DEPARTMENT OF MICROBIOLOGY, IMMUNOLOGY AND PARASITOLOGY, FACULTY OF MEDICINE, Addis AbabaUNIVERSITY Human Papillomavirus Infection and Genotype Distribution in Relation to Cervical Cytology Abnormalities and HIV-1 Infection at TikurAnbesa Teaching Hospital, AA, Ethiopia By MULUKEN DERESE A THESIS SUBMITTED TO GRADUATE PROGRAMME OF ADDIS ABABAUNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE IN MEDICAL MICROBIOLOGY December, 2010 TITLE: “Human Papillomavirus Infection and Genotype Distribution in Relation to Cervical Cytology Abnormalities and HIV-1 Infection in at TikurAnbessaTeaching Hospital, AA, Ethiopia Advisers 1. Dr.Solomon G/Sillasie DMIP, Faculty of Medicine, Addis AbabaUniversity, Addis Ababa, Ethiopia. 2. Dr. YirgueGebrehiwot __ Department of Gynecology and Obstetrics, Faculty of Medicine, Addis Ababa University, Addis Ababa, Ethiopia. COLLABORATORS: 1. Dr. DawitWolday (MD, MSc, PhD), Medical Biotech Laboratories (MBL), Addis Ababa, Ethiopia. 2. Dr. BekureTsegaye Medical Biotech Laboratories (MBL), Addis Ababa, Ethiopia. 3. Dr. WondwossenErgete Department of Pathology, Faculty of Medicine, Addis AbabaUniversity, Addis Ababa, Ethiopia. 4. Prof. ShlomoMaayan, MD, Professor of Infectious Diseases&Director, Hadassah AIDS Center, Hadassah University Hospital, Jerusalem, Isreal Acknowledgment • Above all I would like to acknowledge the School of Graduate Studies, the Department of Microbiology, Parasitology and Immunology, Addis AbabaUniversity for making my graduate study possible and funding this MSc research. • My special thanks also goes to HadassahUniversityHospital ,Jerusalem ,Israel for sponsoring my thesis work in providing chance to work in their virology laboratory and to use equipments, in addition to providing a proper training on molecular laboratory techniques . • My appreciative gratitude and respect also goes to my advisors Dr. YirgueGebrehiwot and Solomon G/Sillasie,for their invaluable comments, scientific criticisms and continuous evaluations throughout my study. • I would like to express my heartfelt thanks to SrYitayish H/mariam and her family for their hospitality and who have recreated the communication opportunity between me and Prof.ShlomoMaayan • I would like also to express my heartfelt thanks to Prof. ShlomoMaayan, who has organized this project in general and specifically partially funded the laboratory expenses and created the training opportunity at HadassahUniversityHospital. • I would also like to extend my gratitude to Prof. Dana Wolf ,Ms Orit Caplan and Mr Leonid Levinson of Hadassah University Virology laboratory unit, for organizing and providing me training on basic molecular techniques. • I would also like to extend my appreciation to Dr. DawitWolday, Dr. BekureTsegaye, Dr. WondwossenErgete for collaboration in this project. • I am very much thankful to all my family members, my mother, SrAmarchTesfaye, AtoBizuayhuMamo, my children, my sister and my brother for their unconditional love, unreserved moral, support and encouragement. • Finally I would like to express my love and my deepest gratitude to my husband DrAssaminewGirma who has been there for me from the beginning of this study and funded this project, which includes covering partial laboratory expenses at Hadassah University Hospital and who has covered all expenses for my round trip ticket to Israel , lodging and daily expenses during my stay at Israel. Table of Contents Acknowledgement…………………………………………………………………………… Table of Content……………………………………………………………………………… List of Tables and Figures…………………………………………………………………… Acronyms……………………………………………………………………………………. Abstract……………………………………………………………………………………… 1. Introduction……………………………………………………………………………. 1.1 Human Papilloma virus virologic characteristic ………………………………. 1.2 Epidemiology of HPV Infection and Cervical Cytology Abnormality ………… 1.3 HPV/HIV Co-Infection………………………………………………………… 1.4 Pathogenesis of HPV Infection on Cervical Cytology Abnormality……………. 1.4.1 Pathogenesis of Oncogenic Proteins………………………………….. 1.5 Clinical Significance of Cervical HPV Infection……………………………….. 1.5.1 Intraepithelial lesion of the uterine cervix (IEL)……………………… 1.5.2 Cervical Cancers……………………………………………………… 1.6 Diagnosis………………………………………………………………………… 1.6.1 Cytology Tests………………………………………………………… 1.6.2 Histopathology………………………………………………………… 1.6.3 HPV DNA Detection………………………………………………….. 1.6.4 HPV Serological Assay……………………………………………….. 1.7 Treatment………………………………………………………………………… 1.8 Prevention ………………………………………………………………………. 2 Significance of the Study……………………………………………………………….. 3 Objectives of the Study…………………………………………………………………. 3.1 General Objective……………………………………………………………….. 3.2 Specific Objectives……………………………………………………………… 4 Material and Method…………………………………………………………………… 4.1 Study Design and Period………………………………………………………… 4.2 Source Population and Study Area………………………………………………. 4.3 Ethical Consideration……………………………………………………………. 4.4 Sample Size Determination……………………………………………………… 4.5 Enrollment Visit and Study Period ……………………………………………… 4.6 Laboratory Investigation………………………………………………………… 4.7 Data Management……………………………………………………………….. 5 Result……………………………………………………………………………………. 5.1 Socio Demographic Characteristics…………………………………………….. 5.2 HPV Prevalence…………………………………………………………………. 5.3 Cytology ………………………………………………………………………… 5.4 HPV, HIV, Cytology Result……………………………………………………… 6 Discussion ……………………………………………………………………………… 7 Conclusion……………………………………………………………………………… 8 Recommendation……………………………………………………………………….. 9 Limitations………………………………………………………………………………. 10 References………………………………………………………………………………. 11 Annexes i iii iv v vii 1 4 6 10 12 12 14 15 16 18 18 19 20 22 23 23 26 26 26 26 27 27 27 27 28 28 29 32 34 34 36 42 48 52 58 59 60 61 72 List of Tables and figures List of Figures page Figure 1: Graphs on overall distribution of HPV --------------------------------------------------36 Figure 2: Graph on frequency of HR HPV infection in study population-----------------------37 Figure 3: Graph on frequency of LR HPV infection in study population-----------------------38 Figure 4:Graph frequency of HPV infection in HIV positive women stratified by age-------41 Figure 5: Graph on HPV frequency in HIV negative women stratified by age --------------41 Figure 6: Pie chart on proportion of cervical cytology abnormality by CIN stages----------- 42 Figure 7 Frequency of Abnormal Genitourinary Symptoms-----------------------------------45 Figure 8: Graph on CD4 count ranges for HIV positive study subjects------------------------ 48 List of Tables page Table 1: Socio demographic characteristic of study participants --------------------------------35 Table 2: Factors associated with HPV status: Bivariate analysis: -------------------------------39 Table 3:: Factors associated with HPV: Multivariate analysis: -----------------------------------39 Table 4: Factors Associated with Cervical Cytology Abnormality: Bivariate analysis:-------43 Tble 5: Factors associated with cervical cytology abnormality, Multivariate analysis --------44 Table 6: HIV Status and Risk of Cervical Cytology Abnormality -------------------------------46 Table 7: HIV status and the most frequent HR HPV genotypes distribution in association with cervical cytology abnormality stages-----------------------------------------------------------47 Table 8: PaP smear result in association with CD4 count in HIV positive women ------------50 List of Appendices page Appendix I(Consent )---------------------------------------------------------------------------------------72 Appendix II (Questionnaire) -------------------------------------------------------------------------79 Acronyms AIDS Acquired Immuno-Deficiency Syndrome ARV Anti-Retroviral ART Anti-Retroviral Therapy ASCUS Atypical Squamous Cells of Undetermined Significance CC Cervical Cancer CIN Cervical Intraepithelial Neoplasia DNA Deoxyribo Nucleic Acid DIMP Department of Immunology , Microbiology and Parasitology EIA Enzyme Immuno Assay GUT Genitor Urinary Tract GYN Gynecology HIV Human Immunodeficiency Virus HPV Human Papilloma Virus HR HPV High Risk Human Papilloma Virus HSIL High grade Squamous Intraepithelial lesion IEN Intraepithelial Neoplasia LCR Long Control Region LDL Lower Detection Limit LR HPV Low Risk Human Papilloma Virus LSIL Low grade Squamous Intraepithelial Lesion MoH Ministry of Health NAs Nucleic Acids OBS Obstetrics OI Opportunistic Infection OPD Out Patient Department ORF Open Reading Frame Pap Papanicolaou Smear PCR Polymerase Chain Reaction RFLP Restriction Fragment Length Polymorphism RNA Ribonucleic Acid SIL Squamous Intraepithelial Lesion STI Sexually Transmitted Infection UNAIDS Joint United Nations Programme on HIV/AIDS VCT Voluntary Counseling and Testing VL Viral Load VLP Virus Like Particles WHO World Health Organization . Introduction Human Papillomavirus (HPV) is one of the most common causes of sexually transmitted viral diseases in both men and women worldwide. The prevalence of HPV infection in the general population is estimated to be between 9 and 13 percent worldwide and varies between 1.6 and 25.6 percent by country (Dan et al., 2008). Papillomaviruses are ubiquitous and have been detected in a wide variety of animals as well as in humans and are specific for their respective hosts. More than 200 types of HPV have been recognized on the basis of DNA sequence data showing genomic differences (KyungWon et al., 2007). Of the 200 HPVs approximately 40 types are associated with lower genital tract infection. HPV can infect basal epithelial cells of the skin or inner lining of tissues and are categorized as cutaneous types or mucosal types. Cutaneous types of HPV are epidermitrophic and target the skin of the hands and feet. Mucosal types infect the lining of the mouth, throat, respiratory tract, or anogenital epithelium. Based on their association with cervical cancer and precursor lesions, genital HPVs can also be grouped in to High-risk (oncogenic HPVs) and Low-risk HPV types. Low-risk HPV types include types 6, 11, 42, 43, and 44 and High-risk HPV types include types 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66,73 and 82 (Folashade et al., 2007). The 200 genotypes are defined by nucleotide sequence variation of more than 10% compared with other known HPV types in the E6, E7 and L1 open reading frames. Those differing by 210% variations are referred to as subtypes whereas intratype variants may vary up to 2% in the coding region and 5% in the non-coding region when compared to that of prototype, variations are commonly caused by deletions or insertions of nucleotides. The degree of polymorphism varies from type to type in all HPV types. These variants are generally classified and named according to their geographic relatedness. Interest in HPV variants is growing rapidly, as increasing evidence suggests that HPV variants may differ biologically and etiologically (Shailja et al., 2008; Jose et al.,2003). Cervical cancer is the second most common cancer among women world wide (Baseman et al., 2005; Eduardo et al., 2001) and the leading cause of cancer related morbidity and mortality in Ethiopian women with or without HIV infection (WHO 2010). Several epidemiological investigations have documented that more than 90% of cervical cancers and precancerous lesions are attributed to infection with oncogenic HPVs (Baseman et al.,2005). The natural history of cervical cancer involves reversible changes in the cervical tissue from a normal state, in which no neoplastic changes are detected in the squamous epithelium, to varying states of cellular abnormalities that ultimately lead to cervical cancer. This sequence forms the premise on which cytologic screening for cervical cancer is based and corresponds to an underlying multistep carcinogenic process in the development of cervical intraepithelial neoplasia (CIN). Low-grade squamous intraepithelial lesions (LSILs) may progress to high grade SILs (HSILs) and invasive cervical cancer or may regress to a normal state. In Ethiopia, although there is no national cancer registry, data collected from retrospective analysis of medical records have clearly indicated that cervical carcinoma is the most frequent cancer among women of reproductive age group (Dawit et al., 2001; France et al.1999). In Tikur Anbessa Teaching hospital the prevalence of abnormal cervical cytological findings among women presenting for Pap screening was 15-20% (Wondwosen E & Bekure T, Unpublished data). HIV-positive men and women are at increased risk of anogenital and oral HPV infection. The risks for HPV-associated high-grade intra-epithelial neoplasia and cancer are also increased. The prevalence of oral, anal, and cervical HPV infection in HIV-positive individuals compared with HIV-negative individuals increases with progressively lower CD4+ levels, as does incident high grade intraepithelial lesions. Studies have shown that HIV-associated attenuation of HPVspecific immune responses allow for persistence of high-grade intraepithelial lesion and sufficient time for accumulation of genetic changes that are important in progression to cancer. Some have also speculated that HIV infection may increase the oncogenicity of high-risk HPV types; HIV also has a negative impact on recurrence of infection and CIN after treatment (Ferenczy et al., 2003; Suzanne et al., 1999). 