority of the population is exposed to HBV by the age of 5 years and the prevalence rate slightly increases thereafter through close contact within households, medical procedures, traditional scarification, when children first attend school, and again when they become sexually active. A large proportion of these children become chronic carriers of HBV, and in adulthood, transmits HBV in exactly the same manner as HIV (Vardas et al., 1999; Burnett et al., 2005; Kotzee et al., 2006). Previous studies conducted in the 1980s and early 1990s found that vertical (in utero) and perinatal HBV transmission did not play a major role in the region, because of the relatively low carriage (between 0% to 18.6%) of hepatitis B e antigen (HBeAg) in pregnant HBV carriers in sub-Sahara Africa. However, a study in South Africa by Vardas and others in 1999 suggests that transmission in early infancy may be on the increase and that maternal HBV antibodies are not being passively transferred to the same extent as in the past. Early epidemiological studies did not show an increased prevalence of HBV among HIV- positive individuals because they were conducted when HIV prevalence were limited by small sample sizes. The prevalence of HBV in HIV-infected adults in sub-Saharan Africa was found to be nearly two times that of HIV-negative patients (Kotzee et al., 2006). Current studies across sub-Saharan Africa countries however have provided a wide range of prevalence estimates and demonstrate the need for ongoing surveillance activities (Kapembwa et al., 2011). University of Ghana http://ugspace.ug.edu.gh 9 In their review, Kapembwa and colleagues in 2011 estimated HBsAg seropositivity in 9.9% of HIV-infected adults in Zambia and cited a number of studies in various parts of Africa with varying HBV-HIV prevalence rates (Table 1). In Ghana, a HBV-HIV co- infection prevalence of 16.7% (140/839) was found among a Ghanaian HIV-positive cohort hospitalized at a tertiary care institution in Kumasi (Geretti et al., 2010). However, though Ghana like most African countries is affected by both HIV and hepatitis B infections, there is a deficit of information on hepatitis B co-infection in people living with HIV infection. Table 1: Surveys of Hepatitis B Virus surface Antigen (HBsAg) Prevalence in HIV- infected Persons in sub-Saharan Africa, 1996-2011 Country Author Publication Year Population Sample size Prevalence of HBsAg (%) Botswana Wester et al. 2006 Outpatient 160 10.6 Cote d‟Ivoire Rouet et al. 2004 Antenatal 501 9.0 Ethiopia Shimelis et al. 2008 Outpatient 305 3.9 Kenya Harania et al. 2008 Outpatient 378 6.1 Malawi Ahmed et al. 1998 Antenatal 50 13.0 Malawi Nyirenda et al. 2008 Inpatient 226 17.5 Malawi Moore et al. 2010 Outpatient 300 6.7 Nigeria Otegbayo et al. 2008 Outpatient 1779 11.9 Rwanda Pirillo et al. 2007 Antenatal 82 2.4 Senegal Diop-Ndiaye et al. 2008 Outpatient 363 16.8 South Africa Fimhaber et al. 2008 Outpatient 502 4.8 South Africa Hoffman et al. 2008 Outpatient 537 19.7 South Africa Lukhwareni et al. 2009 Outpatient 192 22.9 South Africa Di Bisceglie et al. 2010 Outpatient 502 4.8 Tanzania Nagu et al. 2008 Outpatient 260 17.3 Uganda Pirillo et al. 2007 Antenatal 164 4.9 Zambia Oshitani et al. 1996 Antenatal 340 7.1 Zambia Kasolo et al. 2003 Inpatient 214 31.3 Zambia Kapembwa et al. 2011 Outpatient 323 9.9 Source: Kapembwa et al., (2011) University of Ghana http://ugspace.ug.edu.gh 10 Prevalence rates of HBV infection in rural and urban populations have shown marked differences. Kew (1999) reported HBV chronic carriers in South Africa‟s rural former Transkei (Eastern Cape) to be 15.5%, while that in urban areas, Durban was 7.4% and Soweto was 1.3%. 2.3 The status of HIV epidemic in Ghana Since the first case of AIDS was reported in Ghana in 1986, there has been a rise in the number of cases. By the end of 2011, the HIV prevalence among antenatal clients ranged from 0.0% in Adibo, a rural site to 9.6% in Cape Coast, an urban site (NACP, 2011). The HIV epidemic in Ghana continues to be a generalized epidemic with a prevalence of more than 1% in the general population. The HIV prevalence in Ghana varies with geographic areas, gender, age and residence. In 2011, the Central region of Ghana for the first time reported the highest HIV prevalence of 4.7% followed by the Eastern region with 3.6%. 2.4 The status of Hepatitis B in Ghana Hepatitis B is a major public health concern in Ghana. There has never been a nationwide seroprevalence survey for hepatitis B in Ghana and clearly there are no policies and guidelines to streamline its testing among the general population according to the newly created Viral Hepatitis Control Unit of the Ghana Health Service. Prevalence studies in Ghana have been done in a limited number of districts and mainly among blood donors, which has a biased age group. Based on these studies, the hepatitis B prevalence has been University of Ghana http://ugspace.ug.edu.gh 11 estimated to be approximately 30% among blood donors and chronic hepatitis B among the general population is estimated at ≥8% (GHS, 2011).Hepatitis B vaccines were introduced into the country in 2002 to protect children. It is included in the pentavalent vaccine protecting against Haemophilus influenza type B, diphtheria, pertussis, tetanus and hepatitis B virus (GHS, 2002). 2.5 The natural history of HBV disease in HIV co-infected patients There is a body of evidence to show that that the natural history of both HIV and hepatitis B infection appear to be influenced by the other, but depends on which infection was acquired first. The common scenario in sub-Saharan Africa is where children are first infected with HBV and subsequently infected with HIV as adults. When this happens, a number of studies have established that HIV-positive patients with previous cleared HBV infections may lose their protective HBV antibodies due to HIV immunosuppression, and may therefore be at an increased risk of re-infection with HBV. In addition HIV-induced immunosuppression may cause reactivation of recovered (previously anti-HBs positive) as well as „silent‟ chronic infections (Kotzee et al., 2006) 2.6 Impact of HIV on Hepatitis B HIV has a significant impact on the natural history of HBV infection. The presence of HIV prior to HBV infection increases the risk of developing chronic HBV and prolonged alanine aminotransferase (ALT) elevation (Hadler, 1991). HBV-HIV co-infection reduces the rate of spontaneous HBeAg and HBsAg seroconversion, leading to a higher prevalence of HBeAg-positive disease (Thio, 2003). There is also an association between University of Ghana http://ugspace.ug.edu.gh 12 HIV and reactivation of HBV and elevated HBV DNA levels, although serum ALT elevations are milder compared with HBV mono-infected individuals (Colin, 1999; Thio, 2003). Despite this, liver damage progresses more rapidly, condensing the period from HBV acquisition to cirrhosis in individuals with HBV-HIV co-infection (Thio, 2003). Co-infected patients have a poorer response to interferon therapy for hepatitis B. Patients with HBV-HIV co-infection have an increased risk of liver-related complications and death. Since the introduction of HAART in 1996, a major reduction in the incidence of opportunistic infections has led to the emergence of liver disease as one of the leading causes of death in patients with HIV (Thio et al., 2002). Liver-related mortality was more common than death from cardiovascular diseases (9.4%) and non-AIDS malignancies (11%). Active HBV was an independent predictor of liver-related death, with an adjusted relative risk of 3.73 (Oren et al., 2007). 2.7 Impact of Hepatitis B on HIV In contrast, there is no clear evidence that HBV impacts on HIV disease progression. In a cohort of 80 men who have sex with men (MSM), a more rapid progression to AIDS was shown in 32 patients carrying antibodies to the core antigen (Eskild et al., 1992). A theoretical effect of HBV on HIV transcription that might enhance HIV replication and lead to a more rapid reduction in CD4 cell counts in HBV-HIV co-infected patients has been described but there is little evidence to substantiate this (Soriano et al., 2005). Recent studies have shown no convincing evidence that HBV speeds progression to AIDS (Kotzee and colleagues in 2006). They recommended the need for more research University of Ghana http://ugspace.ug.edu.gh 13 documenting progression in endemic African populations since almost all previous studies were conducted in developed countries which differ significantly in populations. Contrary to this assertion, Nikolopoulo and colleagues (2009) found that HBV coinfection increases overall mortality among HIV-positive patients by their meta- analysis of existing publications and reiterated the significance of a comprehensive management of HIV-positive individuals that includes prevention, regular screening for HBV infection, and administration of potent anti-HBV therapy to co-infected patients according to current guidelines. 2.8 Serological Markers of HBV Infection Due to the complexity of HBV diagnosis and test interpretation, it is important to also test for the markers hepatitis B e Antigen (HBeAg), hepatitis B core antibodies (HBcAb) and hepatitis B surface antibodies (HBsAb) besides HBsAg to establish the clinical significance of the infection. HBsAg is usually detectable between week four and week 10 in acute infection. Chronic HBV infection is defined by the persistence of HBsAg for more than six months. Antibody to surface antigen (HBsAb) is a protective antibody that develops with the resolution of acute infection or following successful vaccination against HBV. Antibody to core antigen (HBcAb) is not found as a discrete protein in the serum, however, unlike HBsAb, HBcAb is not a protective antibody. HBcAb remains positive for life following exposure to HBV and is good measure for previous HBV infection. Hepatitis B e antigen (HBeAg) is produced during active viral replication and may act as an immunogen or a tolerogen, leading to persistent infection. Antibody to University of Ghana http://ugspace.ug.edu.gh 14 hepatitis e antigen (HBeAb) is not a protective antibody but it is used as an end-point for treatment. 