University of Ghana http://ugspace.ug.edu.gh HUMAN PAPILLOMAVIRUS (HPV) INFECTION AND ITS ASSOCIATION WITH CERVICAL DYSPLASIA AMONG HIV POSITIVE WOMEN ATTENDING ART CLINIC, KORLE-BU TEACHING HOSPITAL, ACCRA By DODZI KOFI AMELOR (10325595) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF PhD PUBLIC HEALTH DEGREE JULY, 2019 University of Ghana http://ugspace.ug.edu.gh DECLARATION I hereby declare that this submission is my own original and novel work towards a Doctor of Philosophy Degree (PhD.) and that, to the best of my knowledge, it contains no material previously published by another person, nor material which has been accepted for the award of any other degree of the University, except where due acknowledgement has been made in the text. i University of Ghana http://ugspace.ug.edu.gh ABSTRACT Globally, about 35 million people are living with HIV of which 16 million are women aged 15 years and above, 80% of whom live in the Sub-Saharan African region These women living with HIV (WLHIV), especially those having low CD4 T-cell counts are more likely to be infected with human papillomaviruses (HPV), which are the major cause of cervical cancer and it’s precursor lesions. But less is known about the prevalence of HPV infection and the specific types involved in this particular population of women in Ghana. A cross-sectional study was carried out to establish the distribution of genital HPV genotypes among an unscreened population of WLHIV attending a special HIV outpatient clinic at the Korle-Bu Teaching hospital (KBTH) Accra, Ghana. Cervical swabs were available for 538 eligible women. A nested multiplex PCR (NMPCR) assay was conducted for the detection and typing of HPV genotypes 6/11, 16,18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68. Cervical smears were also prepared and examined independently for cytologic abnormalities by two cytologists and confirmed by a pathologist. HPV DNA was detected in 52.0% of all cervical swabs tested, the prevalence of LR (Low Risk) HPV was 10.2% (95% CI: 11.3 – 17.6). while that of HR (High Risk) HPV was 29.7% (95% CI: 27.4 – 35.7). The commonest HR types were, HPV-35 (14.1%), HPV-18 (13.0%;), HPV-58 (7.4%;), HPV-52 (7.2%;), HPV-45 (4.8%;) and HPV 42, a low-risk type, was also common (7.8%;). The frequency of HPV 16 was 2.4%. Cytologicaly, 2.8% had ASCUS, 3.9% LGSIL and 2.8% HGSIL, None of the women was found to have atypical glandular cells or adenocarcinoma. There is a high burden of HPV in women living with HIV attending clinic at the KBTH. I recommend that screening for cervical dysplasia should be done for WLHIV routinely as part of their comprehensive care. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT Blessed be the Lord Jesus Christ, who hath blessed us with all spiritual blessings in heavenly places in Christ (Eph 1 3): I am deeply indebted to my supervisors, Professor E. K Wiredu, Dr Priscilla Nortey and Dr R. H. Asmah for their guidance, remarks and excellent suggestions throughout this successful academic journey. I am privileged to be mentored by these great academic advisors. My appreciation also goes to all the lecturers of the Department of Epidemiology and Disease Control of the School of Public Health, University of Ghana for the knowledge and experiences they shared during my period of study. A special thank you also goes to Dr Samuel Oppong and his team of the Department of Gynaecology, Korle-Bu Teaching Hospital for their immense assistance during the sample taking period. A special thank you also goes to Prof Margaret Lartey of SMD, UG for her support and encouragement. My profound gratitude goes to Dr Timmy Donkoh and his family for their special contribution and support God richly bless you. I am also grateful to the entire staff and patients of Fevers Unit, Korle-Bu Teaching Hospital for their immense contributions toward the success of this project. Last but not least, I will like to specially thank my wife Mrs Bertha Serwaa Amelor for her wonderful support and encouragement during the period of writing this thesis. iii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ............................................................................................................................. I ABSTRACT ................................................................................................................................... II TABLE OF CONTENTS ............................................................................................................. IV LIST OF TABLES ..................................................................................................................... VII LIST OF FIGURES ..................................................................................................................... IX APPENDICES ................................................................................................................................X CHAPTER 1 INTRODUCTION ........................................................................................... 1 1.1 BACKGROUND ............................................................................................................ 1 1.2 PROBLEM STATEMENT ............................................................................................. 4 1.3 CONCEPTUAL FRAMEWORK ................................................................................... 7 1.4 JUSTIFICATION ........................................................................................................... 7 1.5 RESEARCH QUESTIONS .......................................................................................... 10 1.6 GENERAL OBJECTIVE .............................................................................................. 11 1.6.1 SPECIFIC OBJECTIVES ......................................................................................... 11 CHAPTER 2 LITERATURE REVIEW ............................................................................. 12 2.1 HISTORY OF PAPILLOMAVIRUSES (PVS) IN CANCER ...................................... 12 2.2 DIVERSITY OF PAPILLOMAVIRUSES AND EPITHELIAL TROPISMS .............. 13 2.2.1 Classification of HPVs based on disease association .............................................. 14 2.2.2 Epithelial Tropisms .................................................................................................. 15 2.3 MOLECULAR BIOLOGY OF HUMAN PAPILLOMAVIRUSES (HPV) ................. 19 2.3.1 Organization of the HPV Genome ............................................................................ 19 2.3.2 Structure of Human Papillomaviruses ..................................................................... 21 2.4 SOME DISEASES ASSOCIATED WITH MUCOSAL HPV IN HIV PATIENTS ..... 25 2.4.1 Benign HPV-associated cervical lesions .................................................................. 25 2.4.2 Pre-malignant HPV-associated lesions .................................................................... 26 2.4.3 Cervical Cancer ....................................................................................................... 27 2.4.4 Other anogenital cancers ......................................................................................... 28 2.4.5 Head and neck cancer .............................................................................................. 29 2.5 EPIDEMIOLOGY OF HPV INFECTION .................................................................... 30 2.5.1 Global Prevalence of HPV DNA .............................................................................. 31 iv University of Ghana http://ugspace.ug.edu.gh 2.5.2 Prevalence of HPV Genotypes in Cervical Cancer .................................................. 32 2.5.3 Transmission and Natural History of Cervical HPV Infection ................................ 33 2.6 EPIDEMIOLOGY OF CERVICAL CANCER............................................................. 35 2.6.1 Globally and in Africa .............................................................................................. 35 2.6.2 Ghana ....................................................................................................................... 37 2.7 EPIDEMIOLOGY OF HIV/AIDS IN GHANA ............................................................ 38 2.8 HUMAN PAPILLOMAVIRUSES AND HIV/AIDS ................................................... 39 2.8.1 Effect of HIV infection on HPV Acquisition ............................................................. 39 2.8.2 Prevalence of HPV in HIV infected women.............................................................. 41 2.8.1 HIV and HPV type distribution ................................................................................ 42 2.8.2 Persistence of HPV in HIV Positive Women ............................................................ 43 2.8.3 HIV and Cervical Cancer ......................................................................................... 43 2.9 PREVENTION OF CERVICAL DISEASE IN HIV .................................................... 44 2.9.1 Primary Prevention with Available Prophylactic Vaccines ..................................... 44 2.9.2 Universal/ Targeted Screening ................................................................................. 45 2.9.3 Cervical Cancer Screening among HIV Positive Women ........................................ 45 2.10 SCREENING FOR CERVICAL DISEASE ................................................................. 46 2.10.1 Morphological Examination ................................................................................ 46 2.10.2 DNA Testing for the presence of high-risk HPV types ......................................... 47 2.10.3 Indications for Cervical HPV DNA Testing ......................................................... 48 CHAPTER 3 MATERIALS AND METHODS .................................................................. 49 3.1 STUDY DESIGN AND SETTING ............................................................................... 49 3.2 STUDY LOCATION .................................................................................................... 49 3.3 STUDY POPULATION AND SAMPLING ................................................................ 50 3.3.1 Study population ....................................................................................................... 50 3.3.2 Sampling Technique ................................................................................................. 51 3.3.3 Selection of study participants.................................................................................. 52 3.4 INCLUSION CRITERIA .............................................................................................. 53 3.5 EXCLUSION CRITERIA ............................................................................................ 53 3.6 SAMPLE SIZE DETERMINATION............................................................................ 53 3.7 DATA COLLECTION TOOLS: PRETESTING AND ADMINISTRATION ................................ 54 3.8 SPECMEN COLLECTION AND LABORATORY ANALYSIS/TESTS ................... 55 v University of Ghana http://ugspace.ug.edu.gh 3.8.1 Smear Preparation, Fixation and Transportation .................................................... 55 3.8.2 Staining and Screening of Cytological Slides .......................................................... 55 3.8.3 Schedule for Repeat Cytology .................................................................................. 56 3.8.4 DNA Sample Collection ............................................................................................ 57 3.8.5 HPV-DNA Extraction, Detection and Genotyping ................................................... 57 3.8.6 Analysis of Amplification Products .......................................................................... 58 3.9 DATA MANAGEMENT AND STATISTICAL ANALYSIS ...................................... 58 3.10 ETHICS APPROVAL .................................................................................................. 60 CHAPTER 4 RESULTS ....................................................................................................... 62 4.1 DEMOGRAPHIC PROFILE OF STUDY POPULATION ......................................................... 62 4.2 PROPORTION OF WLHIV WITH CERVICAL CYTOLOGY ABNORMALITIES .................... 66 4.3 PREVALENCE OF HPV GENOTYPES AMONG UNSCREENED WOMEN LIVING WITH HIV68 4.4 CORRELATES OF ABNORMAL CYTOLOGY AND THEIR IMPLICATIONS FOR DISEASE PREVENTION ............................................................................................................................... 83 4.5 CORRELATES OF HIGH-RISK HPV INFECTION AND THEIR IMPLICATIONS FOR DISEASE PREVENTION ............................................................................................................................... 91 CHAPTER 5 DISCUSSION ................................................................................................. 97 CHAPTER 6 CONCLUSIONS .......................................................................................... 115 6.1 RECOMMENDATIONS ............................................................................................ 118 REFERENCES ........................................................................................................................... 120 APPENDIX 1 .............................................................................................................................. 133 APPENDIX 2 .............................................................................................................................. 141 APPENDIX 3 .............................................................................................................................. 144 APPENDIX 4 .............................................................................................................................. 146 APPENDIX 5 .............................................................................................................................. 147 vi University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 4.1-1: Socio-demographic characteristics of Women Living with HIV (WLHIV) at the Korle Bu Teaching Hospital, Accra, ............................................................................................................... 63 Table 4.1-2: Reproductive characteristics of WLHIV at the Korle Bu Teaching Hospital, Accra, .............. 64 Table 4.1-3: Clinical characteristics of WLHIVat the Korle- Bu Teaching Hospital, Accra, ...................... 65 Table 4.2-1: Summary of Papanicolau Test Results for WLHIV at the Korle Bu Teaching Hospital, Ghana, ........................................................................................................................................................ 67 Table-4.3-1: Prevalence of human papillomavirus (HPV) in WLHIV at the Korle Bu Teaching Hospital, Accra, Ghana,. ................................................................................................................................ 69 Table 4.3-2: Prevalence of high-risk HPV Genotypes in WLHIV at the Korle Bu Teaching Hospital, Accra, Ghana, ............................................................................................................................................. 70 Table 4.3-3: Prevalence of low-risk HPV Genotypes in WLHIV at the Korle Bu Teaching Hospital, Accra, Ghana, ............................................................................................................................................. 71 Table 4.3-4: Study population demographics and HPV prevalence in WLHIV at the Korle-Bu Teaching Hospital, Accra, Ghana, .................................................................................................................. 72 Table 4.3-5: Sexual history and HPV prevalence in WLHIV at the Korle-Bu Teaching Hospital, Accra, Ghana, ............................................................................................................................................. 74 Table 4.3-6: HPV type distribution stratified according to concurrent cytology outcome and number of detected HPV genotypes HPV Prevalence and cytological outcomes in WLHIV at the Korle Bu Teaching Hospital ........................................................................................................................... 77 Table 4.3-7: Prevalence of LR HPV types by cytological diagnosis at the Korle-bu Teaching Hospital,Accra, Ghana,. .................................................................................................................. 78 Table 4.3-8: Prevalence of HR HPV types by cytological diagnosis at the Korle-Bu Teaching Hospital, Accra, Ghana, ................................................................................................................................. 79 Table 4.3-9: Distribution of low risk, high risk and any type HPV in concurrent cervical cytology outcomes for 538 WLHIV screened at Korle Bu Teaching Hospital, Accra, ................................................. 80 Table 4.3-10: Distribution of HPV genotypes among 538 WLHIV screened at Korle Bu Teaching Hospital, Accra, .............................................................................................................................................. 82 Table 4.6-1: Odds ratios (ORs) for cervical dysplasia and matching 95% confidence intervals (CIs) according to socio-demographic characteristics in WLHIV at Korle Bu Teaching Hospital, Accra, ........................................................................................................................................................ 84 Table 4.6-2: Summary of Model Characteristics for Predicting Cytology Outcome .................................... 86 vii University of Ghana http://ugspace.ug.edu.gh Table 4.6-3: Odds ratios and 95% CI for Abnormal Cytology According to Best Fit Predictive Model ..... 86 Table 4.6-4: Odds ratios (ORs) for cervical dysplasia and corresponding 95% confidence intervals (CIs) according to behavioural and clinical characteristics in WLHIV at the Korle Bu Teaching Hospital, Accra, .............................................................................................................................................. 88 Table 4.6-5: Summary of Model Characteristics for Predicting Cytology Outcome based on behavioural and clinical responses ..................................................................................................................... 90 Table 4.6-6: Odds ratios and 95% CI for Abnormal Cytology According to Best Fit Predictive Model based on behavioural and clinical responses ............................................................................................. 90 Table 4.7-1: Odds ratios (ORs) for high risk HPV infection and corresponding 95% confidence intervals (CIs) according to socio-demographic characteristics in WLHIV at the Korle Bu Teaching Hospital, Accra, .............................................................................................................................. 92 Table 4.7-2: Odds ratios (ORs) for high risk HPV infection and corresponding 95% confidence intervals (CIs) according to behavioural and clinical characteristics in WLHIV at the Korle-Bu Teaching Hospital, Accra, .............................................................................................................................. 95 Table 6.1-1: Combinations of human papillomavirus (HPV) types in women with multiple infections .. 147 viii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1.2-1: Conceptual framework of the interactions of socio-demographic parameters, smoking history, sexual history, clinical history and HPV infection on the pathogenesis of cervical cancer and its precursor lesions among women living with HIV. ........................................................................... 6 Figure 2.2-1: The human papillomaviruses types found in humans fall into five genera, with the Alpha and the Beta/Gamma genera representing the largest groups. The evolutionary tree is based on alignment of the E1, E2, L1, and L2 genes (Doorbar et al., 2012) ................................................. 17 Figure 2.2-2: The alpha-papillomavirus genus of the papillomavirus phylogenetic tree. Oncogenic types closely related to HPV 16 and 18 are highlighted HPV 16 is most closely related to HPV 31. HPV 18 is most closely related to HPV 45. Source: Rose and Stoler, (2006). ........................................ 18 Figure 2.3-1: A schematic diagram of a representative HPV 16 genome. It has three main regions namely, an upstream regulatory region (URR), an early region, which contain open reading frames (ORFs;e.g E1, E2, E3, E4,E5,E6 and E7). (Shah et al., 2020) ........................................................ 22 Figure 2.3-2: A model of the virus capsid depicting superficial pentamers. These conformational epitopes on the major coat or capsid protein L1 displayed on the outer surface of the intact virus particle can be recognized by the immune system and forms the basis of HPV vaccines. Source: Stanley et al., 2006. ............................................................................................................................................... 24 Figure 2.4-1: Estimates of the global burden of HPV related diseases in women. The graph shows on the left hand the clinically apparent conditions related to HPV infections. The right hand side shows the diagnoses that occur in populations where screening is used. The body of the pyramid shows current estimates of the worldwide number of cases, rounded for convenience. Adapted from (WHO/ICO, 2010a). ............................................................................................................... 28 Figure 2.6-1: Global estimates of cervical cancer incidence. The highest adjusted incidence rates occur in Sub-Saharan Africa where fewer studies have been carried out compared to most of Europe and America which have recorded the least number of cases. Adapted from (WHO/ICO, 2010a). ...... 36 ix University of Ghana http://ugspace.ug.edu.gh APPENDICES Appendix 2: ................................................................................................................................................. 141 Appendix 3 .................................................................................................................................................. 144 Appendix 4 .................................................................................................................................................. 146 x University of Ghana http://ugspace.ug.edu.gh ABBREVIATIONS ACIP Advisory committee on immunization practices AIDS Acquired Immune Deficiency Syndrome ART Antiretroviral Therapy ART Antiretroviral Therapy ASC Atypical Squamous Cells ASC-H Atypical Squamous Cells, cannot rule out HSIL ASC-H Atypical squamous cells, cannot rule out HSIL ASCUS Atypical Squamous cells of Undetermined Significance ASCUS Atypical squamous cells of undetermined significance cART Combination antiretroviral therapy CC Cervical Cancer CIN Cervical squamous Intraepithelial Neoplasia dATP Deoxyadenosine Triphosphate dCTP Deoxycytidine Triphosphate dGTP Deoxyguanosine Triphosphate DNA Deoxyribonucleic acid dNTPs Deoxynucleotide Triphosphate ECA Epithelial Cell Abnormality HAART Highly Active Antiretroviral Therapy HIV Human immunodeficiency virus HPV Human papillomavirus HR HPV High risk human papillomavirus HSIL High-grade Squamous Intraepithelial Lesion IARC International Agency of Research on Cancer ICC Invasive Cervical Cancer KBTH Korle-Bu Teaching Hospital LCR Long Control Region LR HPV Low risk human papillomavirus xi University of Ghana http://ugspace.ug.edu.gh LSIL Low-grade Squamous Intraepithelial Lesion NCR Noncoding Region ORF Open Reading Frame SSA Sub-Saharan African STI Sexually transmitted infection URR Upstream Regulatory Region VIA Visual inspection with acetic acid VILI Visual inspection with lugols iodine WLHIV Women living with HIV YLL Years of life lost xii University of Ghana http://ugspace.ug.edu.gh Introduction Chapter 1 INTRODUCTION 1.1 BACKGROUND Globally, about 35 million people are living with HIV of which 16 million are women aged 15 years and above, 80% of whom live in the Sub-Saharan African region (HIV). Low- and middle-resource countries, where women have been hit hardest by the Acquired immune deficiency syndrome (AIDS) epidemic, have historically also had a very high prevalence of human papillomavirus (HPV) infection (Franceschi and Jaffe, 2007). HIV cases in Africa is still on the rise among women due to cultural and heterosexual activities (Awofala and Ogundele, 2018). In 2015, approximately 390,000 new infections of HIV were recorded among young women between the ages of 15 to 24 representing about 60% of all new HIV infection all over the world in this age group (HIV/AIDS, 2018) A number of studies have established that, women living with HIV, especially those having low CD4 T-cell counts are more likely to be infected with human papillomaviruses (HPV) (Burd, 2003b; Strickler et al., 2005), which are the major cause of cervical cancer and it’s precursor lesions (Tornesello et al., 2014) Women living with HIV also have a higher rate of infection with high-risk HPV types, multiple HPV infections and there is also the tendency for the infection to persist for a longer period thus, the likelihood of developing cancer of the cervix (Burd, 2003b; Strickler et al., 2005; Clifford et al., 2006d; Odida et al., 2011; Akarolo-Anthony et al., 2013). It is also well known that, HIV infection itself increases the risk of developing cervical squamous intraepithelial neoplasia (CIN) and cancer of the cervix (Franceschi and Jaffe, 2007) Human papillomaviruses (HPVs) are DNA viruses that infect epithelial (skin and mucosal) cells and they are the most common sexually transmitted infection in both males and females worldwide. They are a heterogeneous group of viruses with circular double-stranded DNA genomes about 8,000 nucleotides in size (SCHEURER et al., 2005) All human papillomavirus 1 University of Ghana http://ugspace.ug.edu.gh Introduction genomes include 3 general regions: (1) an upstream regulatory region (URR), which contains sequences that control viral transcription and replication; (2) an early region, which contains open reading frames (ORFs; e.g. E1, E2, E4, E5, E6 and E7) involved in multiple functions including trans-activation of transcription, transformation, replication, and viral adaptation to different cellular milieus, and (3) a late region, which codes for the L1 and L2 capsid proteins which form the structure of the virion and facilitate viral DNA packaging and maturation. All HPVs described to date contain an E1, E2, E4, L1, L2 and some E6/E7-like functions (Burk et al., 2009). HPV is a major cause of sexually transmitted diseases in both sexes worldwide, with varying degree of prevalence among groups and regions of habitation (De Sanjosé et al., 2007). Infections are mostly asymptomatic and are normally found among sexually active individuals at a point in their lives (Trottier and Franco, 2006; Luchters et al., 2010). Women are at risk and are commonly infected by multiple strains of the virus at young age which makes HPV infection more prevalent among such group as they become sexually active. The prevalence then reduces gradually as they increase in age which may likely be due to the building up of immunity against the virus and reduction in sexual intercourse among them (Khan et al., 2005). Human papillomavirus have two main groups: those found on the surface of the skin and those found in the anogenital tract and the mucosa of the mouth, throat and respiratory tract (Burd, 2003b). Some HPVs causes genital warts, while others are responsible for premalignant squamous intraepithelial lesions of different grades and bad lesions, which are major risk factor for invasive cervical cancers (Burd, 2003b) However, only a few HPV- infected individuals progress to invasive cervical cancer. Most infected individuals eliminate the virus without developing recognized clinical manifestation. 