1.1. Human Papilloma Virus Virologic Characteristics Human Papilloma viruses are members of the Papovaviridae family, are small non enveloped viruses with ~55- nm-diameter has icosahedral capsids composed of 72 capsomers. It contain double-stranded circular DNA genomes of approximately 8,000 bp, replication takes place within the nuclei of infected host cells. (Jose et al., 2003; Simon et al., 2004; Shailja et al., 2008). In virions, the HPV DNA is found associated with cellular histones to form chromatinlike complexes. The viral genomes carry on average eight major ORFs, and these are expressed from polycistronic mRNAs transcribed from a single DNA strand, only one of the two strands of the circular papillomavirus DNA genome is actively transcribed. The genome can be divided into three major portions: a 4-kb early (E) region with 7-8 genes (E1-E7) that encode nonstructural proteins which contains the viral DNA replication functions (E1, E7), transcription control (E2), and cellular transformation (E5, E6, and E7) functions, a 3-kb late (L) region that encodes primary and secondary capsid proteins, and a 1-kb noncoding hyper variable long control region (LCR) that contains a variety of cis elements, which regulate viral replication and gene expression (Karl et al, 2004; Neeta et al.,2005). The life cycle of HPV is linked to the differentiation program of the infected host cell, with production of mature virion particles restricted to differentiated suprabasal cells. Infection by Papilloma viruses is thought to occur through micro wounds of the epithelium that expose cells in the basal layer to viral entry. Heparin sulfate which mediates the initial attachment of the virions to the cell and two molecules of the integrin family, α6β1 and α6β4, expressed on the surface of basal cells, are identified as HPV receptors (Maria Alice et al., 2004). Cells in the basal layer consist of stem cells and transit-amplifying cells that are continuously dividing and provide a reservoir of cells for the suprabasal regions. HPV infection of these cells leads to the activation of a cascade of viral gene expression which results in the production of approximately 20 to 100 extra chromosomal copies of viral DNA per cell (episomal form). This average copy number is stably maintained in undifferentiated basal cells throughout the course of the infection. Among the first viral proteins to be expressed are the replication factors, E1 and E2 (68 and 50 kilodalton respectively). These proteins form a complex that binds to sequences at the viral origin of replication and acts to recruit cellular polymerases and accessory proteins to mediate replication. The E1 and E2 replication factors are also expressed from the early promoter, the ability of E2 to activate and repress expression contributes to the control of viral copy number in undifferentiated cells. The E4 and E5 proteins are involved in regulation of late viral functions. The E6 and E7 proteins of the High-risk HPV types are highly polymorphic, they act as viral oncoproteins, but no such functions are associated with the corresponding proteins from the Low-risk types (Michelle et al., 2004; Karl et al., 2004) . As HPV infected basal cells divide, viral genomes are partitioned into daughter cells, one of which detaches from the basal layer, migrates toward the stratum granulosum, and undergoes differentiation. In normal uninfected epithelia, cells exit the cell cycle as they leave the basal layer, and this often results in the loss of nuclei in suprabasal cells. As infected cells leave the basal layer, they remain active in the cell cycle due to action of the E6/E7 protein. Cells reenter the S phase in highly differentiated cells and activate the expression of cellular replication factors required for viral replication. The L1 and L2 proteins are assembled late and spontaneously form icosahedral capsids. Following virion assembly; mature viruses are released from the uppermost layers of the epithelium. The major capsid protein, L1, can spontaneously self-assemble into 72pentamer virus-like particles (VLPs) which are used to develop vaccine. The minor capsid protein, L2, is important for Papillomavirus infectivity and for efficient establishment of infection (Karl et a.l, 2004). 1.2. Epidemiology of HPV Infection and Cervical Cytology Abnormality Genital infection with Human Papilloma Virus prevalence vary by country and studied population, The incidence of HPV infection varies from 10\100,000 in many developed countries to 40\100,000 in developing countries (Dan et al.,2008), in addition the Age- standardized HPV prevalence varied nearly 20 times between populations, from 1·4% in Spain to 25·6% in Nigeria (Clifford et al., 2005), part of this difference is accounted for the lack of screening program, lack of good medical care and difference in sexual behavior ( Marjon et al., 2006). Transmission of HPV occurs primarily by skin-to-skin contact. Epidemiologic studies clearly indicate that the risk of contracting genital HPV infection and cervical cancer is influenced by sexual activity. An individual is at greater risk of becoming infected with HPV if he or she has had multiple sexual partners at any time or the partner of someone who has had multiple sexual partners. Sexual activity at an early age <20 years also places an individual at increased risk, as does a history of other sexually transmitted diseases, genital warts, cervical or penile cancer in an individual or sexual partner which are known to be risk factors(Nobuo et al.,2004; Burd et al., 2003). In addition to sexual activity, age is an important determinant of risk of HPV infection. Most cervical cancers arise at the squamocolumnar junction between the columnar epithelium of the endocervix and the squamous epithelium of the ectocervix (The cervical transformation zone). At this site, there are continuous metaplastic changes. The greatest risk of HPV infection coincides with greatest metaplastic activity. Greatest metaplastic activity occurs at puberty and first pregnancy and declines after menopause. HPV infection is most common in sexually active young women, 18 to 30 years of age its prevalence ranges from 20-46% in various countries. There is a sharp decrease in prevalence after 30 years of age. Epidemiological studies have shown that acquisition of HPV infection occurs within 1 to 2 years of onset of sexual activity in adolescent females and, in this group, the prevalence of HPV by polymerase chain reaction can be as high as 64%. However, cervical cancer is more common in women older than 35 years suggesting infection at a younger age and slow progression to cancer (Ian et al., 2007; Hugo et al., 2006; Noor et al., 2006). Sexual intercourse with an uncircumcised man can also increase a woman’s risk of HPV infection it is because such kind of men were more likely to contract penile HPV infection, thereby placing their sexual partners at higher risk for HPV infection and cervical cancer (Nobuo et al., 2004). The frequency of individual high-risk HPV types worldwide has been shown to vary in respect to major global regions such as Asia, Europe, North America, South America, and Sub-Saharan Africa. HPV type 16 and 18 are more common in Europe and North America and other HPV types are more common in African populations. The eight most common high-risk types (16, 18, 45, 31, 33, 35, 52 and 58 ) account for 89% of all cervical cancer cases worldwide. HPV 16 is the cause of about 54% of invasive cervical cancers and HPV 18 is the cause of about 17% globally (Muñoz et al., 2003; Clifford et al.,2006). In some African countries the prevalence of the HPV genotype distribution is different when compared with other areas. A study in Mozambique in 72 women with suspected cervical cancer, HPV prevalence was found to be 92% and the most common HPV types were 16 and 18, being present in 69% of tumors (Naucler et al., 2004), A study from Cameroon also showed 41 out of 61 samples tested (67.2%) to have any HPV subtypes positive. The most common high risk types encountered were HPV 45 (24.6%) and HPV 58 (21.5%) (Desruisseau et al., 2009). Little is known about the association of cervical cytology abnormality, cervical cancer and HPV infection, including HPV genotypes in Ethiopia. In a retrospective study (Fanta.2005) conducted in Gondar, Northwestern Ethiopia, reported that out of 284 formalin-fixed and paraffinembedded cervical biopsy specimens, HPV was found in 92.6% of the samples. Of all the squamous cell carcinomas (n=153), adenocarcinomas (n=10), pre-invasive carcinomas (CIN, n=34), inflammatory lesions (n=79) and normal histology (n=8), 99.3%, 60%, 88.5%, 89.8% and 50% were positive for HPV, respectively. HPV type 16 was identified to be the most frequent genotype accounting for more than 76% of all HPV types. HPV type 18 was the second most frequent HPV type accounting for 13.7%. Of all the HPV-positive squamous cell carcinomas, 75% harbored HPV type 16 while 20% were positive for HPV type 18. In another study conducted in Gondar (Abate et al.,2005) reported that among 135 paraffin-embedded cervical tissue blocks 83.7% were positive for HPV. The genotype distribution was, however, not determined. In a study conducted in Addis Ababa (Mihret et al., 2005), they identified 88.2% HPV positivity rate among cervical biopsy specimens from women with documented cervical cancer. Of all the HPV positive specimens, the prevalence of HPV type 16, 18, 26, 31 and 35 was 74.3%, 5.7%, 2.9%, 5.7% and 5.7%, respectively. A study over 4 years at Jimma hospital south west Ethiopia, on women with cervical cancer showed HPV prevalence to be 67.1% and HRHPV genotypes identified were, HPV16, 18, 56, 45, 39, 52, 31, 35, 58, 33 and 59 accounted 55.7%, 8.2%, 8.2%, 4.1%, 2.5%, 1.6%, 1.6%, 1.6%, 0.8%, 0.8%, 0.8% respectively (Bekele et al.,2010) Globally, cervical cancer is the second most common cause of cancer death in women, with an estimated 510,000 newly diagnosed cervical cancer cases and 288,000 deaths. In developing countries cervical cancer is often the most common cancer in women where 80 percent of cases occur. The highest age-standardized incidence rates of cervical cancer have been reported in Malaysia, Southern Africa, Central America, Eastern Africa, and South America. In all of these regions, the rates were over 40 per 100,000 women and the age-standardized mortality rate from cervical cancer in these countries is 9.6 per 100,000 women, twice the rate in developed countries (PATH.2000; WHO report, 2010). In sub Saharan African countries, the age-standardized incidence of cervical cancer is 30 to 67 per 100,000, which is two to ten times higher than that in developed countries (Kahesa et al., 2008; Nobuo et al., 2004; Debbie et al., 2007). A full understanding of the burden of cervical cancer in Africa is limited by scarce national registry data. However, it is clear that high level of exposure to HPV, absence of screening programs and poor access to appropriate treatment have resulted in the highest cervical cancer rates. East Africa has notably high rates of cervical cancer incidence with a crude incidence rate of 25.7 and the annual number of new cancer cases is 33,903/100,000 as well has a mortality rates of 34.6 (WHO .2010). Ethiopia has a population of 20.90 millions women ages 15 years and older who are at risk of developing cervical cancer even though there is no national cancer registry, reports have shown every year 7619 women are diagnosed with cervical cancer and 6081 die from the disease(Parkin et al., 2005). Cervical cancer ranks as the 1st most frequent cancer among women in Ethiopia, and the 1st most frequent cancer among women between 15 and 44 years of age with a crude incidence rate of 23/100,000. Data is not yet available on the HPV burden in the general population of Ethiopia. However, in Eastern Africa, about 33.6% of women in the general population are estimated to harbor cervical HPV infection at a given time (WHO.2010). Low socio-economic status, poor hygiene, early marriage or coitus, multiparity and polygamy have been reported to be associated factors (Azbaha, 1983). 