2.9 Knowledge of health care workers (HCWs) regarding HBV-HIV co-infection According to several researchers, individuals co-infected with HIV and HBV face a higher risk of liver-related death than those who are infected with only HIV (Highleyman, 2002). In order to provide adequate treatments to patients infected with HBV or HBV-HIV, knowledge of the effect of various treatments modalities on each virus as well as of the complex interactions between these viruses is key to the proper and effective management of patients. In many developed and developing countries, however, it has been reported that there is relatively poor awareness about hepatitis B infection and HBV-HIV co-infection among health care providers. In the United States, for example, provider guidelines for hepatitis screening, prevention, treatment and follow up have been in place for decades and are upgraded regularly (CDC, 2005). Current studies of provider knowledge about chronic viral hepatitis B, however, have reported the lack of awareness about prevalence of chronic viral hepatitis B, proper methods and target population for screening. In addition, HCWs and their patients are at risk for exposure to infected blood and body fluids and so far several outbreaks of hepatitis B in health care setting have been reported in recent years in the United States (Fabrizi et al., 2008; CDC 2008 and 2009). This situation is also similar in developing countries where many HCWs have inadequate knowledge of University of Ghana http://ugspace.ug.edu.gh 15 hepatitis B infection and poor safe practices to prevent its transmission among them and their patients (Samuel et al., 2009). In China, for example, the highly educated health providers and public health professionals, especially those in rural areas still have a deficient HBV knowledge (Chao et al., 2010). The Hepatitis Control Unit of Ghana in a 2012 annual review meeting shared findings of a survey and alluded to the limited knowledge of hepatitis generally among health staff and the general population (DSD/EPI Annual Review, 2012). On the contrary, there is an observed high awareness of HIV among HCW in Ghana and this may be probably due to the continuous HIV training workshops by the NACP for different categories of HCW. The lack of education, misconception and lack of equipment have been identified as the major barriers that explain the high prevalence rate of HBV among HCWs and their patients (Lee, 2009). It is quite clear that lack of knowledge among HCWs which are regarded as key players in the prevention and management of hepatitis B infection and HBV-HIV co-infection will give rise to dramatic consequences, some of which include: continuing transmission of the infection, missing of opportunities for prevention of the disease, early diagnosis and medical care and poor health outcomes in infected people. It is important therefore that providers in different countries work with key stakeholders to develop hepatitis B as well as HBV-HIV co-infection education programmes for health workers and social service providers. University of Ghana http://ugspace.ug.edu.gh 16 CHAPTER THREE METHODS 3.1 Study Design The study was a cross-sectional study; using quantitative survey methods complemented by in-depth interviews of Persons Living with HIV and AIDS (PLHIV) and Health Care Workers regarding hepatitis B and HIV co-infection. 3.2 Study Setting The Eastern region was purposively selected because it had consistently reported the highest HIV prevalence rates for both urban and rural sites in the HIV Sentinel Surveillance (HSS) from 2003 to 2010 (6.1 % in 2003, 6.5 % in 2004, 4.7 % in 2005, 4.9 % in 2006, 4.2 % in 2007 to 2009 and 3.2 % in 2010), though there are also areas in the region with low HIV prevalence rates. It is the sixth largest region in Ghana with a projected population of 2,420,927 and growth rate of 1.4% (Census Bureau, 2010). The region has 30 hospitals, 58 health centres, 126 clinics and 315 functional Community- Based Health Planning and Service (CHPS). Out of the 30 hospitals, 20 have well established antiretroviral (ART) clinics (Appendix I) with trained health care workers. Five ART clinics, three rural and two urban in the region were selected for the study (Figure 1). University of Ghana http://ugspace.ug.edu.gh 17 Figure 1: Map of Eastern Region of Ghana showing ART Study Sites 3.3 Study Population Persons Living with HIV and AIDS (PLHIV) seeking care and treatment at selected Antiretroviral therapy (ART) sites in the Eastern region and health care workers including HIV counsellors, nurses, physicians, laboratory technicians, and pharmacists manning the same clinics constituted the study population. University of Ghana http://ugspace.ug.edu.gh 18 3.4 Variables 3.4.1 Dependent Variables:  Presence or absence of HBsAg in PLHIV  Specificity and sensitivity of hepatitis B rapid test kits  Knowledge levels of staff of ART clinics with regards to HBV-HIV co-infection. 3.4.2 Independent Variables:  Socio-demographic characteristics of PLHIV: age, sex, educational level, occupation, marital status, income level, ethnicity and community of residence.  PLHIV medical data (CD4+ T-lymphocyte count, ALT and Aspartate aminotransferase (AST) levels, haemoglobin concentration) and their knowledge of hepatitis B virus infection and its predisposing factors  Profession, sex, number of years working experience and age of health care worker at antiretroviral clinic. 3.5 Sampling Methods 3.5.1 Sample Size Calculation Geretti and colleagues in 2010 estimated a HBV-HIV co-infection prevalence of 16.7% (~17%) in a cohort of PLHIV at a tertiary hospital in Ghana. This prevalence was approximated to 17% (0.17) for the sample size calculation using the Utilities function in Epi-Info version 3.5.1 based on the formula: n = z2pq d2 University of Ghana http://ugspace.ug.edu.gh 19 where n = sample size, z = the standard normal deviation, set at 1.96 corresponding to the 95% CI (risk of type 1 error), p= expected prevalence in the target population, q=1-p and d= absolute precision Adjusting for a non-response rate of 15%; where there could be 5% drop out rate and a 10% inadequate quantity and/or contamination rate of blood serum resulted in 249.5, which was approximated to a minimum of 300 study participants. 3.5.2 Study Sites: The study sites were sampled based on the probability proportional to size (PPS) sampling technique in order to ensure that all individuals in the HIV population in the Eastern region had the same probability of selection irrespective of the size of their cluster. ART clinics in the Eastern region with their respective population sizes were listed in alphabetical order (Appendix I) and the cumulative sum of their population sizes was calculated to give a total ART population of 20043. The sampling interval (SI) of 4009 was calculated by dividing the total population by the number of study sites (5). A random start (RS) of 1000 population size was then chosen between 1 and the SI. The first site, Asesewa Government Hospital was contained in this cumulative population of 1000, and selected for sampling. Selections of the other sites were based on the calculations of the series as follows: RS = 1000; RS + (1xSI) = 5009; RS + (2xSI) = 9017; RS + (3xSI) = 13026 and lastly RS + (4xSI) = 17034, where RS = random start (705) and SI = sampling interval (4009) University of Ghana http://ugspace.ug.edu.gh 20 Consequently, the four other sites were selected based on the cumulative population contained in any of the serial numbers calculated above. However, the last hospital in the series, St. Martin‟s Hospital was dropped and replaced with District Hospital, Begoro because it happens to be in the same area as the second ART site, Atua Government Hospital. Thus, the selected sites were Atua Government Hospital, District Hospital in Begoro, both rural sites and Koforidua Regional Hospital and St. Dominic‟s Hospital in Akwatia, two urban sites. 3.5.3 Persons Living with HIV and AIDS (PLHIV): At each site, numbers were written on pieces of paper, folded and placed in a box for one to be randomly picked by a member of the ART team to obtain a sampling interval. Based on this number, participants were recruited to make up for the sample size of 60 for each site. The interview was conducted in a counselling room by an HIV counsellor to collect data on the socio-demographic characteristics and possible risk factors including intravenous drug use, hepatitis B vaccination record, history of blood transfusion and number of sexual partners (Appendix II). Questions that were inappropriate for children were not included in their questionnaire (Appendix III). 3.5.4 Health care workers in ART clinics: Health care workers in ART clinics at each site that were available during the period of study were purposively selected to self-administer a questionnaire (Appendix IV). The questions were derived from the Guidelines for ART in Ghana as published in September 2011 by NACP. The questionnaire was divided into two subsections; i.e HBV and HIV University of Ghana http://ugspace.ug.edu.gh 21 general knowledge and HBV-HIV management practices. The HCW were made up of clinicians, HIV counsellors, laboratory scientists, pharmacy staffs and PLHIV volunteers. Eleven questions in each section were then used to compile a “general knowledge” index and “management practice” index respectively. The responses were [Yes], [No] and [Don‟t know]. The maximum possible score for each item was 100% and the minimum possible score was 0%. Each index was then calculated by finding the average of the total sum of correct percentages for each question. Knowledge in each index was graded as follows: Poor Knowledge (general knowledge/management practice) = 0%-50% Fair Knowledge (general knowledge/management practice) = 51%-70% Good Knowledge (general knowledge/management practice) = 71%-100% 3.6 Inclusion criteria: Any ART client reporting to any of the selected clinics was eligible to participate irrespective of previous testing for hepatitis B. Informed consent was obtained from each eligible client and/or parent of children. 3.7 Exclusion criteria: Failure to obtain informed consent, children less than 18 months old who had not tested for HIV (PCR DNA) and persons who were too ill to understand the procedures involved in the study were excluded from the study. University of Ghana http://ugspace.ug.edu.gh 22 3.8 Specimen and Data Collection Techniques and Tools Each consenting participant was interviewed by a counsellor trained for the purpose of the study using a structured questionnaire. Blood specimen (8 ml for adults and 5ml for children) was collected into a specimen tube and centrifuged to separate the serum from the cells. To avoid haemolysis, the serum was separated from cells immediately after centrifugation and discharged into two well-labelled cryovials bearing unique identification, sex and age of participant. Grossly haemolytic, lipidic or turbid samples were discarded. The specimen were then kept frozen at the study sites for not more than two weeks and transported under cold chain (vaccine carrier with ice packs) to the Koforidua Regional Hospital to be kept under –20 °C. The specimen from all sites were then transported under cold chain (2–80C) to the NPHRL and Virology Department of the University of Ghana Medical School (UGMS) for serological investigations. A laboratory check list (Appendix II) was attached to each questionnaire and used to collect specimen data from participants. 