2 University of Ghana http://ugspace.ug.edu.gh Introduction Infection with HPV is also an indication of the risk of cervical and anal cancer in females and anal cancer in homosexual males (Chaturvedi, 2010; Doorbar et al., 2012). Infection in the cervix is found in the transformation zone. The virus must reach the basal cell layer for infection to be initiated, this process normally takes approximately 24 hours to occur.(Crosbie et al., 2013) In the year 2000, sub-Saharan Africa experienced an estimated 57, 000 cases of cervical cancer (22% of cases of cancer among women) and an age-standardized incidence of 31cases per 100,000 women (Zeier et al., 2012). Furthermore, the cervical cancer incidence has not decreased in any region of sub-Saharan Africa in recent decades; in fact, significant upward trends have been reported in several areas (Yakasai et al., 2012; Kayumba, 2014; Wanyoike- Gichuhi et al., 2014; Donkoh et al., 2019b). Because both HPV and HIV infection are sexually transmitted, the two infections are often found together (Clifford et al., 2006b; Park et al., 2014). In addition, as a result of HIV-induced immune impairment, there is also an increased probability that HPV infection will become persistent in HIV-infected women (Park et al., 2014) and evolve into precancerous and cancerous lesions of the cervix uteri (Mbulaiteye et al., 2006). As expected, the relative risk of invasive cervical cancer among women living with HIV infection varies from country to country on the basis of the extent to which premature death due to other causes or early detection of cancer prevents progression of pre invasive lesions to the invasive stage (Franceschi and Jaffe, 2007; Sloan and Gelband, 2007). Effective screening and early treatment of precancerous cervical lesions are, thus, key factors in preventing cervical cancer in HIV-infected women (Humans et al., 2007). High-resource countries have been able to prevent cervical cancer among women with HIV/AIDS to different 3 University of Ghana http://ugspace.ug.edu.gh Introduction extents. In the United States, for example, a high percentage of women (about 81%) who receive treatment for HIV undergo annual Papanicolaou smears (Mbulaiteye et al., 2011) and the vast majority (94%) of cases of cervical carcinoma among these women are detected at the in-situ stage (Frisch et al., 2000). 1.2 PROBLEM STATEMENT Cytological screening of the cervix using the Pap smear test and the early detection of HPV play an important role in the secondary prevention of ICC, thereby reducing HPV-associated mortality (WHO/ICO, 2010b). However, due to a lack of effective screening programs in low- and middle-income countries, including Ghana, cervical cancer precursor lesions most often go undetected resulting in development of ICC with its attendant high mortality rates, in these settings. As both HIV infection and HPV infection are sexually transmitted, the two infections are often found together in populations with a high risk for sexually transmitted infections (Sahasrabuddhe et al., 2007; Bernard et al., 2010). Although most women who develop natural HPV infections have a capacity to clear the infections in a few months, HIV-infected women remain at a continued substantial risk for cervical cancer, even if they receive ART because these treatment programs improve their survival long enough for cervical cancer precursors to manifest and progress to invasive cancer (Mbulaiteye et al., 2006; Sinayobye et al., 2014). With the introduction of highly active ART (HAART), women living with HIV are expected to live longer – an infected 25 year-old-woman has an estimated average survival age of 73.9 years therefore if this woman lives that long, HPV persistence may lead to her developing 4 University of Ghana http://ugspace.ug.edu.gh Introduction cervical abnormalities resulting in cervical cancer, to prevent this, other strategies like regular cervical screenings are urgently needed (Ghebre et al., 2017). Ghana does not have a national cervical cancer screening program presently and therefore Pap smears are routinely ordered for differential diagnosis in cases of unusual vaginal bleeding rather than as part of a routine gynaecologic investigation or screening program (Adanu, 2002). As such, data on cervical intraepithelial neoplasia and invasive cancer among women living with HIV and enrolled/unenrolled on HAART is routinely not available. Unfortunately, knowledge on the epidemiology of HPV virus among women living with HIV in Ghana is limited. It is also well known that there are regional differences in prevailing HPV genotypes, (Clifford et al., 2003; de Sanjose et al., 2010; Forman et al., 2012; Pirog et al., 2014) therefore, there is the need to know the specific prevailing HPV genotypes among women living with HIV in Ghana. No policy on the prevention of cervical cancer has been established among women living with HIV therefore it is very important that the local differences and distribution of HPV genotypes among this group of women be known to help inform policy makers for implementing monitoring and evaluating a cervical cancer prevention program in Ghana. 5 University of Ghana http://ugspace.ug.edu.gh Introduction DERMOGRAPHICS Age Occupation Gravida PRESENCE OF CERVICAL ABNORMALITIES SMOKING HISTORY HPV GENOTYPES Ever smoked (ASCUS, LGSIL, Currently smoking HGSIL OR CERVICAL CANCER SEXUAL HISTORY Normal Cytology Contraceptive use Age at first sex Lifetime sexual partners CLINICAL HISTORY ART Usage CD4 Count Figure 1.2-1: Conceptual framework of the interactions of socio-demographic parameters, smoking history, sexual history, clinical history and HPV infection on the pathogenesis of cervical cancer and its precursor lesions among women living with HIV. 6 University of Ghana http://ugspace.ug.edu.gh Introduction 1.3 CONCEPTUAL FRAMEWORK Figure 1.2.1 looks at the factors associated with acquiring HPV infection and the relationship to progression to precancerous lesions and cervical cancer in women living with HIV. Infection with HPV is a necessary and often sufficient cause of cervical cancer. However, not all HPV infections lead to observable cervical lesions as most viral infections clear naturally over the course of time usually spanning 6-18 months (Castle et al., 2009). In most cases (90% of) infections clear spontaneously within 24 months (Schiffman et al., 2007). Once an infection is cleared, it is undetectable by cytological assessment. Evidence from natural history studies reveal that persistent infection with human papillomavirus (HPV) is a necessity for the development of ICC and precursor lesions (Kjaer et al., 1996; Walboomers et al., 1999). Apart from persistent HPV infection, other genetic, viral and population factors have been suspected to play a role in the development of cervical cancer and the development of precancerous lesions (Schlecht et al., 2001; Trottier et al., 2006b; Bosch et al., 2008; Wang et al., 2010). Non-viral factors like a woman’s age, menstrual status and immunosuppression are also likely to influence the outcome of an HPV infection (Bosch et al., 2006). Cytological screening of the cervix using the Pap smear test and the early detection of HPV in cross-sectional studies can reveal HPV genotypes associated with the spectrum of cytological disease. 1.4 JUSTIFICATION The attribution of individual HPV types to cervical intraepithelial lesions varies ethno- geographically (Vaccarella et al., 2006). Since the genotype profile of HPV infection differ from high-risk population to another, the epidemiology of human papillomaviruses have been found to differ from one region to another as well. Among populations of HIV infected women and 7 University of Ghana http://ugspace.ug.edu.gh Introduction commercial sex workers, diverse HPV genotypes may be prevalent depending on several potential confounders. In a previous study evaluating the concordance of HPV infection among heterosexually active couples and the impact of HIV coinfection on the prevalence of HPV, HIV coinfection in one partner had a significant impact on the prevalence of HPV infection in the other partner (SCHEURER et al., 2005). Concordance of the same HPV genotypes was more commonly found among couples where one or both partners were HIV-infected, compared with HIV-uninfected couples (SCHEURER et al., 2005). The majority of HIV-infected women were infected by their male sexual partners in Korea (SCHEURER et al., 2005). As such, HPV genotyping studies among HIV cohorts allow us to establish and compare HPV genotype distributions in populations with different risk factor profiles, to investigate associations with cervical pre-cancerous and (to a lesser extent cancerous lesions) and to appreciate the behaviour of individual HPV genotypes in this population. Furthermore, HPV typing is of importance for characterizing study populations and for monitoring the efficiency of HPV-targeted therapies and vaccines in high-risk populations (van den Brule et al., 2002). Issues related to the epidemiology of HPV infection in this population of women will provide the basis for evaluating the potential risk of spillover of HPV genotypes into the general population and policy decisions regarding HPV vaccination as a national strategy for cervical cancer prevention, and to determine the specific socio-demographic groups that would need to be aggressively targeted for vaccination to obtain optimum protection. Reliable knowledge of the distribution of specific HPV types along the spectrum of cervical abnormalities will allow us to estimate the burden due to HPV types that are vaccine preventable after taking into account the extended cross-protection offered by available vaccines, and as a 8 University of Ghana http://ugspace.ug.edu.gh Introduction matter of fact, the effect of vaccination in Ghana. It has been strongly inferred that HPV testing would be indispensable in an era of HPV vaccination (Franco et al., 2006). HIV-infected women in various settings were found to have higher rates of persistent HPV infection, more abnormal Pap tests, more cervical intraepithelial neoplasia (CIN) lesions, and aggressive cervical cancer, compared with the general population, and these numbers are increasing (Lörincz and Richart, 2003; Burk et al., 2009). Up to 20 % of HIV coinfected patients develop HPV-induced premalignant lesions of the uterine cervix within three years of HIV diagnosis (Jamieson et al., 2002). Progression of an untreated HPV-induced dysplastic lesion can lead to invasive cervical cancer, an AIDS defining illness (Chaturvedi et al., 2009) The high rate of cervical disease reported in HIV-infected women and the extended life expectancy due to better access to antiretroviral therapy (ART) have led to recommendations for aggressive screening and prompt treatment of cervical lesions; yet, the most appropriate strategy to screen HIV infected women, particularly in resource-constrained settings, remains unclear (Cuzick et al., 2003; Zielinski et al., 2004; Clifford et al., 2006b). Moreover, the existing evidence on the impact of ART on the incidence and evolution of CIN is still unclear, thus reinforcing the recommendation of systematic screening of all HIV-infected women, even those on ART (Burk et al., 2009; Unaids, 2010). Overall, the evidence assessing the extent and magnitude of HPV infection and the burden of cervical abnormalities and cancer among HIV-infected women is sparse and remains largely undocumented. The paucity of literature in this population especially in the HAART era motivated this study. 9 University of Ghana http://ugspace.ug.edu.gh Introduction Thus the need for data on HPV prevalence and genotype distribution in Ghana based on sensitive and specific molecular biomarkers of infection, coupled with the need to investigate the effects of ethno-geographic and demographic variables on the prevention of cervical cancer in Ghana, particularly the impact of vaccination, motivated the present work. It is the first study to report the type-specific distribution of HPV in cervical disease in the Greater Accra region of Ghana, using a reliable and highly practicable molecular assay. It is envisaged that the outcome of this novel study would provide evidence-base information and incidence data that will impact future vaccination programme in Ghana and perhaps influence cervical prevention and treatment. 1.5 RESEARCH QUESTIONS 1. What is the proportion of women living with HIV with cervical abnormalities and cervical cancer? 2. What is the proportion of women living with HIV with HPV infection? Which HPV genotypes are commonly found among women living with HIV in Ghana? 3. What is the relationship between HPV genotypes and the presence of cervical abnormalities and cancer if any among women living with HIV? 4. What are the risk factors for abnormal pap smears among women living with HIV? 5. What are the risk factors for HPV positivity among women living with HIV? 10 University of Ghana http://ugspace.ug.edu.gh Introduction 1.6 GENERAL OBJECTIVE This study aims to determine the prevalence and distribution of HPV genotypes and associated cervical disease in an immunodeficiency care setting in Accra, in the Greater Accra Region of Ghana. 1.6.1 SPECIFIC OBJECTIVES Specific Objective 1: To determine the proportion of HIV positive women with cervical cancer and precursor cell abnormalities at the Korle-Bu Teaching Hospital, Accra. Specific Objective 2: To assess the proportion of HIV positive women with HPV infection at the Korle-Bu Teaching Hospital, Accra. Specific Objective 3: To determine the association between high-risk HPV genotypes and abnormal cytology among HIV positive women attending clinic at the Korle-Bu Teaching Hospital, Accra. Specific Objective 4: To identify the factors associated with abnormal cytology among HIV positive women attending clinic at the Korle-Bu Teaching Hospital. Specific Objective 5: To identify the factors associated with high-risk HPV genotype infection among HIV positive women attending clinic at the Korle-Bu Teaching Hospital. 11 University of Ghana http://ugspace.ug.edu.gh Introduction Chapter 2 LITERATURE REVIEW 2.1 HISTORY OF PAPILLOMAVIRUSES (PVs) IN CANCER As early as 1842, an Italian physician became curious of the increase in mortality as a result of cancer of the cervix that were being recorded in married women, widows and prostitutes living in Verona during the period 1760-1839 after he reviewed their death certificates (Gasparini and Panatto, 2009). Death as a result of cervical cancers was not common in virgins and nuns. This led him to conclude that, whatever was responsible for the cervical cancer that were killing these married women, widows and prostitutes and not virgins and nuns was sexually transmitted. Studies on papillomavirus started in the early twenties when in 1933 it was isolated as the possible causal agent of warts in rabbits. Since then, papillomaviruses have been accepted as a natural infectious agents for the development of warts in various mammals including man (Franceschi and Jaffe, 2007). The warts in rabbits were described as having the potential for malignant transformation by Rous in 1935 and the first visualization of human papillomavirus by electron microscopy was reported in 1949 by Strauss et al. In 1950, the carcinogenic potential of HP virus was established. The viral genomic structure of HPV was first demonstrated in 1963 (Crawford & Follett, 1967). Harald cited that Herpes simplex virus type 2 was initially the causative agent for cervical cancer (Awofala and Ogundele, 2018). It was not until the early 1970s that studies on the possible role of HPV’s in cancer was started. Determination of HPV causation of cervical cancer started in 1972 when it was reported in anecdotal reports in the medical literature of rare malignant conversion of genital warts (Condylomata acuminata) into squamous cell carcinomas (zur Hausen, 1977). It was observed that, as many as 70% of 12 University of Ghana http://ugspace.ug.edu.gh Introduction cervical dysplasia might be associated with the so-called koilocytotic atypia which was a hallmark of HPV infection (Basnet et al., 2018). In November 1991, a workshop convened by the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO) officially concluded that, based on the epidemiological and laboratory data, the association between HPV and cervical cancer was beyond reasonable doubt, and infection with the virus should be considered as cause to the development of cervical cancer (Bosch et al., 1992) 2.2 DIVERSITY OF PAPILLOMAVIRUSES AND EPITHELIAL TROPISMS More than 200 papillomaviruses have been identified and have been completely sequenced based on DNA sequence homology instead of conventional serology (de Villiers, 2013). Out of this, approximately 150 are human papillomaviruses (HPV) (Bernard et al., 2010). In recent years, sensitive detection methods have permitted the identification of a large number of new HPV types (primarily Beta and Gamma types) from swabs taken from cutaneous epithelium (de Villiers, 2013). By convention, but without official seal, genotypes are considered distinct if they share less than 90% homology in the DNA sequence of the open reading frame (ORF) coding for the major (L1) capsid protein (Manhart and Koutsky, 2002; Yu et al., 2022). The traditional papillomavirus types a species share between 71 and 89% nucleotide sequence identity within the complete L1 ORF and subtypes have between 90% and 95% homology, and variants between 96% and 98% (De Villiers et al., 2004; Brancaccio, 2020). 13 University of Ghana http://ugspace.ug.edu.gh Introduction The taxonomy of papillomaviruses, which was initially based on genomic cross- hybridizations and restriction patterns is now a system based on phylogenetic algorithms of sequence homology because papillomavirus genomes are thought to be very static, and sequence mutations or recombination are very rare events: mutational changes are thought to occur at frequencies similar to those of the DNA genomes of the infected host organism (Cogliano et al., 2005; McBride, 2021). Phylogenetically, papillomaviruses are recognized as belonging to the family Papillomaviridae having 18 genera(Fang et al., 2020). The approximately 40 types able to infect the human ano-genital tract (De Villiers et al., 2004; Garutti et al., 2018), collectively called the genital HPVs, belong to the alpha genus. Clusters of lower order are known as species; they are closely related phylogenetically and have similar biological properties but not necessarily oncogenic potential (Bosch et al., 2006). 2.2.1 Classification of HPVs based on disease association The difference in the ability of HPVs to cause malignant transformation forms the bases for their classification into Low and High risk types(Georgescu et al., 2018). This idea was conceived from the observation that, some HPV types were more commonly found in cancers than in benign lesions(de Sanjose et al., 2018). HPVs, designated “low-risk” or “non-oncogenic,” such as HPV-6 and HPV-11, induce benign condylomata acuminata and are very rarely found in genital malignancies.(Nicolàs Pàrraga, 2018) These were later followed by a series of studies which established that these risk categories reflect the inherent and differential abilities of the viruses to interfere with the proliferation and 14 University of Ghana http://ugspace.ug.edu.gh Introduction stability of the genome of the affected cells. E6 and E7 proteins from low-risk HPVs, inactivate cellular p53 and pRb tumour suppressor proteins less efficiently than do E6 and E7 proteins from high-risk HPVs (Münger and Howley, 2002). 2.2.2 Epithelial Tropisms In order for infection to occur, there must be changes in the conformation of the capsid protein(McBride, 2021), which may also influence the tropisms of individual HPV types(Cosper et al., 2021). Therefore, tropisms of the papillomaviruses are thought to be primarily controlled at the level of viral gene expression, where regulatory elements within the Long Control Region (LCR) are an essential determinant (Kranjec et al., 2016; Ina et al., 2021). Furthermore, all papillomaviruses contain well-conserved core genes involved in replication and packaging with greater diversity in the E6, E7, E5, and E4 genes which have roles in driving cell cycle entry, immune evasion, and virus release (Doorbar et al., 2012) and therefore the tropisms of particular types. Notably, Beta papillomaviruses lack a recognizable E5 ORF, located downstream of E2 in Alpha papillomaviruses, and which along with E6 and E7, is involved in immune evasion and in optimizing genome amplification efficiency. The E4 protein (which plays a role in virus escape from the epithelial surface), like E5, shows considerable sequence heterogeneity between types as well. All this diversity is thought to reflect the different tropisms and transmission routes of different papillomaviruses (DiMaio and Petti, 2013),(Doorbar, 2013) Perhaps more surprisingly, given its importance in genome amplification, the HPV E6 protein is absent 15 University of Ghana http://ugspace.ug.edu.gh Introduction in HPV 101, 103, and 108 (Gamma genera (Nobre et al., 2009),(de Villiers, 2013). Mucosal HPV are a group of HPVs with high affinity for mucous membrane and belong to the alpha genus, including the high–risk HPV types 16 and 18 and are associated with more than 99% of cervical cancers(Gheit, 2019). Most cutaneous HPV types express E6 and E7 gene products that are structurally similar to those of the mucosal types but their genome does not contain an identifiable E5 Open- Reading Frame (ORF)(Gheit, 2019).Epidemiological studies have clearly demonstrated that a subset of cutaneous HPV types classified into the beta genus are commonly and consistently found in non-melanoma skin cancers. These skin tumours arise mostly at sites exposed to the sun and, contrary to mucosal types, the EV-HPV DNA copy number appears to be much lower than one copy/cell (Asgari et al., 2008). 16 University of Ghana http://ugspace.ug.edu.gh Literature Review Figure 2.2-1: The human papillomaviruses types found in humans fall into five genera, with the Alpha and the Beta/Gamma genera representing the largest groups. The evolutionary tree is based on alignment of the E1, E2, L1, and L2 genes (Doorbar et al., 2012) 17 University of Ghana http://ugspace.ug.edu.gh Literature Review Figure 2.2-2 : The alpha-papillomavirus genus of the papillomavirus phylogenetic tree. Oncogenic types closely related to HPV 16 and 18 are highlighted HPV 16 is most closely related to HPV 31. HPV 18 is most closely related to HPV 45. Source: Rose and Stoler, (2006). 18 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.3 MOLECULAR BIOLOGY OF HUMAN PAPILLOMAVIRUSES (HPV) Papillomaviruses are diverse and have been found in many different animals as well as in humans and are specific for their respective hosts. More than 200 types of HPV have been identified on the basis of DNA sequence data showing genomic differences(Cullen et al., 2015). They are one of the oldest, largest, and most diverse of the known virus families. HPVs, like all papillomaviruses, target the stratified squamous epithelia of the body(McBride, 2017). Genital Human papillomaviruses (HPVs) are sexually transmitted viruses, the high-risk types are well known to be associated with the development of cancer of the cervix and some cytological precursors which are totally preventable conditions. About 70% of cervical cancer cases are caused by HPV genotypes 16 and 18 and the eight most common genotypes (HPV 16, 18, 31, 33,35,45,52 and 58) contribute to 90% of cervical cancer cases(Zhao et al., 2017). 2.3.1 Organization of the HPV Genome The HPV genome is functionally recognisable as three regions with the coding potential of about six early genes (E1 to E7; there is no E3), two late genes (L1 and L2) and an upstream regulatory region (URR) or noncoding region (NCR) of about 850 base pairs separating the first two regions(Azzawi, 2013). In general, each open reading frame (ORF) in a papillomavirus genome is often referred to as a gene. However, a gene, in molecular terms, is defined as the entire nucleic acid sequence that is necessary for the synthesis of a functional transcript. According to this definition, a gene is not equivalent to an ORF. In eukaryotes and many viruses, a gene usually contains exons and introns(Sieber et al., 2018). An ORF encoding a polypeptide is usually spread across multiple exons from various parts of the genome which are combined into a full-length ORF through RNA splicing(Dhamija and 19 University of Ghana http://ugspace.ug.edu.gh Literature Review Menon, 2018). This is particularly true in papillomaviruses even though only one ORF (E1^E4) in papillomaviruses spans two separate exons. The three regions in all papillomaviruses are separated by two polyadenylation (pA) sites: early pA (AE) and late pA (AL) sites (Zheng, 2014). The early region of papillomavirus genomes occupies over 50% of the virus genome from its 5′ half and encodes six common open reading frames (E1, E2, E4, E5, E6 and E7) (Doorbar et al., 2012)that translate individual proteins as briefly described below. Two other ORFs, E3 and E8, were also assigned to this region initially, but have now been recanted. The late region of all papillomavirus genomes, covering almost 40% of the virus genome, lies downstream of the early region and encodes L1 and L2 ORFs for translation of a major (L1) and a minor (L2) capsid protein that encapsulate the viral DNA (Zhu et al., 2017) and are useful in the setting of vaccine design (zur Hausen, 2002). All eight viral open reading frames are arrayed in an overlapping linear fashion on one of the double-stranded circular DNA genome. Protein is encoded by both the early and late gene regions, but the URR does not encode (Park et al., 1995). The URR possesses the origin of replication, numerous binding sites for many repressors and activators of transcription including cis-elements required for regulation of gene expression, replication of the genome, and its packaging into virus particles, implying that it may help in defining the hosts for exact HPV types (Turek, 1994). Functionally, the early genes (E1 to E7) are necessary for the replication of the viral DNA and for the assembly of newly produced virus particles within the infected cells. The late genes are expressed just before the egress of virions. Notably, the viral genome does not encode a unique DNA polymerase, which is required for DNA replication, and therefore the virus must depend on 20 University of Ghana http://ugspace.ug.edu.gh Literature Review the replication machinery of the host-cell for the generation of viral progeny (Munoz et al., 1996). 2.3.2 Structure of Human Papillomaviruses Despite the different diseases they cause, HPVs are small, rounded, non-enveloped DNA tumour viruses with a common non-enveloped icosahedral structure, normally between 50- 60 nm in diameter (Zheng and Baker, 2006). The HPV virion consists of a single molecule of circular, double-stranded DNA about 8 kilobase pairs in length that is bound to cellular histones and contained within a symmetric icosahedral protein coat, the capsid, which is made by the spontaneous assembly of the L1 major and L2 minor capsid proteins (Winer et al., 2003). It is important to note that HPV virus-like particles (VLPs) can be produced by the expression of L1, alone or in combination with L2, in mammalian or non-mammalian expression systems. 