1.3. HPV/HIV Co- Infection Given the overlap in risk factors for HIV and HPV infection, HIV positive women are often coinfected with both of these viruses. Several mechanisms may explain the increased prevalence and more aggressive course of HPV-associated disease in HIV positive individuals. These include direct interactions between the two viruses, attenuated immune response and chromosomal instability (Palefsky et al.,2006). Infection with HIV and Human Papilloma Virus and development of precancerous lesions or cervical cancer are both major public health problems in developing countries. There is evidence to show that HIV positive women have a significantly higher rate of cervical cytology abnormalities than their counterparts and are more likely to progress to invasive carcinoma 10 to 15 years earlier than HIV negative women (Cynthia et al., 2009). It is because HIV positive women nearly have 5 times risk to harbor HR HPV infection and to be infected with multiple genotypes compared to HIV negative women which reflect increased number of sexual exposure (Ellerbrock et al., 2000; Ferenczy et al., 2003). There is scarce data on the epidemiology of HPV and HPV-related malignancies, and the effect of HIV on these diseases in Ethiopia, HIV prevalence in Ethiopia is 2.1% in adults aged 15-49 and prevalence of HPV in normal cervical cytology and precancerous lesion in HIV infected population is not known (WHO 2010). A detailed understanding of the epidemiology and HPV diversity among women presenting with cervical cytological abnormalities with HIV infection is important to implement a proper prevention and treatment program and even before considering the use of the available HPV based vaccines. Studies conducted in Africa showed presence of interrelation between cervical neoplasia and HIV and HPV infections, showing strong associations between HPV and neoplasia and between HIV and neoplasia and pointing out an interaction between HPV and HIV infections which may lead to a conclusion that HIV seems to be a cofactor in the association between HPV and cervical neoplasia. Unlike developed countries no increase in invasive cervical cancer has been noted among HIV-positive women in Africa, where both HIV infection and cervical cancer are endemic. The reasons for this epidemiological discrepancy are unclear; however, they may be related in part to the rather short lifespan of untreated HIV-positive women in comparison to the 10 years on average required for CIN to progress to invasive cervical cancer (Ferenczy et al., 2003; Palefsky et al., 2003). Study conducted in 106 women presenting to a sexually transmitted infections clinic in Kampala, Uganda showed that HPV prevalence was 46.2% and HIV prevalence was 34.9%. High risk genotypes 52, 58, and 16 were the genotypes of all women infected with HPV. 18% were found to have genotypes 16 and/or 18. Seventy-three percent of HIV-positive women versus 16% of HIV-negative women had abnormal Pap smears. Among HIV-positive women, abnormal Papanicolaou (Pap) smears were associated with the presence of high risk HPV genotypes. The majority of the women infected with HPV were infected with high risk HPV genotypes other than 16 and 18 (Blossom et al., 2007). In another study undertaken in Lusaka, Zambia, 145 HIVinfected non-pregnant women were screened at a tertiary care centre for HPV and genotyping test. Among high-risk types, HPV 52 (37.2%), 58 (24.1%) and 53 (20.7%) were more common overall than HPV 16 (17.2%) and 18 (13.1%) in women with high-grade Squamous intraepithelial lesions or Squamous cell carcinoma on cytology. High-risk HPV types were more likely to be present in women with CD4 cell counts <200 (OR: 4.9, 95% CI: 1.4-16.7, P=0.01) and in women with high-grade or severe cervical cytological abnormalities (OR: 8.0, 95% CI: 1.7-37.4, P=0.008) (Sahasrabuddhe et al., 2007). Likewise, a study conducted in Burkina Faso, HPV was detected among 66% of women with abnormal cervical cytology. The most frequent HPV subtypes were HPV52 (14.7%), HPV35 (9.4%), HPV58 (9.2%), HPV51 (8.3%) and HPV16 (5.9%). Prevalence of HIV was 36.5% and HIV positivity was associated with increased rates of high-risk HPV infection and high-grade squamous intraepithelial lesion cases (Didelot et al.,2005). A survey in Kenya on female sex workers showed, one third of the women to be HIV infected (283/803; 35.2%) and these women were more likely to have abnormal cervical cytology than HIV-negative women (27%, 73/269, versus 8%, 42/503; P < 0.001). Of HIV-infected women, 73.3% had high-risk HPV (200/273) and 35.5% had HPV 16 and/or 18 (Stanley et al., 2010). 1.4. Pathogenesis of HPV Infection on Cervical Cytology Abnormality One of the key events of HPV-induced carcinogenesis is the integration of the HPV genome into a host chromosome. HPV genome integration often occurs near common fragile sites of the human genome. Expression of the viral E6 and E7 genes is consistently maintained; whereas other portions of the viral DNA are deleted or their expression is disturbed. Loss of expression of the HPV E2 transcriptional repressor is significant, as it may result in deregulated HPV E6 and E7 expression. There is also evidence for increased HPV E6/E7 mRNA stability after integration, and specific alterations of host cellular gene expression have been detected upon HPV genome integration. Cells that express E6/E7 from integrated HPV sequences have a selective growth advantage over cells with episomal HPV genomes. In general HR HPV integration has been reported with different frequencies in all spectrums of cervical neoplasias from LSIL to HSIL and Cervical Cancer, the frequency of HR HPV viral integration increased in parallel with the severity of cervical lesions. These reports have suggested that viral integration could represent a risk for tumor progression. In addition, HPV integration has been associated with poor clinical outcome (Karl et al., 2004; Noor et al., 2006; Hugo et al., 2006). 1.4.1. Pathogenesis of Oncogenic Proteins The E6 proteins of both high- and low-risk types are approximately 150 amino acids in size. Many of the first insights into the action of E6 have come by studying its interactions with p53. To overcome the proapoptotic activities of p53 and allow for cell cycle progression, E6 binds to p53 in a ternary complex with ubiquitin ligase called E6AP. Formation of this complex result in the ubiquitination of p53 and subsequent degradation by the 26S proteasome, leading to a reduction in the half-life of p53 from several hours to less than 20 min. E6 can also indirectly down regulate p53 activity through its association with p300/CBP, which is a coactivator of p53. Since p53 regulates both the G1/S and G2/M checkpoints of the cell cycle, its rapid turnover results in abrogation of these controls, leading to chromosomal duplications and centrosomal abnormalities. Interestingly, the binding of E6 to E6AP also results in the self-mediated ubiquitination of E6AP(Karl et al.,2004; Michelle et al.,2004; Kyung et al.,2007). High-risk HPV E6 proteins also have p53-independent transforming activities. These HPV E6 proteins contain a carboxyl terminal PDZ binding domain which enables it to interact with PDZ proteins. The binding of PDZ family members MUPP-1, hDLG, and hSCRIB to the extreme C terminus of high-risk E6 proteins results in the degradation of the PDZ protein. The ability of high-risk HPV E6 proteins to associate with PDZ host proteins is relevant to cellular transformation by eliminating trophic sentinel signaling .In combination with E7, high-risk HPV E6 proteins contribute to immortalization of primary human epithelial cells through the induction of telomerase activity. High-risk E6 proteins induce hTERT expression at a transcriptional level (Karl et al., 2004; Michelle et al.,2004; Stephanie et al., 2007) HPV E7 proteins are low-molecular-weight proteins of approximately 100 amino acids. The HPV E7 proteins interact with the retinoblastoma tumor suppressor protein (pRB) and the related “pocket proteins” p107 and p130 through a conserved LXCXE sequence within CR2 sequences. The binding of E7 sequesters Rb away from E2F/DP1 complexes, resulting in the constitutive activation of the corresponding genes. In addition to binding Rb, E7 mediates its degradation through the ubiquitin proteosome pathway. Furthermore, Rb family members are major regulators of the cell cycle exit that occurs during epithelial differentiation. The abrogation of Rb function by E7 thus allows for productive replication in differentiated suprabasal cells (Karl et al., 2004; Michelle et al.,2004). In addition to binding to Rb family members, HR HPV E7 proteins associate with cyclins A and E as well as cyclin-dependent kinase (cdk) inhibitors p21 and p27. Since the cyclins and the associated kinases drive cell cycle progression by phosphorylating the Rb protein, it is not surprising that the E7 protein would act to enhance the activity of these proteins hence contributing to the maintenance of a replication-competent cellular milieu in differentiated host epithelial cells (Scott et al., 2006). The third group of proteins bound to high-risk E7 is the histone deacetylases (HDACs). In HPV negative cells, Rb binds to HDACs and recruits them to E2F inducible promoters. Recently, E7 proteins were shown to bind HDACs independently of their binding to Rb, and this association is important for the role of E7 in immortalization as well as episomal maintenance (Michelle et al., 2004). HPV16 E7 binds to an active cullin 2 ubiquitin ligase complex and that association correlates with the ability of HPV E7 to transform cells and to dysregulate G1/S cell cycle checkpoints (Kyung et al., 2007).One of the intriguing properties of the high-risk E7 proteins is their ability to induce genomic instability. Many HPV positive cancers contain consistent patterns of aneuploidy, suggesting that changes in chromosome number are important events in progression. Expression of E7 alone was shown to be sufficient to induce an increase in abnormal centrosome numbers in primary human keratinocytes. Centrosomes are major microtubule-organizing centers and coordinate segregation of chromosomes into daughter cells during cell division (Karl et al., 2004; Michelle et al., 2004; Peter et al., 2004; Stefan et al., 2002). 