3.9 HBV (HBsAg ELISA) Laboratory Procedure Baseline participants‟ samples (serum) were first of all tested for HBsAg by commercially available ELISA assay; HBsAg (Foresight®, Acon Laboratories, Inc. USA). Samples that were HBsAg positive were followed up with testing for hepatitis B serologic markers, HBcAb, HBeAb and HBeAg using ELISA kits (Foresight, Acon Laboratories, Inc., USA). University of Ghana http://ugspace.ug.edu.gh 23 The frozen sera were completely thawed and well mixed prior to testing. The ELISA reagent was also allowed to reach room temperature prior to testing. The materials that were provided in the kit were Microwell Plates, Conjugate, Concentrated Wash Buffer, Substrate A (citrate-phosphate buffer containing hydrogen peroxide), Substrate B (tetramethylbenzidine), Stop Solution (0.5M Sulfuric acid), Negative Controls, Positive Control and Plate Sealers. Other materials required but not provided by manufacturer were freshly distilled water, sodium hypochlorite solution, absorbent paper, incubator, calibrated automatic microwell plate washer, disposable gloves, calibrated micropipettes with disposable tips, timer, vortex mixer, calibrated microplate reader. These items were either prepared or provided for by the virology laboratory or bought from the open market. In accordance with the manufacturer's instructions, the procedure for testing was strictly followed. A working wash buffer was prepared by pouring the concentrated wash buffer in a graduated cylinder and filling it with freshly distilled water to 1000ml. Well A1 was left as the blank well after which 100µl of Negative control was added to wells B1 and C1 followed by 100µl of Positive control in wells D1 and E1. 100µl of each participant‟s specimen were assigned wells starting at F1 to H12. 50µl of Conjugate was added to each well except for the blank. The contents of the microwell plate were then mixed gently by swirling on the flat bench for 30 seconds, after which the plate was covered with a plate sealer and incubated at 37.5oC for 120 minutes. After incubation, the plate sealer was removed and each well was washed five times with 350µl of working wash buffer per well. The buffer was removed by turning the microwell plate upside down on absorbent tissue for 10 seconds. It was ensured that all the wells had been University of Ghana http://ugspace.ug.edu.gh 24 completely washed and dried. 50µl of substrate A was then added to each well followed by 50µl of substrate B to develop a blue colour in wells containing reactive specimen while the non-reactive specimen remained clear. The contents of the microwell plate were mixed gently again by swirling and the plate covered with a plate sealer for incubation at 37.5oC for 30 minutes. The plate sealer was removed and 50µl of stop solution was added to each well. A yellow colour was developed in wells containing reactive specimen (Fig. 2) which was read at 450 nm with a spectrophotometer. Figure 2: Microwell Plate showing HBV ELISA Results (Source: Virology Lab; UGMS) 3.9.1 Principle of the Foresight HBsAg ELISA kit assay The Foresight HBsAg ELISA Kit is a solid phase enzyme immunoassay based on a sandwich principle for the in vitro detection of Hepatitis B surface antigen (HBsAg) including the subclass of HBsAg (ad, ay.) in human serum. The microwell plates were pre-coated with monoclonal antibodies specific for HBsAg. During the course of the assay, the positive control, negative control and samples were added to the microplate wells and incubated at 37.50C. The microtiter plate wells were then thoroughly washed to University of Ghana http://ugspace.ug.edu.gh 25 remove other unbound components of the sample. A standardized preparation of horseradish peroxidase (HRP) conjugated antibody specific for HBsAg (substrate A) was added to each well to “sandwich” the antibodies immobilized during the first incubation. After washing again to remove any unbound HRP conjugate, a TMB (3.3‟, 5,5' tetramethyl-benzidine) substrate solution (substrate B) was added to each well to produce a blue colour, indicating the amount of HBsAg present in the specimen. The enzyme (HRP) and substrate were then allowed to react over a 10-minute incubation period. The enzyme-substrate reaction was then terminated by the addition of sulfuric acid solution and the colour change from blue to yellow was measured spectrophotemetrically at a wavelength of 450nm. Wells containing HBsAg and HRP conjugate exhibited a change in color. The intensity of this color change was proportional to the concentration of HBsAg in the specimen. 3.9.2 Validation and Quality Control of ELISA Testing As shown in Table 2, the mean absorbance of negative control was then calculated. To ascertain the validity of the test results obtained, it was ensured that: 1. The blank well had absorbance of less than 0.050 when read at 450 nm 2. The mean absorbance after subtraction of blank absorbance was less than 0.100 for the negative control 3. The mean absorbance after subtraction of blank absorbance was greater than 1.000 for the positive control. University of Ghana http://ugspace.ug.edu.gh 26 Table 2: Negative Control Calculation Item Absorbance Negative Control: Well B1 0.023 Negative Control: Well C1 0.021 Total Absorbance of Negative Control 0.023 + 0.021 = 0.044 Mean Absorbance of Negative Control 0.044/2= 0.022 Blank Absorbance: Well A1 0.002 NCx*: Mean Absorbance of Negative Control – Blank Absorbance 0.022 – 0.002 = 0.020 Participants‟ specimen with absorbance less than the cut-off value were non-reactive and considered negative for HBsAg. Specimen with absorbance greater than or equal to the cut-off value was considered initially reactive for HBsAg. Reactive specimens were retested with First Response HBsAg rapid test kit for confirmation before final interpretation. Specimens that were reactive in at least one of the re-tests were presumed to be repeatedly reactive and were confirmed by further screening for additional hepatitis B infection serological markers to aid in determining the clinical significance of infection. Specimen with values within ± 10% of the cut-off value were retested in duplicates for final interpretation. The results were read and recorded by two independent laboratory scientists under the supervision of the technical head (Appendix V). University of Ghana http://ugspace.ug.edu.gh 27 3.10 Data Quality Control Measures 3.10.1 Pretesting of questionnaires The questionnaires were pretested by the principal investigator and the research assistants at the ART clinic of Nsawam Government Hospital to ensure that participants understood and interpreted the questions in the same way and it did not take too long to complete. The questionnaires were also tested on the consistency of questions and whether they yielded useful data. Some questions were revised and others dropped after the pretesting. 3.10.2 Training of HIV counsellors and laboratory personnel Two counsellors and two laboratory personnel at each study site were trained on-site on the sampling technique and administration of the study‟s questionnaire as well as generating unique identification numbers for each participant. 3.10.3 Double data capture The data was coded, entered and analysed twice to compare the results to exclude any differences. The results were found to be identical. 3.11 Laboratory Quality Control Measures Internal quality control measures that were employed to minimize variability of assay results included: 3.11.1 Running of control samples Standard controls contained in the reagent pack and internal control samples across the analytical range of the assay (low, medium and high controls) and negative controls in University of Ghana http://ugspace.ug.edu.gh 28 previous assays were obtained from the virology laboratory and included in specimen assays. 3.11.2 Replication of Assay To validate the test results, 32 (10%) samples were picked at random and ran alongside other assays in the virology laboratory and compared with their initial results. 3.11.3 Test of storage conditions Temperature checks were periodically done by laboratory personnel and recorded on a chart pasted on the fridges and freezers. The temperature was monitored regularly during supervisory visits by the principal investigator and research assistants. 3.11.4 Supervision of laboratory testing Assays were conducted by the principal investigator with the assistance of two research assistants under supervision from the technical heads of the NPHRL and the Virology Department of the UGMS to address quality control measures such as calibration of pipettes, specimen collection and aliquot, incubation at correct temperatures and correct calculation of cut-off values. 3.12 Data Processing and Analysis Data was entered into Epi Data version 3.1 software (EpiData Corp. Odense, Denmark) and exported into SPSS version 16 software package for analysis. Univariate analysis was expressed as frequencies and percentages for the various categorical variables and data described in person, place and time. Secondly, bivariate analyses were conducted to University of Ghana http://ugspace.ug.edu.gh 29 assess the associations between HBsAg serological status and study sites and socio- demographic characteristics of study participants. Pearson‟s chi-square test was used to determine statistically significant differences in prevalence between the different study sites, between the sexes, and between the different age groups. Finally, Logistic regression model was used to analyse predisposing factors associated with HBV infection A p value <0·05 was considered significant. HBV-HIV knowledge and management practice indexes were calculated. 3.13 Ethical Considerations Ethical approval to undertake the research was sought from the Research and Ethical Review Committee of the Ghana Health Service (Ethical Clearance ID No: GHS-ERC 15/01/12) (Appendix VI). Permission was sought and obtained from the management of the selected health facilities. Participants were fully informed about the purpose, procedures, risks and benefits of participating in this study. Those who agreed to participate signed or thumb printed the informed consent form (Appendix II). We also sought consent from the parents or guardians of participants who were below 18 years and the assent of children aged 7-17 years before recruitment into study (Appendix III). To ensure privacy and confidentiality, HIV counsellors in the study sites known by the participants were trained to conduct interviews in HIV counselling rooms. Participants benefitted from free ELISA screening for HBsAg and its markers and those with infection were referred to clinicians at the ART sites for treatment. Each participant was assured that their responses would be kept confidential and data collected will be kept for the purpose of this study only. University of Ghana http://ugspace.ug.edu.gh 30 CHAPTER FOUR RESULTS 4.1 General Overview The results are based on interviews and testing for the presence of HBV (assessed as HBsAg) in blood specimen obtained from Persons Living with HIV and AIDS (PLHIV) who attended five ART clinics in the Eastern region of Ghana from March to June 2012. Health care workers (54) in the same ART clinics also answered questions pertaining to their general knowledge of HBV infection and HBV-HIV co-infection management practices. The ART clinics are located in five towns in the Eastern region namely Akwatia, Asesewa, Atua, Begoro and Koforidua. 