21 University of Ghana http://ugspace.ug.edu.gh Literature Review Figure 2.3-1: A schematic diagram of a representative HPV 16 genome. It has three main regions namely, an upstream regulatory region (URR), an early region, which contain open reading frames (ORFs;e.g E1, E2, E3, E4,E5,E6 and E7). (Shah et al., 2020) Their genomes are made up of double stranded circles (episomes) of close to 8000 base pairs, which contain eight or nine Open Reading Frames (ORFs). Although gene number is limited by the small size of the papillomavirus genome, the number of encoded protein is much greater, as gene expression involves the use of multiple promoters and complex pattern of splicing (Zheng & Baker, 2006)(Shah et al., 2020). The virus coat contains 360 molecules of L1 proteins organized into 72 capsomers, each containing of 5L1 molecules (Schiller & Lowy, 2012). HPV particles also contain different number of L2 molecules which are not fully exposed on the surface of the virion (Rubio et al., 2011). During 22 University of Ghana http://ugspace.ug.edu.gh Literature Review infection, L2 becomes available for binding to the extracellular matrix and is cleaved by furin (Kines, Thompson, Lowy, Schiller, & Day, 2009). The main exposed surface region of L1 are made up of a series of hypervariable amino acid loops that are unique between different papillomavirus types, in response to host immune selection pressure with antibodies raised to one HPV type binding to distantly related types occurs only poorly. The virus genome also encodes regulatory proteins that stimulate cell cycle entry and cell proliferation as well as proteins that mediate virus genome replication and transmission. Many of these genes are found within the early region of the virus but the L2 gene product also has key immediate early function in viral genome delivery within the cell (Wang & Roden, 2013). 23 University of Ghana http://ugspace.ug.edu.gh Literature Review L1 Figure 2.3-2: A model of the virus capsid depicting superficial pentamers. These conformational epitopes on the major coat or capsid protein L1 displayed on the outer surface of the intact virus particle can be recognized by the immune system and forms the basis of HPV vaccines. Source: Stanley et al., 2006. 24 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.4 SOME DISEASES ASSOCIATED WITH MUCOSAL HPV IN HIV PATIENTS HPVs are highly ubiquitous and also distributed throughout the body, but with different anatomic predilections that allow three major groups of HPV-associated diseases to be distinguished: (1) cutaneous warts, such as plantar, common, and flat warts; (2) epidermodysplasia verruciformis, a rare autosomal recessive genetic hereditary skin disorder in which the affected individual is abnormally sensitive to HPV (mostly 5 and 8) and has a high- risk of carcinoma of the skin; and (3) genital or mucosal lesions including conditions such as genital warts, laryngeal papillomas, as well as pre-cancers (dysplasias or intraepithelial neoplasias) and cancers of the uterine cervix, vagina, vulva, penis, anus, and of the head and neck region (Bosch et al., 2006). 2.4.1 Benign HPV-associated cervical lesions Condyloma acuminatum or cervical warts results when HPV infect the ano-genital region with low-risk HPV types such as HPV 6 and 11, although many other genotypes can also be found, including HPV 2, 16, 18, 30–33, 35, 39, 41–45, 51–56, and 59 (McKee et al., 1981; Clifford et al., 2005a; Petry et al., 2012). They present as papules, nodules or soft, filiform, pinkish, sessile or pedunculated growths. In men, genital condylomas more commonly involve the coronal sulcus, the glands penis, and the penile shaft. In women, lesions commonly affect the external genitalia and the cervix (Chuang, 1987) They may be diagnosed by cytologic evaluation of a Papanicolaou (Pap) smear or at the time of colposcopic evaluation for an abnormal Pap smear. The subtle appearance of thickened, raised, and whitish epithelium can be easily concealed by cervical mucus (Bosch et al., 2006). These warty lesions may present at multiple sites, affecting not only the external cervix (exocervix), but potentially extending up 25 University of Ghana http://ugspace.ug.edu.gh Literature Review into the cervical canal to the squamo-columnar junction (Bosch et al., 2006). The HPV types that cause benign genital warts can also cause problematic papillomas at oral sites, which can be difficult to treat because of their location. Focal epithelial hyperplasia is a rare HPV-related disease of the oral mucosa that is more common in children and women. Lesions are mainly located in the lower lip, but less frequently may affect the upper lip, tongue, oral mucosa, oropharynx, palate, and floor of mouth. HPV 13 and 32 are the most common cause (Vera-Iglesias et al., 2007). 2.4.2 Pre-malignant HPV-associated lesions Pre-cancerous lesions are defined biologically as lesions that have, in principle, a capacity to progress potentially to invasive cervical cancer if left untreated (Cuschieri et al., 2004). The pre-malignant changes characteristic of cervical cancer represent a spectrum of histological abnormalities ranging from low-grade squamous intraepithelial lesion (LSIL)/CIN1 through high-grade squamous intraepithelial lesion (HSIL)/CIN2 to HSIL/CIN3 carcinoma-in-situ (Woodman et al., 2007). The severity of neoplasia reflects the extent to which basal-like cells (i.e. poorly differentiated cells with a high nuclear/cytoplasmic ratio) extend toward the epithelial surface and the extent of suprabasal cell division. Low-grade lesions typically show evidence of productive viral infection with the presence of koilocytes in the suprabasal cell layers being regarded as a key manifestation of CIN1/LSIL. HPV is detectable in 90–100% of cervical abnormalities, ranging from incipient cytological abnormalities and dysplasia (Bedoui et al., 2009)to cervical cancer (Solomon et al., 2002; Jenkins, 2007)[50–(Muñoz et al., 2003) 26 University of Ghana http://ugspace.ug.edu.gh Literature Review HSILs are mostly found in association with HR-HPV while LSILs are frequently associated with LR-HPV-6 and -11 (Lacey et al., 2006). 2.4.3 Cervical Cancer It is now universally acknowledged that malignant squamous and glandular neoplasms of the cervix are caused by infection of cervical epithelium by specific HPV types (Bosch et al., 1995a; Munoz et al., 2003a). Persistent intraepithelial neoplasias may eventually evolve into invasive carcinoma (Cuschieri et al., 2004). Almost 90% of cervical cancers are squamous cell carcinomas. The fraction of cervical cancer attributable to HPV-16 is 53.5% (Munoz et al., 2004). Taken together, HPV-16 and HPV-18 account for about 70% of all cervical squamous cell carcinomas. Furthermore, the projected HPV-16/18 fraction is higher in more advanced (72– 77%) than in less advanced (65–72%) regions. Approximately 41–67% of high-grade squamous intraepithelial lesion (HSIL), 16–32% of low-grade squamous intraepithelial lesion (LSIL) and 6–27% of atypical squamous cells of undetermined significance (ASCUS) are also projected to be HPV-16/18-positive (Munoz et al., 2004). Taken together, HPV is responsible for 5% of all human cancers, over 10% of cancers in women and up to 15% of cancers in women in developing countries (Winer and Koutsky, 2004). 27 University of Ghana http://ugspace.ug.edu.gh Literature Review Figure 2.4-1: Estimates of the global burden of HPV related diseases in women. The graph shows on the left hand the clinically apparent conditions related to HPV infections. The right hand side shows the diagnoses that occur in populations where screening is used. The body of the pyramid shows current estimates of the worldwide number of cases, rounded for convenience. Adapted from (WHO/ICO, 2010a). 2.4.4 Other anogenital cancers These include cancers of the vulva, vagina, penis, and anus. Most vulvar cancers (92%) are solitary, keratinizing squamous cell carcinomas (SCC). HPV prevalence is 90% in vulvar intraepithelial neoplasia and basaloid or warty cancers, but is found in only 6% of keratinizing SCC (Hording et al., 1996) (Toki et al., 1991). HPV 16 is the most prominent type in vulvar cancer, with HPV 18, 21, 31, 33, and 34 detected at lower frequencies. In addition, HPV is responsible for 85% of vaginal cancer, with HPV 16 being detected in 60% of invasive tumors. 28 University of Ghana http://ugspace.ug.edu.gh Literature Review HPV is also detected in basaloid and warty cancers of the penis, but only rarely in keratinizing SCC and verrucous cancers of the penis. In invasive penile cancer, HPV 16 is the most prevalent type (40–70%), followed by HPV 6 (22%), 52 (15%), and 11 (4%) (Rubin et al., 2001). HPV is present in 80–96% of anal cancer with HPV 16 being the most prevalent type (De Vuyst et al., 2009) Anal cancer is more common in men who have sex with men, individuals with a history of anal warts, and in immunosuppressed populations. 2.4.5 Head and neck cancer HPV is recognized as a major risk factor for the development of head and neck squamous cell cancers (HNSCC). A recent meta-analysis showed that HPV prevalence in HNSCC increased significantly from 41% in 2000 to 72% in 2004 (Mehanna et al., 2013). HPV prevalence is significantly higher in oropharynx SCC than in the oral cavity with the tonsil having higher prevalence than other anatomic sites (Herrero et al., 2003). These HPV-associated cancers display clinical and molecular features distinct from other HNSCCs. The patients with HPV- positive cancer have at least a 50% improvement in overall survival at 5 years, which is equivalent to an approximate 30% difference in absolute survival. HPV association is now part of routine diagnostic procedures when assessing the prognosis of HNSCC. HPV 16 is the most common type found in HNSCC, but other HPV types such as 18, 31, 33, and 35 can also be detected (Mehanna et al., 2013; Awuah, 2014). 29 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.5 EPIDEMIOLOGY OF HPV INFECTION HPV, the most common infection acquired sexually contribute to approximately 5% of all human cancers and 12% of all female cancers (zur Hausen, 2002). With improving methods of detection, HPV DNA has been found in almost all cervical cancer cases worldwide (Bosch et al., 1995b; Walboomers et al., 1999). In addition to cervical cancer, high-risk HPV infections of the penis, vulva, and vagina can lead to cancer at these sites. Though these cancers are not as common as cervical cancer in developing countries, an estimated 85% of anal cancers, 50% of the cancers of the vulva, vagina, and penis, 20% of oropharyngeal cancers, and 10% of laryngeal and oesophageal cancers are attributable to HPV (zur Hausen, 1996; WHO, 1999). Numerous studies have stated the prevalence of HPV DNA in both ambiguous and negative cytologic results as well as in cancer (IARC, 1995; Gjooen et al., 1996; Ferenczy et al., 1997; Bosch and de Sanjose, 2003; Baseman and Koutsky, 2005; Herrero et al., 2005; Domfeh et al., 2008) . Crude prevalence estimates of HPV infection (based on HPV testing of asymptomatic women in the general population) range from 2 to 44%. This wide variation in prevalence estimates could also be explained by age differences among population samples studied, and by differences in the molecular sensitivity of the various HPV DNA assays used to detect viral DNA (Bosch and de Sanjose, 2003). The designated “high-risk” group comprises fifteen members (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) (Munoz et al., 2003b). High-risk HPV DNA has been shown to be present in (Bosch et al., 1995b; Walboomers et al., 1999) and implicated in the 30 University of Ghana http://ugspace.ug.edu.gh Literature Review development of cervical pre-malignant lesions and invasive cervical cancer worldwide (Kjaer et al., 1996; Liaw et al., 1999; Nobbenhuis et al., 1999; Wallin et al., 1999). Recent evidence suggests that so-called high-risk types account for almost 90% of cervical infections (Aral and Holmes, 1999). Twelve are classified as “low-risk” types (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and CP6108) and have the least propensity to persist and induce malignant lesions (Munoz et al., 2003b). Three are classified as “probable high-risk” types (26, 53, and 66) (Munoz et al., 2003a) being less frequently found in cancers but are often found in squamous intraepithelial lesions (SILs). Some authors refer to these HPV types as intermediate-risk (Burd, 2003a). 2.5.1 Global Prevalence of HPV DNA Genital HPV infection is the most common STI among women (Coutlee et al., 2002). An estimated 291 million women are expected to be infected with HPV-DNA worldwide. Out of this, approximately 105 million women worldwide will have an HPV-16 or -18 infection at least once in their lifetime (Burchell et al., 2006). There have been studies on the prevalence of HPV DNA in cervical specimens from women with all possible cytological and histological outcomes. The diversities in methodology limit comparability of findings from these studies. In this regard, the International Agency for Research on Cancer’s (IARC) data from 15 areas in 4 continents regarding women aged 15 to 74 years provides a suitable reference point for HPV epidemiology. In this large-scale study, the age-standardized prevalence ranged from less than 5% in some Mediterranean and South East Asian countries to more than 15% in several countries in Latin America and among a few African populations (Clifford et al., 2005b). In another comprehensive review of studies that used standardized inclusion criteria 31 University of Ghana http://ugspace.ug.edu.gh Literature Review and controlled for variables that may have challenged the comparability of the studies, prevalence estimates of HPV infection among women with negative cytology results ranged from 10% to 15%. Age-specific prevalence estimates showed HPV DNA to be more prevalent among young women, with a decline in young adult women and a variable pattern afterwards (Kjaer et al., 2001). In some countries, notably in the Americas, the prevalence increased again in postmenopausal age groups. In Europe, a plateau in the middle-age groups was maintained whereas in other high-prevalence countries in Asia and Africa the prevalence remained fairly constant across all age groups (Kjaer et al., 2001). A meta-analysis of 78 published studies estimated HPV prevalence among women with normal cytology (Gellin et al., 2000; Womack et al., 2000). The adjusted global prevalence of 10.41% (95% confidence interval, CI: 10.2– 10.7%) compares well with the IARC finding, with considerable variation by region. Only 8 out of the total 78 studies came from Africa, representing 4% of the total number of women tested. Africa had the highest age-adjusted prevalence of 22.12%. The WHO estimates that 21.5% of women from Western Africa have an infection at a given time (WHO/ICO, 2010a). 2.5.2 Prevalence of HPV Genotypes in Cervical Cancer A collective look at 12 studies carried out across 25 countries using standardized protocol and GP5+/6+ HPV-DNA testing by means of PCR assessed HPV prevalence in 3085 women with cancer of the cervix (Munoz et al., 2004). The general HPV-DNA occurrence was 96% and the 15 common types observed, in descending order were, HPV-16, -18, -45, -31, -33, -52, - 58, -35, -59, -56, -39, -51, -73, -68 and -66. HPV-16 and -18 accounted for 70% and the eight most found types (HPV-16, -18, -45, -31, -33, -52, -58 and 35) which contributed to 89% of all cervical cancer cases worldwide (Clifford et al., 2006a). A greater prevalence of HPV-16 32 University of Ghana http://ugspace.ug.edu.gh Literature Review was observed in Northern Africa, Europe and North America, of HPV type 45 was observed in sub-Saharan Africa and of HPV type 31 in Latin America (Munoz et al., 2004). A complete meta-analysis of greater than 14,500 cases from studies put out up to January 2006 has been done (Curtis and Sutherland, 2004) the result was similar the results of the pooled study. The common HPV types seen were, HPV-16, -18, -31, -33, -35,-45, -52, and -58. HPV-16 prevalence was different from 52% in Asia to 58% in Europe, and HPV-18 was not the same 13% in South/Central America to 22% in North America (Curtis and Sutherland, 2004). 2.5.3 Transmission and Natural History of Cervical HPV Infection HPV is the most common sexually transmitted disease among women (Coutlee et al., 2002). Available experimental evidence suggests that a cervical HPV infection, as distinguished from HPV infections at more superficial sites which may follow vertical or horizontal transmission, is only acquired as a result of penetrative vaginal sexual intercourse (Ley et al., 1991; Koch et al., 1997; Quint et al., 2012). Barnabas and colleagues (de Sanjose et al., 2007) estimated the probability of male-to-female transmission for HPV-16 as 60% per partner making it more transmissible than other viral STIs but comparable to bacterial STIs (Fleury et al., 2008) such as gonorrhoea (50%) and syphilis (60%) (Cutts et al., 2007). In order for the HPV to be able to enter and start an infectious process there is a requirement for a loss of continuity of the epithelium, so that the virus can be in contact with the permissive cells, which are the basal cells of the epithelium.(Miller and Torday, 2017) Once they have infected the target cells, viral replication starts. The assembly of the virions is carried out in 33 University of Ghana http://ugspace.ug.edu.gh Literature Review the upper strata of the epithelium when the cells have differentiated (granular cells), since this is a prerequisite for this maturity event and cell differentiation. During sexual activity, micro-trauma of the genital epithelium, particularly in the transformation areas of the cervical epithelium, allows exposure of the basal cells to active proliferation of the various types of HPV, allowing union between the receptor of the basal cells with the protein from the viral capsid L1. Once the virus is joined to the cell surface, it beds into the host cell cytoplasm, a process that has been identified as endocytosis (Bousarghin et al., 2003). Cervical HPV infection is usually only probable after most women begin their first sexual relationship (Collins et al., 2002). Due to the usually transient nature of infection, the virus might be detected only intermittently and the concurrent or sequential detection of different HPV types is common (Ho et al., 1995). The median duration of a newly detected cervical HPV infection is reported to be approximately 9 to 12 months, with only 10% of infections still detectable after 24 months (Woodman et al., 2001). In practice, it must be noted that there is often an overestimation of the median duration of infection since infections clear quickly (e.g.,<3–4 months) and escape most study designs (Ho et al., 1995). HR-HPV infections, particularly HPV 16, infections tend to last longer than those of LR-HPV types (Castellsague et al., 2001; Woodman et al., 2001). 2.5.3.1 Multiple Infections Co-infection with multiple HPV types is found in 20% to 50% of HPV-infected women (Liaw et al., 2001; Clifford et al., 2005b) and is currently arousing much research interest. Although the tendency of multiple HPV types to cluster within women has been frequently observed 34 University of Ghana http://ugspace.ug.edu.gh Literature Review (Thomas et al., 2000; Liaw et al., 2001; Rousseau et al., 2001; Mendez et al., 2005), just 1 out of 12 designated carcinogenic variants must necessarily be present to cause cervical cancer (Walboomers et al., 1999). Results from longitudinal studies in HPV-co-infected women propose that the existence of more than one HPV genotypes does not support persistence of HPV infections (Molano et al., 2003; Plummer et al., 2007). It is believed that the excess infections are independent events occurring because all HPV infections generally share the same transmission route and are all associated with the same risk factors (Thomas et al., 2000; Mendez et al., 2005). 2.6 EPIDEMIOLOGY OF CERVICAL CANCER 2.6.1 Globally and in Africa World-wide, cervical cancer is the second commonest cause of female cancer after breast cancer, accounting for up 13% of all female cancers. Each year, across the world, approximately 490,000 women are newly diagnosed and 274,000 die from invasive cancer of the uterine cervix induced by oncogenic types of human papillomavirus (HPV) (Franceschi et al., 2006; Ginsburg et al., 2017) The overwhelming majority of women affected by this completely preventable disease live in resource-constrained nations where access to screening services and health insurance coverage is limited or non-existent (Franceschi et al., 2006; Louie, De Sanjose, & Mayaud, 2009; Wabinga et al., 2014). In the so called developed countries less than 0.5% of women will develop cervical cancer before age 65 but in developing countries, 2% of women are likely to get it before age 65 (Parkin and Bray, 2006; 35 University of Ghana http://ugspace.ug.edu.gh Literature Review WHO/ICO, 2010a). The highest number of cases per region is reported in sub-Saharan Africa, South America, South-Central Asia and South-Eastern Asia (Parkin and Bray, 2006). In West Africa, estimated age-standardized rates per 100,000 women per year range from 26.9 in Cote D’Ivoire, through 28.6 in Burkina Faso, 30.0 in Togo, 33.0 in Nigeria, 39.5 in Ghana to 56.3 in Guinea as compared to 15.2 in the world ( Figure 2.6-1) (WHO/ICO, 2010a). Figure 2.6-1: Global estimates of cervical cancer incidence. The highest adjusted incidence rates occur in Sub-Saha ran Africa where fewer studies have been carried out compared to most of Europe and America which have recorded the least number of cases. Adapted from (WHO/ICO, 2010a). 36 University of Ghana http://ugspace.ug.edu.gh Literature Review Mortality rates are substantially lower than incidence. Worldwide, the ratio of mortality to incidence is 55% (Parkin and Bray, 2006). Because cancer of the cervix is found cervical young women, it is a leading cause of years of life lost (YLL). In Latin America, the Caribbean and Eastern Europe, cervical cancer contributes greatly to YLL than other diseases such as tuberculosis, maternal conditions or AIDS. It also makes the major input to YLL from cancer in the highly populated regions of sub-Saharan Africa and South-Central Asia (Parkin and Bray, 2006). 2.6.2 Ghana Women aged 15 years and above make up 6.57 million of Ghana’s population and this age group can get cervical cancer (WHO/ICO, 2010a). There is little information on the actual prevalence and incidence of cervical cancer in Ghana owing to lack of a national population- based cancer registry. The country has several cancer registries which are hospital-based data collected from hospital cases. It is estimated that Ghana has one of the highest rates in the world being a third world country with no organized cervical cancer prevention programme (Parkin et al., 2003; Parkin and Bray, 2006). In one hospital-based study, cervical cancer accounted for 58.3% of all gynaecologic cancers (Agorastos et al., 2005). In 2006, Wiredu and Armah (2006) reported a hospital based review of human tisues and mortality records of a 10 year period in which cervical cancer was the fourth most common cause of cancer 37 University of Ghana http://ugspace.ug.edu.gh Literature Review mortality in females after malignancies of the breast (17.24%), haematopoietic organs (14.69%), and liver (10.97%). Based on data collected elsewhere in sub-Saharan Africa, the WHO projected that cervical was the leading cause of female mortality among the cancers with over 2,000 deaths in the year 2010 alone (IARC and WHO, 2008; WHO/ICO, 2010a). The Kumasi Cancer Registry was established in Ghana in 2012 to provide information on cancer cases seen in the city of Kumasi. Recently, the registry has published data from the year 2012. Among females, the most common cancers were breast cancer (33.9%), cervical cancer (29.4%), ovarian cancer (11.3%) and cancer of the Endometrium (4.5%) (Laryea et al., 2014). 2.7 EPIDEMIOLOGY OF HIV/AIDS IN GHANA The HIV epidemic in Ghana is described as a widespread epidemic with constant prevalence of more than 1%in the general population (Ghana, 2012) Using the annual sentinel surveys carried out among antenatal attendants, the HIV epidemic in Ghana seems to have a downward trend, from 3.6% in 2003 through to 2.2% in 2008 and to 2.0% in 2011(Ampofo, 2009; Agyei- Mensah and Aikins, 2010). The estimated deaths that occur annually for 2010 and 2011 were 17,230 and 15,263 respectively. Sentinel survey data for 2012 gave a prevalence of 1.7% and 11,655 AIDS death. In 2013 sentinel report, the prevalence of HIV further decreased to 1.3% with an estimated death of 10,074.Recent data from the 2014, 2015 and 2016 sentinel reports depicts an upsurge of HIV prevalence. The HIV prevalence rate for2014 was 1.6% (up from 1.3% in 2013), then went up to 1.8% in 2015 and then 2.4% in 2016 38 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.8 HUMAN PAPILLOMAVIRUSES AND HIV/AIDS 2.8.1 Effect of HIV infection on HPV Acquisition Human papillomavirus (HPV) is the most common sexually transmitted infection and at least 50% of sexually active people will get HPV infection at some time in their lives (Bernard et al., 2010). HPV infection can lead to genital warts and (pre)malignant lesions. It is estimated that 80% of women get infected by hrHPV in their lifetime. However, most of these infections are sub-clinical or transitional and in normally cleared after 12-30 months in 70-90% of infected women (Alba, Cararach, & Rodríguez-Cerdeira, 2009). For both hrHPV and low risk viruses, there are reports that the infection lasts for about 4-5 months (Alba et al., 2009). Though majority of infections can be cleared within 2 years by the human immune system, some infections persist in infected cells and for people with a compromised immune system this leads to a rapid transformation of the infected cells to precancerous and cancerous lesions (Hawkins et al., 2013). A number of studies have shown that HIV-infected women are at a higher risk for HPV infection and progression of HPV lesions to cervical cancer compared to HIV-negative women (Palefsky et al., 1999);Banura et al., 2011). Underlying this observation is that HIV infected women seem to have a higher HPV DNA persistence rate than HIV negative women (Banura et al., 2010) and are also infected with multiple HPV types than HIV-negative women (Chakravarty et al., 2016; Williamson, 2015). In a study carried out in South Africa, a high prevalence of multiple HPV infections was observed (Mbulawa, Coetzee, & Williamson, 2015). Invasive cervical cancer samples were found to have far more multiple HPV infections 39 University of Ghana http://ugspace.ug.edu.gh Literature Review (PR =1.75, 95% confidence intervals: 1.18 to 2.58) in a meta-analysis carried out among women infected with HIV in Africa (Clifford et al., 2016). In a recent meta-analysis, it was found that women living with HIV have a higher chance of getting infected with the human papillomavirus (Liu, Sharma, Tan, & Barnabas, 2018). In cross-sectional studies HIV seropositivity is associated with increased prevalence of cervical HPV infection, greater prevalence of precancerous cervical lesions, and with grade of dysplasia. Prospective studies have demonstrated that low CD4+ T-cell count and/or high HIV viral load are associated with the incident detection and duration of HPV infection. A review of available literature on the HPV type-specific risk among HIV-seropositive women (Clifford et al., 2006b) revealed that the proportion of HIV-positive women with HPV16 rose with increasing severity of cervical lesions. Nevertheless, HPV16 remained underrepresented in HIV positive women with HSIL, who were significantly more likely to be infected with HPV types 11, 18, 33, 51, 52, 53, 58 and 61 compared with the general female population with HSIL. Regardless of the type of treatment, there is a higher failure rate for cervical neoplasia in women with HIV, although the subsequent lesions are typically low grade. The rationale for treatment is to eliminate pre-cancerous lesions and ensure the lowest possible risk of cancer through constant surveillance. In addition, the large numbers of individuals infected with other HR-HPV genotypes (Banura et al., 2011) suggests that these other HR-HPV types should be considered in the development of the next generation of vaccines. 