1.5. Clinical Significance of Cervical HPV Infection Infection with one of approximately 15 oncogenic types of Human Papillomavirus has been established as a necessary but insufficient cause of cervical cancer, which is particularly common in young sexually active women. Results of cross-sectional studies have revealed that although up to 40% of such women are infected with an HR-HPV, fewer than 10% of infected women develop persistent HPV infections whereby most individuals (70% in adults to 90% in adolescent) eliminate the virus 12 to 24 months after initial diagnosis (Herbert et al., 2008) and only 28% will develop high-grade intraepithelial dysplasia of the cervix within 5 – 6 years of the initial infection, it was also observed that usually it takes 10-20 years between infection and the development of cancer (MariaAlice et al.,2004; Svetlana et al.,2005; Jill et al., 2008). 1.5.1. Intraepithelial lesion of the uterine cervix (IEL) IEL are precursors of cervical cancer which are considered to be the earliest morphological changes associated with cancer. HPV infection and precancerous lesions may have different evolutions such as regression, persistence or progression. ‘‘HPV viral persistence’’ is defined as three consecutive high-risk HPV DNA test results in women greater than or equal to age 30 yrs (Herbert et al., 2008). Risk factors for infection persistence and progression with HR HPV are viral load, continuous virus production, multiple HPV genotype infection, HPV integration, intratypic HPV polymorphism, immunosuppression, host HLA types, diet with low anti oxidants, old age, smoking, oral contraceptive use for more than five years, low socioeconomic status, 5 full-term pregnancies, presence of vulvar condylomata and site of infection especially the squamous columnar junction and the squamous epithelium of the cervical transformation zone are factors that induce persistence. The magnitude of the effect of HPV persistence was stronger with longer duration of infection, wider HPV testing intervals, and lack of screening program (Herbert et al., 2008; Jill et al., 2008). In addition studies suggest that whether a woman will develop cervical cancer depends on a variety of additional factors that act in concert with cancer-associated HPV types in the process that leads to cervical cancer. The primary immune response to HPV infection is cell mediated; therefore, conditions that impair cell-mediated immunity such as renal transplantation or human immunodeficiency virus disease (low CD4 count, advanced stage of HIV infection, high HIV viral load) increase the risk of acquisition and progression of HPV. HIV-infected subjects show an increased prevalence, incidence and severity of infections and lesions (Sadeep et al., 2007). The regression of HPV infection commonly takes place within 3 years, where they clear their HPV some 3 months before cytological regression (Anna et al.,2005), There is evidence that the host’s humoral or cellular immune response is responsible for the regression of HPV infection, since immune vigilance affects susceptibility to HPV-related lesions and their regression. Compelling evidence indicated that a small but definite fraction of the infected population is at risk for developing invasive cervical cancer after many years or decades of a long latency period of primary infection indicating that additional genomic alterations may also be necessary for progression to cancer (Maria Alice et al., 2004; Shang-Lang et al.,2006). Infection of the genital tract by HPVs can initially result in low-grade lesions termed dysplasias or cervical intraepithelial neoplasia grade I (CIN I). These lesions exhibit only mildly altered patterns of differentiation, and many of them are cleared by the immune system in less than a year. CINI /LSIL reflect active transient HPV replication and has high rates of occurrence; it is generally managed clinically by follow-up rather than immediate treatment because it regresses rapidly. Studies have shown that the pooled estimate of high risk (oncogenic) HPV DNA positivity among women with LSIL was 76.6% (Thomas et al., 2007). CIN I is caused by a variety of HPV types, about 25% (15-32%) by either HPV16 or HPV18, and about 5% by HPV 6 or HPV 11(Debbie et al., 2007). Some of these LSIL, however, are not cleared by the immune system and can persist for periods as long as several decades progressing to more advanced lesions, the precancerous lesions CIN II and CIN III are about 41-57% attributable to HPV16 and HPV18 and detection of HPV infection is a better predictor for CIN II-III than CIN I. In these cases medical intervention is needed to prevent these lesions from progressing to invasive carcinoma (Michelle et al., 2004; Debbie et al., 2007; Thomas et al., 2007). 1.5.2. Cervical Cancers Squamous Cell Carcinoma Cervical cancer is an abnormal growth of the cells of the cervix and cancers are characterized by the cells that they originally form from. The most common type of cervical cancer is called Squamous Cell Carcinoma; it comes from cells that lie on the surface of the cervix known as squamous cells. Squamous cell cervical cancer compromises about 80% of all cervical cancers, it develops gradually through well-characterized precursor lesions (SILs). Most cervical cancers arise at the squamo-columnar junction and transformation zone between the columnar epithelium of the endocervix and squamous epithelium of the ectocervix where continuous metaplastic changes occur (Neeta .2005; Debbie et al., 2007; Baak et al.,2006). The stepwise development of invasive cancer (HPV acquisition, HPV persistence, development of cancer precursors, and invasion) takes 10-20 years on average without apparent clinical sign symptoms and with the longest amount of time from high-grade lesions to invasive cancer, although there are cases that develop more rapidly. The relatively slow development of cancer from the time of initial infection has contributed to the success of screening programs. If a cervical cancer does develop, it may initially not cause any symptoms or it may cause abnormal vaginal discharge or bleeding. This can include bleeding between menstrual periods, bleeding after sexual intercourse, or bleeding after menopause. This bleeding may be no more than a spot of blood (Eileen.2003; Neeta et al., 2005; Debbie et al., 2007). Adenocarcinoma The second most common form is Adenocarcinoma; it comes from cells that make up glands in the cervix and its precursor lesions, are frequently located high in the endocervical parts of the transitional zone, which may make them less accessible to the brush and less prone to be represented in a standard specimen of exfoliated cells. Global estimates indicate that adenocarcinomas now comprise up to one quarter of cervical cancer cases in some Western countries. Postulated explanations include an increasing specificity of subtype, the capability to diagnose the disease, an inability of cytologic screening to reduce adenocarcinoma, and heterogeneity in cofactors related to persistent human papillomavirus infection (Freddie et al., 2005; Allan et al., 2006; Xavier et al.,2006). Some evidence indicates that cofactors that contribute to the progression of HPV infected cervical cells to Adenocarcinoma are distinct from those that contribute to the progression to Squamous cell carcinoma. For example, smoking and high parity have been associated with increased risks of squamous cell carcinoma, but they have no association with Adenocarcinoma, and obesity seems to be a risk factor for Adenocarcinoma but not for squamous cell carcinoma. Adenocarcinoma and Adenocarcinoma in situ are associated more with acquisition of HPV-18 than squamous cell carcinomas. More recent studies are estimating the proportion of adenocarcinomas harboring high-risk HPV types to be close to 100% and for proper detection of adenocarcinoma or its precursor by screening it is advisable to use improved endocervical sampling techniques (Freddie et al., 2005; Allan et al.,2006; Xavier et al .,2006). 1.6. Diagnosis To date, HPV cannot be cultured in vitro, and immunological tests are inadequate to determine the presence of HPV cervical infection. Indirect evidence of anogenital HPV infection can be obtained through physical examination and by the presence of characteristic cellular changes associated with viral replication in Pap smear or biopsy specimens. Alternatively, biopsies can be analyzed by nucleic acid hybridization to directly detect the presence of HPV DNA, or molecular techniques can be used to diagnose HPV presence and to determine the specific genotype or types. Acceptable specimens include Specimens collected using cervical brushes or spatulas used to take exfoliated cells cervical swabs (Digene cervical Sampler Digene Cervical Brush and Specimen Transport medium), Specimens collected in liquid medium (Cytyc Preserv Cyt Solution for ThinPrep Pap test slides) or cervicovaginal lavage or freshly collected cervical biopsies 2-5mm in cross section (Eileen.2003). 1.6.1. Cytology Tests Conventional Cytology The primary method for detection of presence of cervical cytology abnormality is still the Papanicolaou-stained (Pap) smear. This method was named for pathologist George Papanicolaou, who introduced the method in 1949 before the cause of cervical cancer was known. Since its introduction, the Pap smear has helped to reduce cervical cancer incidence and mortality rates by roughly half to two-thirds. The Pap smear is a screening tool that looks for changes in cells of the transformation zone of the cervix. The Pap smear reporting and classification system is the Bethesda system. The Bethesda System 2001 classifies squamous cell abnormalities into four categories which promote the concept of a disease continuum from precursor lesions to invasive cancer. The Pap smear procedure has some limitations and its average sensitivity is 60% and its specificity is 95%. False negative rates as high as 20 to 30% have been reported (Eduardo et al.,2003 ;Eileen.2003; Folashade et al.,2007). Monolayer Cytology In these methods, the specimen is collected in a preservative solution rather than being spread directly on the microscope slide by hand. Cellular structure is better preserved because the cells are immediately fixed. In addition, a cervical brush is used to collect the specimen, which provides almost twice as many epithelial cells as do other collection devices. The uniform monolayer created by these methods is easier for a technician to read, and the process prevents drying artifacts and removes most contaminating mucus, protein, red blood cells, bacteria, and yeast. The preserved sample is enriched using density gradient centrifugation to remove inflammatory cells and non diagnostic debris which will be followed by pap smear staining. Another way to improve the Pap smear diagnosis is to stain directly for HPV using probe sets that detect HPVs (Schmitt et al., 2008). 