4.2 Socio-Demographic Characteristics of Study Participants (PLHIV=320) Table 3 shows the total number of participants for the study. Out of 320 PLHIV, 232 (72.5%) were females. The ages of participants ranged from 4 to 76 years with a median age of 40.0 years SD ±12.7. The mean ages of females and males were 39.17 years (SD ±11.7) and 43.9 years (SD ±14.3) respectively, and the difference was statistically significant (p=0.008). Most study participants, 119 (37.2%) and females, 100 (31.3%) were aged 31-40 years, though most males, 27 (8.4%) were aged 41-50 years. Adults (defined as ≥18years) were 307 (95.9%) and children (defined as <18years) were 13 (4.1%) as shown in Table 3. University of Ghana http://ugspace.ug.edu.gh 31 Table 3: Age and Sex Distribution of Study Participants, ART Clinics, Eastern Region, 2012 (n=320) Age Group (years) Male n (%) Female n (%) Total n (%) 1 – 10 4 (1.3) 2 (0.6) 6 (1.9) 11 – 20 3 (0.9) 6 (1.9) 9 (2.8) 21 – 30 4 (1.3) 36 (11.3) 40 (12.5) 31 – 40 19 (5.9) 100 (31.3) 119 (37.2) 41 – 50 27 (8.4) 50 (15.6) 77 (24.1) 51 – 60 25 (7.8) 27 (8.4) 52 (16.3) 61 – 70 3 (0.9) 8 (2.5) 11 (3.4) 71 – 80 3 (0.9) 3 (0.9) 6 (1.9) Age Category Adults (≥18yrs) 82 (25.6) 225 (70.3) 307 (95.9) Children (<18yrs) 6 (1.9) 7 (2.2) 13 (4.1) Total 88 (27.5) 232 (72.5) 320 (100.0) Most study participants were married, 141 (44.1%) and were engaged in trading or business, 132 (44.6%). Nearly half of the respondents, 155 (48.4%) belonged to the Krobo ethnic group and income levels was low among respondents with 193 (60.3%) earning less than GH¢100 per month. Most of them had not registered or renewed their health insurance and a significant number of participants, 197 (60.6%) paid for ART services and drugs out-of-pocket (Table 4). University of Ghana http://ugspace.ug.edu.gh 32 Table 4: Socio-Demographic Characteristics of Study Participants, Eastern Region, 2012 (n=320) Characteristic Number Percentage 95% Confidence Interval Ethnicity Akyem Krobo 7 others Missing values Total 44 155 106 15 320 13.8 48.4 33.1 4.7 100.0 1.82-1.90 1.46-1.57 1.61-1.72 Education None Primary Secondary Tertiary Missing values Total 76 149 74 6 15 320 23.8 46.6 23.1 1.9 4.7 100.0 1.71-1.80 1.47-1.58 1.72-1.81 1.96-1.99 Marital Status Single Married Separated Divorced Widowed Co-habiting Missing values Total 33 141 13 61 45 13 14 320 10.3 44.1 4.1 19.1 14.1 4.1 4.4 100.0 1.86-1.93 1.50-1.61 1.93-1.98 1.76-1.85 1.82-1.89 1.93-1.98 Occupation Trading/Business 8 others Missing values Total 132 164 24 320 41.3 51.3 7.5 100.0 1.53-1.64 1.43-1.54 Level of income < GH¢100 GH¢101 – 250 GH¢251 – 500 > GH¢500 Missing values Total 193 49 11 7 60 320 60.3 15.3 3.4 2.2 18.8 100.0 1.34-1.45 1.80-1.88 1.94-1.98 1.96-1.98 Payment of GH¢5.0 for ART services Out-of-pocket Employer Relatives NHIS PLHIV-group Others Missing values Total 194 37 1 63 10 3 12 320 60.6 11.6 0.3 19.7 3.1 0.9 3.8 100.0 1.33-1.44 1.92-1.97 1.99-2.00 1.75-1.84 1.94-1.98 1.98-2.00 University of Ghana http://ugspace.ug.edu.gh 33 4.3 Clinical Characteristics of Study Participants Clinical data, including CD4+ T-lymphocyte counts, haemoglobin concentration (HB), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was extracted from the folders of study participants (Table 5). Table 5: Clinical Characteristics of Study Participants and HBV Infection, Eastern Region, 2012 (n=320) Clinical Data n (%) 95% CI Mean count p-value CD4 count (n=191) <350 ≥350 119 (62.3) 72 (37.7) 409.7-525.2 467 cells/µl 0.283 Haemoglobin Conc. (n=226) <12g/dl ≥12g/dl 155 (68.6) 71 (31.4) 10.5-15.4 12.91 g/dl 0.964 ALT level (n=167) 9.5-39.2 U/L(females) < 9.5 U/L(females) >39.2U/L (females) 11.6-53.1U/L (males) <11.6U/L (males) >53.1U/L (males) 92 (55.1) 3 (1.8) 27 (16.2) 32 (19.2) 2 (1.2) 11 (6.6) 30.4-41.7 36.07 U/l 0.002 AST level (n= ) 15.5-46.5 U/L(females) < 15.5 U/L(females) >46.5U/L (females) 18.7-65.0U/L (males) <18.7U/L (males) >65.0U/L (males) 87 12 23 34 5 7 34.0-46.9 40.53 U/l 0.061 University of Ghana http://ugspace.ug.edu.gh 34 The mean counts for CD4 count, HB, ALT and AST were 467 cells/µl, 12.9 g/dl, 36.07 U/l and 40.53 U/l respectively. There was found to be a statistically significant association between increasing serum ALT level and HBsAg positivity (p=0.002) as shown in Table 5. 4.4 Laboratory Analysis 4.4.1 HBV Status (assessed as HBsAg ELISA) of Study Participants Of 320 study participants‟ sera that were tested by ELISA, 28 were positive for HBV, giving a HBV-HIV co-infection prevalence of 8.8%. Most of them, 14 (4.4%) were aged 31-40 years followed by participants aged 41-50 years, with 6 (1.9%) being HBV positive. There were however no HBV positives in the age groups 21-30, 61-70 and 71- 80 years respectively. Most HBV positive participants were adults, 24 (7.5%) and children were only 4 (1.3%). HBV infection was significantly associated with being an adult (p=0.004) (Table 6) Majority of the participants were drawn from rural sites (i.e. 58.75%). Of this number, 15 (53.6%) were HBsAg positive but the difference in the prevalence rates between rural and urban populations was not statistically significant (p=0.560) (Table 7). University of Ghana http://ugspace.ug.edu.gh 35 Table 6: Age Distribution of Study Participants and HBV Infection (HBsAg ELISA), Eastern Region, 2012 (n=320) Age Group (Years) No. of Participants n(%) HBsAg Positive n(%) HBsAg Negative n(%) p-value 1 – 10 6 (1.9) 1 (0.3) 5 (1.5) 0.401 11 – 20 9 (2.8) 3 (0.9) 7 (2.2) 21 – 30 40 (12.5) 0 (0.0) 40 (12.5) 31 – 40 119 (37.2) 14 (4.4) 105 (32.8) 41 – 50 77 (24.1) 6 (1.9) 70 (21.9) 51 – 60 52 (16.3) 4 (1.3) 48 (15.0) 61 – 70 11 (3.4) 0 (0.0) 11 (3.4) 71 – 80 6 (1.9) 0 (0.0) 6 (1.9) Age Category Adults (≥18yrs) 307 (95.9) 24 (7.5) 283 (88.4) 0.004 Children (<18yrs) 13 (4.1) 4 (1.3) 9 (2.8) Total 320 28 (8.8) 292 (91.2) Table 7: HBV Infection of Study Participants by Location of ART Site, Eastern Region, 2012 (n=320) Study Site Participants n (%) HBsAg Positive n(%) HBsAg Negative n(%) p-value Rural 188 (58.8) 15 (4.7) 173 (54.0) 0.560 Urban 132 (41.2) 13 (4.1) 119 (37.2) Total 320 (100) 28 (8.8) 292 (91.2) OR= 0.794 (95% CI=0.36-1.72) University of Ghana http://ugspace.ug.edu.gh 36 4.4 Distribution of HBsAg Positive Study Participants in ART Sites, Eastern region Figure 3 shows the prevalence of HBV-HIV co-infection in the different ART study sites. Asesewa Government Hospital reported the highest prevalence of 13.6% (8/59), followed by St. Dominic‟s Hospital with 11.4% (8/70) and Atua Government Hospital reporting the least. The median prevalence was 8.3% (95% CI: 0.095-0.41, Interquartile range=7.1- 11.4) Figure 3: HBV Prevalence in ART Study Sites, Eastern Region (n=320) 4.5 Hepatitis B Serologic “Markers” in HBV Positive Study Participants Of the 28 HBsAg positive participants, 26 (i.e 92.9%) had antibodies to hepatitis B core antigen (HBcAb) signifying current or previous exposure to HBV. Hepatitis B e antigen University of Ghana http://ugspace.ug.edu.gh 37 (HBeAg) was present in 9 (32.1%) signifying active viral replication and 10 (35.7%) had antibodies to the hepatitis B e antigen (HBeAb) signifying HBeAg seroconversion or end-point for treatment (Table 8). Table 8: HBV Serologic 'Markers' among HBV Positive Study Participants, Eastern Region, 2012 (n=28) HBV Marker No. (%) 95% CI HBeAg 9 (32.1) 1.49-1.86 HBeAb 10 (35.7) 1.49-1.86 HBcAb 26 (92.9) 0.99-1.23 4.6 Characteristics of ART Staff (HCW) in Study Sites Fifty-four HCWs in ART clinics were recruited into the study. There were seven different categories of professionals made up 28 (51.9%) nurses, 11 (20.4%) laboratory scientists/biomedical scientists, 6 (11.1%) medical officers, 5 (9.3%) pharmacists and 4 (7.4%) biostatisticians. Their ages were grouped into four categories of 10 year intervals beginning with 20-29 years. Most of them, 24 (44.4%) were aged 20-29 years. Their number of years of experience working at the ART clinic ranged from 1 to 29 years with median of 3.0 years. For the purposes of analysis, the knowledge items in the questionnaire were divided into subsections addressing the following issues (a) general knowledge related to hepatitis B and HIV infections and (b) knowledge of HBV-HIV co- infection management practices as derived from ART Guidelines 2011. University of Ghana http://ugspace.ug.edu.gh 38 4.6.1 General Knowledge of Hepatitis B and HBV-HIV co-infection Most study participants, 53 (98.1%) had heard about HBV infection and also knew that a person could be infected with both HBV and HIV. However, more than half of them, 28 (51.8%) thought that HBV was another name for liver disease and a significant number, 33(61.1%) did not think that HBV could be transmitted as a hospital-acquired infection (Table 9). Table 9: HCW 'General Knowledge' Items Frequency Distribution, Eastern Region, 2012 (n=54) Knowledge Item Correct Score n (%) Incorrect Score n (%) Don’t Know n (%) Ever heard of hepatitis B Yes 53 (98.1) No 1 (1.9) 0 (0) HBV is another name for liver disease No 19 (35.2) Yes 28 (51.8) 7 (13.0) Hepatitis B infection is caused by a RNA virus No 17 (31.5) Yes 29 (53.7) 8 (14.8) Hepatitis B can be transmitted as nosocomial infection Yes 33 (61.1) No 18 (33.3) 3 (5.6) HBV can be transmitted like other STDs Yes 52 (96.3) No 2 (3.7) 0 (0) A person can be infected with both HIV and HBV Yes 53 (98.1) No 1 (1.9) 0 (0) Possible routes of transmission of HBV 1. blood and blood products 2. Sexual intercourse 3. needles and sharps 4. faeco-orally 5. handshake with infected person 6. contaminated water Correct Scores 48.5 (89.7) Incorrect Scores 5.5 (11.3) Don‟t Know 0 (0) Possible ways of preventing HBV infection i. Avoid eating meat ii. Vaccination iii. Sex without condom iv. Avoid food not well cooked v. Proper disposal of sharps, needles and blood vi. Avoid holding hands with infected person 50.2 (92.8) 3.8 (7.2) 0 (0) Mean Score* 84.2% * An average of the sum of correct percentages for each general knowledge question University of Ghana http://ugspace.ug.edu.gh 39 Knowledge of HBV routes of transmission and ways to prevent hepatitis B infection (general knowledge items) were 89.7% and 92.8% respectively. Six (11.1%) study participants thought that shaking hands with an infected person could transmit HBV and 5 (9.3%) did not know that sexual intercourse was a possible route of HBV transmission (Table 4.7). Overall, the average score for the “general knowledge” index was 84.2% (SD ±20.53, 95% CI: 89.8-98.1) 4.7.2 Knowledge of HBV-HIV Co-infection Management Practices Most ART staff (94.4%) knew that screening all PLHIV for HBV was part of baseline tests but only 27 (50.0%) knew when to start HAART before vaccination in adults and only 26 (48.1%) knew for children (Table 4.12). More than half of ART staff 33 (61.1%) knew that a positive HBsAg repeat test indicated chronic infection and 26 respondents (48.1%) knew the period within which to monitor a PLHIV client with HBV co-infection. However, only 7 (13.0%) of them knew about other hepatitis B serologic markers besides HBsAg. The mean