40 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.8.2 Prevalence of HPV in HIV infected women The prevalence of HPV among women living with HIV varies between parts of the globe, 80% in Zambia and 90-100% in Uganda (Banura et al., 2011). In another related study carried out in Brazil, an HPV prevalence of 65.6% was observed among HIV positive men undergoing antiretroviral treatment (Guimarães et al., 2011). A prevalence of 64% was observed in a meta- analysis carried out on women living with HIV in Kenya (Menon et al., 2016). It is reported that, HIV infection increases the prevalence of HPV among women (Liu, Sharma, Tan, & Barnabas, 2018). In spite of the variation in prevalence of human papillomavirus infection among women living with HIV from different parts of the globe, some reports indicate a twofold higher prevalence equalled to their HIV negative counterparts. Sub-Saharan Africa reported 62% HPV prevalence compared to 24%, 30% compared to 5% in North America and 30% compared to 14% in Europe, in HIV positive and HIV negative women respectively (Bruni et al., 2010; Clifford et al., 2017). In a study carried out in India, a prevalence of 32.2% HPV infection was observed among HIV positive women compared to 9.1% among HIV negative women (Sarkar et al., 2011). A meta-analysis of data from United States of America showed HPV prevalence of 46% among women living with HIV compared to 29% in women from the general population (Park et al., 2016). In a study carried out in Cape Town on 1371 HIV-negative women and 8050 HIV-negative women, there was an increase in the prevalence of high-risk HPV (HR-HPV) among HIV positive women (52.4%) than among HIV negative women (20.8%) (McDonald et al., 2014). 41 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.8.1 HIV and HPV type distribution HIV infection is a strong independent risk factor for cervical cancer (Schlecht et al., 2001) and it is not surprising that the association between cervical cancer and HIV infection is reflected by a corresponding correlation between HPV infection and HIV infection (Ferenczy et al., 2003). Actually the high incidence of cervical cancer among HIV positive women may be explained by increased HPV persistence (Minkoff et al., 1998; Ahdieh et al., 2001; Moscicki et al., 2004), susceptibility to a broad range of HPV genotypes (Goncalves et al., 1999; Baay et al., 2004; Chaturvedi et al., 2005), especially the highly oncogenic types 16 and 18, and to infection with multiple HPV types (Sun et al., 1997). A review of available literature on the HPV type-specific risk among HIV-seropositive women revealed that the proportion of HIV-positive women with HPV16 rose with increasing severity of cervical lesions. There is mounting evidence that other HPV types such as HPV 33, 35, 45, 52 and 58 can be more prevalent than HPV 16 and 18 in some African countries (Munoz et al., 2004). For example, the proportion of HPV infection caused by HPV-16/18 was lower in HIV- positive women in Cameroon after HPVs 45, 58 (Desruisseau et al., 2009). Also, HPV 35 was slightly more common than HPV 16 in Mozambique both in women with normal cytology and in those with HSIL or worse (Castellsague et al., 2001). HPV 52 was found slightly more frequently than HPV 16 or HPV 35 in Kenya (De Vuyst et al., 2003), in Zambia (Sahasrabuddhe et al., 2007), in Burkina Faso (Didelot-Rousseau et al., 2006) in colposcopically normal women in Zimbabwe (Gravitt et al., 2002) and in women presenting to a sexually transmitted infections clinic in Uganda (Blossom et al., 2007). In Senegal, HPV 42 University of Ghana http://ugspace.ug.edu.gh Literature Review 16 and 58 were the most common types overall and in women with cervical lesions (Xi et al., 2003). 2.8.2 Persistence of HPV in HIV Positive Women Aside increasing prevalence, immunodeficiency has also been a major cause of HPV persistence among both males and females worldwide mostly as a result of HIV infection A study carried out in Nigeria established that, HIV positive women have higher persistence of HPV infection than their HIV negative counterparts (Adebamowo et al., 2016; Adebamowo et al., 2017){Gupta, 2015 #1248 A seven-fold increase in the rate of persistence of human papillomavirus infection was observed over a twelve-month period in a cohort of HIV infected young women in South Africa (Adler et al., 2015). A couple of other studies have also established that there is evidence of persistence of HPV infection among women living with HIV (Kelly et al., 2017){Miranda, 2013 #1261}(Dalstein et al., 2003; Lörincz and Richart, 2003; Cogliano et al., 2005; Kriek et al., 2016). 2.8.3 HIV and Cervical Cancer Cervical cancer is the major disease caused by human papillomavirus infection in women living with HIV. A recent systematic review and meta-analysis showed that, HIV positive women had increased incidence of Low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions ( HSIL) which was largely due to increased HPV persistence(Liu et al., 2018) The odds of having CIN2+ was 6.55 times ( 95% CI 4.44-9.67) greater in HIV positive women was observed in a study in Malawi (Kohler et al., 2016). In 43 University of Ghana http://ugspace.ug.edu.gh Literature Review another study, increased cervical cancer was documented among HIV positive women than in the general population with standardized incidence ratios (SIRs) of 4.2 to 8.9 (Chaturvedi, Madeleine, Biggar, & Engels, 2009) 2.9 PREVENTION OF CERVICAL DISEASE IN HIV 2.9.1 Primary Prevention with Available Prophylactic Vaccines It is well understood that population-based prophylactic immunization based on an efficacious vaccine could potentially prevent the occurrence a greater proportion of cervical cancer and precancerous lesions were it to achieve wide- spread coverage (de Sanjosé et al., 2011). Gardasil TM (Merck &CO;Inc), Cervarix TM (Glaxosmithkline) and Gardasil 9TM ( Merk&Co;Inc) are the names of the three vaccines available for use against HPV infections (Herweijer et al., 2016; Joura et al., 2015). Each of these three vaccines fights against the acquisition of HPV 16 and HPV 18 infections which are known to cause over 70% of cervical cancer. The quadrivalent type has HPV 6 and HPV 11 added to fight against genital warts infection and finally the 9-valent vaccine has HPVs 31,33,45,52 and 58 added. Efficacy of these vaccines among HIV negative women is 98% for preventing type specific Cervical Intraepithelial Neoplasia (CIN) and 100% of all genital warts (Joura et al., 2015) Vaccine efficacy studies are needed on HIV positive women to establish the benefits of the vaccine to such a population. It has been recommended that HIV positive adolescents and adults be vaccinated between the ages of 13 to 25 (Masur et al., 2014). The right antibody response to the vaccine is needed to establish lifetime immunity among women living with HIV to prevent precancerous lesions and therefore cervical cancer. A study on sero-conversion among women 44 University of Ghana http://ugspace.ug.edu.gh Literature Review aged 13 to 45 showed highest conversion in those with CD4 count greater than 200 cells/µl compared to those lower than 200 cells/μl (Kojic et al., 2014). In another study, 90-100% sero- conversion rates were recorded in HIV infected boys and girls aged 7-12 years (Weinberg et al., 2012). 2.9.2 Universal/ Targeted Screening The value of all cervical cancer screening techniques is in their ability to detect precursor cancer lesions in the cervix that can readily be remedied to avert cancer. Thus, screening is not a test for cancer. The paradigm of cervical cancer screening with use of cervical cytology (Pap smear) is the prime example of such a system. In advanced countries, strategic and universal cytology screening has massively reduced risk for cervical cancer. Regardless of the promise of universal vaccination, it is unlikely that screening programmes could be discontinued or even scaled-down for several reasons: immunization will only protect against HPV types that are targeted by the vaccine; protection will not be absolute and its longevity is uncertain; as yet, the possibility of genotype replacement cannot be excluded; and older women not covered by vaccination programmes will continue to be at risk. 2.9.3 Cervical Cancer Screening among HIV Positive Women It is well known that, HPV infections are higher and can persist in women living with HIV and because of its persistence there is the need for regular screening to detect early any abnormality for prompt treatment to prevent the development of cancer of the cervix among this vulnerable group of women. It is recommended that such groups of women undergo Pap 45 University of Ghana http://ugspace.ug.edu.gh Literature Review test yearly for three years and if consecutively normal then the test is recommended every three years (Frellick, 2017). The screening strategies being used are cytology based method with Papanicolaou test (Pap test), HPV DNA testing and visual inspection with acetic acid (VIA), visual inspection with Lugols iodine (VILI). In the less developed regions where there is limited resources and no instituted national cervical cancer control policy, a “see and treat” tactic is recommended by the World Health Organization (WHO). This provides an immediate treatment with cryotherapy without colposcopy and biopsy reducing loss to follow up rate. VIA has been found to show a good performance with a sensitivity of 48-76% for CIN2+ (Chibwesha et al., 2016; Firnhaber et al., 2013). Women living with HIV need exceptional precautions when using a VIA based screening model but testing for HPV provides a number of advantages which includes easy and a short time for sample collection compared to VIA and the likelihood of self-sampling. 2.10 SCREENING FOR CERVICAL DISEASE 2.10.1 Morphological Examination Epithelial cell abnormalities usually develop without recognizable symptoms. Therefore the rationale for screening for cervical cancer has always been to detect abnormal cervical cells: including precancerous cervical lesions, as well as early cervical cancers at an early stage where remedial measures may be taken at reasonable cost. However, most women in countries with poorly-resourced healthcare systems do not have access to reliable screening programs 46 University of Ghana http://ugspace.ug.edu.gh Literature Review and consequently report to hospital only when lesions are at an advanced invasive stage. Women living with HIV are not excluded from this accessibility challenge. Overall screening and diagnosis is a major challenge in developing countries. The estimated coverage of cancer screening in developing countries is 19% compared to 63% in developed countries. However, in some areas, a screening rate of 1% or less has been observed in countries such as Bangladesh, Ethiopia, and Myanmar (Wabinga et al., 2000; Clifford et al., 2003). 2.10.2 DNA Testing for the presence of high-risk HPV types HPV genomes and viral oncoproteins, which are present in all affected cells, represent convenient markers for a transient or persistent infection. The rationale for designing HPV DNA detection tests, also known as HPV testing, is based on the knowledge that persistent infection with high-risk HPV is a necessary cause of cervical cancer (Schiffman et al., 1993; Walboomers et al., 1999). The definitive diagnosis of current human papillomavirus (HPV) infection is made by detection of viral nucleic acids using molecular detection methods (Woodman et al., 2001; de Sanjose et al., 2003). Several methods of HPV testing have been developed to be able to do this effectively. In general, these tests can be grouped into (1) those reporting a pooled “high-risk” positive/negative result versus genotype specification, and (2) those targeting viral DNA versus viral mRNA or oncoprotein. 47 University of Ghana http://ugspace.ug.edu.gh Literature Review 2.10.3 Indications for Cervical HPV DNA Testing HPV DNA testing has also been recommended to replace or be combined with conventional cytological screening procedures but the former may be prohibitively expensive in settings where it is needed most. DNA testing is already employed as an adjunct to cervical cytology. Reflex HPV DNA testing is recommended in all cases of ASC-US. If high-risk HPVs are absent, the patient is returned to routine screening cervical cytology a year later. If high-risk HPVs are present, the patient is referred to immediate colposcopy with biopsy. If competently performed, these techniques clearly improve the detection rate of high-risk HPV DNA in women, and also avoid some false-negative diagnoses (Schlecht et al., 2001). In women who have had negative cytology but positive high-risk HPV DNA, repeat testing with both tests after a year may reassure the woman. Joint Pap and HPV testing is advocated to increase the sensitivity of either screening method for obstinate cervical lesions. According to some reports (Arbyn et al., 2006a), a small percentage of patients testing positive for high- risk HPV types with normal cytology will go on to develop HSIL as early as six months on follow-up. Other unique scenarios supporting repeat HPV DNA testing are in the case of a persistent ASC-US Pap with negative colposcopic evaluation. Finally, those women that have had both negative cytology and negative high-risk HPV DNA on an annual screen may be candidates for combined testing every three years. 48 University of Ghana http://ugspace.ug.edu.gh Materials and Methods Chapter 3 MATERIALS AND METHODS 3.1 STUDY DESIGN AND SETTING A cross-sectional observational study was undertaken among women living with HIV and attending clinic at the Korle-Bu Teaching Hospital. Women presenting for routine clinic were selected from May 2011 to December 2014 to participate in a study to characterize the cytological lesions and the prevalence of human papillomaviruses (HPV) as well as to screen for predictive factors for cervical abnormalities and high-risk infection among this population. 3.2 STUDY LOCATION The study site was the HIV Clinic at the Korle-Bu Teaching Hospital (Latitude 5 degrees, 36 minutes north; Longitude 0 degrees, 10 minutes east). Korle-Bu Teaching Hospital is located at Guggisberg Avenue (10 minutes’ drive under normal traffic from the central business centre of Accra, which is the capital town of the Greater Accra Region of Ghana. Accra has projected population of 4 million, making it the eleventh largest metropolis in Africa. Accra is sited in the coastal savannah ecological zone of Ghana with a land size of about 173 km2. The average daily temperature is around 30oC (86oF) and receives an annual rainfall of between 600 mm and 800 mm. Korle-bu Teaching Hospital was established on October 9, 1923 and it has grown from an initial 200 bed capacity to about 2,000. It is currently the third largest hospital in Africa and the leading national referral centre in Ghana (KBTH, 2017). It has currently 17 clinical and diagnostic departments/units and an average daily attendance of 1,500 patients with about 250 daily patient admissions. It has three centres of excellence, the National Cardiothoracic Centre, the National Reconstructive Plastic Surgery and Burns Centre and the National Radiotherapy 49 University of Ghana http://ugspace.ug.edu.gh Materials & Methods Centre (KBTH, 2017). In addition, it is the leading tertiary hospital in Ghana and serves as a research and training facility for students and researchers of the University of Ghana. The HIV Clinic is located in the Fever's Unit under the Department of Medicine, KBTH. Currently the KBTH HIV Clinic has a 24-beds and runs the largest cohort of HIV patients in Ghana. Presently about 20,000 patients attend the clinic of which about 11,000 are on ART (KBTH, 2017). The Korle-Bu Teaching Hospital has been providing ART services to PLHIV since December 2003. The hospital runs the HIV Clinic three days in a week (Mondays, Wednesdays and Fridays) and operates an institutionalised electronic database which stores all clinic visits as well as medications, laboratory investigations and other patients’ data. Currently the staff strength at the clinic is 5 clinicians, 30 nurses, 3 public health nurses, 8 pharmacists, 4 biomedical scientists, 6 volunteers, 8 counsellors and 5 data room staff. The fever's unit also houses cases of rabies and tetanus (3-bed capacity) (KBTH, 2017). 3.3 STUDY POPULATION AND SAMPLING 3.3.1 Study population The study population is the 20,000 HIV-positive patients who attend the HIV clinic and the sampling frame is the electronic register of patients (KBTH, 2017). The patient population is made up of about 11,000 on ART with the rest on care. Each patient is scheduled to attend the clinic once every three months (at a minimum) for both clinical assessment and dispensing of medication (for patients on ARVs) (KBTH, 2017). Some patients have been on treatment for the past 11 years. At HIV clinic enrolment, patients routinely present with baseline clinical and laboratory evaluations like physical examinations, full blood count, liver enzymes, serum creatinine, and 50 University of Ghana http://ugspace.ug.edu.gh Materials & Methods CD4+ T-cell count. Follow-ups are done every 3 months whilst occasional viral load is requested for when treatment failure is suspected since it is expensive and patients are unable to afford as a routine laboratory investigation. The recommended baseline CD4 + T-cell count for initiating ART was a count of 250 cells/μL or less and WHO clinical stage III/IV regardless of CD4+ count. This changed later to 350 cells/ μL or less in the 2011 guidelines and is presently pecked at 500 cells/ μL (NACP, 2014). Patients receive comprehensive care at a minimum cost of GH¢15 ($3.00) or receive free care when registered under the National Health Insurance Scheme. Irrespective of these requirements, patients not meeting the criteria are not excluded from receiving continuous care (KBTH, 2017). The care includes medical review, laboratory examinations, adherence counselling and general education on the disease and its management. Adherence is reviewed at every clinic visit and documented and is considered satisfactory when patients’ self-report of compliance tallies with pill count (NACP, 2014). 3.3.2 Sampling Technique A sample of 538 women attending HIV clinic at the Korle Bu Teaching Hospital were randomly selected to be part of the study. Any patient attending the clinic during the study period was given a chance to participate in the study after fulfilling the inclusion criteria. Sampling was carried out on clinic days. List of clients attending clinic on each of these days was obtained and a sample size of 10/day was selected by computer generated random digit numbers. A maximum of 10 clients per clinic day was selected because of time constraint in taking the smears by the gynecologist within the clinic operating hours. Informed consent was 51 University of Ghana http://ugspace.ug.edu.gh Materials & Methods obtained from all women recruited for this study and clients were given the option of refusing participation without any effect on their treatment at the clinic. 3.3.3 Selection of study participants A randomised selection of study participants was carried out as follows: The sampling unit was made up of individuals attending the HIV clinic which operates 3 days in a week (Mondays, Wednesdays and Fridays) and attends to about 150 patients on each of these days. Samples were picked from patients booked for each of the clinic days. Patients visit the clinic every three months which indicates that, a patient is likely to present to the clinic at least once every 36-clinic days. Sampling was done to give every patient an equal chance of being selected to be part of the study; hence, computer generated random sequence of eight unique code numbers was generated from the sampling frame for each clinic day for a 36 clinic day cycle (before the same group of patients returned for review). On each clinic day, five (5) patients were recruited into the study and sampled. Every patient in a particular day's list (sampling frame) was given a unique code number (extracted from folder number). Previously sampled patients were excluded from subsequent sampling frame based on date of last clinic attendance. After recruitment, patients were taken through the consent process and sampled only after satisfying the study inclusion/selection criteria. Recruited patients who did not satisfy the inclusion/selection criteria or who refused to take part in the study on any particular study day were not replaced but the enrolment procedure continued until the sample size estimated was reached. A total of 109 clinic days were used for the recruitment of study participants. 52 University of Ghana http://ugspace.ug.edu.gh Materials & Methods 3.4 INCLUSION CRITERIA All clients 18 years and above presenting to the clinic with a verifiable diagnosis of HIV infection were qualified to be included in the study. . 3.5 EXCLUSION CRITERIA Individuals who failed to give consent were not allowed to participate in this study. Pregnant women and women who had undergone hysterectomy or conisation, or could not undergo an interview or a speculum vaginal examination were excluded from the study. 3.6 SAMPLE SIZE DETERMINATION In the present study, a sample size of 538 women was considered ideal to afford adequate statistical power to achieve all the objectives and meet underlying statistical assumptions for inferential statistics. For objectives 1 and 4, to meet the objective of estimating the prevalence of a categorical dependent variable (presence of Pap smear abnormality) with two outcomes in a population of women attending clinic, a cohort of 334 individuals was calculated to give adequate power to the study. The minimum required sample size was calculated using StatCalc application of EpiInfo 3.5.3 using the formula N= z2 p (1-p)/d2 at 95% confidence interval. In this equation, N= desired sample size, z= the standard normal deviation (set at 1.96 corresponding to the 95% CI), p= the proportion in the target population estimated to have the outcome of interest, d= Precision or degree of accuracy desired (set at 0.05). In a previous study, the prevalence of Pap smear abnormalities among women attending reproductive health services was 44% (Dames et al., 2009). For Objective 2, the aim was to estimate the occurrence of a categorical dependent variable (presence of high-risk HPV infection) with two outcomes 53 University of Ghana http://ugspace.ug.edu.gh Materials & Methods in the WLHIV. The prevalence of HR-HPV infection in the population was established at 23% (Donkoh, 2015). Similarly, after correcting for population size, it was determined that 270 subjects was a suitable sample size to estimate the prevalence of high-risk HPV infection among participants in this setting with precision (d=0.05) at 95% confidence level. For objectives 3 and 5, the minimum required sample size was calculated using the formula N= z2 p (1-p)/d2 at 95% confidence interval. In this equation, N= desired sample size, z= the standard normal deviation (set at 1.96 corresponding to the 95% CI), p= the proportion in the target population estimated to have the outcome of interest, d= Precision or degree of accuracy desired (set at 0.05). In previous studies in sub-Saharan Africa, prevalence rates have ranged from 40-86% in women living with HIV (Yar et al., 2015; Menon et al., 2016). From this, a 50% prevalence of HPV was assumed yielding a maximum sample size estimate of 450. Taking all these sample requirements into consideration, five hundred and fifty women were selected in order to retain a dependable sample in the event of attrition and unforeseen participant absenteeism. Twelve patients were excluded for not giving consent and pre- analytical errors, leaving a final sample of 538 women. 3.7 Data Collection Tools: Pretesting and Administration An in-depth, semi-structured, interviewer-administered questionnaire (Appendix 1) and all other data collection and screening tools were pre-tested for a week in a non-study area. Lessons learnt from the exercise provided a rational basis to restructure the format and presentation of interview guides and questionnaire for optimum achievement of study aims and benefits. The pre-tested questionnaire, which was in English, was administered in Twi by the investigator in the presence of the nurses as required. It was ensured that participants understood the questions first, before answering them. Information was sought on demographics and socioeconomic characteristics. 54 University of Ghana http://ugspace.ug.edu.gh Materials & Methods 3.8 SPECMEN COLLECTION AND LABORATORY ANALYSIS/TESTS 3.8.1 Smear Preparation, Fixation and Transportation Papanicolau (Pap) smear was done on all participants. Epithelial cells were obtained from the cervix with Pap Pak® cytology kit (Medical Packaging Corporation, Camarillo, CA, USA) using guideline provided by the producers of the product with minor alteration. Briefly, pre- labelled cervical smears were taken by performing vaginal speculum examination of the cervix, inserting the tip of the cervical spatula up through the cervical external os and rotating it around the ecto- and endo-cervix with special emphasis on the squamo-columnar junction. The sample on the spatula was rapidly but lightly stroked, thinly and evenly across the surface of the slide without any delay and immediately covered the slide with a mixture of 95% ethanol and 5% polyethylene glycol (carbowax) (BD-TriPath Imaging) according to the manufacturer’s instructions. Endocervical smears were taken with brush applicator by rotating slightly in endocervical canal. Sampled cervical material was spread out evenly onto appropriate sections on slides by rolling endocervical brush across the glass slide from the frosted end towards the opposite end. The preparation was immediately fixed as before. Finally, slides were allowed to dry, packaged in mailing pouches and taken to the Cytology Laboratory accompanied with completed cytology request forms. The request forms accompanying the specimen were completed with the patient’s name, a unique study and hospital code, age, date of collection of specimen, date of last menstrual period and other pertinent clinical information. 