1.6.2. Histopathology Patients with abnormal Pap smear findings who do not have a gross cervical lesion are usually evaluated by colposcopy and colposcopy-directed biopsy. Following application of a 3% acetic acid solution, the cervix is examined with a bright filtered light under 10- to 15-fold magnification. Aceto-whitening and the vascular patterns characteristic of dysplasia or carcinoma can be seen. Colposcopy can detect low-grade and high-grade dysplasia but does not detect microinvasive disease. Biopsy can be used to confirm most diagnoses by observing characteristic pathologic features of HPV infection such as epithelial hyperplasia (acanthosis) and degenerative cytoplasmic vacuolization (koilocytosis) in terminally differentiated superabasal cells with atypical nuclei (Eileen.2003; Julia et al.,2006). In addition, stains can be used which detect HPV antigens or HPV nucleic acids. Monoclonal and polyclonal antibodies are available that detect HPV common antigen, a linear epitope in the middle of the major capsid protein, which is broadly expressed among the different HPV subtypes. HPV DNA or RNA can be demonstrated in biopsy tissues by in situ hybridization with probes labeled with either radioisotopes or chemically reactive ligands which are detected by autoradiography, fluorescence, or a detection of color reaction. Characteristics of the signal (confluent versus punctuate) may reflect either the episomal or integrated form of the viral target DNA. Intensity of the signal may reflect copy number. Target-amplified or signal-amplified in situ techniques have been developed to immuno enzymatically detect a small number of HPV nucleic acid sequences with high sensitivity by using bright-field microscopy (Eileen.2003) 1.6.3. HPV DNA Detection HPV DNA testing has been shown to be more sensitive than cytology and improves patient management when used in addition to cytology testing as a primary screening tool in women aged 30 years or older, since viral infections in this age group are less likely to be of a transient nature than those in the younger women. The negative predictive value of HR-HPV DNA testing is very high (99.0%)which may allow screening intervals to be increased for women found to be negative by both cytology and HPV DNA testing (Eduardo et al.,2003; Francesca et al.,2007; Philip.2008). In addition to its role in the studies of etiology and the natural history of cervical cancer, HPV DNA testing has been used screening or management-related purposes defined as follows (Eduardo et al.,2003; George.2008). i) Primary screening—for the detection of cases of cervical cancer or of its precursor lesions among asymptomatic women -as true population screening. ii) Secondary triage—for the detection of cases of cervical cancer or of its precursor lesions among women who were initially found to have an abnormal Pap smear that requires further evaluation. iii) Follow-up of treated cases—for improved surveillance of recurrent cervical lesions after treatment. A positive HR HPV DNA test 6 months after treatment of CIN II/CIN III is more predictive for recurrence than an abnormal cytology. iv) To monitor the impact of vaccine implementation strategies. v) To determine type-specific persistence, to measure viral load and to evaluate the clinical significance of co-infection with multiple HPV types (George.2008; Jian et al., 2006). General primer PCR: The majority of studies using PCR to date have used consensus primers to amplify a broad spectrum of HPV types in a single PCR amplification. These primers target conserved regions of the HPV genome such as the L1 capsid gene. The MY09 plus MY11 primers target a 450-bp fragment within the HPV L1 ORF. The GP5+ plus GP6+ primers target a fragment within the region targeted by MY09 and MY11 with an analytical sensitivity of 0.5 to 10 fg (10–200 copies). In addition a PCR-based detection system which uses a general primer set, designated SPF (short PCR fragment) (SPF1,SPF2, SPF10) that amplifies a short (65-184)bp segment of the L1 region of the HPV genome are used. Amplicons are detected using ELISA, Line blot assay, by hybridization techniques PCR-reverse hybridization technique, direct sequence analysis or RFLP (Sadeep et al.,2007; Thomas et al.,2003) Type Specific PCR: Several genotyping methods have been developed in order to identify highrisk HPV in liquid-based cytology samples and tissue samples. Type-specific PCR assays are based on the sequence variations present in the E6 and E7 genes of HPV subtypes that target approximately 100 bp in the E7 ORF. Genotyping of HPV can be done using the target products amplified by PCR which will subjected to sequence analysis, restriction fragment length polymorphism (RFLP) analysis, and hybridization with type-specific probes, Reverse line blot assays have also been developed and validated for further analysis of genotypes (Eileen.2003; Thomas et al.,2003; Morie et al.,2008). Real-time PCR assay based on fluorescence resonance energy transfer (LightCycler) based on fluorigenic 5-nuclease chemistry (TaqMan) and a semiquantitative consensus GP5+/6+PCR enzyme immnoassay is a new, highly sensitive, real-time general PCR that will allow quantification and typing of more than 50 HPV genotypes. This method can also be used for urine samples, permitting mass screening of HPV genital infections (Hesselink et al.,2005; Christopher et al.,2007). Liquid Hybridization The Hybrid Capture (Digene, Beltsville, Md.) assay is the only kit currently approved by the FDA for the detection of HPV DNA in cervical samples for clinical diagnosis. The Hybrid Capture assay has been used in many studies, and the second generation Hybrid Capture II version of the assay is now widely used in clinical diagnostic laboratories. It is an antibody capture/solution hybridization/signal amplification assay that uses chemiluminescence detection to qualitatively detect the presence of HPV. In this assay, the DNA in the patient samples is first denatured and mixed with an RNA probe pool in a buffered solution in a tube. Two RNA probe pools are used; the assay can be performed using both probe pools together or separately. The probe A pool recognizes low-risk HPV-6, -11,-42, -43, and -44, and the probe B pool recognizes high-risk HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68. The assay dose not distinguish among HPV types (Eileen.2004; Thomas et al.,2003). 1.6.4. HPV Serological Assay Virus-like particle serology is a very useful epidemiologic tool for defining past infection with HPV. The assays are type specific and are usually negative in never-infected individuals. Overall, frequency and titer of several types of serum antibodies generated against HPV show a great variability that is dependent on the HPV type specificity, on the recognized epitopes, on the type of samples, and on the sensitivity of the assay. It was observed that the modest antibody responses measured in several studies reflect lack of sensitivity of the assay or a deficient immune response to HPV, particularly in the case of cancer series where integration of HPV genomes impairs the expression of capsid antigens. Moreover, a weak immune response is often observed in HPV infections because these viruses do not cause viremia. As a consequence, one would expect misclassification as an important problem when considering VLP serology to estimate the cumulative prevalence of HPV infection. It was also observed that there is a high cross reactivity between different genotypes which limits its specificity (Thomas et al., 2003). 1.7. Treatment Treatment of women for different stage of the disease is different at each stage. For women diagnosed with ASC-US and LSIL follow up with HPV DNA testing, pap smear or colposcopy with in certain time interval are management options. For patients with HSIL and invasive lesions a number of factors such as size, stage, and histologic features of the tumor, lymph node involvement, risk factors for complications from surgery or radiation, and patient preference determine the course of treatment. In general, noninvasive intraepithelial lesions identified only microscopically are treated with superficial ablative procedures such as cryotherapy, cold knife conization or laser therapy. These are outpatient office procedures, and fertility is maintained. Loop electrosurgical excision procedures are now considered to be the preferred treatment for noninvasive squamous lesions. In these procedures, an electrically charged wire is used to excise the transformation zone and distal endocervical canal. It is less expensive than laser therapy and preserves the excised tissue for histologic examination of margin status (Eileen.2004; Folashade et al., 2007). . Surgery may be used to remove cancerous tissue. If the cancer has spread, surgery to remove the cervix and uterus, called a hysterectomy, may be necessary. Sometimes the fallopian tubes, ovaries, and lymph nodes from the pelvis are removed at the same time. Radiation therapy is often prescribed for cervical cancer that has spread beyond the cervix. In radiation therapy, highenergy rays are used to kill cancer cells (Eileen.2004). 1.8. Prevention Strategies for preventing HPV infection include sexual abstinence, being in a monogamous relationship with an uninfected person, condom use, or vaccinations where women already infected with high-risk types of HPV will not benefit from. Prevention of HPV infection is difficult, Infections are often asymptomatic and so individuals may transmit the infection to multiple partners without ever becoming aware of the infection. Condom use has been shown to lower the risk of transmission, however, are not totally reliable, since HPV may be contracted by contact with other parts of the body, such as the labia, scrotum, or anus, that are not protected by a condom. In addition it is also possible to prevent cervical cancer when cytologic and HPV DNA detection screening programs are employed that facilitate the detection and treatments of precancerous lesions early hence increase the relative survival rate of individuals. Health education about the HPV infection, the disease it causes and how it can be prevented is very important (Bethany.2006; Herbert et al., 2008; Ginocchio et al., 2008). HPV vaccines are usually composed of virus-like particles (VLPs), which are empty virus capsids containing the major HPV capsid antigen and possibly the minor capsid antigen but lacking viral DNA. The vaccines are produced by expressing the L1 or L1 and L2 ORFs in eukaryotic cells. These proteins then self-assemble into VLPs which are highly immunogenic stimulating B and T cell responses and generating high titers of neutralizing antibody. Data from clinical trials indicate that antibody levels fall after the booster injection but persist for at least 18-24 months post vaccination. There are some data that the immune response to vaccination against type16 and 18 provides some protection against type 45 and 31, both important in the etiology of cervical cancer , thus increasing the projected protection from vaccination to 75-80% (Meenakshi et al.,2007; Lynette .2009). The quadrivalent HPV vaccine, the first to be developed and approved on June 8, 2006—was designed to protect against HPV types 6, 11, 16, and 18 which account for 70% of cervical cancers and 90% of genital warts. Phase 3 trials of this quadrivalent vaccine demonstrated 100% efficacy in preventing HPV type 16 and type 18 associated cervical cancers, adenocarcinoma in situ, and CIN. If these vaccines become widely available, they promise to dramatically reduce the burden of HPV-associated disease, as well as the cost and psychosocial burdens associated with HPV infection. Study showed the bivalent HPV vaccine that protects against HPV types 16 and 18, offers potential elimination of up to 70% of invasive cervical cancers, 60% of high grade CIN and 90% of genital warts. The quadrivalent HPV vaccine can be administered to females aged 9 to 26 years and 9-15 for males (Eileen.2004). It is administered in three Intra muscular injections, 0.5 ml each, at intervals 0, 2, and 6 months. For sexually naïve females, vaccination will prevent infection with HPV types 6, 11, 16, and 18. In females who have already had sexual contact, vaccination will prevent infection with HPV types to which they have not already been exposed. In a study it was shown that these vaccines provide high-level protection against persistent HPV infection and HPVrelated CIN II–III up to 6.5 years after immunization (Melissa et al., 2008). Thus, the study of cervical cancer prevention within the context of vaccines requires accurate detection of typespecific incidence and persistent HPV infections associated with cancer and precancerous lesions (Neeta .2005; Bethany.2006). 