score for the “management practice” index was 53.12% (SD ±35.06; 95% CI: 13.0-88.9) as shown in Table 10. University of Ghana http://ugspace.ug.edu.gh 40 Table 10: Frequency Distribution of ART Staff Knowledge Items for HBV-HIV Co- infection Management Practices, Eastern Region, 2012 (n=54) Knowledge Item Correct Score n(%) Incorrect Score n(%) Don’t know n(%) PLHIV tested for HBV as part of baseline tests True 51(94.4) False 1(1.9) 2(3.7) Adults recommended to start HAART before vaccination True 27(50.0) False 22(40.7) 5(9.3) Children with CD% indicating severe immunosuppression recommended to start vaccination before HAART False 26(48.1) True 16(29.6) 12(22.2) Months required to repeat HBV test for PLHIV who test positive today 6 months 46(85.2) Any other 5(9.3) 3(5.6) Repeat test for HBsAg still positive after the period indicated above defined as: Chronic 33(61.1) Any other 12(22.3) 9(16.7) PLHIV remains HBV+ after the period, needs to i. assess for liver damage ii. prevent liver damage from other causes like alcohol and drugs iii. prevent transmission to others iv. vaccinate him/her against the infection True 53(98.1) False 0(0) 1(1.9) True 51(94.4) False 2(3.7) 1(1.9) True 50(92.6) False 2(3.7) 2(3.7) False 40(74.1) True 10(18.5) 4(7.4) Most effective blood test indicator of liver damage ALT 32(59.3) Any other 19(35.2) 3(5.6) HBV markers tested besides HBsAg include i. HbsAb True 4(7.4) False 50(92.6) 0(0) ii. HbcAb True 7(13.0) False 47(87.0) 0(0) iii. HBeAg True 7(13.0) False 47(87.0) 0(0) iv. HBeAb True 3(5.6) False 51(94.4) 0(0) v. HBV DNA True 3(5.6) False 51(94.4) 0(0) Persons with HBV-HIV co-infection monitored at least once within a period of 6 months 26(48.1) Any other 25(43.6) 3(5.6) Mean score ** 53.12% ** An average of the sum of correct percentages for each management practice question University of Ghana http://ugspace.ug.edu.gh 41 4.8 Demographic and HBV Risk Factor Analysis among Study Participants A logistic regression model was used to assess the effect of potential risk factors of HBV infection and socio-demographic characteristics of study participants. Only 4/311 (1.3%) reported to have vaccinated against HBV (Figure 4), out of which only 2 (50%) completed the full course. Of 309 study participants, 49 (15.9%) reported intravenous drug use (IDU) and 48/311 (15.4%) reported or had scarification marks and/or tattoos on their bodies. Figure 4: HBV Vaccination Status of Study Participants (n=307) Lack of knowledge about hepatitis B infection, not testing previously for HBV, no vaccinations against HBV, a household member with hepatitis B infection, not using condom always for sexual intercourse, intravenous drug use, receiving blood transfusion(s), having scarification marks or tattoos on body, number of sexual partners in past 12 months and having a previous sexually transmitted infection were identified as risk factors but their associations were not statistically significant with HBV infection. University of Ghana http://ugspace.ug.edu.gh 42 Having tested previously for hepatitis B (p=0.020) and a partner testing positive for hepatitis B in the past (p=0.010) were found to be significant factors for hepatitis B infection. PLHIVs with HBV positive partners were about eight times more likely to be positive for HBsAg and those who reported having tested for HBV previously were likely to also test positive for HBsAg (Table 11). Out of the six (6) people who reported testing for hepatitis B previously, three (3) tested positive with ELISA and another three (3) out of 33 who reported testing negative previously for hepatitis B, also tested positive with ELISA. Study participants reporting these two variables were found to be mutually exclusive (p=0.020). Table 11: Multivariate Analysis of HBV Infection and Risk Factors of Study Participants, Eastern Region, 2012 (n=320) Risk Factor Odds Ratio 95% CI p-value Lower Upper Heard about hepatitis B 0.8 0.36 1.72 0.55 Partner positive for hepatitis B 7.9 1.26 50.36 0.01 Had previous hepatitis B vaccinations 3.5 0.35 35.24 0.25 Household member with hepatitis B 1.1 1.05 1.13 0.59 Condom use always 1.2 0.47 2.98 0.70 Intravenous drug use 0.7 0.19 2.35 0.53 Had blood transfusion (s) 1.2 0.46 3.16 0.68 Scarification marks/tattoos on body 1.0 0.33 3.14 0.95 Had a sexually-transmitted infection 0.9 0.33 2.62 0.90 Tested for hepatitis B in the past 0.02 Number of sexual partners in past 12 months 0.51 University of Ghana http://ugspace.ug.edu.gh 43 CHAPTER FIVE DISCUSSION 5.1 Introduction HBV and HIV are endemic in sub-Saharan Africa and co-infections with these two viruses are common due to shared routes of transmission. Epidemiological studies across sub-Saharan Africa countries have provided a wide range of prevalence estimates.This clearly demonstrate the need for ongoing surveillance activities in different geographical areas of the continent (Kapembwa et al., 2011). Considering its public health importance, Ghana‟s ART guidelines were reviewed to include the management of the co-infection and therefore aim of this study was to determine the HBV co-infection prevalence among HIV-seropositive clients attending ART clinics in the Eastern region of Ghana and to assess the knowledge of staff of the ART Clinics with regards to the management of the co-infection as derived from the guidelines. 5.2 Characteristics of Study Participants This study‟s population had similar demographics to the ART population in the Eastern region, which has a preponderance of females attending ART clinics. More females, 232 (72.5%), adults, 307 (95.9%) and those aged 31-40 years, 119 (37.2%) were recruited into the study and this can be explained by a number of factors including social, biologic, cultural and active sexual lifestyles. In 2011 for example, females on HIV care and antiretroviral therapy were 3349 (67.2%) and 1669 (70.7%) respectively, whereas children on care and treatment were only 373 (7.5%) and 166 (7.0%) respectively for the University of Ghana http://ugspace.ug.edu.gh 44 same year (Eastern Regional STI/AIDS Control Programme, 2012). Almost half, 149 (44.1%) of the study participants had primary level education and only 6 (1.9%) had some kind of tertiary education. The number of them engaged in trading or some kind of business was 132 (41.3%) and this is a reflection of the population in the Eastern region where most people have only basic education and engaged in trading (Ghana Districts, 2006). In spite of the trading or business activities, most of them, 193 (60.3%) had income levels below GH¢100 and it was expected that they would embrace a social intervention such as the national health insurance scheme to mitigate the high cost of health care services, but only 63 (19.7%) had registered for the scheme. HBV vaccination among the study participants (PLHIV) was very low, 4 (1.3%) and this situation is similar for the general population and even HCWs, according to the Ghana Health Service, due to the limited knowledge of the disease among health staff and the population (DSD Annual Report, 2012). 5.3 Prevalence of HBV-HIV Co-infection among Study Participants In this study, the HBV prevalence (assessed as HBsAg) among PLHIV attending antiretroviral clinics in rural and urban sites in the Eastern region was 8.8% (28/320), which is in keeping with Puoti et al.‟s (2008) estimate of worldwide HBV-HIV prevalence of 5–10%. Ghana is reported to have a hepatitis B prevalence rate of 8–15%, thus placing the country in the bracket of Hepatitis B endemic countries as per WHO‟s classification. For such countries with intermediate and high HBV endemicity, HBV-HIV University of Ghana http://ugspace.ug.edu.gh 45 co-infection prevalence rates have been found to be 10-20% (Lee, 2008; Nyirenda, 2008; Diop-Ndiaye, 2008), but that found in the current study was slightly lower. This study‟s prevalence is also lower than what has been found in a study in Ghana among a cohort of HIV-positive individuals reporting a prevalence of 16.7% at the Komfo Anokye Teaching Hospital (KATH), Kumasi (Geretti et al, 2010). This could be attributed to the fact that KATH, being a tertiary referral hospital for the middle belt of the country received more clients, most of whom may have serious conditions such as AIDS and liver diseases. In a another study at the Korle-Bu Teaching Hospital (KBTH), the premier tertiary referral hospital in the country for example, a HBV prevalence of 42.5% was found among patients diagnosed with cirrhosis (Blankson et al, 2005). There are varying HBsAg prevalence estimates across the continent. Kapembwa and colleagues (2011) had a prevalence estimate of 9.9% in Zambia, while a 10.6% prevalence was quoted for Botswana (Wester et al, 2006), 16.8% for Senegal (Diop- Ndiaye et al, 2008), 17.3% for Tanzania (Nagu et al, 2008), and 19.7% for South Africa (Hoffman et al, 2008). This study‟s prevalence is also higher than reported prevalence rates of 3.9% (outpatients) in Ethiopia (Shimelis et al, 2008), 6.1% (outpatients) in Kenya (Harania et al, 2008), 6.7% (outpatients) in Malawi (Moore et al, 2010), 2.4% (antenatal clients) in Rwanda (Pirillo et al, 2007) and 4.8% (outpatients) in South Africa (Fimhaber et al, 2008; Di Bisceglie et al, 2010). University of Ghana http://ugspace.ug.edu.gh 46 Prevalence rates between rural and urban populations have showed marked differences in Africa. In 1996, Kew reported a prevalence rate of 15.5% in South Africa‟s rural former Transkei, while those of urban Durban and Soweto were much lower, that is 7.4% and 1.3% respectively. This study also reported a similarly high prevalence of 13.6% in Asesewa, a rural site; however another rural site, Atua, reported zero HBV-HIV co- infection prevalence. The highest prevalence for an urban site was 11.4% for Akwatia. Overall, the rural prevalence compared to urban was 53.6% but the difference in the rates between rural and urban populations was not statistically significant (p=0.560; OR=0.794 rural/urban). 