3.8.2 Staining and Screening of Cytological Slides The modified Papanicolaou method that uses a standard nuclear stain, hematoxylin and two cytoplasmic counterstains (Orange-G-6 & Eosin Azure) has been recommended for the staining of cervical cytology slides (Koss and Melamed, 2006). Consequently, in order to 55 University of Ghana http://ugspace.ug.edu.gh Materials & Methods remove the carbowax protective layer, fixed smears were placed in 95% ethanol overnight. , Slides were then passed through a second alcohol wash to ensure complete removal of carbowax and hydrated under running tap water for 4 minutes. Slides were stained in Mayer’s haematoxylin nuclear stain and blued in alkaline running water for 3 minutes each after arranging them in racks. Slides were then primed by washing them in 95% ethanol for 2 minutes after which they were stained in Orange G stain for 2 minutes and rinsed with 95% ethanol. Slides were then stained with Eosin Azure. Finally, slides were washed in two changes of absolute ethanol to dehydrate them and cleared in xylene. DPX mountant that is compatible with the clearing agent, transparent and with a refractive index similar to the glass slides and specimen was used for binding the coverslip onto the slide (GCC Diagnostics, Gainland Chemical Co., Sandycroft Flints, UK). Slides were placed on the bench to air dry after which they were screened for any intra-epithelial lesions. Slides were examined independently by two experienced cytotechnologists at the Korle-Bu Teaching Hospital. All positive cases, unsatisfactory smears and 10% random selection of all negative cases were reviewed by a senior pathologist for final interpretation, reporting and quality control. 3.8.3 Schedule for Repeat Cytology Repeat sampling for all cases with unsatisfactory smears were performed after 8 weeks. This is because the scraped surface may not have re-epithelialized and the chance of a false negative result is increased before 6 weeks (Chan et al., 2002). 56 University of Ghana http://ugspace.ug.edu.gh Materials & Methods 3.8.4 DNA Sample Collection Exfoliated cervical cells wre collected with the cytobrush after preparation of the Pap smears and washed into a pre-labelled tube containing DNA Guard (Biometrica, San Diego, USA), to preserve cellular DNA at room temperature (for up to three months) until DNA extraction using the QIAamp DNA Mini kit. 3.8.5 HPV-DNA Extraction, Detection and Genotyping DNA was extracted using a commercial kit (Qiagen Ltd, Maryland, USA) using the manufacturer’s instructions as shown in Appendix 2. Briefly, cervical cells were lysed by incubation at 56oC with 20µl QIAGEN Proteinase K in a proprietary buffer AL (300 µl). Next, 400 µl ethanol (96–100%) was added to the extracted DNA to prime and bound to a special matrix in 2ml spin columns. The sample was washed with buffer AW1 and AW2 by centrifuging at 8000 rpm for 1 min and 14,000 rpm for 3 min respectively. The bound, pureDNA was eluted into 50µl Buffer AE and stored at -70oC in duplicate until further processing. HPV-DNA was detected and identified by nested multiplex PCR (NMPCR) (Sotlar et al., 2004). A forward primer (GP-E6-3F) and two backward primers (GP-E7-5B and GP-E7-6B) constituted general primer PCR. The PCR reaction mix of 50µl contained 10X PCR buffer, 2.5 mM MgCl2 200µM of each of the four deoxyribonucleoside triphosphates (dNTP), 15pmols of each E6/E7 consensus primers and 1.25 units of Taq polymerase enzyme. Four microlitres (4µl) of DNA amplification reactions were carried out using extracts as templates. . This was done using a thermal cycler (BIOER GenePro thermocycler, BIOER Co., USA). The cycling parameters for the first round PCR with E63F/E75B/E76B consensus primers was 94o C for four minutes, followed by 40 cycles of 94o C for one minute, 40o C for two minutes, 72o C for two minutes and a single final elongation step of 72o C for 10 minutes. In the second 57 University of Ghana http://ugspace.ug.edu.gh Materials & Methods round PCR, 2µl of first round PCR product, 15pmols of forward and reverse primers for genotyping were used. Primers for the identification of high-risk genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 and low-risk genotypes 6/11, 42, 43, and 44 were used in four cocktails, each containing four to five different primer pairs (Appendix 3). For the second run, the parameters used in the first run PCR mix were kept but the cycling parameters were: : 94o C for four minutes followed by 35 cycles of 94o C for 30 seconds, 56o C for 30 seconds, 72o C for 45 seconds and a single final elongation step of 72o C for four minutes (Sotlar et al., 2004). 3.8.6 Analysis of Amplification Products The amplified products were run by gel electrophoresis on 2% agarose gel and stained with 0.5µg/ml ethidium bromide. Ten microlitres of each sample was added to 2µl of orange G (5X) gel loading dye. The DNA marker used was a hundred base pair molecular weight (Sigma, MO, USA) which was run alongside the PCR products. The gel was prepared and run in 1X TAE buffer using a mini gel system at 100 volts for one hour and photograph taken over UV illuminator (Sotlar et al., 2004). 3.9 DATA MANAGEMENT AND STATISTICAL ANALYSIS The data obtained through respondents’ questionnaires was checked for accuracy and entered immediately into the computer using Microsoft® Excel® (Microsoft Corporation, Redmond Campus, Washington DC, USA) microcomputer software. Data was analysed and managed using SYSTAT 12 for Windows (2007) (SYSTAT Software Inc., Florida, USA) and GraphPad Prizm 5.31 for Windows (2007) (GraphPad Software, San Diego California USA). All HPV types were classified as low or high risk for high-grade cervical disease or as uncharacterized. HPV assignments were as described elsewhere (Munoz et al., 2003a). HPV types grouped within the high-risk category included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 58 University of Ghana http://ugspace.ug.edu.gh Materials & Methods 59 and 68. HPV types grouped within the low-risk category included 6/11, 42, 43, 44 and 66. Women with multiple HPV types were considered to be in each risk category on the basis of the detection of any 1 of the high-risk or low-risk, HPV types. Analyses were not restricted to women with only single HPV infections. A goal of the statistical analysis was to describe the relationship of sex history and age to the detection of high-risk, low-risk, and uncharacterized HPV types, as well as to evaluate additional subject characteristics for their association with the detection of these HPV groups after controlling for sex history and age. Sex history included number of lifetime sex partners, number of sex partners in the past year, age at first intercourse, number of years since first sexual intercourse, and sex history of the current sex partner. Additional subject characteristics included age, ethnicity, marital status, education level, age at menarche, parity, smoking history, history of use of condoms, and birth control pills. Defining the relationship among HPV detection, sex history, and age was done with simple contingency tables. Initial analysis was done to acquire descriptive statistics. Categorical variables were compared using Chi square test and student t-test was used for quantitative variables. For hypotheses comparing frequencies among groups, X2 tests was performed; comparisons of subgroups (e.g., gender, race) was assessed by logistic regression. The multivariate binary logistic regression model was used to determine the odds of the predictors on the dependent variable. A backward stepwise regression selection was adopted for the outcome and the predictor variables. Several models were fitted. For Objective 4, the question to be addressed was, which of the explanatory variables are predictive of abnormal cytology? Investigation of the determinants of abnormal cytology proceeded in two steps. First, unadjusted relationships between abnormal cytology and potential predictor variables were examined singly with margin of error set at .30. Then, the regression 59 University of Ghana http://ugspace.ug.edu.gh Materials & Methods procedure was repeated to create the best model for predicting the effect of potential variables on cytology outcome. In the multivariate logistic regression model, age, educational status, occupation, marital status, income, ethnicity, age at first pregnancy and age at sexual debut were initially entered in one step. Then non-significant variables were removed in stepwise fashion to give the best fit model. The final model based on educational status, income, and age at sexual debut. These steps were repeated for behavioural and clinical characteristics of women. In the initial model, smoking history, current tobacco use, condom use, HIV duration, ART use, ART duration, discharge, sores and CD4 count were loaded in one step. The final model was based on ART use and CD4 count. For Objective 5, the question to be addressed was, which of the explanatory variables were predictive of high-risk HPV infection? Investigation of the determinants of high-risk HPV infection proceeded in two steps. First, unadjusted relationships between high-risk HPV infection and potential predictor variables were examined singly with margin of error set at .30. In univariate analysis, formal education and estimated income had significant main effects for high-risk HPV infection and therefore were associated with high-risk infection. This relationship did not disappear after adjusting for potential confounders such as woman’s age, younger age at sexual debut and recent sexual activity. Recent sexual activity is defined for a woman who reported having a sexual partner in past six months. The final regression model was based on age, educational status, occupation, marital status, income, ethnicity, age at first pregnancy and age at sexual debut. 3.10 ETHICS APPROVAL Approval was sought and obtained from the Korle Bu Teaching Hospital Ethical Committee and the College of Health Sciences ethics review board (MS-Et/M-P4.6/2012-2013). Study participants were sufficiently told of the reasons, nature, processes, risks and dangers of the 60 University of Ghana http://ugspace.ug.edu.gh Materials & Methods procedure. Importance was placed on anonymity, secrecy and the free will to decide not to be part of the study any time without any consequences. Informed consent: All study subjects consented by signatures.t. 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 an informed consent sheet. Confidentiality: Each participant’s responses were kept confidential and data collected were kept for the purpose of this study only. Safety procedures: Cytology samples were taken strictly by gynaecologists who have been taking samples for more than one year. Women showing signs of sexually transmitted infection e.g. (cervicovaginal discharge) were referred for treatment and asked to return to the clinic after two weeks for study screening and enrolment. 61 University of Ghana http://ugspace.ug.edu.gh Results Chapter 4 RESULTS 4.1 Demographic Profile of Study Population The presentation of the demographic and behavioral profile of the population is important because this study investigated the association of some demographic and behavioural characteristics with the detection of 18 low-risk and high-risk genital HPVs in HIV-infected women recruited at the Korle-Bu Teaching Hospital for cervical screening. Cervical samples were obtained from 538 women aged 20 to 69 years. The mean age of the participants was 40.7 years (SD=8.4years). Age and all other continuous variables studied were found to follow a Gaussian distribution according to D’Agostino-Pearson normality test. Percentages were calculated based on the total number of respondents to each demographic parameter. The four major ethnic groupings in Ghana were represented in the study as follows: Akan (48.8%), Mole-Dagbani (16.9%), Ewe (19.5%) and Ga (19.3%). Illiteracy was reported by 23.8% of the women. The greater fraction of the study population had completed primary school (57.4%), while 4.6% had accessed tertiary level education. Married women formed 38.3% and 23.4% were widowed. Single women accounted for less proportion of 2.0% of the study participants. With regards to employment, majority of the women were involved in non-formal work, mostly petty trading, and accounting for 78.8%. 62 University of Ghana http://ugspace.ug.edu.gh Results Table 4.1-1: Socio-demographic characteristics of Women Living with HIV (WLHIV) at the Korle Bu Teaching Hospital, Accra, Variable Frequency Percentage Valid responses Age (years) 538 <25 6 1.1 25-34 118 21.9 35-44 248 46.1 45-54 137 25.5 55-64 29 5.4 Formal education 538 None 128 23.8 Primary 309 57.4 Secondary 76 14.1 Tertiary 25 4.7 Occupation 538 Civil servant 35 6.5 Unemployed 79 14.7 Non-formal work 424 78.8 Marital status 5 Divorced 96 17.8 Married 206 38.3 Single 110 20.5 Widowed 126 23.4 Income level (Gh¢/month) 532 <100 138 26.0 100-249 254 47.7 250-500 41 7.7 >500 27 5.1 Unsure 72 13.5 Ethnicity 532 Akan 230 43.2 Ewe 105 19.7 Ga 104 19.5 Mole-Dagbani 91 17.1 Others 2 0.4 63 University of Ghana http://ugspace.ug.edu.gh Results Table 4.1-2: Reproductive characteristics of WLHIV at the Korle Bu Teaching Hospital, Accra, Variable Frequency Percentage Valid responses Only wife 507 Yes 494 97.4 No 13 2.6 Other wives 13 1 11 84.6 2 2 15.4 Recent sexual partners 538 Yes 265 49.3 No 273 50.7 Lifetime sexual partners 263 <3 63 24.0 3-6 185 70.3 >6 15 5.7 Age at coitache 531 <16 81 15.2 16-18 247 46.5 >18 203 38.2 Age at first pregnancy 511 <18 91 17.8 18-25 333 65.2 >25 87 17.0 Number of pregnancies 511 <2 73 14.3 2-4 252 49.3 >4 186 36.4 Number of miscarriages 511 None 386 75.5 1 73 14.3 2 29 5.7 >2 23 4.5 Induced abortions 511 none 255 50.0 1 96 18.8 2 72 14.1 >2 88 17.2 Number of still-births 511 None 484 94.7 1 24 4.5 64 University of Ghana http://ugspace.ug.edu.gh Results 2 3 0.6 Condom use 538 Never 256 47.6 Sometimes 137 25.5 Always 145 27.0 Table 4.1-3: Clinical characteristics of WLHIVat the Korle- Bu Teaching Hospital, Accra, Variable Frequency Percentage Valid responses HIV duration (months) 535 <48 118 22.1 48-71 119 22.2 72-95 104 19.4 96-119 106 19.8 >119 88 16.4 ART use 511 Yes 429 84.0 No 82 16.o ART duration (months) 424 <30 148 35.0 30-59 95 22.4 60-89 128 30.2 >89 53 12.5 Vaginal discharge 537 Yes 35 6.5 No 502 93.5 Presence of sores 537 Yes 10 1.9 No 527 98.1 65 University of Ghana http://ugspace.ug.edu.gh Results The proportion of women involved in polygamous marriages were 2.4% and half of them (50.7%) had not been sexually active within the last six months prior to the time of administering the questionnaire. For those who responded to the question on “lifetime sexual partners” 34% had between three to six partners and 2.8% had above six partners. Approximately half of the women (45.9%) had their first sexual encounter between the ages of 16 and 18 years whiles 15.1% had theirs below 16 years of age. A greater proportion of the women (61.9%) had their first conception between the ages of 18 and 25 years and 46.8% had between 2 and 4 conceptions but the average number of lifetime pregnancies was 4.10 (SD=2.71) and a few reported from 10 to 32 pregnancies. Majority (71.7%) had no miscarriages and 47.4% had not had any abortions. Almost all the women (90.0%) had never had a still birth and approximately half had never used condoms before whiles 27.0% always use condoms. Women on ART accounted for 79.9%, there was no vaginal discharge in 93.3% and no vaginal sore (98.0%). 4.2 Proportion of WLHIV with Cervical Cytology Abnormalities Out of the 538 women for whom a Pap smear was available for evaluation, 433 (80.5%) were negative for intraepithelial lesion or malignancy. In addition, 26 slides were unsatisfactory for evaluation due to various reasons and 27 slides had neither transformation zone components nor features of dyskaryosis/intraepithelial lesions (Table 4.2-1). Although these were considered negative, false negatives are likely to be included since sampling of the transformation zone is not assured. In all, the proportion of women with abnormal cytology was 9.7% (52 out of 538 women). Fifteen women (2.8%) showed atypical squamous cells of undetermined significance (ASCUS), 21 (3.9%) low-grade squamous intraepithelial lesions, and 15 (2.8%) high-grade squamous intraepithelial lesions. None of the women was 66 University of Ghana http://ugspace.ug.edu.gh Results suspected to have invasive squamous cell carcinoma and none was found to have atypical glandular cells or adenocarcinoma. Other infections were detected. Among all women screened, 69 (12.8%) had bacterial vaginosis alone, 30 (5.6%) had vaginitis alone, 14 (2.6%) had candidiasis alone and 1 (0.2%) had Trichomonas vaginalis infection. Four representing 0.7% of the women had bacterial vaginosis and Trichomonas vaginalis together. Table 4.2-1: Summary of Papanicolau Test Results for WLHIV at the Korle Bu Teaching Hospital, Ghana, Cytology Result according to Bethesda System Frequency Percentage (%) (N) Abnormal cervical cytology ASCUS 15 2.8 ASC-H 1 0.2 LGSIL 21 3.9 HGSIL 15 2.8 Normal cervical cytology NILM* 433 80.5 No TZC 27 5.0 Unsatisfactory 26 4.8 Total 538 100.0 NILM: Negative for intraepithelial lesions or malignancy, ASCUS: Atypical squamous cells of unknown significance, ASC-H: Atypical squamous cells, cannot rule out HSIL, LGSIL: Low-grade squamous intraepithelial lesions, HGSIL: high-grade squamous intraepithelial lesions. 67 University of Ghana http://ugspace.ug.edu.gh Results 4.3 Prevalence of HPV Genotypes among Unscreened Women Living with HIV Cervical swabs were obtained from 538 HIV-positive women with no history of Pap smear screening and analysed for 18 HPV types. Table 4.3-2a shows the prevalence of HPV types detected in smears from these women. The prevalence of low-risk HPV (LR-HPV), types 6/11, 42, 43, 44 and 66, is shown in Table 4.3-2b and that of high-risk HPV (HR-HPV), types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 in Table 4.3-2c. The occurrence of HPV DNA was 52.0% (280/538 women, 95% CI: 47.7-56.3). The prevalence of LR-HPV without taking note of other types was 22.3% (120/538 women; 95% CI 18.8-26.1) while that of HR HPV without accounting for other types was 41.8% (225/538 women; 95% CI 37.6-46.1). LR HPV only infection was seen in 10.2% (55/538 women, 95%CI 7.8-13.1). HR HPV only infection was seen in 29.7% (160/538 women, 95% CI 25.9-33.8). Both HR and LR were seen in 12.1% (65/538 women, 95%CI 9.4-15.1). . 68 University of Ghana http://ugspace.ug.edu.gh Results Table-4.3-1: Prevalence of human papillomavirus (HPV) in WLHIV at the Korle Bu Teaching Hospital, Accra, Ghana,. Parameter Frequency Percentage HPV present (N=538) %a Yes 280 52.0 No 258 48.0 Type of HPV detected (N=280) %b HR HPV only 160 57.1 LR HPV only 55 19.6 Both HR & LR HPV 65 23.2 Number of HPV genotypes detected (N=280) %b Single 151 53.9 Multiple 129 46.1 a Percentages calculated using total number of cases (N= 538) as common denominator. b Percentages calculated using number of positive cases (N= 280) as common denominator. α Women infected with only LR HPV. Low-risk HPV types include 6/11, 42, 43, 44 and 66. β Women infected with only HR HPV. High-risk HPV types include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. All 18 HPV genotypes screened were detected. The most common HR-HPV types were (Table 4.3-2), HPV-35 with 14.1% (76 out of 538 women, 95%CI 11.3-17.4), HPV-18 (13.0%; 70 out of 538 women, 95%CI 10.3-16.1), HPV-58 (7.4%; 40 out of 538 women, 95% CI 5.4-10.0), HPV-52 (7.2%; 39 out of 538 women), and HPV-45 (4.8%; 26 out of 538 women, 95% CI 3.2-7.0). HPV 42 with 7.8% (39 out of 538 women, 95% CI 5.2-9.8) was the commonest LR-HPV type detected (Table 4.2-3). The frequency of HPV 16 (the main HR-HPV type linked with cervical cancer worldwide) was 2.4% (13 out of 538 women, 95%CI 1.3-4.1). 69 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-2: Prevalence of high-risk HPV Genotypes in WLHIV at the Korle Bu Teaching Hospital, Accra, Ghana, HPV type n* %a %b %c HPV-52 39 7.2 14.1 17.3 HPV-56 9 1.7 3.2 4.0 HPV-35 76 14.1 27.4 33.8 HPV-18 70 13.0 25.3 31.1 HPV-58 40 7.4 14.4 17.8 HPV-68 16 3.2 5.8 7.1 HPV-51 12 2.2 4.3 5.3 HPV-39 18 3.3 6.5 8.0 HPV-45 26 4.8 9.3 11.6 HPV-16 13 2.4 4.7 5.8 HPV-59 7 1.3 2.5 3.1 HPV-33 5 0.9 1.8 2.2 HPV-31 9 1.7 1.1 4.0 HPV-44 20 3.7 7.2 8.9 HPV-66 3 0.6 1.1 1.3 * Because of multiple infections, women may be counted more than once. a Percentages calculated using total number of cases (N= 538) as common denominator. b Percentages calculated using number of positive cases (N= 277) as common denominator c Percentages calculated using number of HR cases (N= 225) as common denominator. 70 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-3: Prevalence of low-risk HPV Genotypes in WLHIV at the Korle Bu Teaching Hospital, Accra, Ghana, HPV type n* %a %b %c HPV-42 45 8.4 16.2 37.8 HPV-43 28 5.2 10.1 23.5 HPV-66 3 0.6 1.1 2.5 HPV-6/11 32 5.9 11.6 26.9 HPV-44 20 3.7 7.2 16.8 * Because of multiple infections, women may be counted more than once. a Percentages calculated using total number of cases (N= 538) as common denominator. b Percentages calculated using number of positive cases (N= 277) as common denominator c Percentages calculated using number of LR cases (N= 119) as common denominator. Study population demographics and human papillomavirus (HPV) genotype prevalence estimates are presented in Table 4.3-4 for all infected women. Row percentages are computed to show within-group HPV prevalence estimates. The estimated age-related prevalence rates ranged from 0.7% in women aged below 25 years to 49.7% in women between the ages of 35 and 44 years. Women who had primary level education had low-risk HPV prevalence of 35.2% compared to 63.6% in those with tertiary level education. The prevalence of high-risk HPV was also 63.6% in those with tertiary level and 87% in women with primary level education. The prevalence of HPV across the different ethnic groups was approximately the same however, the Mole-Dagbani had the highest prevalence of 56.0%. 71 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-4: Study population demographics and HPV prevalence in WLHIV at the Korle- Bu Teaching Hospital, Accra, Ghana, Prevalence of HPV, %# High-risk Characteristic No. (%*) Low-risk HPV HPV Any HPV Age (N=532) 25-34 118 (22.2) 40.4 77.2 46.6 35-44 248 (46.6) 44.0 80.6 54.0 45-54 137 (25.8) 39.4 81.7 51.1 55-64 29 (5.5) 50.0 81.2 55.2 Education (N=538) Primary 309 (57.4) 35.2 87.0 51.8 Secondary 76 (14.1) 50.0 72.7 56.6 Tertiary 25 (4.6) 63.6 63.6 44.0 None 128 (23.8) 52.4 71.4 49.2 Marital status (N=538) Divorced 96(17.8) 41.7 77.1 49.0 Married 206 (38.3) 36.5 82.3 46.1 Single 110 (20.4) 51.7 75.9 52.7 Widowed 126 (23.4) 53.8 83.3 61.1 Ethnicity (N=530) Akan 230 (43.4) 38.2 85.5 47.0 Ewe 105 (19.8) 48.2 76.8 52.4 Ga 104 (19.6) 37.5 78.6 53.8 Mole-Dagdani 91 (17.2) 47.2 74.5 56.0 Parity Gravidae (N=511) Less than 2 73 (14.3) 46.2 76.9 53.4 2 to 4 252 (49.3) 46.5 75.6 50.0 Greater 4 186 (36.4) 36.4 86.9 52.2 # Row percentages are computed to show within-group HPV prevalence estimates. *Percentage fraction of respondents. 72 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-4 continued Prevalence of HPV, %# Characteristic No. (%*) Low-risk HPV High-risk HPV Any HPV Abortion (N=256) 1 96 (37.5) 47.6 76.2 43.8 2 72 (28.1) 43.2 84.1 61.1 >2 88 (34.4) 40.4 84.6 56.8 Miscarriage (N=125) 1 73 (58.4) 42.5 82.5 53.4 2 29 (23.2) 22.2 88.9 31.0 >2 23 (18.4) - 100.0 43.5 Age at first pregnancy (years) (N=511) ≤17 91 (17.8) 44.2 79.1 47.3 18-25 333 (65.2) 41.0 80.9 52.6 >25 87 (17.0) 47.7 77.3 50.6 # Row percentages are computed to show within-group HPV prevalence estimates. *Percentage fraction of respondents. 73 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-5: Sexual history and HPV prevalence in WLHIV at the Korle-Bu Teaching Hospital, Accra, Ghana, Prevalence of HPV, %# Low-risk High-risk Characteristic No. (%) HPV HPV Any HPV Age at maiden sexual encounter (N=531) < 16 81 (15.2) 42.9 76.2 51.9 16-18 247 (46.5) 41.1 82.3 49.0 >18 203 (38.2) 43.5 79.6 53.2 HIV duration (months)a (N=531) <48 118 (22.1) 32.4 81.1 61.9 48-71 119 (22.2) 51.0 78.4 41.2 72-95 104 (19.4) 46.3 75.9 51.9 96-119 106 (19.8) 45.6 82.5 53.8 >119 88 (16.4) 42.9 83.3 47.7 lifetime sex partners (N=263) <3 63(24.0) 50.0 80.0 63.5 3-6 185 (70.3) 43.4 78.7 64.9 >6 15 (5.7) 50.0 87.5 53.3 ART(N=511) Yes 429 (83.9) 43.4 80.3 52.7 No 82 (16.1) 27.8 91.7 42.7 ART duration (months) (N=424) <30 148 (34.9) 39.7 84.6 52.0 30-59 95(22.4) 49.1 67.9 54.7 60-89 128(30.2) 48.3 81.7 46.9 >89 53(12.5) 39.4 84.8 62.3 Wife of monogamous relationship a(N=269) No 117 (43.5) 44.4 77.8 69.2 Yes 152 (56.5) 35.3 82.4 43.1 # Row percentages are computed to show within-group HPV prevalence estimates. *Percentage fraction of respondents. a Prevalence of HPV (any type) varies significantly across categories. 74 University of Ghana http://ugspace.ug.edu.gh Results Prevalence of HPV, %# Characteristic No. (%*) Low-risk HPV High-risk HPV Any HPV Condom use (N=538) Always 145 (27.0) 49.3 73.1 44.8 Never 256 (49.2) 36.4 86.0 50.0 Sometimes 137 (25.5) 46.4 77.4 61.8 Ever smoked tobacco (N=536) Yes 6 (1.1) 25.0 100.0 66.7 No 530 (98.9) 42.9 80.0 51.3 Vaginal Discharge (N=537) Yes 35 (6.5) 40.0 84.0 68.6 No 502 (93.5) 42.9 79.9 50.2 Skin Ulcers(N=537) Yes 10 (1.7) 75.0 87.5 80.0 No 527 (98.1) 41.7 80.1 50.9 # Row percentages are computed to show within-group HPV prevalence estimates. *Percentage fraction of respondents. a Prevalence of HPV (any type) varies significantly across categories. 75 University of Ghana http://ugspace.ug.edu.gh Results Tables 4.3-6, 4.3-7 and 4.3-8 shows HPV type distribution in women living with HIV at the Korle-Bu Teaching Hospital, Accra, stratified according to cytological outcome (presence or absence of cervical cytology abnormality) and number of detected HPV genotypes. The number of women with an LR-HPV genotype infection (irrespective of the presence of other HPV types) was calculated using 538 as the denominator and separated under two categories: single and multiple infections. The results are presented based on the presence or absence of cervical cytological dysplasia. There were 26 unsatisfactory slides out of which17 had HPV infection: 7 had multiple HPV infections and 10 had single HPV infections (the distribution of HR- and LR-HPV in rejected slides are given in Table 4.3-9). The majority of women 86.2% (464/538 women, 95% CI 83.0-89.0 :) had normal cervical findings. Only 9.7% (52/538 women, 95% CI 7.3-12.5 (9.7%) had a concurrent abnormal Pap smear result. Among women who had abnormal cervical results, the prevalence of HPV infection was 50.0% (26/52 women, 95% CI 35.8-64.2) compared to 51.5% (239/464 women, 95%CI 46.8-56.1) who had normal cytology results. Women who had abnormal Pap smear findings did not have a disproportionate prevalence of HR-HPV infection (χ2=0.01, df=1, P=0.91). The prevalence of HR HPV infection was 80.8% among women with abnormal simultaneous pap result compared to 79.9% among women with normal Pap smear findings. Again, women with atypical pap smears did not show a greater tendency to harbour multiple HR-HPV infections compared to women with normal pap smears (χ2=0.57, df=1, P=0.45). Multiple infections were more likely to be associated with HR-HPV genotypes than with LR-HPV infection (χ2=122.3, df=2, P=0.00). 76 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-6: HPV type distribution stratified according to concurrent cytology outcome and number of detected HPV genotypes HPV Prevalence and cytological outcomes in WLHIV at the Korle Bu Teaching Hospital Pap test Normal Abnormal Total HPV type Single Multiple count (%a,b) Single Multiple count (%c,d) Single Multiple Total (%) n= 464 (86.2) n= 52 (9.7) n= 538 HPV negative 225 (48.5a) 26 (50.0 c) 251 (46.7 e) HPV positive 129 110 239 (51.5a) 12 14 26 (50.0 c) 141 124 265 (49.3 e) HR HPV positive α 81 110 191 (79.9 b) 8 13 21 (80.8 d) 89 123 212 (75.7 f) LR HPV positive β 48 54 102 (42.7 b) 4 9 13 (50.0 d) 52 63 115 (41.1 f) α All women harbouring at least one HR HPV infection. Cases with more than one HR infection are counted once. β All women harbouring at least one LR HPV infection. Cases with more than one LR infection are counted once. %a: out of 464 women with normal cytology %b: out of 239 HPV positive cases with normal cytology %c: out of 52 women with abnormal cytology %d: out of 26 HPV positive cases with abnormal cytology %e: out of 538 WLHIV %f: out of 280 HPV positive WLHIV, not excluding 22 unsatisfactory pap smears absent from table 4.4-1 77 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-7: Prevalence of LR HPV types by cytological diagnosis at the Korle-bu Teaching Hospital,Accra, Ghana,. Pap test Normal Abnormal Total HPV type Single Multiple Total (%#) Single Multiple Total (%#) Single Multiple Total (%#) Low-risk infections 42 a 17 16 33 (73.3) 3 5 8 (17.8) 20 21 41 (91.1) 43 8 17 25 (89.3) 0 3 3 (10.7) 8 20 28 (100.0) 66 0 2 2 (66.4) 0 1 1 (33.3) 0 3 3 (100.0) 44 13 5 18 (90.0) 1 1 2 (10.0) 14 6 20 (100.0) 6/11 8 20 28 (87.5) 1 2 3 (9.4) 9 22 31 (96.9) sub-total 46 60 106 (82.8) 5 12 17 (13.3) 51 72 123 (96.1) # Row percentages are computed to show within-group HPV prevalence estimates. May not add up to 100% because of 22 unsatisfactory cytological slides a Prevalence of HPV (any type) varies significantly across categories. 