2. Significance of the Study A detailed understanding of the epidemiology of HPV genotype diversity among women presenting with cervical cytological abnormalities with or without HIV infection is warranted before consideration is given in the future use of available licensed quadrivalent HPV vaccine which contains VLP’s of genotypes 6, 11, 16 and 18, which is not adequately polyvalent to induce protective immunity against all HR HPV types. This study will help to determine which genotype is most prevalent in Ethiopia. The finding would also help policy makers to put in place strong screening program and give appropriate health education for those at risk and then vaccinate the target population that were not exposed for the above HR HPV types, in order to reduce the prevalence of cervical cancer. The study of and clear understanding of the effect of HIV on cervical cancer would help clinicians to properly manage patients with cervical cancer and precancerous lesions. 3. Objectives of the Study 3.1. General Objective The general objective of this study was to determine the prevalence of HPV infection and HPV genotype distribution with cervical cytology abnormalities and HIV-1 infection on women attending the Gynecology and Obstetrics out patient clinic at Tikur Anbessa Teaching Hospital. 3.2. Specific Objectives The specific objectives were: (i) To determine the prevalence of HPV infection in the study population. (ii) To identify the prevalent genotypes among the study participants. (iii) To determine the association of HIV -1 infection and CD4 count level on HPV prevalence and cervical cytology abnormality. (iv) To assess risk factors associated with HPV infection and cervical cytology abnormality development. 4. Materials and Methods 4. 1. Study Design and Study Period The study design was a cross-sectional study in women attending the Gyn & Obs out-patient clinic at (Tikur Anbessa Teaching Hospital) Faculty of Medicine in Addis Ababa, Ethiopia. The study period was from January 2009 to December 2010. 4.2. Source Population and Study Area Consecutive and consenting study participants were recruited from women attending the Gyn & Obs out-patient clinic, Faculty of Medicine in Addis Ababa University (Tikur Anbessa Teaching Hospital).The hospital is the only specialized center for cancer treatment handling referral cancer patients from all over Ethiopia. At the Gyn/Obs Clinic, individuals who came for any Gynecological problem were offered a verbal explanation about the nature of the study for the study enrolment, where HIV testing was included. 4.3. Ethical Consideration The study was reviewed and approved by the Research and ethics committee of the DMIP, GYN and OBS Department and the IRB of the Medical Faculty, Addis Ababa University and the IRB of Hadassah University Hospital. The study was also reviewed and approved by the National Ethical Review Committee of the National Science and Technology Ministry. Written informed consent was obtained from all the study participants (Appendix 1). Subjects who conceded for HIV test were given written informed consent and were signed by the study participant and by the assigned counselor. Finger prints of study participants were taken for those who can not write. The consent form was prepared in Amharic, the local language which was attached on the participant’s medical records. At the Gyn/Obs Clinic, all women were offered HIV counseling and testing based on the Ministry of Health’s National Guidelines. Counselors assigned at the clinic provided pre-and post-test information for study participants. Results of HIV test were kept confidential and all individuals tested for HIV were provided with follow-up post-test counseling based on the national guideline. The importance of testing the sexual partner was emphasized. Those HIV-positive individuals were linked to HIV care and treatment services at the hospital. 4.4. Sample Size Determination The sample size was determined using the two-proportion formula, n= Z 2 α /2 p 1 (1 - P1 ) - Z β 2 p 1 (1 - P1 ) p 2 (1 - P2 ) P1 − P2 Assuming, Zα/2 = confidence limit = 95% P1 = 0.15 P2 = 0.32 β= power of 80% d = degree of precision within 5% N= 391 (123 HIV positives and 268 HIV negatives). 4.5. Enrolment Visit and Study Procedure At the enrolment visit, detailed socio-demographic information, as well as a clinical history and information on behavioral risk factors, including past history of STIs, were collected using standardized questionnaire (Appendix 2). The criteria for enrollment were: 1. Women willing for Pap screening. 2. Willing to undergo counseling and HIV testing. 3. Not pregnant. 4. Anti-retroviral treatment naïve. 5. Age > 18 years. 6. Willing to participate in the study. The exclusion criteria were: 1. Pregnant/lactating women. 2. Women on menstrual cycle 3. Virgin girls. 4. Age <18 years. 5. Unwillingness to participate in the study. Clinical examination was done and two cervical specimens were collected: one smeared onto a glass slide used for routine cytological examination and a second cervical swab was used for HPV detection and swab was placed in 1 ml of normal saline and then immediately placed in ice before transfer to the laboratory. In addition, blood specimens were collected for HIV-1 serology and CD4+ cell count. HIV tests were done at Tikur Anbessa Teaching Hospital at GYN /OBS outpatient clinic by the investigator and result were available on the day of the test. All cervical swabs were transported to MBL within three hours of collection and stored at -20oC until analyzed. Cervical swabs were shipped in dry ice to Hadassah Virology laboratory in Jerusalem, Israel for training purposes for HPV PCR and genotype determination. The investigator was provided with the necessary training in Israel on how to perform DNA extraction, PCR amplification of HPV, performing genotyping as well as quality control issues, including the interpretation of results. After training all the assays were performed at Hadassah University Hospital virology laboratory by the investigator from August 2010 to October 2010. 4.6. Laboratory Investigations Serological testing for HIV: HIV testing was performed based on National Algorithms for VCT. Briefly, HIV screening was done by rapid KHB test (Shangi, KEHUL) and reactive samples were tested again by Stat Pack (Chemo Biomedical). Discordant samples were subjected to a tiebreaker test using UniGold. CD4 cell determinations: CD4+ T-cell counts were determined by FACScan (Becton Dickinson). MBL participates in External Quality Assurance Scheme with QASI, Health Sante, Canada, for CD4 cell count enumeration. Cervical cytology: Cervical material was taken with a wooden applicator, smeared on a slide, immediately fixed with 95% ethanol, and allowed to air dry. The smears were stained with Pap stain at MBL and reported by pathologists according to the criteria of Bethesda classification system (2001). All smears were reviewed by two pathologists independently at MBL and Tikur Anbessa Teaching Hospital, who were unaware of the clinical or other laboratory findings of the study participants to avoid bias. HPV detection and genotyping: The cervical swabs were kept at room temperature to defreeze. DNA was extracted using the Mag NA Pure LC 1.0 extraction instrument (Roche.cat.no.12 236 931 001) and its reagents. The instrument is a robotic work station which is used for automated isolation of nucleic acids (DNA, total RNA, mRNA) from different kinds of crude sample materials (whole blood, culture cells, tissue ,bacteria or fungi ). It uses a specially designed Mag NA Lc reagent kits that contain, buffers that are important for removal of PCR inhibitors, salts and proteins. It also contains lysis and binding buffers used for cell lysis and binding of total nucleic acids. A proteinase K containing buffer used for digestion of proteins. A magnetic particles suspension was used for binding of total nucleic acids. An elution buffer was used which is important for elution of total nucelic acids, dilution of elutes and for reconstitution of Proteinase K. A 200µl of the thawed cervical specimen was pipetted in to the sample cartilage which can hold 32 samples and put in to the instrument after all the extraction reagents are ready in the instrument .The total time for automated purification of 32 samples including positive and negative controls was ~90 minutes. After obtaining the extracted DNA, The general methodology of primary PCR amplification of a 450 bp segment of the HPV L1 gene with a pair of consensus MY09/MY11 primers was done followed by nested PCR with a pair of GP5+/GP6+ general primers for HPV DNA amplification. A 268 bp sequence of human beta-globin gene using GH20 and PC04 primers were used as internal control of specimen adequacy. Negative (Phosphate Buffer Saline) and positive (known positive sample) external controls were used to see the quality of the procedure. For Taq PCR amplifications, the reaction mixture of 45 µL contained (a master mix) (NH4)2SO4, 20 mM Tris-HCl pH 8.0, 25 mM MgCl2, triton x-100, 2.5 mM of each dNTP and 2.5 units of ORNAT Taq polymerase (LAROVA GmbH.Germany). The mixture was prepared as a Taq master mix (Red Load Taq Master) in presence of 10% glycerol to facilitate loading. 1.5µl of each consensus primer (MY09/MY11) and 5 µL of the extracted HPV DNA were added in an Eppendorf tube for the first PCR run. For the second PCR run 2µl of the first PCR product was added in Eppendorf tube with 48µl of the master mix and the second primer set GP5+/GP6+. The reaction mixture was subjected to 35 cycles of amplification in a thermocycler. Each cycle consisted of a denaturing step at 94°C for 0.5 min, an annealing step at 55°C for 1 min, and a chain elongation step at 72°C for 1 min and the whole Nested PCR process needed 7 hours for 32 specimens as a batch. Following amplification, 10 µl of PCR products were electrophoresed through 2% agarose gel after staining with ethidium bromide and examined under UV transillumination for expected amplicons of 450 bp (for HPV DNA) and or 268 bp (for beta-globin). The virology unit of Hadassah University Hospital has an annual external quality control program with QCMD Human Papilloma Virus EQA (Glasgow G20 OSP Scotland, UK). For DNA sequencing, Nested PCR products were cleaned up with an EXoSAP-IT Kit that contains nucleases to digest short sequences (amersham pharmacia biotech). One µL of the cleaned Nested PCR products, 1µL of 5 µmolar GP6+ primer (as the sequencing primer), 1 µL of the BigDye® Ter minator (v 1.1/Sequencing Standard Kit), 3.5 µL 5 ×buffer, and 13.5 µL water in a total volume of 20 µL were used according to the protocol supplied by the manufacturer (Applied Biosystems), for 20 enzymatic primer extension/termination reaction cycles. The reaction mixture was loaded in automated ABI thermocycler Model 3730 a four capillary genetic analyzer for direct sequencing. Sequence alignments were performed against various standard HPV genotype sequences stored in the GenBank database by online BLAST analysis to arrive at specific genotyping. 