5.4 Risk Factors for HBV infection in Study Participants One of the major determinants of HBV prevalence rates is the age of acquiring the infection. In this study, there was a statistical significant association showing higher prevalence among adults, which is a widely observed fact, due to increasing risk of exposure with time. Similar findings were observed by Pereira (2006) in Brazil and Nagu (2008) in Tanzania. In Kotzee‟s (2006) review of HIV-HBV co-infection in Southern Africa, he observed that a vast majority of the region‟s population is exposed to HBV by the age of 5 years and thereafter prevalence rates increase slightly when children first attend school, and again when they become sexually active. The highest prevalence for HBV, (50%) was found in the age group 31–40 years, which is also similar to the study by Pereira (2006) in the state of Mato Grosso, Brazil. More females were observed to be University of Ghana http://ugspace.ug.edu.gh 47 HBV positive which is in contrast with many population studies including Burnett (2005) who observed that though both sexes are equally exposed to HBV, HBV carriage was higher in sub-Saharan males with male:female ratios ranging from 1.5:1 to 4:1. The occurrence in this study could probably be due to the very high number of female participants (72.5%) that were enrolled, a reflection of the ART population in the Eastern region, which has more females than males attending ART clinics. Though the HBV-HIV co-infection prevalence was quite high, classic predisposing factors for blood-borne diseases such as having scarification marks/tattoos on body (p=0.958), intravenous drug use (0.539), and having previous HBV vaccination (p=0.251) were not statistically significant in the logistic model. This could probably be due to the small number of participants reporting for these factors. Having tested for or a partner testing for HBV in the past few years were however statistically significant predisposing factors to HBsAg infection and the most likely postulate could be that those who responded in the affirmative might have presented with signs and symptoms of hepatitis B infection to the hospital and were made to test for the presence of HBV. To reduce the incidence of hepatitis B infection in PLHIV, the WHO and the guidelines for ART in Ghana, like many others, recommend vaccination against HBV for all PLHIV that are HBsAg negative. Despite this, only 1.3% of those interviewed reported previous vaccination against HBV and this came as no surprise because less than half of them had never heard about hepatitis B infection. A number of studies in sub-Saharan Africa have observed low HBV vaccination coverage for PLHIV and even for HCWs. In Ziraba et University of Ghana http://ugspace.ug.edu.gh 48 al‟s (2010) study, he observed that as low as 6.2% of HCWs in a tertiary hospital in Uganda reported vaccination against HBV. HBV carrier state, defined as HBsAg positive and HbcAb negative was found in only one (3.6%) of HBsAg positive individuals. Antibodies to hepatitis B core antigen were present in 86.7% of HBsAg positive individuals indicating previous or ongoing infection with HBV in an undefined time frame. Medical data of PLHIV abstracted for the purpose of this study showed that there was a statistically significant association of increasing serum ALT levels with HBsAg infection, though that of serum AST level was only marginal (p=0.61). This occurrence could be due to the fact that both variables were all reported in the same subjects, who formed only 53.1% of the total study population. 5.5 Assessment of Health Care Workers Knowledge of HBV-HIV Co-infection HCWs are key players in the prevention and management of hepatitis B and HIV infections. The WHO (2004) reported that although hepatitis B is a leading infectious cause of death and claims the lives of millions each year, it remains virtually unknown to the general public, at-risk populations, and policymakers; even health-care providers lack knowledge and awareness about this infection. As a consequence, most of the people living with hepatitis B do not know that they are infected, placing them at greater risk for severe, even fatal, complications from the disease and increasing the likelihood that they will spread the virus to others. University of Ghana http://ugspace.ug.edu.gh 49 This study‟s results showed that, overall 84.2% ART staff had indicated the right knowledge with regards to general knowledge of HBV infection. It was surprising however, that a number of them, 11.1% thought that shaking hands with an infected person could transmit HBV and 9.3% did not know that sexual intercourse was a possible route of transmission for HBV. A HCW with this kind of belief will be unwilling to touch HBV positive patients for fear of getting infected. It is believed that knowledge is usually the first step towards the modification of a desirable behaviour and it is therefore imperative for all HCWs to have adequate knowledge in this area to help them to provide the right management of HBV co-infection in PLHIV. Again, HCWs are expected to educate and counsel their clients with the correct preventive and disease transmission information and they can only do that effectively if they are better informed themselves. The low “management practice” index in this study may be due to the fact that the guidelines on management of HBV co-infection in HIV-positive individuals are relatively new and time is needed for ART staff to be conversant with them. 5.6 Limitations of Study 1. Only study participants who were HBsAg positive were further screened for other hepatitis B serologic markers. This was due to the high cost of ELISA reagents for the HBV markers. Testing all study participants for HbcAb (antibodies to hepatitis B core Antigen) for example would have detected persistent, low-level carriers and those in the window period of acute infection. Testing for HBc IgM would have detected participants with occult HBV infection (HBc IgM positive but HBsAg negative). University of Ghana http://ugspace.ug.edu.gh 50 2. The risk factors for HBV infection were self-reported by study participants and could be subjective. University of Ghana http://ugspace.ug.edu.gh 51 CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions This study found 8.8% prevalence of hepatitis B virus co-infection among persons living with HIV in the Eastern region with varying site specific prevalence ranging from 0% to 13.6% in Atua and Asesewa respectively. Although, the prevalence was higher in rural populations than in urban populations the difference was not significant. The hepatitis B vaccination coverage and hepatitis B related knowledge among PLHIV were found to be low. The study did not establish any relationships between classical predisposing factors and hepatitis B infection except for individuals who had previously tested for the disease or whose partners had previously tested positive for hepatitis B. Generally, staff of ART clinics exhibited a high level of hepatitis B related knowledge, though there were some areas of concern. Knowledge of HBV-HIV co-infection management practices as derived from the new ART guidelines of 2011 was however quite low, especially knowledge of hepatitis B serologic markers and their clinical significance with regards to HBV infection. University of Ghana http://ugspace.ug.edu.gh 52 6.2 Recommendations On the basis of existing literature and the findings of this study, it is recommended to the Ministry of Health through:  The Expanded Programme for Immunization (EPI) to consider including HbsAg negative PLHIV in their HBV vaccination programme for children.  The National Public Health Reference Laboratory (NPHRL) to evaluate the performances of a specified number of imported rapid test kits and share the results with importers and health facilities to make informed decisions before importing into the country and purchasing them for use respectively.  The Health Promotion Unit to implement continuous education and training of health workers on hepatitis B infection and HBV-HIV co-infection. 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Journal of Clinical Infectious Diseases. 48: 1763-1771 Norder, H., Courouce, A. M., Coursaget, P., Echevarria, J. M., Lee, S.D., Mushahwar, I. K., Robertson, B. H., Locarnini, S. and Magnius, L.O. (2004). Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes. Intervirology. 47: 289–309. Nagu, T.J., Bakari, M. and Matee, M. (2008).Hepatitis A, B and C viral co-infections among HIV-infected adults presenting for care and treatment at Muhimbili National Hospital in Dar es Salaam, Tanzania. BMC Public Health.8:416. Nyirenda, M., Beadsworth, M. B., Stephany, P., Hart, C. A., Hart, I. J., Munthali, C., Beeching, N. J. and Zijlstra, E. E. (2008). Prevalence of infection with hepatitis B and C virus and co-infection with HIV in medical inpatients in Malawi. Journal of Infection. 57: 72-77. University of Ghana http://ugspace.ug.edu.gh 57 Oren, K Fix, Lorcarnini, Stephen A, Peters, Marion, Stephen G. (2007). Virology And Clinical Management of Hepatitis B And HIV Coinfection. Physicians’ Research Network, Vol. 11. Owiredu, W. K. B. A., Osei-Yeboah, J., Amidu, N. and Laing, E. F. (2012). Residual Risk of Transmission of Hepatitis B Virus through Blood Transfusion in Ghana: Evaluation of the performance of Rapid ICT Assay with Enzyme Linked Immuno- sorbent Assay. Journal of Medical and Biomedical Sciences. 1: 17-28. Palella, F. J., Baker, R. K., Moorman, A. C. (2006). Mortality in highly active antiretroviral therapy era: changing causes of death and disease in the HIV Outpatient Study. Journal Acquired Immune Deficiency Syndrome. 43: 27 – 34. Parkin, D. M. (2006).The global health burden of infection-associated cancers in the year 2002. International Journal of Cancer. 118: 3030–3044. Pol, S. (2005). Epidemiology and natural history of hepatitis B. Journal Review Practical. 55: 599-606. Randrianirina, F., Carod, J. F., Ratsima, E., Chrétien, J. B., Richard, V. and Talarmin, A. (2008).Evaluation of the performance of four rapid tests for detection of hepatitis B surface antigen in Antananarivo, Madagascar. Journal of Virological Methods. 151: 294- 297. Samuel, S. O., Aderibigbe, S. A., Salami, T. A. T. and Babatunde, O. A. (2009).Health workers‟ knowledge, attitude and behavior towards hepatitis B infection in Southern Nigeria. International Journal of Medicine and Medical Sciences. 1: 418-424. Scheiblauer, H., El-Nageh, M., Diaz, S., Nick, S., Zeichhardt, H., Grunert, H.P. and Prince, A. (2010).Performance evaluation of 70 hepatitis B virus (HBV) surface antigen (HBsAg) assays from around the world by a geographically diverse panel with an array of HBV genotypes and HBsAg subtypes.Vox Sang. 98: 403–414. Soriano, A., Lozano, F., Oliva, H., García, F., Nomdedéu, M., De Lazzari, E., Rodríguez, C., Barrasa, A., Lorenzo, J. I.,Del Romero, J. , Plana, M., Miró, J. M., Gatell, J. M., Vives, J. and Gallart, T.(2005). Polymorphisms in the interleukin-4 receptor alpha chain gene influence susceptibility to HIV-1 infection and its progression to AIDS. Immunogenetics. 57: 644-654. University of Ghana http://ugspace.ug.edu.gh 58 Sulkowski, M. S., Thomas, D. L., Mehta, S. H., Chaisson, R. E. and Moore, R. D. (2002). Hepatotoxicity associated with nevirapine or efavirenz-containing antiretroviral therapy: Role of Hepatitis C and B infections. Hepatology. 35: 144-148. Thio, C. L, Seaberg, E. C. and Skolasky, R. (2002).HIV-1, hepatitis B virus, and risk of liver-related mortality in the Multicenter Cohort Study (MACS). Lancet. 360: 1921-1926. Thio, C. L. (2003). Hepatitis B in the human immunodeficiency virus-infected patient: epidemiology, natural history, and treatment. Seminars in Liver Disease Journal. 23: 125-136. Thio, C. L. (2009).Hepatitis B and Human Immunodeficiency Virus co-infection. Hepatology. 49: 1-8. Thompson, N. D., Perz, J. F., Moorman, A. C., and Holmberg, S. D. (2009).Nonhospital Health Care–Associated Hepatitis B and C Virus Transmission: United States, 1998– 2008. Annals of Internal Medicine. 