78 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-8: Prevalence of HR HPV types by cytological diagnosis at the Korle-Bu Teaching Hospital, Accra, Ghana, Pap test Normal Abnormal Total Single Multiple Total (%#) Single Multiple Total (%#) Single Multiple Total (%#) High-risk infections 52 9 24 33 (84.6) 0 4 4 (10.3) 9 28 37 (94.9) 35 20 44 64 (84.2) 3 6 9 (11.8) 23 50 73 (96.1) 18 11 49 60 (85.7) 1 5 6 (8.6) 12 54 66 (94.3) 58 8 24 32 (80.0) 0 5 5 (12.5) 8 29 37 (92.5) 56 4 4 8 (88.9) 0 1 1 (11.1) 4 5 9 (100.0) 39 8 7 15 (83.3) 1 2 3 (16.7) 9 10 19 (100.0) 51 3 6 9 (75.0) 0 2 2 (16.7) 3 8 11 (91.7) 59 1 5 6 (85.7) 0 0 0 (0.0) 1 5 6 (85.7) 45 6 16 22 (84.6) 0 4 4 (15.4) 6 20 26 (100.0) 16 2 9 11 (84.6) 0 2 2 (15.4) 2 11 13 (100.0) 68 7 20 27 (81.8) 1 2 3 (9.1) 8 22 30 (90.9) 33 1 4 5 (100.0) 0 0 0 (0.0) 1 4 5 (100.0) 31 2 6 8 (88.9) 1 0 1 (11.1) 3 6 9 (100.0) sub-total 82 218 300 7 33 40 89 252 341 (95.7) # Row percentages are computed to show within-group HPV prevalence estimates. May not add up to 100% because of unsatisfactory cytology slides a Prevalence of HPV (any type) varies significantly across categories. 79 University of Ghana http://ugspace.ug.edu.gh Results The present study investigated the relationship between detection of HPV genotypes and the presence of specific diagnostic cytological abnormality in women living with HIV at the Korle-Bu teaching hospital, Accra. Table 4.3-9 presents the distribution of all types of HPV, low risk and high-risk HPVs and the corresponding diagnostic category for these women. Prevalence of HPV infection ranged from 25.9% (95% CI 11.1-46.3) among women with no TZC to 56.3% (95% CI32.3-83.7) among women with ASC and 65.4% (95% CI 44.3-82.8) in women with unsatisfactory smears. The cross-tabulation procedure in SPSS helps to evaluate differences in distribution of various cytology outcomes and the prevalence of low risk, high-risk and any type HPV among these women. In this regard, no significant difference was noted for the any type HPV infection, high-risk HPV, or low-risk HPV and Pap smear outcome (Table 4.3-9). Table 4.3-9: Distribution of low risk, high risk and any type HPV in concurrent cervical cytology outcomes for 538 WLHIV screened at Korle Bu Teaching Hospital, Accra, Prevalence of HPV, %* Characteristic No. (%#) Any HPVa High-risk HPV Low-risk HPV No TZC 27 (5.0) 7 (25.9) 5 (18.5) 2 (7.4) NILM 433 (80.5) 230 (53.1) 184 (42.5) 99 (22.9) ASCUS 15 (2.8) 9 (60.0) 9 (60.0) 2 (13.3) LSIL 21 (3.9) 9 (42.9) 6 (28.6) 7 (33.3) ASC-H 1 (0.2) 1 (100.0) 1 (100.0) 0 (0.0) HSIL 15 (2.8) 7 (46.7) 5 (33.3) 4 (26.7) Unsatisfactory 26 (4.8) 17 (65.4) 15 (57.7) 6 (23.1) (χ2, df=5, P-value) 10.2; 0.12 4.91; 0.56 8.36; 0.21 # Percentage fraction of respondents. * Row percentages are computed to show within-group HPV prevalence estimates. a Prevalence of HPV (any type) varies significantly across categories (Pearson x2 test). 80 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-10 shows the exclusive distribution of HPV genotypes in the 538 women according to concurrent cytological diagnosis. Each genotype found in multiple infection was treated as an isolated event and tallied exclusively. The commonest HPV genotypes detected among women with ASCUS were HPV-18 (20% (95%CI 4.3-48.1) of all ASCUS cases), 35 (20% (95%CI 4.3- 48.1) of all ASCUS cases) and 31, 39, 45, 51, 52, 58, 68 (6.7% (95%CI 0.2-31.9) of all ASCUS cases each). Among women with LSIL most prevalent were HPV-52 (28.6% (95%CI 11.3-52.2) % of all LSIL cases), HPV-35 (14.3% (95%CI 3.0-36.3) of all LSIL cases), and HPV 16,18, 58,39 (9.5% (95%CI 1.2-30.4) of all LSIL cases). HPV-52, 35 (20% (95%CI 4.3-48.9) of all HSIL cases) and 58,45 (13.3% (95%CI 1.7-40.5) of all HSIL cases) were top three in that order for high grade intraepithelial/severe dysplasia lesions. HPV-35 (14.8% (95%CI 11.6-18.5) of all normal cases), HPV-18 (13.2% (95%CI 10.1-16.7) of all normal cases), HPV-52 (7.6% (95%CI 5.3-10.5) of all normal cases), HPV-58 (7.2% (95%CI 4.9-10.0) of all normal cases) and HPV-68 (5.8% (95%CI 3.8-8.4) of all normal cases) was more commonly detected in women without any precancerous lesions. 81 University of Ghana http://ugspace.ug.edu.gh Results Table 4.3-10: Distribution of HPV genotypes among 538 WLHIV screened at Korle Bu Teaching Hospital, Accra, HPV ASCUS LSIL HSIL ASC-H NILM genotype N=15 (%) N=21 (%) N=15 (%) N= 1 (%) N=433 (%) High-risk infections 52 1(6.7) 6 (28.6) 3(20.0) - 33(7.6) 35 3(20.0) 3(14.3) 3(20.0) - 64(14.8) 18 3(20.0) 2 (9.5) 1(6.7) 57(13.2) 58 1(6.7) 2 (9.5) 2(13.3) - 31(7.2) 56 - - 1(6.7) - 7(1.6) 39 1(6.7) 2 (9.5) - - 14(3.2) 51 1(6.7) - 1(6.7) - 9(2.1) 59 - - - - 6(1.4) 45 1(6.7) 1(4.8) 2(13.3) 1(100.0) 20(4.6) 16 2 (9.5) - - 10(4.0) 68 1(6.7) 1(4.8) 1(6.7) - 25(5.8) 33 - - - - 5(1.2) 31 1(6.7) - - - 7(1.6) Low-risk infections 42 2(13.3) 3(14.3) 3(20.0) - 33(7.6) 43 - 2 (9.5) 1(6.7) - 25(5.8) 66 1(6.7) - - - 2(0.5) 44 1(6.7) 1(4.8) - - 17(3.9) 6/11 1(6.7) 2(9.5) - - 26(6.0;) 82 University of Ghana http://ugspace.ug.edu.gh Results 4.4 Correlates of abnormal cytology and their implications for disease prevention Tables 4.6-1 and 4.6-2 show the crude and adjusted odds ratios (ORs) for Pap smear abnormality and corresponding 95% confidence intervals (CIs) according to socio-demographic and reproductive characteristics among women. However, after adjusting for potential confounders such as woman’s age and age at first pregnancy, younger age at sexual debut had significant main effect for abnormal Pap smear result later in the life of women in the study. Then, the regression procedure was repeated to create the best model for predicting the effect of potential variables on cytology outcome. The final model- shown here in Table 4.6-3 was based on educational status, income, and age at sexual debut, significantly predicted Pap smear results (x2 =28.816, df= 8, N=504, p=0.002) and explained up to 10% (Nagelkerke R2=0.096) of the variability in and cytology outcome. 83 University of Ghana http://ugspace.ug.edu.gh Results Table 4.4-1: Odds ratios (ORs) for cervical dysplasia and matching 95% confidence intervals (CIs) according to socio-demographic characteristics in WLHIV at Korle Bu Teaching Hospital, Accra, Parameter Pap smear abnormality N(%) #n(%) χ2 (p-value) β (p-value) OR 95% CI µβ (p-value) µOR 95% CI Age* 13.2 (0.11) 0.29 -0.33 (0.143) 0.967 0.93-1.01 <25 6 (1.1) 2 (33.3) 1 25-34 118 (21.9) 16 (13.6) -1.13 (0.21) 0.32 0.06-1.91 35-44 248 (46.1) 21 (8.5) -1.65 (0.07) 0.19 0.03-1.11 45-54 137 (25.5) 13 (9.5) -1.49 (0.10) 0.27 0.04-1.40 55-64 29 (5.4) 0 (0.0) -20.51 (0.99) 0.00 Education 6.7 (0.35) 0.21 0.175 Primary 309 (57.4) 35 (11.3) 1 1 Secondary 76 (14.1) 2 (2.6) -1.55 (0.04) 0.21 0.05-0.90 -2.20 (0.033) 0.110 0.02-0.84 Tertiary 25 (4.6) 2 (8.0) -0.43 (0.57) 0.65 0.15-2.90 0.45 (0.687) 1.565 0.18-13.79 never attended 128 (23.8) 13 (10.2) -0.13 (0.71) 0.88 0.45-1.73 -0.19 (0.605) 0.824 0.40-1.71 Occupation 2.7 (0.62) 0.93 0.999 Formal sector 35 (6.5) 3 (8.6) 1 1 Unemployed 79 (14.7) 7 (8.9) 0.10 (0.88) 1.11 0.27-4.60 -19.32 (1.00) - - Non-formal sector 424 (78.8) 42 (9.9) 0.21 (0.74) 1.22 0.36-4.18 0.06 (0.957) 1.061 0.9.32 Marital status 8.7 (0.79) 0.06 0.112 Divorced 110 (20.4) 11 (10.0) 1 1 Married 96 (17.8) 16 (16.7) -0.97 (0.01) 0.38 0.12-0.81 -1.02 (0.016) 0.360 0.16-0.83 Single 206 (38.3) 15 (7.3) -0.60 (0.15) 0.55 0.24-1.25 -0.62 (0.203) 0.539 0.21-1.40 widowed 126 (23.4) 10 (7.9) -0.85 (0.05) 0.43 0.18-0.99 -0.66 (0.149) 0.515 0.21-1.27 84 University of Ghana http://ugspace.ug.edu.gh Results Parameter Pap smear abnormality # N(%) n(%) χ 2 (p-value) β (p-value) OR 95% CI µβ (p-value) µOR 95% CI Income 10.1 (0.26) 0.15 0.093 <100 138 (25.9) 7 (5.1) 1 1 100-249 254 (47.7) 33 (13.0) 1.07 (0.01) 2.87 1.24-6.69 1.16 (0.01) 3.20 1.33-7.71 250-500 41 (7.7) 3 (7.3) 0.42 (0.57) 1.52 0.38-6.19 0.73 (0.35) 2.07 0.45-9.46 >500 27 (5.1) 2 (7.4) 0.42 (0.61) 1.52 0.30-7.78 -0.70 (0.65) 0.50 0.03-9.96 can't tell 72 (13.5) 7 (9.7) 0.76 (0.17) 2.13 0.72-6.36 20.33 (1.00) - - *Ethnicity 15.9 (0.04) 0.45 0.524 Akan 230 (43.2) 26 (11.3) 1 1 Ewe 105 (19.7) 8 (7.6) -0.43 (0.31) 0.65 0.23-1.45 -0.41 (0.36) 0.66 0.28-1.59 Ga 104 (19.5) 6 (5.8) -0.76 (0.10) 0.47 0.19-1.17 -0.76 (0.13) 0.47 0.18-1.24 Mole-Dagbani 91 (17.1) 11 (12.1) 0.08 (0.84) 1.08 0.51-2.30 0.07 (0.87) 1.07 0.46-2.5 Age at maiden pregnancy 5.0 (0.54) 0.50 -0.00 (0.63) 0.99 0.99-1.00 <18 91 (16.9) 9 (9.9) 1 18-25 333 (61.9) 36 (10.8) 0.09 (0.82) 1.09 0.50-2.37 >25 87 (16.2) 5 (5.7) -0.64 (0.27) 0.53 0.17-1.65 n/a 27 (5.0) 2 (7.4) -0.34 (0.68) 0.71 0.14-3.53 Age at sexual debut 7.1 (0.13) 0.12 -0.09 (0.04) 0.97 0.84-0.99 <16 81 (15.3) 12 (14.8) 1 16-18 247 (46.5) 23 (9.3) -0.57 (0.14) 0.56 0.27-1.20 >18 203 (38.2) 15 (7.4) -0.84 (0.04) 0.43 0.19-0.97 a Predictor variable has significant overall effect on outcome variable. * Prevalence of abnormal cytology varies significantly across categories (Pearson x2 test). # Row percentages computed to show within-group prevalence for cervical dysplasia. Multivariate regression: Variable(s) removed in step 2: occupation. Variable(s) removed on step 3: age at first pregnancy. Variable(s) removed on step 4: ethnicity. Variable(s) removed on step 5: marital status. Variable(s) removed on step 6: age. Variables in the equation: educational status, income and age at sexual debut. 85 University of Ghana http://ugspace.ug.edu.gh Results Table 4.4-2: Summary of Model Characteristics for Predicting Cytology Outcome Model x2 df P-value Nagelkerke R2 Variables educational status, income and age at sexual debut, age, marital status, Step 1 35.295 19 0.013 0.141 ethnicity, age at first pregnancy, occupation educational status, income and age at Step 2 35.000 18 0.009 0.140 sexual debut, age, marital status, ethnicity, age at first pregnancy educational status, income and age at Step 3 34.747 16 0.004 0.139 sexual debut, age, marital status, ethnicity educational status, income and age at Step 4 31.210 15 0.008 0.125 sexual debut, age, marital status educational status, income and age at Step 5 25.948 11 0.007 0.104 sexual debut, age educational status, income and age at Step 6 23.816 8 0.002 0.096 sexual debut Table 4.4-3: Odds ratios and 95% CI for Abnormal Cytology According to Best Fit Predictive Model Parameter Pap smear abnormality µ Variable β p-value µOR 95% CI Education 0.172 Primary 1 secondary -1.671 0.026 0.188 0.04-0.82 Tertiary -0.093 0.930 0.911 0.11-7.29 never attended -0.170 0 170 0.844 0.42-1.68 Income 0.085 <100 1 100-249 1.249 0.005 3.488 1.46-8.32 250-500 0.945 0.204 2.574 0.60-11.07 >500 0.454 0.684 1.575 0.18-14.02 can't tell 1.068 0.063 2.911 0.94-8.98 Age at sexual debut -0.117 0.005 0.890 0.82-0.97 Constant 0.830 0.282 0.436 Dependent variable: Cytology outcome 86 University of Ghana http://ugspace.ug.edu.gh Results Tables 4.6-4 and 4.6-5 show the crude and adjusted odds ratios (ORs) for Pap smear abnormality and corresponding 95% confidence intervals (CIs) according to behavioural and clinical characteristics of women. In univariate analysis HIV duration (months) (p=0.02), and CD4 count (p=0.04) were significantly associated with abnormal cytology outcome. The outcome model- shown here in Tables 4.4.6-6 was based on ART use and CD4 count, significantly predicted Pap smear results (x2 =8.46, df= 2, N=344, p=0.015) and explained up to 6% (Nagelkerke R2=0.06) of the variability in and cytology outcome. 87 University of Ghana http://ugspace.ug.edu.gh Results Table 4.4-4: Odds ratios (ORs) for cervical dysplasia and corresponding 95% confidence intervals (CIs) according to behavioural and clinical characteristics in WLHIV at the Korle Bu Teaching Hospital, Accra, Parameter Pap smear abnormality N(%) #n(%) χ2 (p-value) β (p-value) OR 95% CI µβ (p-value) µ OR 95% CI *Ever smoked 9.9 (0.04) 0.58 0.15 No 6 (1.1) 1 (16.7) 1 1 Yes 530 (98.7) 50 (9.4) 0.61 (0.58) 1.83 0.21-16.00 -1.69 (0.15) 0.18 0.02-1.84 Current tobacco use 1.2 (0.55) 0.34 No 534 (99.3) 51 (9.6) 1 1 Yes 4 (0.7) 1 (25.0) 1.10 (0.34) 3.01 0.31-29.50 18.72 (0.99) - - Condom use 6.7 (0.15) 0.67 0.18 Always 145 (27.0) 12 (8.3) 1 1 Never 256 (47.6) 25 (9.8) 0.24 (0.52) 1.27 0.62-2.61 0.43 (0.54) 1.54 0.39-6.10 Sometimes 137 (25.5) 15 (10.9) 0.36 (0.38) 1.44 0.65-3.19 1.10 (0.11) 3.00 0.78-11.51 a HIV duration (months) 12.8 (0.12) 0.02 -0.01 (0.48) 0.994 0.98-1.01 <48 118 (22.1) 10 (8.5) 1 48-71 119 (22.2) 21 (17.6) 0.83 (0.04) 2.30 1.03-5.15 72-95 104 (19.4) 7 (6.7) -0.28 (0.59) 0.76 0.28-2.08 96-119 106 (19.8) 6 (5.7) -0.45 (0.40) 0.64 0.22-1.82 >119 88 (16.4) 8 (9.1) 0.06 (0.91) 1.06 0.40-2.81 ART use 6.2 (0.21) 0.35 0.25 No 82 (16.0) 11 (13.4) 1 1 Yes 429 (84.0) 41 (9.6) -0.34 (0.35) 0.71 0.35-1.45 0.76 (0.25) 2.14 0.59-7.79 a Predictor variable has significant overall effect on outcome variable. * Prevalence of abnormal cytology varies significantly across categories (Pearson x2 test). # Row percentages computed to show within-group prevalence for cervical dysplasia. 88 University of Ghana http://ugspace.ug.edu.gh Results Table 4.6-4 continued Pap smear abnormality Parameter N(%) #n(%) χ2 (p-value) β (p-value) OR 95% CI µβ (p-value) µOR 95% CI *ART duration 15.5 (0.02) 0.01 -0.01 (0.49) 0.99 0.97-1.02 <30 148 (34.9) 24 (16.2) 1 30-59 95 (22.4) 4 (4.2) -1.51 (0.01) 0.22 0.07-0.66 60-89 128 (30.2) 11 (8.6) -0.77 (0.04) 0.46 0.22-0.98 >89 53 (12.5) 2 (3.8) -1.64 (0.03) 0.19 0.08-0.85 Vaginal discharge 0.8 (0.65) 0.43 0.83 No 502 (93.5) 50 (10.0) 1 1 Yes 35 (6.5) 2 (5.7) -0.58 (0.43) 0.56 0.13-2.40 0.18 (0.83) 1.20 0.24-6.04 Ulcers/Sores 1.7 (0.78) 0.31 0.12 No 527 (98.0) 50 (9.5) 1 1 Yes 10 (1.9) 2 (20.0) 0.82 (0.31) 2.28 0.47-11.00 -1.33 (0.12) 0.27 0.05-1.44 CD4 count -0.00 (0.04) 0.99 0.99-1.00 -0.00 (0.18) 0.99 0.99-1.00 a Predictor variable has significant overall effect on outcome variable. * Prevalence of abnormal cytology varies significantly across categories (Pearson x2 test). # Row percentages computed to show within-group prevalence for cervical dysplasia. 89 University of Ghana http://ugspace.ug.edu.gh Results Table 4.4-5: Summary of Model Characteristics for Predicting Cytology Outcome based on behavioural and clinical responses Model x2 df P-value Nagelkerke R2 Variables Ever smoked, current tobacco use, condom use, HIV duration, ART use, Step 1 17.56 10 0.063 0.123 ART duration, discharge, sores, CD4 count. Ever smoked, current tobacco use, Step 2 17.52 9 0.041 0.122 condom use, HIV duration, ART use, ART duration, sores, CD4 count. Ever smoked, condom use, HIV Step 3 17.19 8 0.028 0.120 duration, ART use, ART duration, sores, CD4 count. Ever smoked, condom use, HIV Step 4 16.81 7 0.019 0.117 duration, ART use, sores, CD4 count. Condom use, HIV duration, ART use, Step 5 15.18 6 0.019 0.106 sores, CD4 count. Condom use, ART use, sores, CD4 Step 6 13.54 5 0.019 0.095 count. Step 7 10.14 4 0.038 0.072 ART use, sores, CD4 count. Step 8 8.46 2 0.015 0.060 ART use, CD4 count. Table 4.4-6: Odds ratios and 95% CI for Abnormal Cytology According to Best Fit Predictive Model based on behavioural and clinical responses Parameter Pap smear abnormality µ Variable β p-value µOR 95% CI ART use 1.093 0.025 2.98 1.15-7.77 CD4 count -0.002- 0.043 0.99 0.99-1.00 constant -1.86 0.000 1.56 Dependent variable: Cytology outcome 90 University of Ghana http://ugspace.ug.edu.gh Results 4.5 Correlates of high-risk HPV infection and their implications for disease prevention In this section, the question to be addressed is which of the explanatory variables are predictive of the response, high risk HPV infection? Since the outcome was a binary categorical variable, the logistic regression model helps to determine this relationship. Therefore, investigation of the determinants of abnormal cytology proceeded in two steps. First, unadjusted relationships between high-risk HPV infection and potential predictor variables were examined singly. Table 4.7-1 shows the crude and adjusted odds ratios (ORs) for high-risk HPV infection and corresponding 95% confidence intervals (CIs) according to socio-demographic and reproductive characteristics among women. In univariate analysis, formal education and estimated income had significant main effects for high-risk HPV infection and therefore were associated with high-risk infection. This relationship did not disappear after adjusting for potential confounders such as woman’s age, younger age at sexual debut and recent sexual activity. Recent sexual activity is defined for a woman who reported having a sexual partner in past six months. The regression model based on age, educational status, occupation, marital status, income, ethnicity, age at first pregnancy and age at sexual debut (Table 4.7-1), significantly predicted high risk HPV infection (x2 =38.545, df= 15, N=427, p=0.001) and explained up to 12% (Nagelkerke R2=0.116) of the variability in high-risk HPV infection. 91 University of Ghana http://ugspace.ug.edu.gh Results Table 4.5-1: Odds ratios (ORs) for high-risk HPV infection and corresponding 95% confidence intervals (CIs) according to socio-demographic characteristics in WLHIV at the Korle Bu Teaching Hospital, Accra, Parameter High risk HPV infection N(%) #n(%) χ2 (p-value) β (p-value) OR 95% CI µβ (p-value) µ OR 95% CI Age 1.9 (0.59) 0.70 0.41 25-34 118 (21.9) 55 (46.6) 1 1 35-44 248 (46.1) 134 (54.0) 0.26 (0.26) 1.30 0.83-2.03 0.52 (0.12) 1.69 0.87-3.29 45-54 137 (25.5) 70 (51.1) 0.21 (0.41) 1.24 0.75-2.04 0.60 (0.12) 1.83 0.86-3.90 55-64 29 (5.4) 16 (55.2) 0.31 (0.46) 1.37 0.60-3.12 0.38 (0.57) 1.47 0.37-5.58 aFormal education 0.3 (0.56) 0.08 0.03 Yes 410 (76.2) 214 (52.2) 1 1 never attended 128 (23.8) 63 (49.2) -0.37 (0.08) 0.69 0.45-1.05 -0.63 (0.03) 0.53 0.30-0.96 Marital status 7.4 (0.06) 0.09 0.89 Divorced 96 (17.8) 47 (49.0) 1 1 Married 206 (38.3) 95 (46.1) -0.01 (0.97) 0.99 0.60-1.63 0.17 (0.74) 1.19 0.44-3.19 Single 110 (20.4) 58 (52.7) 0.06 (0.83) 1.06 0.61-1.86 0.22 (0.59) 1.25 0.56-2.78 Widowed 126 (23.4) 77 (61.1) 0.53 (0.05) 1.70 0.99-2.91 0.28 (0.44) 1.33 0.65-0.72 92 University of Ghana http://ugspace.ug.edu.gh Results Parameter High risk HPV infection N(%) #n(%) χ2 (p-value) β (p-value) OR 95% CI µβ (p-value) µOR 95% CI a*Income 21.2 (0.00) 0.00 0.00 <100 138 (25.7) 93 (67.4) 1 1 100-500 295 (54.8) 140 (47.5) -0.96 (0.00) 0.38 0.25-0.58 -0.76 (0.00) 0.47 0.29-0.76 >500 27 (5.0) 14 (51.9) -1.46 (0.02) 0.23 0.09-0.56 -1.62 (0.02) 0.20 0.05-0.79 Ethnicity 2.8 (0.58) 0.99 0.57 Akan 230 (42.8) 108 (47.0) 1 1 Ewe 105 (19.5) 55 (52.4) 0.00 (0.99) 1.00 0.63-1.61 -0.26 (0.43) 0.77 0.41-1.46 Ga 104 (19.3) 56 (53.8) 0.06 (0.81) 1.06 0.66-1.70 0.22 (0.49) 1.25 0.66-2.36 Mole-Dagbani 91 (16.9) 51 (56.0) 0.04 (0.88) 1.04 0.63-1.70 0.39 (0.28) 1.47 0.73-3.0 Age at sexual debut 0.62 (0.43) 0.73 0.75 <18 328 (61.0) 163 (49.7) 1 1 18 and over 203 (38.2) 108 (53.2) 0.06 (0.73) 0.73 0.75-1.52 0.07 (0.75) 1.08 0.67-1.74 Recent Sexual activity 1.9 (0.39) 0.41 No 273 (50.7) 146 (53.3) 1 1 Yes 264 (49.1) 130 (49.2) -0.25(0.16) 0.77 0.55-1.10 -0.34 (0.41) 0.71 0.32-1.60 a Predictor variable has significant overall effect on outcome variable. * Prevalence of abnormal cytology varies significantly across categories (Pearson x2 test). # Row percentages computed to show within-group prevalence for cervical dysplasia. Multivariate regression: Variables in the equation: age, formal education, marital status, income status, ethnicity, age at sexual debut and recent sexual activity. 93 University of Ghana http://ugspace.ug.edu.gh Results Table 4.7-2 shows the crude and adjusted odds ratios (ORs) for high-risk HPV infection and corresponding 95% confidence intervals (CIs) according to behavioural and clinical characteristics of women. However, in multivariate analysis, after adjusting for age, log CD4 count and other confounders, HIV duration (p=0.04) and vaginal discharge (p=0.01) had significant main effects on cytology outcome. ART duration was not included in multivariate analysis because it was significantly correlated with HIV duration and could result in misleading variable coefficients. The final equation was based on 507 women. The resulting logistic regression equation shown here in Table 4.7-2 significantly predicts high risk HPV infection (x2 =20.64, df= 8, N=507, p=0.008) and explained up to 5% (Nagelkerke R2=0.05) of the variability in high-risk HPV infection. 94 University of Ghana http://ugspace.ug.edu.gh Results Table 4.5-2: Odds ratios (ORs) for high-risk HPV infection and corresponding 95% confidence intervals (CIs) according to behavioural and clinical characteristics in WLHIV at the Korle-Bu Teaching Hospital, Accra, Parameter high risk HPV infection # N(%) n(%) χ 2 (p-value) β (p-value) OR 95% CI µβ (p-value) µOR 95% CI *Condom use 5.31 (0.02) 0.02 0.07 Always 145 (27.0) 49 (33.8) 1 1 176 -0.40 (0.13) 0.67 0.44-1.03 393 (73.0) -0.46 (0.02) 0.63 0.42-0.94 Never/Sometimes (44.8) aHIV duration (months) 7.92 (0.09) 0.09 0.04 <48 118 (22.9) 60 (50.8) 1 1 48-71 119 (22.1) 40 (33.6) -0.71 (0.00) 0.49 0.29-0.83 -0.83 (0.00) 0.44 0.24-0.77 72-95 104 (19.3) 41 (39.4) -0.46 (0.09) 0.63 0.37-1.07 -0.60 (0.04) 0.55 0.31-0.97 96-119 106 (19.8) 47 (44.3) -0.26 (0.33) 0.77 0.46-1.30 -0.28 (0.34) 0.76 0.42-1.35 >119 88 (16.4) 35 (39.8) -0.45 (0.12) 0.63 0.37-1.12 -0.58 (0.64) 0.56 0.31-1.03 ART use 0.16 (0.69) 0.69 0.60 183 Yes 429 (79.7) -0.09 (0.69) 0.91 0.56-1.46 -0.14 (0.60) 0.87 0.52-1.45 (42.7) ART duration 4.33 (0.23) 0.23 <30 148 (27.5) 66 (44.6) 1 30-59 95 (17.7) 36 (37.9) -0.28 (0.30) 0.76 0.45-1.28 60-89 128 (23.8) 49 (38.3) -0.26 (0.29) 0.77 0.48-1.25 >89 53 (9.9) 28 (52.8) -0.33 (0.30) 1.39 0.74-2.61 a*Vaginal discharge 5.15 (0.02) 0.03 0.01 Yes 35 (6.5) 21 (60.0) -0.79 (0.03) 0.45 0.23-0.91 -1.06 (0.01) 0.35 0.15-0.79 2.64 -0.34 (0.24) 0.71 0.40-1.26 logCD4 count (0.36) a Predictor variable has significant overall effect on outcome variable. * Prevalence of high-risk HPV prevalence varies significantly across categories (Pearson x2 test). # Row percentages computed to show within-group prevalence for cervical dysplasia. Categorical variables are shown as count (%). 95 University of Ghana http://ugspace.ug.edu.gh Results Continuous variables are shown as mean (SD). Multivariate regression: Variables in the equation: age, condom use, HIV duration, ART use and occult vaginal discharge. 96 University of Ghana http://ugspace.ug.edu.gh Discussion Chapter 5 DISCUSSION Antiretroviral therapy (ART) for PLHIV provides a golden opportunity to increase cervical screening through the integration of ART services with frequent screening of women for cervical cancer (Torre et al., 2016). Here, data is presented on the burden of cervical cancer and precancerous lesions among women living with HIV and attending specialized care in Accra, Ghana. HIV is known to be an important independent risk factor for development of precursor lesions and cervical cancer (Getinet et al., 2015). Although the overall prevalence of cervical epithelial abnormalities (9.3%) in this population is higher than what has previously been reported in the general population in Ghana (Odida et al., 2011), no cases of squamous cell carcinoma or adenocarcinoma were detected cytologically in this population. Additionally, the prevalence rates of LSIL and HSIL are 3.9 and 2.8%, respectively. Equivocal smears (ASC-) accounted for approximately 3% of women. These rates are higher than some estimates reported in both low-resource populations in Ghana (Handlogten et al., 2014; Donkoh, 2015) and other countries with an absence of screening programs, such as Cameroun (HIV/AIDS, 2018), South Africa (Fonn et al., 2015) and India (Burd, 2003b). High-resource populations with well-established screening programs and lower HIV endemicity, often have lower rates of epithelial cell abnormality (ECA). Indeed lower rates of ECA than what we found in this study have been reported among Australian (Soccio et al., 2015) and Turkish women (Açikgöz and Ergör, 2010). Italian women (Meloni et al., 2014) and Chinese (Tao et al., 2014). In general, studies among the general population in resource-limited countries have reported high prevalence of cervical neoplasia that coincide with estimates from HIV populations. Inadvertent inclusion of women with HIV-infection in general population studies frequently 97 University of Ghana http://ugspace.ug.edu.gh Discussion occurs in HIV endemic populations leading to variation in estimates of squamous intraepithelial lesions (Getinet et al., 2015). Clarifying the HIV status of study participants greatly helps to appreciate and compare estimates in this regard. Studies from Nigeria report ECA prevalence estimates ranging from 7.6% (Thomas et al., 2012), 11.2% (Ajah et al., 2015), 8.9% (De Lemos et al., 2012), to 22.6% (Duru et al., 2015). Since very little is known about the prevalence and incidence of cervical abnormalities among HIV infected women and most studies in the general population reporting on cervical abnormalities do not disclose HIV status, it is plausible that high rates of ECA may reflect HIV endemicity even in general population studies. Unusually high prevalence of epithelial cell abnormalities may also be an indicator for the inclusion of HIV-infected women in the population studied (Getinet et al., 2015). Previous studies with large samples of women from South African found higher rates of ECA than what is reported here: 15.2% (Allan et al., 2008) and 17.3% (Richter et al., 2013). Additionally, similar studies from Rwanda, 20% (Kayumba, 2014), Senegal, 21.0%, (Xi et al., 2003), Guinea, 9.5% (Keita et al., 2009), Gabon, 13.0%, (Assoumou et al., 2016) and Ethiopia, 14.1%, (Getinet et al., 2015) have all reported high prevalence of precursor cervical lesions. Immunosuppression from HIV infection is a -known risk factor for development of precursor cervical lesions and cervical cancer and as a result, cervical lesions are expected to be more aggressively present in immunocompromised patients (Strickler et al., 2005). However, the present rate of precancerous lesions falls comparably within the lower range of estimates for other high-risk (HR) populations of women. Several studies indicate that among African women, being HIV-positive was associated with a high risk of presenting squamous intraepithelial lesions of the cervix, with ORs ranging from 4.4 to 17 (Didelot-Rousseau et al., 2006; Hawes et al., 2006). 