4.7. Data Management and Statistical Analysis: Questionnaire responses, cervical cytology results, HPV results, HIV results and other laboratory data were entered onto SPSS 16.0 database. Through-out the study, double-data entry were undertaken in order to assure quality of the data. Descriptive/Univariate analysis: Proportions and actual number of cases were used to describe frequency outputs for categorical variables. Mean and Standard deviation were used to describe continuous variables. Results of the descriptive analysis were presented using tables, graphs and numerical summaries. Bivariate analysis: Cross-tabulations were used to explore and display the relation between two categorical variables. Chi-square statistics and Fisher’s exact test (when the requirements for chisquare test were not met) were used to identify the existence of association between two categorical variables in the cross-tabulation. Binary logistic regression analysis (unadjusted odds ratio) was also used to identify the strength of association of the various potential determinant factors with presence of HPV infection and cervical cytology abnormality. Multivariate analysis: Multiple logistic regression analysis (using the Enter method) was carried out in order to control the effects of suspected confounding factors, especially the effects of level of CD4 cell count and HIV-1 co-infection while investigating the association of other factors with HPV and cervical cytology abnormality status. P value of less than 0.05 was considered to be statistically significant. In this multivariate analysis the general associations of interest were: 1. The association of HPV infection with HIV status,CD4 count level, age, number of sexual partners, condom use, marital status and history of sexually transmitted infections; and 2. The association of abnormal cervical cytology development with age, HIV status, CD4 counts level, use of oral contraceptives, and parity of women. More specifically, we were interested to investigate: 1. The effect of age in HPV infection and cervical cytology abnormality. 2. The effect of age in HIV negative and HIV positive women on cervical cytology abnormality. 3. The effect of HIV status in HPV prevalence and cervical cytology abnormality presence. 4. The association of CD4 count (stratified) in HPV infection and cervical cytology abnormality presence. 5. Result 5.1. Socio Demographic Characteristics A total of 391 women participated in the study, out of whom 360(92.1%) had a complete data available, including an interpretable Pap smear, a positive β-globin DNA PCR and known behavioral and demographic characteristics while 31(7.9%) were excluded from the study. According to the sample size determination, 123 were HIV positive women. The age ranged from 19 to 75yrs and the mean age was 40.5yrs with SD of 12.6. The mean age of HIV positive women was 33.4yrs and that of HIV negative women was 43.6 yrs. The socio-demographic characteristics of the study participants are given in table 1, by HIV sero-status of study subjects. There was no significant statistical difference between HIV negative and HIV positive women in their socio demographic characteristics except their age. Of the total 360 women 51.9% were married, 57.5% employed and 42.5% were house wives. HIV negative women were slightly older than HIV positives (28.2 % of women to be greater than or equal to 50 years old). The mean parity of HIV negative women was higher compared to HIV positives (3.3 versus 1.9) respectively. The majority of the study participants (68%), especially most of HIV positive women (87.7%) were from Addis Ababa. Most of HIV positive women had more than one sexual partner compared with HIV negative women (47% versus 37.5%) respectively. In both groups presence of history of STI was very low accounting only 13.1%. Only 6.7% of the women have ever used condom and 30.6% of the women used Oral Contraceptive in their life in both study groups, and 7.7% used Oral Contraceptive pills for more than 5 years. Table 1. Socio-Demographic Characteristics of Study Participants, Tikur Anbessa Teaching Hospital, AA Ethiopia,2010 Characteristic HIV Negative No(245) (No & %) HIV Positive No(115) (No & %) Total No(360) 47 (40.9) 63 (54.8) 5 (4.3) 100 186 74 33.4 40.5 Percentage (%) Age-(yrs) <30 30-50 >50 Mean Age 53 (21.6) 123 (50.2) 69 (28.2) 43.6 27.8 51.7 20.5 Parity – 0 1-5 >5 Address 45 (18.4) 119 (48.6) 81 (33) 31 (26.9) 79 (68.7) 5 (4.4) 76 198 86 21.1 55 23.9 Addis Ababa 144 (57.8) 101 (87.8) 245 68.1 Out of Addis Ababa Marital Status 101 (41.2) 14 (12.2) 115 31.9 Married Divorced Single Widowed No. of life time sexual partner 1 >1 History of STI 148 (60.4) 26 (10.6) 9 (3.7) 62 (25.3) 39 31 14 31 187 57 23 93 51.9 15.8 6.4 25.9 61 (53) 54 (47) 214 146 59.4 40.6 24 (20.9) 91 (79.1) 47 313 13.1 86.9 Yes NoUse of Oral Contraceptive Never Ever Condom Use Never Ever 153 (62.5) 92 (37.5) 23 (9.4) 222 (90.6) (33.9) (26.9) (12.3) (26.9) 176 (71.8) 69 (28.2) 74 (64.3) 41 (35.7) 250 110 69.4 30.6 238 (97.1) 7 (2.9) 98 (85.2) 17 (14.8) 336 24 93.3 5.2. HPV Prevalence Over all, Human Papillomavirus was detected in 64.4% (232/360) of the study participants. A broad diversity of HPV genotypes were detected on the direct sequencing assay, 33 HPV genotypes were identified among the 232 HPV positive specimens. The mean age of women with HPV infection was 45.5yrs. Women without HPV infection accounted for 35.6% (128/360), their mean age was 34.8yrs (figure 1). HPV Status of 360 Participants Number of Women 200 150 181 128 100 51 50 0 HPV Negative HR HPV Positive LR HPV Positive HPV Status Figure 1: Overall Distribution of HPV Infection in Study Participants: Tikur Anbessa Teaching Hospital, AA, Ethiopia, 2010 Overall, HR HPV accounted for 78% (181/232), and the most abundant HR-HPV types were HPV 16, HPV 35, HPV 56, HPV 45 and HPV 18 in descending order. HPV-16 was detected in 46.4% (84/181) of the HR HPV positive specimens. In contrast, HPV types 31, 33, 39, 51, 52, 58, 59 and 70 were detected in smaller proportions of samples, ranging from 1% to 2% of the all HR HPVs (figure 2). HR HPV Genotypes Prevalence (Total 181) 50 46 P e rc e n t 40 30 20 6 10 4 8 1 3 7 7 1 2 4 2 1 1 4 2 1 0 HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV 16 18 31 33 35 39 45 51 52 56 58 59 66 68 70 73 82 HR HPV Genotypes Figure 2: Prevalence of HR HPV in the Study Population: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010. Low risk HPV accounted for 22% (51/232) of all HPV infected women and the most frequent LR HPV types being HPV 53, 6,11, 54, 62 and 84 accounting from 12% to 8 % of all LR HPV types (figure 3). p e rc e n t LR HPV Genotypes Prevalence(Total 51) 14 12 10 8 6 4 2 0 12 8 8 8 8 8 6 6 HPV 81 HPV 83 4 HPV 6 HPV 11 HPV 30 2 2 HPV 32 HPV 40 2 HPV 53 HPV 54 HPV 61 2 HPV 62 HPV 67 HPV 84 LR HPV Genotypes Figure 3: Prevalence of LR HPV in the Study Population: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 In the bivariate analysis there was no significant statistical association between HPV infection or genotype distribution and most of the socio-demographic variables which were considered to be potential risk factors for HPV infection. But there was an association with residence area, HIV status, cervical cytology abnormality, GUT symptoms. Table 2: Factors Associated with HPV Status: Bivariate analysis: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010. Factors Response categories Address Addis Ababa Outside Addis Ababa Non-Reactive Reactive Positive Negative HIV status Cervical cytology abnormality GUT symptoms None At least one Chi-square (Pvalue) 4.7(P=0.03) 4.3 (P=0.038) 15.7(P=0.000) 12.8(P=0.000) However on multivariate analysis using the logistic regression, only cervical cytology abnormality was found to have statistically significant association with the presence of HPV infection. It was found that the likelihood of having HPV among those without cervical cytology abnormality was 0.37 times less as compared to those with cervical cytology abnormality. Conversely, those with cervical cytology abnormalities were 2.7 times more likely to have HPV infection. There was no significant statistical difference in prevalence of HPV infection between HIV positive and HIV negative women. There was no evidence of an association between HIV status and either high risk or low risk HPV types. So presence of HIV infection was not identified as a risk factor for HPV infection in this study. Table 3: Factors associated with HPV Infection: multivariate analysis: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 Factors Response categories Address Addis Ababa Outside Addis Ababa Non-Reactive Reactive Negative Positive None At least one HIV status Cervical cancer GUT symptoms Crude OR CI) 1.00 1.6(1.0, 2.5) 1.00 1.6(1.0, 2.6) 1.00 2.9(1.7, 5.1) 1.00 2.2(1.4, 3.3) (95% Adjusted OR (95% CI) 1.00 1.2 (0.7, 2.2) 1.00 1.7 (0.95, 3.1) 1.00 2.7 (1.4, 5.3) 1.00 1.6 (0.9, 1.6) Overall 50.6% of HPV infection was observed in women aged 30-50 years old; however there was no significant statistical association between age and HPV infection in the study subjects. Prevalence of HPV infection by HIV status, stratified by age is shown in figure 4. Among HIV negative women, HPV prevalence for women aged <30 years, 30–50 years, and >50 years were 50%, 65%, and 66.7% respectively. Among HIV positive women, HPV prevalence for women aged <30 years, 30–50 years, and >50 years were 67.8%, 68.5%, and 80% respectively. This indicates presence of high prevalence of HPV infection in both groups in all age categories hence presence of persistent infection. Type-specific analysis of HPV showed that there is no as such significant difference in HIV positive and HIV negative women. HPV Prevalence by HIV Status Stratified by Age 80 70 60 50 Percent 40 30 20 10 0 68 80 69 65 67 50 HIV Negative HIV Positive HIV Positive <30 HIV Negative 30-50 >50 Age in Years Figure 4: HPV Prevalence by HIV Status Stratified by Age: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 5.3. Cytology The prevalence of cervical cytology abnormalities among the study participants was 26.1% (94/360). Two hundred sixty six women had normal cervical cytology finding and their mean age was 37.6yrs. CIN I was present in 5% (18/360) of women, CIN II was found in 3.9% (14/360), CIN III in 11.7% (42/360) being the most frequent finding and SCC 5.6% (20/360).The detailed description of cervical cytology is shown in figure 5. Figure: 5 Proportion of Cervical Cytology Abnormalities in study population: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 The mean age of women with abnormal cervical cytology finding was 47.7yrs. Presence of cervical cytology abnormality in HIV negative women aged <30 years, 30 30-50years 50years and >50 years were 1.9%, 30.1% and 53.6% respectively and that of HIV positive women were 17.8% among <30 years,16.7% in women aged 30 30-50 50 years and in women above 50 years of age no cervical cytology abnormality was detected. An increased risk of cervical cytology abnormality development with increasing age was observed (P= 0.000). In the bivariate analysis using Pearson Chi-Square test high parity and condom use were statistically associated with cervical cytology abnormality (P=0.000 and P= 0. accounting 51.5% and 73.4% of women with abnormal cervical cytology finding respectively. Presence of abnormal cervical cytology was also statistically associated with marital status, P=0.003, where cervical cytology abnormality was observed most frequently in married women. But risk factors such as history of sexually transmitted infections, use of oral contraceptives for a long period of time (> 5 years) were not statistically associated with cervical cytology abnormality. Table 4:: Factors Associated with Cervical Cytology Abnormality: Bivariate analysis Tikur Anbessa Teaching Hospital, AA, Ethiopia, 2010 Factors Response categories Age 35 or less 35-50 >50 years Addis Ababa Outside Addis Ababa No formal education Attended formal education Ever married Never married Reactive Non-reactive Positive Negative Yes No None At least one 0 1-5 >5 Address Educational status Marital status HIV status HPV status Condom use GUT symptoms Parity of women Chi-square (Pvalue) 40.7(P=0.000) 31.2(P=0.000) 45.7(P=0.00) 9.0(P=0.003) 5.9(P=0.016) 15.7(P=0.000) 5.6(P=0.018) 42.4(P=0.000) 42.3(P=0.000) In multivariate analysis using logistic regression, risk factors that were found to have a statistically significant association with the presence of cervical cytology abnormalities were age of women, residence area of women, HPV status of women and the presence of GUT symptoms. Hence age greater than 50 years, living outside Addis Ababa, HPV