150: 33-39. Torane, V. P. and Shastri, J. S. (2008). Comparison of ELISA and rapid screening tests for the diagnosis of HIV, hepatitis B and hepatitis. Indian Journal of Medical Microbiology. 26:284-285. UNAIDS, (2011).World AIDS Day Report, 2011. Vardas, E., Mathai, M., Blaauw, D., Mcanerney, J., Coppin, A. and Sim, J. (1999).Preimmunization epidemiology of hepatitis B virus infection in Southern African children. Journal of Medical Virology. 58: 111 – 115. Viral Hepatitis Control Unit (2012). Ghana Health Service, Expanded Programme on Immunization (EPI)/Disease Surveillance Department (DSD) Annual Review Meeting, 17-18 January, 2012, Erata Hotel. Ward, J. (2011).The impact of HIV-hepatitis co-infection. AIDS. 18: 33-37. Weber, R., Sabin, C. A., Friis-Moller, N., Reiss, P., El-Sadr, W. M. and Kirk, O. (2006).Liver-related deaths in persons infected with the human immunodeficiency virus: the D:A:D study. Archives of Internal Medicine. 166:1632-1641. World Health Organisation (WHO) (2012).Policy on collaborative TB/HIV activities- Guidelines for national programmes and other stakeholders. Geneva, Switzerland, WHO, 2012. University of Ghana http://ugspace.ug.edu.gh 59 World Health Organization (WHO) (2004).Hepatitis B fact sheet.Available at http://www.who.int/inf-fs/en/fact204.html. (Accessed 15/09/11). Ziraba, A.K., Bwogi, J., Namale, A., Wainaina, C.W., and Mayanja-Kizza, H. (2010).Sero-prevalence and risk factors for hepatitis B virus infection among health care workers in a tertiary hospital in Uganda. BMC Infectious Disease.10:191. University of Ghana http://ugspace.ug.edu.gh 60 APPENDICES APPENDIX I – ART sites in the Eastern region of Ghana ART Site (Eastern Region) Population Cumulative Popn Site Sampled Afram Plains 299 299 Akuse 325 624 Asamankese 302 926 Asesewa 498 1424 1000 Atibie Hosp 270 1694 Atua 5130 6824 5009 District Hospital Begoro 439 7263 District Hospital Kibi 342 7605 Enyiresi 364 7969 Holy Family 960 8929 Koforidua Regional Hosp 2972 11901 9017 New Abirem 133 12034 Nsawam 450 12484 Oda Hosp 483 12967 St. Dominic 1475 14442 13026 St. Joseph 320 14762 St. Martin‟s* 4325 19087 16964 Suhum 264 19351 Tetteh Quarshie 200 19551 VRA 492 20043 * St. Martin‟s Hospital was replaced with District Hospital Begoro (DHB) University of Ghana http://ugspace.ug.edu.gh 61 APPENDIX II – Informed Consent Form (Adult) and Questionnaire 1 CONSENT TO PARTICIPATE IN A RESEARCH PROJECT TITLE: HEPATITIS B VIRUS CO-INFECTION AMONG HIV-SEROPOSITIVE PERSONS IN THE EASTERN REGION OF GHANA Dear Participant, Explanation of Procedures You are being invited to participate in a research project that seeks to determine the prevalence of Hepatitis B virus (HBV) co-infection among Persons Living with HIV (PLHIV) in the Eastern region of Ghana. HIV infection negatively impacts the natural history of hepatitis B leading to increased rates of persistent infection, loss of protective antibody against HBV, increased liver-related mortality, and increased risk of liver cancer at lower CD4_ T cell counts among others. Early detection of HBV infection will help the treatment and may prevent mother-to- child transmission. The approach of this study is through the use of a questionnaire and blood specimen collection. You will be required to answer questions on personal profile and risk factors for HBV acquisition. Afterwards, you will be examined by a ounselor and 8ml of blood specimen will be collected from you. Risks and Discomforts By participating in this research, you are likely to experience some form of discomfort. This includes the discomfort or embarrassment of questioning, and the pain of blood specimen collection. You can refuse to answer any of the questions that may be posed to you. If an untoward event happens, a clinician will provide you with free medical care. Benefits By participating in this study, you could learn about your HBV status which will help your clinician to optimize your clinical management or referral for vaccination against HBV. The study is also expected to provide data on HBV-HIV University of Ghana http://ugspace.ug.edu.gh 62 co-infection in your community for policy makers. Confidentiality All information gathered from the study will remain confidential. Your identity as a participant will not be disclosed to any unauthorized persons; only the researchers, Ghana Health Service and School of Public Health will have access to the research materials, which will be kept in a locked drawer. Any references to your identity that would compromise your anonymity will be removed or disguised prior to the preparation of the research reports and publications. Left-over specimen will be kept for a period of a year after the study but will not be used for any future research of a restricted, specified or unspecified nature without your consent. Withdrawal from Project Participation in this study is voluntary; refusal to participate will involve no penalty. You can withdraw consent and discontinue participation in this project at any time without prejudice from the research team. Costs and/or Payments to subject There will be no costs for participating in the research. Also, you will not be paid to participate in this research project. Any questions concerning the progress of research project and/or in the case of injury due to the project, participants can call Dr. Priscillia Nortey, Department of Epidemiology, School of Public Health (0243303362), Professor E. Afari, School of Public Heath (0208131828), Dr. S.B. Ofori, Koforidua Regional Hospital (0243654771), Mr. Gideon Kye-Duodu of the School of Public Heath (0244465259) or ________________________________________. Questions regarding any rights issues as a person in this research project should be directed to the Prof. Fred Binka (020 8131031) Chairman of the Ethical Review Committee of the Ghana Health Service. University of Ghana http://ugspace.ug.edu.gh 63 Consent to participate in Research I, _____________________________________________ Tick as appropriate (this decision will not affect your ability to enter the study): � Confirm that I have read the written information (or have had the information read to me) for the study Hepatitis B Virus Co-infection among HIV-seropositive Persons in the Eastern Region of Ghana and that the study procedures have been explained to me by study staff during the consent process for this study. � Confirm that I have had the opportunity to ask questions about this study and I am satisfied with the answers and explanations that have been provided. � Understand that I grant access to data to authorized persons described in the information sheet. � Have been given time and opportunity to consider taking part in this study. I consent to participate in the above research study. Signature or Thumbprint of Subject: ____________ Date:______/_____/_______ Signature of Interviewer:____________________ Date:______/_____/_____ Name of Impartial Witness: _______________ Signature of Impartial Witness: _____________ Date:_____/_____/_______ University of Ghana http://ugspace.ug.edu.gh 64 Questionnaire 1 – Antiretroviral Client (Adult) TITLE: PREVALENCE OF HEPATITIS B VIRUS CO-INFECTION AMONG HIV- SEROPOSITIVE PERSONS ATTENDING ART CLINICS IN THE EASTERN REGION OF GHANA Date: |_____|_____|______| SECTION A: IDENTIFICATION/SOCIO-DEMOGRAPHY OF HIV CLIENT Reporting Health Facility/ART Clinic HOSPREP Reporting District/Municipality DISTREP Form Number/ Client ID : PIN Sex 1.Male 2.Female SEX Date of birth DOB Age Year Month AGE Ethnicity ETHNIC What is your educational Level? 1. None 3. Secondary 2. Primary 4. Tertiary EDUC What is your marital Status? 1. Single 4. Divorced 2. Married 5. Widowed 3. Separated 6. Co-habiting 7. Other (please specify)_____ MARITST What is your occupation? OCUPATIN What is your community of residence? COMM What is your level of Income 1. GH¢500 INCOM University of Ghana http://ugspace.ug.edu.gh 65 How do you pay for the GH¢5 monthly bill for ART services and other medical bills? 1. Out-of-pocket 3. Employer 2. Relatives 4. NHIS 5. Other (please specify)____________ PAYMNT SECTION B: KNOWLEDGE ABOUT HIV AND HEPATITIS B VIRUS INFECTIONS 1. Have you tested for HIV? 1. Yes 2. No 3. cannot recall HIVTST 2. Did you test HIV positive? 1. Yes 2. No 3. cannot recall HIVPOS 3. Do you know about Hepatitis B Virus (HBV)? 1. Yes 2. No 3. cannot recall HBVKNO 4. Have you tested for HBV? 1. Yes 2. No 3. cannot recall HBVTST SECTION C: RISK FACTORS FOR HEPATITIS B VIRUS INFECTION 5. Has your partner ever tested positive for HBV? 1. Yes 2. No HBVPART 6. Have you had previous HBV vaccinations? 1. Yes 2. No HBVACC 7. If answer is Yes to Q.20, what was the outcome of the vaccinations? 1. completed course 2. not completed 3. cannot recall HBVCOM 8. Do you always use condoms during sexual intercourse with your partner? 1. Yes 2. No CONDUSE 9. Has anyone in your household tested positive for HBV? 1. Yes 2. No HBVHSH 10. How many sexual partners have you had in the past 12 months? 1. 0 3. 2 2. 1 4. ≥ 3 SEXPART 11. Have you ever used drugs through intravenous means (IDU)? 1. Yes 2. No IDU 12. Have you ever had a blood transfusion? 1. Yes 2. No BLDTRNS 13. If answer is Yes to Q.23, how many times have you had it? 1. Once 3. ≥3 2. Twice BLDNUM 14. Do you have any scarification marks or tattoos? 1. Yes 2. No SCARMKS 15. If answer is Yes to Q.26, where on your body is it? SCARBDY 16. For what purpose was it? SCARPUR 17. Have you ever had a sexual transmitted infection (STI)? 1. Yes 2. No HADSTI University of Ghana http://ugspace.ug.edu.gh 66 SECTION D: MEDICAL DATA 33. Haemoglobin Concentration Level (last) _________g/dl Date ____/____/____ HBCONC 34. CD4 Count (current) ___________ Date ____/____/____ CD4CNT 35. ALT Level (current) ___________ Date ____/____/____ ALTLEV 36. AST Level (current) ___________ Date ____/____/____ ASTLEV SECTION E: LABORATORY INVESTIGATIONS 37. Venous Blood Specimen Taken 1. 8mls (Adults) 2. 