98 University of Ghana http://ugspace.ug.edu.gh Discussion Precancerous cervical lesions and cervical tissue neoplasm in the cervical area were prevalent conditions in Sub-Saharan Africa. For instance, in Rwanda, a cross-sectional study of 293 HIV- infected women recruited at a military hospital found a 20.0% rate of ECA (Kayumba, 2014). Studies conducted in Zambia and Kenya reported higher diagnostic frequencies of cervical epithelial abnormalities among women infected with HIV (Lehtovirta et al., 2008; Memiah et al., 2012). A systematic review and meta-analysis conducted among HIV positive women in Sub-Saharan Africa showed that the prevalence of precancerous cervical lesions ranged from 2.9% to 76% (Strickler et al., 2005). Furthermore, the pooled prevalence of precancerous cervical lesion among the HIV-positive women in Sub-Saharan Africa was 25.6%. In a report by Isaakidis et al., (Isaakidis et al., 2013) Mumbai, cervical cytology was abnormal in 19.0% of women recruited from an antiretroviral therapy (ART) clinic. HIV positive women were four times more likely to have abnormal cytology than HIV-negative women (Blossom et al., 2007). Among HIV-infected women attending Pusan National University Hospital in Korea, HIV- infected women had tenfold higher odds of abnormal cervical cytology and a prevalence of 18.3% (vs 1.8%) (Park et al., 2014). In addition, studies in Cambodia (25.0%) (Marie-Eve et al., 2009), the Bahamas (44.0%) (Dames et al., 2009), among women attending HIV clinics also report an elevated prevalence of precancerous lesions than has been reported by this study. A comparative cross-sectional study was conducted among HIV positive women attending gynecological examination in cervical cancer screening center in Ethiopia. Prevalence of ECA (17.8 %) was observed among HIV positive women (Getinet et al., 2015). Sub-Saharan Africa showed that the prevalence of precancerous cervical lesions ranged from 2.9% to 76% (Strickler et al., 2005). Among HIV-infected women attending Pusan National University Hospital in Korea, HIV positive women had tenfold higher odds of abnormal cervical 99 University of Ghana http://ugspace.ug.edu.gh Discussion cytology and a prevalence of 18.3% (vs 1.8%) (Park et al., 2014). In addition, studies in Cambodia (25.0%) (Marie-Eve et al., 2009), the Bahamas (44.0%) (Dames et al., 2009), among women attending HIV clinics also report an elevated prevalence of precancerous lesions than has been reported by this study. A comparative cross-sectional study was conducted among HIV positive women attending gynecological examination in cervical cancer screening center in Ethiopia. Prevalence of ECA (17.8 %) was observed among HIV positive women (Getinet et al., 2015). HIV is known to be an important independent risk factor for development of precursor lesions and cervical cancer (Getinet et al., 2015). Antiretroviral therapy (ART) for PLHIV provides a golden opportunity to increase cervical screening through the integration of ART services with frequent screening of women for cervical cancer (Torre et al., 2016). Early initiation of HAART and sustained adherence for viral control is likely to reduce incidence and progression of SIL and ultimately incidence of invasive cervical cancer (Memiah et al., 2012; Akarolo-Anthony et al., 2013). The lower threshold of cervical pre-cancer/SIL in the presence of HPV infections among women living with HIV may reflect the high coverage of HAART in this population (80%) even though data on the length of ART was not available, it is expected to be high among women recruited from HIV clinics such as this (Minkoff et al., 2010; Massad et al., 2016). Several African studies (Clifford et al., 2006d; Chaturvedi, 2010; Doorbar et al., 2012; Zeier et al., 2012) provide indication that earlier initiation and effective HAART over a prolonged duration can prevent cervical lesion incidence and progression and promote regression. Recent evidence from two systematic reviews and meta-analyses of studies in African populations and the world over have concluded that early initiation of HAART coupled with a high HAART compliance may be the defining reason for lower-than-expected prevalence of precancerous and cancerous lesions in hospital-based studies among women with HIV (Harms et al., 2006; Akarolo-Anthony et al., 2013). 100 University of Ghana http://ugspace.ug.edu.gh Discussion Although the Pap Smear test has a very good specificity for malignancy and precursor lesions, it suffers a corresponding low sensitivity at the same time (Saslow et al., 2012). The implication of this may be that a high proportion of genuine cases may be missed, usually showing up in cytology reports in the ambiguous atypical squamous cells (ASC) category, with adverse clinical implications. The results of this study confirm that ASC were a common finding (Davey, 2005). The most effective way to manage this outcome is a matter of concern for healthcare services. Recent guidelines recommend that, following two initial normal Pap-smears at a 6- month interval, all HIV-positive women should undergo annual cervical cytologic examination. In addition, it is recommended that all immunosuppressed women with atypical squamous cells undergo colposcopy (Yar et al., 2016). In well-endowed settings, cervical cancer screening strategies integrating HPV testing have been proven to improve disease detection and afford longer screening cycles (Saslow et al., 2012) and could be explored in high-stakes patient populations such as this. This approach benefits from the HPV test’s greater sensitivity in diagnosing women with CIN3+ (Arbyn et al., 2006b). In more advanced cytology screening settings, the practice has been to perform reflex HPV testing for all ambiguous cytology reports (ASCUS) and onward colposcopy evaluation in the event of a positive HPV screen. Another way is to perform cytology and HPV testing simultaneously (Wright et al., 2014). Both approaches have been shown to improve the clinical performance of cytology in other settings. Though several studies have consistently shown a high burden of HPV-associated diseases in HIV positive women even in the era of HAART (Sinayobye et al., 2014), presently, there is limited or non-existent routine screening services for many HIV positive women. There are advantages to providing routine screening to HIV positive women. In Zambia, for example, routine screening prevented one death from ICC for every 46 HIV positive women screened (Yar et al., 2016). 101 University of Ghana http://ugspace.ug.edu.gh Discussion Cervical cancer caused by persistent infection with high-risk human papillomaviruses is a defining condition of HIV/AIDS.(Denny et al., 2012) In the present era of type-specific vaccines and HAART, there is a lot of interest regarding the prevalence and distribution of HPV among women living with HIV. This study presents an estimate on the fraction of individuals carrying a new, persistent, or recurring HPV infection in the population of women presenting to HIV clinic during the study period. Among women enrolled in this study, the overall prevalence of HPV infection was 52.0% and the prevalence of high and low risk HPV infection was 29.7% and 10.2% respectively. Among women on ART the overall prevalence of high-risk HPV infection was 80.3% against 91.7% in those not enrolled on ART. Concordant estimates have been reported in several studies from sub-Saharan Africa. A cross- sectional hospital-based study involving 255 HIV infected women was conducted at an HIV care and treatment clinic in Mwanza, Tanzania. The prevalence of HPV among HIV positive women was 51.4% while the prevalence of high risk types was 48.6% (Mbulaiteye et al., 2006). Among 220 HIV positive women recruited during community cervical cancer screening programme in states of Ogun and Lagos, the prevalence of only high risk HPV infection was 24.5% (Gelband and Sloan, 2007) similar to what was found in the present study. In a cross- sectional observational study among HIV positive women recruited at the University Teaching Hospital in Lagos, Nigeria, the prevalence of HPV among HIV positive women was 44.9% while the prevalence of high risk types was 37.5% (Franceschi and Jaffe, 2007). The present findings (prevalence of 52.0%) are in line with the results from other studies in other countries that reported high HPV prevalence rates in HIV-infected women (Chuang, 1987; De Sanjosé et al., 2007; Vera-Iglesias et al., 2007; Bedoui et al., 2009). Park et al., (2007) retrospectively reviewed women living with HIV attending Pusan National University Hospital 102 University of Ghana http://ugspace.ug.edu.gh Discussion (Korea) for the prevalence and genotype distribution of HPV infection and reported an overall HPV infection rate of 46.7%. Highly elevated rates of HPV infection have been reported in the Ashanti Region of Ghana and in other major cities in African (De Vuyst et al., 2009; Gasparini and Panatto, 2009). Yar et al., (2015) conducted a study on 107 women infected with HIV. Overall HPV positivity was 86.9%. The excess HPV positivity was probably an effect of the expanded number of 28 high- and low- risk HPV genotypes that were tested for compared to 18 HPV genotypes in the present study. High prevalence rates of HR-HPV have been reported among two separate studies in HIV- positive Rwandan women in Kigali, 46% (Trottier et al., 2006a) and 50.8% (De Sanjosé et al., 2007) as well as studies in other double risk populations such as commercial sex workers infected with HIV with reported HR-HPV prevalence rates ranging from 44-73% (Trottier and Franco, 2006; Trottier et al., 2006a; Luchters et al., 2010), In all of these studies, the ART status of participating women was undisclosed and could point to the higher than expected rates. In a cross-sectional study of 498 HIV-positive women in Kenya, 68.7% of women were HPV- positive: and 52.6% had HR HPV. Elevated CD4 count and prolonged HAART use were significantly associated with lower prevalence of high-risk HPV infection in that study (Gasparini and Panatto, 2009). This prevalence of 68.7% is considerably higher than the prevalence rates of 37.2% that was found in women from the general population in Kumasi, Ghana (Donkoh, 2015). It is well established that there is a significantly higher HPV prevalence in HIV positive women compared to HIV negative women. There are several comparative studies to support this claim. A high magnitude of association of HIV infection with high-risk HPV was previously found in a Uganda (Bosch et al., 1992) and Rwanda (Franceschi et al., 2006). Park et al., (2014) retrospectively reviewed HIV-infected women attending a University Hospital for the prevalence and genotype distribution of HPV infection among HIV-infected women in Korea. 103 University of Ghana http://ugspace.ug.edu.gh Discussion The authors of the study reported an overall, HPV infection rate significantly higher and associated with five-fold odds in HIV-positive women than HIV-negative women (46.7% against 14.1%). A prevalence of (77.4% against 41.6%) Yar et al., 2016 and (65.6% against 30.2%) Obiri-Yeboah et al., 2017 in HIV-positive and HIV-negative women respectively were observed in studies in Ghana (Yar et al., 2016; Obiri-Yeboah et al., 2017). Other studies previously showed that HIV-infected women are more likely than those without HIV infection to have persistent HPV (Adler et al., 2015) In general, the significantly higher HPV prevalence among HIV-positive women can be attributed to increased susceptibility, decreased ability to clear infection which may be due to factors directly related to immunosuppression and frequent reactivation of latent infection associated with immunosuppression (Khan et al., 2005). The destruction of CD4 cells by HIV may increase the likelihood of HPV establishing infection (Mbulaiteye et al., 2011). The evidence also suggests that immunosuppression leads to higher probability of HPV reactivation, potentially due to incomplete clearance of HPV DNA (Humans et al., 2007). In one African study HPV was inversely associated with age in HIV negative women as the prevalence of HPV among HIV positive women remained high in all age-groups (Bosch et al., 1992). Owing to advances made in ART and management, women infected with HIV now live longer, fulfilling lives. Cervical cancer caused by HPV is considered to be a needless and preventable cause of death. Knowledge of the distribution of HPV genotypes prevalent among high-risk populations can guide the implementation of successful primary prevention of these cancers in women who eventually develop HIV infection. The commonest HPV types detected here were HPV-35 (14.1%), HPV-18 (13.0%), HPV-58 (7.4%), HPV-52 (7.2%), HPV-45 (4.8%) HPV-44 (3.7%), HPV-39 (3.3%) and HPV-68 (3.2%). The frequency of HPV 16 was 2.4%. HPV 42, a low-risk type, was also common (7.8%). 104 University of Ghana http://ugspace.ug.edu.gh Discussion Although the mechanism by which HIV increases risk of cervical cancer is not completely understood, studies suggest that HIV-induced immunosuppression leads to an inability to control the expression of HPV and the production of HPV oncoproteins E6 and E7 (McKee et al., 1981) and the risk appears to be associated with increased HPV persistence that may result from immunosuppression related to HIV. Furthermore, the risk is greater in women with CD4 counts less than 200 cells per microliter and in those with high plasma HIV RNA levels (Hawes et al., 2006). There is evidence that HIV-1 infection is associated with an increased rate of HPV infection, mainly restricted to HR-HPV types which are the cause of invasive cancer of the cervix (Didelot-Rousseau et al., 2006; Petry et al., 2012). There are not many reports on the molecular epidemiology of HPVs in Ghanaian women. Furthermore, differences in analytical methodology and sensitivity complicates any potential comparisons. A study involving HIV women in Kumasi showed that infections of HPV 58, 35, 31, 68, 53, 52, 18, 16 were most common (Yar et al., 2015). Apart from variability in HPV 31 and 53, the rest of the HPVs detected were confirmed in this study as well to be prevalent among Ghanaian women with HIV infection. The common genotypes observed in this study was also similar to a study conducted in Central Region of Ghana (Obiri-Yeboah et al., 2017) where the authors found (HPV 35, 52, 58, 16 and 18)). HIV-infected women are reported to acquire a broader spectrum of HPV genotypes compared to HIV-naïve women (Clifford et al., 2006c; Didelot-Rousseau et al., 2006; Sahasrabuddhe et al., 2007). Among populations of HIV positive women in the same country, diverse HPV genotypes may be prevalent depending on several potential confounders such as ART regimes and sexual activity of women. In a previous studies evaluating the concordance of HPV infection among heterosexually active couples and the impact of HIV coinfection on the prevalence of HPV, HIV coinfection in one partner had a significant impact on the prevalence of HPV infection in the other partner (SCHEURER et al., 2005). Concordance of the same HPV genotypes was more 105 University of Ghana http://ugspace.ug.edu.gh Discussion commonly found among couples where one or both partners were HIV-infected, compared with HIV-uninfected couples (SCHEURER et al., 2005). The majority of WLHIV got infected by their male sexual partners in Korea (SCHEURER et al., 2005). This can be very significant for cervical cancer prevention in this population. In a comparative cross-sectional analytic observational study among HIV positive women at the Lagos University Teaching Hospital (LUTH) Lagos, Nigeria, the commonest high risk types seen were types 31, 52, 53 and 35 (Franceschi and Jaffe, 2007). In a cross-sectional study involving 106 women at an STI clinic in Kampala the commonest types were HPV 52, HPV 16, and HPV 58; HPV 16/18 combined was 18.4% (Blossom et al., 2007). In a cross-sectional study of 498 HIV-positive women in Kenya, common genotypes found included HPV-35 (12.3%), HPV-16 (10.8%), HPV-52 (7.2 %), HPV-56 (6.4 %), HPV-18, 58 (5.4% each), and HPV-31 (5.2%) (Gasparini and Panatto, 2009). Park et al. (2014) retrospectively reviewed HIV-infected women attending Pusan National University Hospital in Korea and observed that, the most common HR-HPV genotype detected in these women was HPV 16 (10%), followed by 18 (6.7%) and 52 (5%). However, this was a limited sample size. Prevalence of quadrivalent vaccine-preventable types (HPV 6, 11, 16, and 18) was 21.7% and 2.3% in HIV-positive women and HIV-negative women, respectively. The results of the present study suggests that there may be some variability in the distribution of high-risk genotypes in HIV positive women compared to the general population as noted previously (Yar et al., 2015). The detection of all HR types, even HPV16, is higher in HIV positive women in comparison with HIV-negative women. However, the prevalence of HPV16 in HIV positive women is known to be less affected by decreases in CD4+ cell counts than other high-risk HPV types (Wanichwatanadecha et al., 2012). This may occur because clearance of oncogenic HPV infection is less successful in HIV positive women due to a lack of an appropriate immune response (Frisch et al., 2000) which may be especially obvious for high 106 University of Ghana http://ugspace.ug.edu.gh Discussion risk genotypes other than 16 and 18 (Bernard et al., 2010). These two are thought to be sufficiently oncogenic that their persistence in the cervical mucosa is less dependent on the immune system of the host woman. Once the impact of ART in HPV infection and cervical cancer is defined, the dynamics of this variability would be easier to appreciate. The combined prevalence of 15.4% of the highly oncogenic HPV 16 and 18 in this study is similar to estimates in other studies among HIV positive women and in the general population (Münger et al., 1992; Clifford et al., 2006a). The reason may be that HPV 16 and 18 are better at evading the host immune system and are less impacted by HIV-associated immunosuppression (Boshart et al., 1984; Cogliano et al., 2005). In one case, the relative risk of acquiring HPV 16/18 was higher among HIV-positive women, nevertheless HPV 16/18 clearance was not found to differ by HIV status (Bernard et al., 2010; Wanichwatanadecha et al., 2012). Studies have found that HPV 16 in particular is more weakly associated with the immune system compared to other types (Münger et al., 1992; Wanichwatanadecha et al., 2012). This is consistent with studies that find HIV-positive women have a greater diversity of HPV types in normal cytology and low-grade lesions, but the relative prevalence of HPV 16 and 18 increases with severity of lesions and CC and the proportion of cervical cancer attributable to HPV 16/18 is similar between HIV positive and negative women (Jeon et al., 1995; Forslund et al., 2004). Quadrivalent HPV vaccine was immunogenic and well tolerated in HIV-infected young women in a study (Palefsky et al., 1999). HPV vaccine is recommended by the Advisory Committee on Immunization Practices (ACIP) for HIV-infected individuals through age 26 years for those who did not get any or all doses when they were younger (Bernard et al., 2010). Prevalence of vaccine-preventable types (HPV 6, 11, 16, and 18) or types (HPV 6, 11, 16, and 18, 31, 33, 45, 52, 58) in our HIV-infected women was 21.3% and 43.3%% respectively, suggesting that innovative strategies would be needed to effectively prevent these infections. HPV DNA or 107 University of Ghana http://ugspace.ug.edu.gh Discussion serologic screening may be needed before immunization in HIV-infected women in Ghana, although the ACIP does not recommend HPV DNA or serologic screening before immunization (Bernard et al., 2010). It is well established that the contribution of different HPV genotypes to cervical cancer and intraepithelial lesions vary greatly owing to differences in carcinogenic potential (Muñoz et al., 2004). However, HPV infection in general progresses to squamous intraepithelial neoplasia more frequently and rapidly in HIV-infected women than in the general population (Boshart et al., 1984; Münger et al., 1992; Organization and Cancer, 2007). This work presents the relationship between detection of HPV genotypes and the presence of cytological lesions in women living with HIV (Table 4.5-1) to give insight on the impact of HIV-related immunodeficiency on the natural history of high risk HPV in incidental cervical precancerous lesions. Prevalence of HPV infection ranged from 25.9% among women with normal Pap findings to 53.3% among women with ASCUS. No significant differences were noted for the nature of the infection: any type HPV infection, high-risk HPV, or low-risk HPV and Pap smear outcome. The commonest HPV genotypes detected among women with ASCUS were HPV-18, 35 and 31, 39, 45, 51, 52, 58, 68. Among women with LSIL most prevalent were HPV-52, HPV- 35, HPV 16 and HPV 18, 58, 39. HPV-52, 35 and 58, 45 were top three in that order for high grade intraepithelial (HSIL)/severe dysplasia lesions. The genotype-specific distribution detected in women without any precancerous lesions was as follows: HPV-35, HPV-18, HPV- 52, HPV-58 and HPV-68. The result of the present study confirms that there is an increased attributable fraction for non- HPV16 types in precancerous lesions. This relative shift from HPV16 to other HR types has been observed previously for HIV-infected women with normal cytology and cervical intraepithelial lesions (Clifford et al., 2006a), and has significant implications for using vaccine- 108 University of Ghana http://ugspace.ug.edu.gh Discussion based prevention strategies as a sole measure among women with HIV. A meta-analysis confirmed that HPV16 was underrepresented relative to other high-risk HPV types among HIV- positive women, in all grades of cervical diagnoses up to high-grade precancerous lesions (Clifford et al., 2006a). Variation in HPV genotype distribution in ICC is not new as it has been observed in other regions of the world. It is hypothesized that host immunogenetic factors and biologic interplay between different HPV genotypes or variants are most likely responsible (Bousarghin et al., 2003). Studies from sub-Saharan Africa have shown under-representation of HPV-16 in women with abnormal cytology that have been attributed to HIV/AIDS endemicity and the propensity for women with HIV to harbor a greater range of HPVs including multiple infections (Boshart et al., 1984). HPV 35, for instance, was slightly more common than HPV 16 in Mozambique both in women with normal cytology and in those with HSIL or worse (Castellsague et al., 2001). HPV 52 was found slightly more frequently than HPV 16 or HPV 35 in Kenya (De Vuyst et al., 2003) and in women with normal colposcopy in Zimbabwe (Gravitt et al., 2002). In Senegal, HPV 16 and 58 were the most common types overall and in women with cervical lesions (Xi et al., 2003). Indeed, the importance of HPV16 increases with severity of lesion (Muñoz et al., 2004), so that HPV16 remains by far the most common high-risk HPV type, in both HIV-positive and HIV- negative ICC (Boshart et al., 1984). However, the proportion of ICC attributable to HPV16 is marginally lower in HIV-positive than HIV-negative women. This shows that it will be useful to focus preventive effort on other HR types for women with HIV in order to prevent the occurrence of cervical intraepithelial lesions. This effort may not be as beneficial in ICC since the difference in HPV16 proportion in ICC is less dependent on HIV status than that seen among normal cytology and cervical intraepithelial lesions (Clifford et al., 2006a). 109 University of Ghana http://ugspace.ug.edu.gh Discussion Nevertheless, the present data confirms reports that the attributable fraction for other vaccine types such as HPV18, 52 and 45 in cervical pre-cancer is concomitantly higher, so that current expanded prophylactic vaccines against these genotypes may be of greater utility in HIV positive women than earlier HPV16 and 18 based vaccines, which have shown some promise anyway in HIV-infected populations (Frellick, 2017). In general, the high prevalence of high-risk HPV in ASCUS/LSIL and HSIL among HIV positive women in the present study is similar to several reports on HIV-positive women with precancerous lesions. Concomitant reports of a high prevalence of high risk HPV genotypes have been documented in sub-Saharan Africa in cervical/vaginal specimens and HSIL lesions (Blossom et al., 2007; Donkoh, 2017; Donkoh et al., 2019a). In these reports, HIV positive women had more high risk HPV genotypes detected compared to HIV negative women. In a particular report, mean number of HPV genotypes detected was higher among HIV positive compared to HIV negative women (Donkoh et al., 2019a). The high prevalence of non-vaccine preventable genotypes such as HPV-35 in HSIL in this study is an important finding because of implications for disease prevention. Many studies report a high prevalence of HPV 35 in Africa. For instance, it was slightly more common than HPV 16 in Mozambique both in women with normal cytology and in those with HSIL or worse (Castellsague et al., 2001). Data from meta-analysis of 21 African studies reveal that a large part of the non-vaccine-type related ICC remaining fraction in African women is attributable to HPV35, regardless of HIV status (Boshart et al., 1984). Three prophylactic vaccines containing virus-like particles that offer protection against cervical pre-cancers and cancers are available. Studies have indicated that these vaccines are safe, well tolerated and efficacious against the vaccine-related HPV types that cause persistent infection 110 University of Ghana http://ugspace.ug.edu.gh Discussion and cervical disease in young ladies (Harper et al., 2004; Lu et al., 2011; Petrosky et al., 2015). Additionally, the quadrivalent and nonavalent vaccines targeted against HPV 6, 11, 31, 33, 45, 52 and 58 and can prevent 90% of all cases of genital warts (Villa et al., 2005; Petrosky et al., 2015). Mathematical models predict that widespread use of preventive HPV vaccines containing genotypes 16/18 have the potential to reduce deaths from ICC by 50% over several decades (Koutsky and Harper, 2006; Nobre et al., 2009). In sum, the major study finding is that high-risk HPV infection was frequent in women living with HIV and vaccine preventable HPVs are significantly associated with squamous intraepithelial lesions, suggesting that this population follows an epidemiological pattern in common with other HPV-infected women, who remain at high risk for cervical neoplasia from high risk, vaccine preventable genotypes. Additionally, there was a high prevalence of non- vaccine HPV types such as HPV-35, 39 and 68 among women with ambiguous and high grade squamous intraepithelial lesions. As such, early vaccination may protect women who eventually develop vaccine-preventable HPV types but for best results, regular screening of women and colposcopic management of HIV-positive women will continue to be justified for preventing the excess morbidity from non-vaccine types found here. HIV is known to be an important independent risk factor for development of precursor lesions and cervical cancer (Getinet et al., 2015). Several studies conducted in sub-Saharan Africa indicate that among African women, being HIV infected was associated with a high risk of presenting squamous intraepithelial lesions of the cervix, with ORs ranging from 4.4 to 17 depending on the grade of the lesion and other factors (DiMaio and Petti, 2013; Doorbar, 2013). Low- and middle-resource countries, where women have been hit hardest by the AIDS epidemic, have historically also had very high prevalence of human papillomavirus (HPV) infection (Clifford et al., 2006b) and a high incidence of cervical cancer and precancerous 111 University of Ghana http://ugspace.ug.edu.gh Discussion lesions (Parkin et al., 2003). With the adoption of HAART, women living with HIV and persistent HR-HPV are expected to live longer than before and may develop precancerous lesions which, if unchecked, may retrogress to end-stage cancer (Moodley and Mould, 2005). This study investigated the determinants of abnormal cytology among women living with HIV and attending specialized care in Accra, Ghana. After adjusting for potential confounders such as woman’s age and age at first pregnancy, age at sexual debut (mean 18.3 ± 3.0 years) had significant main effect for abnormal Pap smear result later in the life of women. This finding is supported by the report from Ethiopia (Getinet et al., 2015) and Tanzania (Gondos et al., 2005). The final predictive model was based on educational status, income, and age at sexual debut, significantly predicted Pap smear results explained up to 10% of the variability in cytology outcome (Table 4.6-3). Educational status, income, and age at sexual debut (Gondos et al., 2005; Getinet et al., 2015) have been previously reported to be important predictors of precancerous lesions in similar studies. Other important risk factors that have been reported such as marital status (Getinet et al., 2015), polygamy, history of STI (Gondos et al., 2005; Getinet et al., 2015; Schulze et al., 2016), oral contraception and high parity (Getinet et al., 2015) were not significant risk factors in the present study. The reason for this might be that the effect of some variables may be modified by other variables and possibly confound their effect in different populations (Gondos et al., 2005; Getinet et al., 2015). Among women with normal cytology and throughout the range of cytological abnormalities detected in African women HPV genotype distribution often differs from other world regions (WHO/ICO, 2010b). Variation in HPV genotype distribution in cervical lesions results from host immunogenetic factors and biologic interplay between different HPV genotypes (Bousarghin et al., 2003). Since this variation has implications for the efficacy of multivalent preventive HPV vaccines in high-risk populations, there is much interest to decipher the putative 112 University of Ghana http://ugspace.ug.edu.gh Discussion factors predicting high-risk HPV infection in these populations such as HIV positive women in particular. Regarding socio-demographic characteristics, after adjusting for potential confounders such as woman’s age, younger age at sexual debut and recent sexual activity, formal education and income status were associated with high-risk infection (Table 4.7-1). Regarding women’s sexual behavioral characteristics, after adjusting for potential confounders such as age and log CD4 count, duration of HIV infection (p=0.04) and vaginal discharge (p=0.01) were significant predictors of cytology outcome (Table 4.7-2). In a similar study, 498 HIV-positive women were assessed for the cross-sectional association of HPV and CIN with various characteristics, CD4 count and use of combination antiretroviral therapy (cART) among HIV-positive women. High-risk human papillomavirus-positivity did not vary significantly by age but it was negatively associated with education level (Gasparini and Panatto, 2009). The finding that duration of HIV infection is a predictor of HR-HPV infection confirms what has been reported previously and may explain how the young age and HIV positivity may interact in the aetiology of cervical lesions (Palefsky et al., 1999). In a recent systematic review of twenty studies in African women, it was observed that “number of lifetime sexual partners”, “HIV infection” and “age” were the risk factors for high occurrence of HPV infection which is consistent with other studies (Palefsky et al., 1999)The number of sexual partners which has often been cited as a risk factor for high-risk HPV infections (Franceschi et al., 2006) was not associated with high-risk HPV here. Hormonal contraceptive use was also not found to be associated with high-risk HPV infection among women in conformity with other studies in African women (Lazcano-Ponce et al., 2001; Peyton et al., 2001; Wandeler et al., 2016). Rather, a weak association between barrier method of contraception and high-risk HPV became insignificant after adjusting for HIV duration in this study. 113 University of Ghana http://ugspace.ug.edu.gh Discussion ART duration was not an independent predictor of high-risk HPV prevalence but its effects were likely mediated through HIV duration. This was confirmed by (Franceschi et al., 2006) in similar circumstances in Rwanda. Although prophylactic HPV vaccines may prove to be the ultimate cervical cancer prevention strategy, there are already 2-3 generations of at-risk, high- risk HPV-positive women who will not benefit from HPV vaccination, and universal HPV vaccination is decades away. Moreover, in the ART era, HIV positive women will live longer, have a greater high-risk HPV burden, and in the absence of comprehensive screening and treatment, will likely remain at an elevated risk of invasive cervical cancer. Thus, there is still a need to better understand the hallmarks of natural immunity against HPV infection. This may provide therapeutic avenues against HPV and HPV related disease that might be used to prevent HPV-related cancers in those already infected with high-risk HPV 114 University of Ghana http://ugspace.ug.edu.gh Conclusions Chapter 6 CONCLUSIONS The prevalence of precancerous cervical lesions was lower than what has been reported among other cohorts of HIV-infected women in Africa and similar settings. Although the prevalence of precancerous cervical lesion and tissue change reported among HIV-positive women was below regional estimates in this population, it was still very high and represents a significant proportion of the clinic population that would benefit from screening and treatment to prevent invasive cancer. Hence, this finding strongly confirms that HIV-infection is highly convergent with development of a cervical precancerous lesions and has implication for the management of HIV women attending specialized healthcare in the country especially in the era of sustainable ART. It is recommended that screening of HIV-infected women for cervical cancer should be a matter of routine practice. Availability and accessibility of these services can be done through their integration into HIV clinic manuals. Prompt initiation of HAART through an early enrolment into care may reduce the prevalence and progression of cervical precancerous lesions. Furthermore, this finding has implications for future interventions in the implementation of prophylactic HPV vaccines to be the part of care and support programme for HIV-infected women according to recommended guidelines for extended vaccination in sexually active HPV negative women. In conclusion, HIV positive women had a high burden of HPV infection at the time when their life-spans are being prolonged by expanded access to highly active anti-retroviral therapy (HAART) and medical care. Though several studies have consistently shown a high burden of HPV-associated diseases in HIV positive women even in the era of HAART, presently, there is limited or non-existent routine screening services for many HIV positive women. Evidence elsewhere demonstrates that there are advantages to providing routine screening to HIV positive 115 University of Ghana http://ugspace.ug.edu.gh Conclusions women. In Zambia, for example, routine screening prevented one death from ICC for every 46 HIV positive women screened In the United States, CC screening programs have been highly effective in reducing CC incidence and HPV vaccines have been shown to reduce the prevalence of HPV 16 and 18-attributable precancerous lesions. The high prevalence of HPV infection, STIs, and cervical lesions detected among women attending ART clinic in this study demonstrates a need for routine cervical screening of all HIV- infected women in this setting. These patients need to be properly triaged in order to ensure effective use of scarce resources. In the meantime, a simple, preferably one-stop, cervical cancer screening strategy may be a sustainable model for HIV positive women. The results of this study confirms findings that there may be some variability in the distribution of HPV genotypes in HIV positive women compared to the general population. The potential spill-over effects of genotypes from this high-risk population into the general population may be significant for cancer prevention efforts in the Ghanaian population. In this study, HIV positive women had multiple HR-HPV genotypes in excesses of HPV 16/18 consistent with previous studies. This finding is in line with reports that the reason HPV 16 and 18 are underrepresented in precancerous lesions compared to other genotypes may be that, they are better at evading the host immune system and are less impacted by HIV-associated immunosuppression. Consequently, it is clear that the vaccines with expanded genotype coverage may be more beneficial than those containing only HPV 16 and 18 genotypes in preventing precancerous lesions in this population. It is also clear that cervical screening will continue to play a role in cancer prevention in this population. In sum, the major study finding is that high-risk HPV infections were frequently detected in women living with HIV infection. Given the high prevalence of HIV-HPV coinfection, associated cervical lesions represent a major public health burden in the country. Vaccine preventable HPVs are significantly associated with squamous intraepithelial lesions, suggesting 116 University of Ghana http://ugspace.ug.edu.gh Conclusions that this population follows an epidemiological pattern in common with other HPV-infected women, who are at high risk for cervical neoplasia from high risk, vaccine preventable genotypes. HR-HPV 16, 18, 31, 33, 45, 52 and 58 targeted by two licensed preventive HPV vaccines are the most common genotypes found in invasive cervical cancer even among HIV positive women. However, the present data reveals that a large number of women may harbour other HR-HPV genotypes not targeted by the currently available HPV vaccines: there was a high prevalence of non-vaccine HPV types such as HPV-35, 39 and 68 among women with ambiguous and high grade squamous intraepithelial lesions. The indication is that these vaccines could be less effective and leave women open to risk for invasive cancer if used in isolation. Early vaccination may protect women who eventually develop vaccine-preventable HPV typeks but for best results, regular screening of women and colposcopic management of HIV-positive women will continue to be justified for preventing the excess morbibity from non-vaccine types found here. Moreover, in the future, any preventive HPV vaccine(s) should contain more HR- HPV genotypes of significance confirmed by more evidence. In conclusion, it was established that early age at sexual debut was a significant risk factor for precancerous lesions among HIV positive women. Therefore, awareness creation on these risk factors such as early age of sexual debut and those reported in other studies should be intensified as part of preventive effort. The general awareness of risk factors of cervical dysplasia is very low even among well-educated women. Furthermore this study proves that such awareness should be provided to girls at the level of primary education in order to be most effective. The predictive model shown here (Table 4.6-3) has important implications for the management of HIV-positive women and other high-risk populations in the era of HAART. In conclusion, we found that HIV positive women presenting for routine clinical care had a high high-risk HPV prevalence. The present data have important implications for the management of 117 University of Ghana http://ugspace.ug.edu.gh Conclusions this common occurrence of high-risk HPV in HIV-positive women and other high-risk populations in the era of HAART. In the general population, high-risk HPV-based screen and treat, in which all high-risk HPV-positive women are treated immediately, may be a reasonable strategy, despite the lack of adequate colposcopy facilities and pathologists to provide tissue- based diagnosis to guide treatment. However, in the HIV positive population, especially those who are highly immune suppressed and are likely to be at the greatest risk of cervical cancer, the data available here shows that high-risk HPV-based screening may not have the desired specificity to be used alone, especially in a screen-and treat strategy. In the current context, approximately a third of the HIV positive women would test high-risk HPV positive and be treated. In other populations, even greater proportions would be treated Thus, it may be desirable to use a secondary, triaging formula to determine which high-risk HPV-positive women need immediate treatment (triage positive) and which might have treatment deferred (triage negative) until there is evidence of high-risk HPV persistence. Study Limitations This study had limitations mainly due to the cross-sectional design with potential problems with direction of causality. Eighteen HPV genotypes were tested for in this study, we have approximately 40 alpha-papillomaviruses capable of infecting the ano-genital tract of human. We did not sequence the genotypes to check on gene variability. 6.1 RECOMMENDATIONS Epithelial cell abnormalities usually develop without recognizable symptoms. Therefore the rationale for screening for cervical cancer has always been to detect abnormal cervical cells: including precancerous cervical lesions, as well as early cervical cancers at an early stage where 118 University of Ghana http://ugspace.ug.edu.gh Conclusions remedial measures may be taken at reasonable cost. Most women in countries with poorly- resourced healthcare systems do not have access to reliable screening programs and consequently report to hospital only when lesions are at an advanced invasive stage. Women living with HIV are not excluded from this accessibility challenge. Overall screening and diagnosis is a major challenge in developing countries, Ghana inclusive. In the light of the findings of this work, this situation must begin to change for the better. Access to reliable cervical cancer screening must be considered to be a priority for high-risk populations. This work has confirmed previous reports in overseas settings that women living with HIV have a greater prevalence of precancerous lesions by demonstrating a similar trend in a population of women from the largest Ghanaian HIV clinic. It is recommended that focus must be given to treating women with precancerous lesions to prevent their progressing to cervical cancer and death. Further investigations are need to determine the optimal cervical cancer screening strategies, those that balance the benefits and harms of screening, in known HIV-positive women living in Africa. 119 University of Ghana http://ugspace.ug.edu.gh REFERENCES Açikgöz A. and Ergör G. 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Villa L.L., Costa R.L., Petta C.A., Andrade R.P., Ault K.A., Giuliano A.R., Wheeler C.M., Koutsky L.A., Malm C., Lehtinen M., Skjeldestad F.E., Olsson S.E., Steinwall M., Brown D.R., Kurman R.J., Ronnett B.M., Stoler M.H., Ferenczy A., Harper D.M., Tamms G.M., Yu J., Lupinacci L., Railkar R., Taddeo F.J., Jansen K.U., Esser M.T., Sings H.L., Saah A.J. and Barr E. (2005) Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. The Lancet Oncol 6, 271-278. Wabinga H., Parkin D., Wabwire-Mangen F. and Nambooze S. (2000) Trends in cancer incidence in Kyadondo County, Uganda, 1960–1997. British Journal of Cancer 82, 1585. Walboomers J., Jacobs M., Manos M., Bosch F., Kummer J., Shah K., Snijders P., Peto J., Meijer C. and Munoz N. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. 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Zhao X.-L., Hu S.-Y., Zhang Q., Dong L., Feng R.-M., Han R. and Zhao F.-H. (2017) High-risk human papillomavirus genotype distribution and attribution to cervical cancer and precancerous lesions in a rural Chinese population. Journal of gynecologic oncology 28. Zheng Z.-M. (2014) Human papillomaviruses. In Cancers in People with HIV and AIDS, pp. 87-112: Springer. Zheng Z.-M. and Baker C.C. (2006) Papillomavirus genome structure, expression, and post-transcriptional regulation. Frontiers in bioscience: a journal and virtual library 11, 2286. Zhu Y., Wang Y., Hirschhorn J., Welsh K.J., Zhao Z., Davis M.R. and Feldman S. (2017) Human papillomavirus and its testing assays, cervical cancer screening, and vaccination. Advances in clinical chemistry 81, 135- 192. 131 University of Ghana http://ugspace.ug.edu.gh Appendix Zielinski G.D., Bais A.G., Helmerhorst T.J., Verheijen R.H., de Schipper F.A., Snijders P.J., Voorhorst F.J., van Kemenade F.J., Rozendaal L. and Meijer C.J. (2004) HPV testing and monitoring of women after treatment of CIN 3: review of the literature and meta-analysis. Obstetrical & gynecological survey 59, 543-553. zur Hausen H. (1996) Papillomavirus infections--a major cause of human cancers. Biochim Biophys Acta 1288, F55- 78. zur Hausen H. (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2, 342-350. 132 University of Ghana http://ugspace.ug.edu.gh APPENDIX 1 PATIENT QUESTIONNAIRE Project Title: Genotypes of Human Papillomavirus among HIV Positive Women Attending Clinic at the Korle-bu Teaching Hospital, Accra, Ghana Subject No………… Date…………….. Personal Profile 1. Age………………………… 2. Educational Level: None [ ] Primary [ ] Secondary[ ] Tertiary [ ] 3. Occupation…………………………………………………………………. 4. Residence………………………………………………………………….. 5. Region……………………………………………………………………… 6. Ethnicity……………………………………………………………………. 7. Marital status: Single[ ] Married[ ] Separated[ ] Divorced[ ] Widowed 8. How do you pay your medical bills? Self [ ] Relatives[ ] Employer[ ] NHIS Risk Factors for HPV infection Sexual Partners 9. Are you the only wife of your husband? Yes[ ] No[ ] 10. If no how many wives does your husband have? ................ 11. How many permanent sexual partners do you have? ............. 12. How many sexual partners have you had in the past 6months? …… 13. How many lifetime sexual partners have you had? …………… 14. At what age did you first have sex? .............................................. 133 University of Ghana http://ugspace.ug.edu.gh 15. How often do you have sex? ................................................. History on Pregnancy 16. Have you ever been pregnant? Yes [ ] No [ ] If yes, what was your age at first pregnancy? ............................... How many times have you been pregnant? ..................................... How many of your pregnancies resulted in miscarriage or abortion? ................ How many of your pregnancies resulted in still birth? .................................. Smoking status 17. Do you smoke tobacco? Yes [ ] No[ ] If yes, how long have you been smoking? ............................................ How many cigarettes on average do you smoke per week? ...................... 18. Have you smoked before? Yes [ ] No[ ] 19. If yes how long did you smoke? ........................................................... 20. Do you use any other tobacco-related product? (snuff) Yes [ ] No[ ] If yes, how long have you been using it? ............................................ How many tobacco-related products on average do you smoke per week? ....... Condom Use 21. Do you use condoms during sex? Always[ ] Sometimes[ ]Never[ ] 134 University of Ghana http://ugspace.ug.edu.gh Other Sexually Transmitted Infection when were you diagnosed of HIV? ....................................................................... 22. Are you using anti-retroviral therapy? Yes [ ] No [ ] 23. If yes, how long have you been using it?................................................ 24. Do you have any sexually transmitted infection? Yes[ ] No[ ] If yes, state what type it is...................................................................... When was it diagnosed? ........................................................................ 25. Have you been infected with any other sexually transmitted infection before? Yes [ ] No [ ] 26. If yes, state what type it was.................................................................... When was it diagnosed? ........................................................................ Subject number: ……………………………………………………….. CONSENT TO PARTICIPATE IN A RESEARCH PROJECT 135 University of Ghana http://ugspace.ug.edu.gh TITLE OF PROJECT: Genotypes of Human Papillomavirus among HIV Positive Women Attending clinic at the Korle bu Teaching Hospital, Accra, Ghana Before agreeing to participate in this research study, it is important that you read the following explanation of this study. This statement describes the purpose, procedures, benefits, risks, discomforts, and precautions of the program. Also described are the alternative procedures available to you, as well as your right to withdraw from the study at any time. Explanation of Procedures You are being invited to participate in a research project to determine the prevalence and genotypes (the type of Human Papillomavirus found) of HPV infection among women living with HIV in Accra. The approach of the research is through the use of a questionnaire and cervical specimen collection. You will complete the questionnaire that will require answers on personal profile and risk factors for HPV infection (i.e. number of sexual partners, smoking status, contraceptive use etc.). Afterwards, you will be examined by medical officer and cervical specimen will be collected by inserting a speculum into your vagina, which spreads the vagina open and allows access to the cervix. A sample of cells is then collected from the outer opening or os of the cervix by scraping with an Aylesbury disposable spatula. An endocervical brush is rotated in the central opening of the cervix. This procedure will be carried out in a private room with your comfort and safety in mind. Your cell phone numbers will be collected so that we can contact you if need be for treatment or management purposes. Risks and Discomforts 136 University of Ghana http://ugspace.ug.edu.gh By participating in this research, you are likely to experience some form of discomfort. This includes the discomfort of questioning, physical examination, and the pain of cervical specimen collection. 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 Women who are found to have genital warts, Cervical Intraepithelial Neoplasia (CIN) and cervical cancer shall be called back for treatment and management. However, this research is expected to provide data on HPV genotypes and distribution for policy makers when vaccination becomes a mainstream policy in Ghana. 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 Allied Health Science will have access to the research materials, which will be kept under lock. 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. Withdrawal from Project Participation in this study is voluntary; refusal to participate will involve no penalty. You are free to withdraw consent and discontinue participation in this project at any time without prejudice from the research team. 137 University of Ghana http://ugspace.ug.edu.gh Costs and/or Payments to Subject for Participation in Research 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 research project and/or in the case of injury due to the project, participants can call Professor E. K Wiredu, School of Allied health Sciences (0244664184) or Dodzi Kofi Amelor of the School of Public Heath (0266876544). Questions regarding any rights issues as a person in this research project should be directed to the chairpersons of the Ethical Review Committees of the Korle-bu Teaching Hospital and School of Allied Health Science respectively. Consent to participate in Research I, …………………………………………………………………………… � Confirm that I have read the written information (or have had the information read to me) for the study :Genotypes of Human Papillomavirus among HIV Positive 138 University of Ghana http://ugspace.ug.edu.gh Women Attending clinic at the Korle bu Teaching Hospital, Accra, 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 authorised persons described in the information sheet. � Have been given time and opportunity to consider taking part in this study. Thumb of subject Tick as appropriate (this decision will not affect your ability to unable to sign enter the study): I consent to participate in the above research study. Signature of Subject: .......................................... Date............................... Signature of Interviewer: .................................... Date............................... Name of Impartial Witness: …………………………… Signature of 139 University of Ghana http://ugspace.ug.edu.gh Impartial Witness: ……………………………. Date............................... 140 University of Ghana http://ugspace.ug.edu.gh APPENDIX 2 Appendix 1: Protocol: DNA Purification from Cervical Swabs using Qiagen DNA Mini kit (Spin Method) 1. Centrifuge sample at 5000 rpm for 5 min. 2. Add 20 µl QIAGEN Proteinase K and 300 µl Buffer AL to the sample. Mix immediately by vortexing for 15 s. NB: Do not add Qiagen Proteinase K directly to Buffer AL. 3. Centrifuge at 5000rpm for 5 min and separate filtrate into 1.5ml microcentrifuge tubes. 4. Incubate at 56°C for 10 min. 5. Add 400 µl ethanol (96–100%) to the sample and mix again by vortexing. Briefly centrifuge to remove drops from inside the lid. 6. Carefully apply 700 µl of the mixture from step 4 to the QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate.* 7. Repeat step 6 by applying up to 700 µl of the remaining mixture from step 5 to the QIAamp Mini spin column. 8. Carefully open the QIAamp Mini spin column and add 500 µl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube, and discard the collection tube containing the filtrate.* 9. Carefully open the QIAamp Mini spin column and add 500 µl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Recommended: Place the QIAamp Mini spin column in a new 2 ml collection tube and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min. 10. Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube, and discard the collection tube containing the filtrate. 11. Carefully open the QIAamp Mini spin column and add 50 µl Buffer AE. Incubate at room temperature for 5 min, and then centrifuge at 6000 x g (8000 rpm) for 1 min. 12. Aliquot 25 µl portions of eluate into 1.5ml microcentrifuge tubes on ice. Label with sample code and date. Store at -20oC. Precautions: *Flow-through contains Buffer AL or Buffer AW1 and is therefore not compatible with bleach. 141 University of Ghana http://ugspace.ug.edu.gh Adapted from QIAamp DNA Mini and Blood Mini Handbook 04/2010 142 University of Ghana http://ugspace.ug.edu.gh 143 University of Ghana http://ugspace.ug.edu.gh APPENDIX 3 Appendix 2 DNA sequence detail of oligonucleotide primers used for HPV genotyping Adapted from Sotlar et. al., (2004). 144 University of Ghana http://ugspace.ug.edu.gh 145 University of Ghana http://ugspace.ug.edu.gh APPENDIX 4 Appendix 3 146 University of Ghana http://ugspace.ug.edu.gh APPENDIX 5 Table 6.1-1: Combinations of human papillomavirus (HPV) types in women with multiple infections Double Triple Four Five or more HPV cluster frequency HPV cluster frequency HPV cluster frequency HPV cluster frequency 16,18 2 16,42,44 16,18,6/11,35 16,18,35,42,58 2 16,33 18,33,42 16,6/11,43,51 18,35,43,58,68 16,35 18,35,39 18,33,35,52 45,6/11,56,43,35 18,33 18,35,42 2 18,35,42,52 18,6/11,35,42,45,52 18,35 12 18,35,58 2 18,35,42,68 16,18,35,42,45,58,68 18,45 18,35,68 18,42,52,68 18,52 2 18,42,39 18,52,58,35 18,58 2 18,42,45 18,6/11,33,35 18,6/11 4 18,42,58 18,6/11,35,58 2 18,68 18,43,52 35,45,52,56 31,35 18,45,58 42,44,51,66 31,43 18,52,68 45,58,43,51 35,39 18,58,68 2 56,52,35,68 35,45 18,6/11,35 6/11,18,52,68 35,51 31,43,52 3 35,52 3 35,42,39 35,58 2 35,45,52 35,68 2 35,51,58 39,45 2 35,52,68 39,58 43,45,51 42,51 44,66,68 2 Table x continued 147 University of Ghana http://ugspace.ug.edu.gh Double Triple Four Five or more HPV cluster frequency HPV cluster frequency HPV cluster frequency HPV cluster frequency 42,58 2 45,58,44 42,59 2 52,56,43 42,68 58,35,68 43,39 6/11 ,16,35 43,51 6/11,39,43 2 43,58 3 6/11,52,58 43,68 45,52 2 45,58 45,68 2 52,42 2 52,44 52,68 56, 58 59,16 59,51 59,52 6/11 , 45 6/11,31 6/11,43 2 6/11,68 3 Unless otherwise stated in the frequency column these clusters are all single occurrences. 148