5mls (Children) Date ___/___/____ 38. HBsAg Test Result (ELISA) 1. Positive 2. Negative 3. Indeterminate HBVELSA 39. Hepatitis B Profile (if 38 is Positive) Serological Markers 1. HBeAg 2. HBeAb 3. HBcAb 1. Positive 2. Negative HBVPROF 40. HBsAg Test Result (Rapid Test Kit) Rapid Test Kit Wondfo Virucheck One Step 1. Positive 2. Negative HBVRAP D Name of Interviewer:__________________ Date:______________ University of Ghana http://ugspace.ug.edu.gh 67 APPENDIX III – Informed Assent Form (Child) and Questionnaire 2 (7 – 17 YEARS OLD) TITLE: PREVALENCE OF HEPATITIS B VIRUS CO-INFECTION AMONG HIV- SEROPOSITIVE PERSONS ATTENDING ART CLINICS IN THE EASTERN REGION OF GHANA THIS CONSENT FORM IS USED TO DOCUMENT THE PERMISSION OF PARENT(S)/GUARDIAN(S) TO ALLOW A MINOR TO TAKE PART IN A RESEARCH STUDY. AS USED IN THIS CONSENT FORM “YOU” REFERS TO THE MINOR WHO WILL TAKE PART IN THE RESEACRH STUDY. Dear Participant, Explanation of Procedures You are being invited to participate in a research project that seeks to determine the prevalence of Hepatitis B virus (HBV) co-infection among Persons Living with HIV (PLHIV) in the Eastern region of Ghana. HIV infection negatively impacts the natural history of hepatitis B leading to increased rates of persistent infection, loss of protective antibody against HBV, increased liver-related mortality, and increased risk of liver cancer at lower CD4_ T cell counts among others. Early detection of HBV infection will help the treatment and may prevent mother-to-child transmission. The approach of the research is through the use of a questionnaire and blood specimen collection. You will be required to answer questions on personal profile and risk factors for hepatitis B acquisition. Afterwards, you will be examined by a counsellor and 5ml of blood specimen will be collected from you. Risks and Discomforts By participating in this research, you are likely to experience some form of discomfort. This includes the discomfort or embarrassment of questioning, physical examination, and the pain of blood specimen collection. You can refuse to answer University of Ghana http://ugspace.ug.edu.gh 68 any of the questions that may be posed to you. The team will try and decrease your chances of these risks from occurring, but if an untoward event happens, they will provide you with free medical care. Benefits By participating in this study, you could learn about your HBV status which will help your clinician to optimize your clinical management or referral for vaccination against HBV if you test negative. The study is also expected to provide data on HBV- HIV co-infection in your community for policy makers. Confidentiality All information gathered from the study will remain confidential. Your identity as a participant will not be disclosed to any unauthorized persons; only the researchers, Ghana Health Service and School of Public Health will have access to the research materials, which will be kept in a locked drawer. Any references to your identity that would compromise your anonymity will be removed or disguised prior to the preparation of the research reports and publications. Left-over specimen will be kept for a period of a year after the study but will not be used for any future research of a restricted, specified or unspecified nature without your consent. Withdrawal from Project Participation in this study is voluntary; refusal to participate will involve no penalty. You can withdraw consent and discontinue participation in this project at any time without prejudice from the research team. Costs and/or Payments to subject There will be no costs for participating in the research. Also, you will not be paid to participate in this research project. Any questions concerning the progress of research project and/or in the case of injury due to the project, participants can call Dr. Priscillia Nortey, Department of Epidemiology, School of Public Health (0243303362), Professor E. Afari, School of Public Heath (0208131828), Dr. S.B. Ofori, University of Ghana http://ugspace.ug.edu.gh 69 Koforidua Regional Hospital (0243654771), Mr. Gideon Kye-Duodu of the School of Public Heath (0244465259) or ________________________________________. Questions regarding any rights issues as a person in this research project should be directed to the Prof. Fred Binka (020 8131031) Chairman of the Ethical Review Committee of the Ghana Health Service. Consent to participate in Research PARENT/GUARDIAN’S STATEMENT Documentation of Permission I have been given a copy of this form. I have read it or it has been read to me. I understand the information and have had my questions answered to my satisfaction. I agree to take part in this study. I understand that I will be informed of any new findings developed during the course of this research study that may affect my willingness to stay in this research study. __________________ ______________________ |_____|_____|________| *Child‟s Assent Parent/Legal Guardian‟s Signature Date I have fully explained to ________________ [Parent/Legal Guardian/Participant Signature] the nature and purpose of the above-described study and the risks that are involved in its performance. I have answered all questions to the best of my ability. |_____|_____|________| __________________________________ Date Principal Investigator or Representative‟s Signature University of Ghana http://ugspace.ug.edu.gh 70 Questionnaire 2 – Antiretroviral Client (Minor) TITLE: PREVALENCE OF HEPATITIS B VIRUS CO-INFECTION AMONG HIV- SEROPOSITIVE PERSONS ATTENDING ART CLINICS IN THE EASTERN REGION OF GHANA Date: |_____|_____|______| SECTION A: IDENTIFICATION/SOCIO-DEMOGRAPHY OF HIV CLIENT Reporting Health Facility/ART Clinic HOSPREP Reporting District/Municipality DISTREP Form Number/ Client ID : PIN Sex 1.Male 2.Female SEX Date of birth DOB Age Year Month AGE Ethnicity ETHNIC What is your community of residence? COMM How do you pay for the GH¢50 monthly bill for ART services and other medical bills? 1. Out-of-pocket 3. Employer 2. Relatives 4. NHIS 5. Other (please specify)_____________ PAYMNT SECTION B: KNOWLEDGE ABOUT HEPATITIS B VIRUS INFECTIONS 1. Do you know about Hepatitis B Virus (HBV)? 1. Yes 2. No 3. cannot recall HBVKNO 2. Have you tested for HBV? 1. Yes 2. No 3. cannot recall HBVTST 3. What was the result of your HBV test? 1. Positive 2. Negative 3. cannot recall HBVRST University of Ghana http://ugspace.ug.edu.gh 71 SECTION C: RISK FACTORS FOR HEPATITIS B VIRUS INFECTION 4. Have you had previous HBV vaccinations? 1. Yes 2. No HBVACC 5. If answer is Yes to Q.20, what was the outcome of the vaccinations? 1. completed course 2. not completed 3. cannot recall HBVCOM 6. Has anyone in your household tested positive for HBV? 1. Yes 2. No HBVHSH 7. Have you ever used drugs through intravenous means (IDU)? 1. Yes 2. No IDU 8. Have you ever had a blood transfusion? 1. Yes 2. No BLDTRNS 9. If answer is Yes to Q.23, how many times have you had it? 1. Once 3. ≥3 2. Twice BLDNUM 10. Do you have any scarification marks or tattoos? 1. Yes 2. No SCARMKS 11. If answer is Yes to Q.26, where on your body is it? SCARBDY 12. For what purpose was it? SCARPUR SECTION D: MEDICAL DATA 22. Haemoglobin Concentration Level (last) _________g/dl Date ____/____/____ HBCONC 23. CD4 Count (last) ___________% Date ____/____/____ CD4CNT 24. ALT Level (last) ___________ Date ____/____/____ ALTLEV 25. AST Level (last) ___________ Date ____/____/____ ASTLEV SECTION E: LABORATORY INVESTIGATIONS 26. Venous Blood Specimen Taken 5mls (Child) Date ___/____/___ 27. HBsAg Test Result (ELISA) 1. Positive 2. Negative 3. Indeterminate HBVELSA University of Ghana http://ugspace.ug.edu.gh 72 28. Hepatitis B Profile (if 38 is Positive) Serological Markers 1. HBeAg 2. HBeAb 3. HBcAb 1. Positive 2. Negative HBVPROF 29. HBsAg Test Result (Rapid Test Kit) Rapid Test Kit Wondfo Virucheck One Step 1. Positive 2. Negative HBVRAP D Name of Interviewer:_________________ Date:______________ University of Ghana http://ugspace.ug.edu.gh 73 APPENDIX IV - Antiretroviral Clinic Staff Questionnaire 3 Date: |_____|_____|______| SECTION A: GENERAL INFORMATION 1,1 Reporting Health Facility/ART Clinic ________________________________ 1.2 Reporting District/Municipality ________________________________ 1.3 Respondent Number |__________|___________|__________| 1.4 Profession ________________________________ 1.5 Sex Male Female 1.6 Work Experience in ART Clinic __________________ Years 1.7 Age (Tick as appropriate) 20-29 30-39 40-49 50-59 SECTION B: KNOWLEDGE OF ART STAFF ON HEPATITIS B AND HIV CO- INFECTION Question Response Code 2.1 Have you ever heard of Hepatitis B virus (HBV) infection? Yes No P1 2.2 Is HBV another name for HIV? Yes No P2 2.3 What are the possible routes of transmission of Hepatitis B virus? (circle as many answer(s) as possible) 1. Blood and blood products 2. Sexual Intercourse 3. Contaminated Water 4. Needles and Sharps 5. Faeco-oral 6. Others (please specify)_______ P3 University of Ghana http://ugspace.ug.edu.gh 74 (please tick appropriate answer) Yes No 2.4 Is hepatitis B infection caused by a DNA or RNA virus? P4 2.3 Can Hepatitis B be transmitted as a nosocomial (hospital-acquired) infection? P4 2.4. Can Hepatitis B be transmitted like other Sexually-Transmitted Diseases (STDs)? P4 2.5 Can HBV be transmitted from mother to child during childbirth? P5 2.6 Do healthcare workers have a greater risk of Hepatitis B infection by virtue of their work P6 2.7 Is there a vaccine for Hepatitis B? P7 2.8 What are some of the ways of preventing Hepatitis B infection? (please choose as many as possible) 1. vaccination 2. avoid drinking contaminated water 3. avoid touching clients 4. avoid food not well cooked 5. proper disposal of sharps, needles and blood P8 2.9 Can one be infected by both HIV and HBV? P9 SECTION C: KNOWLEDGE OF HEPATITIS B AND HIV CO-INFECTION MANAGEMENT PRACTICES Question Response Code 3.1 All Persons Living with HIV (PLHIV) should be tested for Hepatitis B surface Antigen (HBsAg) as part of baseline tests? True False P9 3.2 If answer is Yes for Q.1, what other recommendation(s) besides vaccination do you give to those who test negative for HBsAg? _____________________________ _____________________________ _______________________ P10 3.3 To ensure good response to Highly Active Antiretroviral Therapy (HAART) for HBsAg negative clients, it is recommended to start vaccination before HAART? True False 3.4 If CD% indicates severe immunosuppression University of Ghana http://ugspace.ug.edu.gh 75 in children, it is recommended to start vaccination before HAART? True False 3.5 What specific counsel or recommendation(s) do you give those who test positive for HBsAg? _____________________________ _________________________ P11 3.6 At what time do you repeat HBsAg test for PLHIV who test positive for hepatitis B? ___________________________ P12 3.7 If the repeat test for HBsAg is still positive after the period above, it indicates an chronic infection True False 3.8 If a PLHIV has chronic HBV infection, there is a need to a. Assess for liver damage by checking blood Alanine transferase (ALT) level b. Prevent liver damage from other causes like alcohol and drugs c. Prevent transmission to others True False True False True False P13 3.9 How do you prevent transmission of infection from HBsAg positive ART clients to others? _____________________________ _________________________ P14 3.10 What is the optimal time for initiating anti- HBV treatment in co-infected persons? ___________________________ P15 3.11 Do you monitor persons with HBV-HIV co- infection by checking ALT levels? Yes No P16 3.12 When do you monitor persons with co- infection of HBV-HIV? __________________________ P17 Name of Field Worker:_____________________ Date:______|______|________ University of Ghana http://ugspace.ug.edu.gh 76 APPENDIX V – Hepatitis B Serologic Markers Assay (ELISA) Researcher and Assistant conducting HBsAg ELISA Assay in Virology Lab., UGMS Foresight HBsAg (Samples 194 – 290) Foresight HBsAg (Samples 97 – 193) Foresight HBsAg (Samples 1 – 96) Foresight HBsAg (Samples 291 – 320) University of Ghana http://ugspace.ug.edu.gh 77 APPENDIX VI-Approved Ethical Clearance University of Ghana http://ugspace.ug.edu.gh