positivity, and presence of GUT symptoms were found to have positive and statistically significant association with presence of cervical cytology abnormalities. Table 5: Factors Associated with Cervical Cytology Abnormality: Multivariate analysis, Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 Factors Response categories Age 30 or less 31-50 >50 years Addis Ababa Outside Addis Ababa Negative Positive None At least one Address HPV status GUT symptoms Crude OR (95% CI) 1.00 3.1 (1.5, 6.2) 5.4 (1.6, 18.6) 1.00 3.9 (2.3, 7.7) 1.00 2.9 (1.7, 5.0) 1.00 5.1 (3.0, 5.6) Adjusted OR (95% CI) 1.00 3.1 (1.5, 6.3) 8.8 (4.1, 19.1) 1.00 2.3 (1.1, 4.8) 1.00 2.3 (1.2, 4.5) 1.00 3.4 (1.8, 6.7) As shown in the above table those women more than 50 years of age are 8.8 times at a higher risk of having cervical cytology abnormality as compared to those women aged below 30 years. It was also found out that women aged 31-50yrs are 3 times at a higher risk of cervical cytology abnormality as compared to those aged equal or less than 30 years. Mean age of women with abnormal cervical cytology finding was 48 while that of women with normal pap smear result was 37.7, T test 7.4 (95% CI 7.73-13.25) P =0.000 indicated presence of statistically significant difference in age among the two groups. Residence of the study subjects was also found to have statistically significant association with cervical cytology abnormalities. In this study it was found that those living outside Addis Ababa were 2.3 times at risk of having cervical cytology abnormalities as compared to those living in Addis Ababa, P=0.000. The prevalence of abnormal cervical cytology in women living outside AA was 73.4%. Those women with at least one GUT symptoms (Dysuria, Abnormal Vaginal bleeding, Dysparunia, and Itching) were found to be 3.4 times more likely to have cervical cytology abnormalities as compared to those women having none of the above mentioned GUT symptoms(P=0.000). Among women with Dysuria and abnormal vaginal discharge the prevalence of cervical cytology abnormality was 50.7% and 54.4% respectively. Prevalence of GUT Symptoms in Study Subjects 40 35 30 25 Percent 20 15 10 5 0 39 23 16 4 Dysuria ABN Vaginal Discharge Itching 7 Ulcer Dysparunia GUT Symptoms Figure 6: Prevalence of Abnormal Genitourinary Symptoms: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 HPV is positively associated with presence of cervical cytology abnormalities which was detected 100% in women with abnormal cervical cytology finding P=0.000.Those women who have HPV infection were found to be 2.3 times at risk of having cervical cytology abnormalities as compared to their counter part women without HPV infection. Among Women with any HPV infection, 59.5% (138/232) had normal cervical cytology finding and 40.5% (94/232) had abnormal cervical cytology finding (table 6). High Risk HPV infection in those with abnormal cervical cytology finding accounted 93.6% (88/94), P=0.000. HPV 16 caused 62.7% of all cervical cytology abnormalities followed by HPV 45 (7.4%), HPV 35 (5.3%), HPV 18 (4.5%) and HPV 31(3.2%). HR HPV infection in women with normal cervical cytology finding was 68.1% (94/138) with HPV 16 being the most prevalent one followed by HPV 56 and 35 respectively. Of women with SCC, HR HPV accounted 100% (20/20), HPV 16 (65%) being the most prevalent genotype. In those with CIN III finding, 95.2% (40/42) had HR HPV infection and the most common HR HPV was HPV 16 (66.7%), and the less prevalent HR HPVs were HPV 18 and 31 accounting 3.2 % each. In women with CIN II finding HR HPV infection was 92.9% and HPV 16 was the most frequent type 66.7% (12/18). In women with CIN I, the prevalence of LR HPV was 22.2% (4/18) and that of HR HPV was 77.8% (14/18), and among the HR HPVs HPV 16 accounted for 55.6% (table 7). High Risk HPV types were statistically significantly associated with cervical cytology abnormalities in the analysis P=0.000. HPV 16 was detected in 62.7% of all samples with abnormal cervical cytology findings and had a statistically significant association with an abnormal pap smear finding P=0.000 and OR=14.9 (95% CI 8.3 - 26.6). Table 6: HIV Status and Risk of Cervical Cytology Abnormality: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010. Cervical Cytology Result Normal CIN I CIN II CIN III SCC Number Tested (N=360) 266 18 14 42 20 HIV Positive (n=115) 96 11 3 5 0 HIV Negative (n= 245 ) 170 7 11 37 20 Table 7: HIV Status and most Prevalent HPV Genotype Distribution in Association with Cervical Cytology Abnormality: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 Genotypes HPV 16 HPV 18 HPV 31 HPV 35 HPV 39 HPV 45 HPV 52 HPV 56 HPV 58 HPV 66 HPV 70 LR HPV Genotypes HPV 16 HPV 18 HPV 31 HPV 35 HPV 39 HPV 45 HPV 52 HPV 56 HPV 58 HPV 59 HPV 66 HPV 70 HPV 73 HPV 82 LR HPV Neg 10 4 3 4 Pap Smear Result in HIV positives CIN I CIN II CIN III SCC 5 1 4 0 1 1 0 0 0 0 0 0 1 0 0 0 Total 20 6 3 5 2 3 0 0 0 1 0 1 0 0 2 5 3 5 0 0 0 0 0 0 0 0 3 5 3 1 0 0 0 4 0 4 17 1 0 2 0 0 0 0 0 0 0 0 0 1 4 19 SCC 13 0 1 1 0 2 1 1 0 0 0 0 0 1 0 64 5 5 10 3 7 1 8 4 4 1 4 3 1 30 Pap Smear Result in HIV Negatives Neg CIN I CIN II CIN III 15 5 8 23 3 0 0 2 2 0 0 2 6 0 0 3 3 0 0 0 2 1 0 2 0 0 0 0 6 0 1 0 4 0 0 0 2 0 0 2 1 0 0 0 3 0 1 0 2 0 0 1 0 0 0 0 26 1 1 2 Total 5.4. HPV, HIV and Cytology Result Out of the total 123 HIV positive women 115 women had a complete set of data. Among these women 31.3% had a CD4 cell count <200 cells/µl. Further exploration in to the HIV positive individuals for their CD4 count indicated that about 62.6% have CD4 count less than 350 cells/µl and only 14.8 % had CD4 count more than 500 cells/µl (figure 7). CD4 Count Range s in HIV Po sitiv e Wo me n 35 29 30 32 26 25 20 Num ber of Wom en 15 10 17 11 5 0 <50 50-200 201-350 351-500 >500 CD4 Count Ranges Figure 7: CD4 Count Ranges for HIV Positive Study Subjects: Tikur Anbessa Teaching Hospital, AA Ethiopia, 2010 The prevalence of HPV infection was significantly high in HIV-1-infected women, 68.7% (79/115), mainly owing to a higher prevalence of HR-HPV 75.9% (60/79), the most prevalent HR HPV being HPV16, 33.3% (20/60) (table 7). The prevalence of LR HPV infection was 24.1% (19/79). There was no statistically significant association b/n HPV infection and age among HIV-1-seropositive women P=0.539. Out of the total HPV infected women, 75.9% (60/79) had normal cervical cytology finding, HR HPV infection accounted 71.7% (43/60). The mean age of these women was 33.5yrs and their mean CD4 count was 530 cells/µl. Women without HPV infection and normal cervical cytology finding accounted 31.3% (36/115), their mean age and CD4 count were 33 and 323 cells/µl respectively. The Prevalence of abnormal cervical cytology finding in HIV positive women was 16.5% (19/115). The mean age of women with abnormal cervical cytology finding was 34 years and their mean CD4 count was 205.9 cells/µl, the prevalence of HR HPV infection was 89.4% (17/19). In this group, there is high prevalence of early stage of disease (CIN I ,57.8 %) compared with other cervical cytology abnormalities; the most frequently detected HR HPV type at this stage was HPV 16, 45.4% (5/11) followed by HPV 18, HPV 35, HPV 58 and HPV 66 each accounting 9.1% (1/11). LR HPV infection was 18.2%, and the mean CD4 count of these women was 200 cells/µl. Women with high grade lesion were less frequent, CIN II finding accounted 15.8% (3/19). HR HPVs responsible at this stage were HPV 16, 18 and 45 each accounting 33.3% (1/3). CIN III accounted 26.4% (5/19) and the most common HR HPV was HPV 16, 80 % (4/5) followed by HPV 45, 20%. No woman was detected having SCC. There was no significant statistical association betweenCD4 count and HPV infection but in the bivariate analysis, CD4 count level had statistically significant association with presence of an abnormal cervical cytology finding, P=0.018; where 54.5% of women with CD4 count less than 50 cells/µl had an abnormal cervical cytology finding (table 8). In bivariate analysis presence of cervical cytology abnormality in women aged <30 yrs was statistically associated with HIV positive women (P=0.006) compared to HIV negative women with the same age. Table 8: Pap Smear Result in Association with CD4 Count in HIV Positive Women: Tikur Anbessa Teaching Hospital, AA, Ethiopia, 2010 Abnormal Cervical Cytology CD4Count Absent Present Total Category (cells/µl) No (%) No (%) <50 5 (45.5) 6(54.5) 11 50-200 24(82.8) 5 (17.2) 29 201-350 29 (90.6) 3 (9.4) 32 351-500 23(88.5) 3 (11.5) 26 >500 15 ( 88.2) 2 (11.8) 17 Total 96 19 115 Out of the total study participants, 245 were HIV negative with a complete data, the mean age was 43.6year. The prevalence of HPV infection of any type was 62.4% (153/245), the HR HPV accounted 78.4% (120 /153) and LR HPV 21.6% (33/153). The most prevalent HR HPV was HPV 16, 54.2% (65/120) followed by HPV 35 and HPV 56 which accounted 8.3% (10/120) and 6.7% (8/120) respectively. From women who had HPV infection 50.9% (78/153) had normal cervical cytology finding, HR HPV infection in women with normal cervical cytology finding accounted 68%. In this study, a higher prevalence of (30.6%, 75/245) cervical cytological abnormalities were observed in HIV negative women owing to HR HPV infection 92% (69/75), (table 6). The mean age of women with cervical cytology abnormality was 51.8yrs. There was also a high prevalence of high grade dysplasia (CINII, CIN III ) accounting 64% (48/75) of all dysplasias in HIV negative women, and HR HPV DNA was detected (95.8%, 46/48), and the most common HR HPV type was HPV16 accounting 64.6% (31/48). CIN I was detected in 9.3% of women with abnormal cervical cytology finding, prevalence of HR HPV was 85.7% (6/7), HPV 16 accounted 71.4% (5/7). Advanced stage of disease i.e. invasive cervical cancer (SCC) was also observed in 20 cases (26.7%) of women with abnormal cervical cytology findings and presence of HR HPV accounted 100% (20/20) and the most frequent HPV genotype was HPV16 65% (13/20) (table 7). Women with abnormal cervical cytology in this group had high parity, the mean parity being 4.8 and 62.7% having more than five live births which has, indicated high parity to be a risk factor among HIV negatives P=0.003. In HIV negative (P=0.021) with abnormal women age was statistically associated cervical cytology finding in women aged more than 50 years compared to HIV positive women with the same age. 6. Discussion This study is one of the first studies conducted to determine the prevalence of cervical HPV infection and its genotype distribution in relation to cervical cytology abnormality and HIV-1 infection in Ethiopia. The study was conducted on patients referred from all regions of Ethiopia for any Gynecological problems to Tikur Anbessa Teaching Hospital GYN/OBS OPD, the only referral hospital for cervical cancer cases, which was thought to concentrate the majority of HPV genotypes circulating in the general population. Though it is difficult to make direct comparisons between studies as HPV DNA detection methods differ, in this study, HPV prevalence was found to be low (64.4%) as compared to other studies conducted in Ethiopia which have detected HPV prevalence to be more than 80% on biopsy samples suspected of cervical cancer (Fanta.,2005; Abate et al.,2005; Mihiret et al.,2005). However it is one of the highest prevalence in unselected populations, compared with the study conducted in rural Ethiopia in Attat hospital on women attending out patient clinic which was 15.9% (Ruland et al 2006), in Kenya 44.3% on women attending family planning (Vuyst et al., 2003), in rural community Gambia 13% (Wal et al., 2005) and Tanzania 34% in antenatal women (Philippe et al., 2003). But the prevalence is consistent with other studies conducted on African women; A study in Ethiopia at Jimma hospital conducted on women with cervical cancer showed 67.1% (Bekele et al., 2010), a study in Zambia ( Christopher et al.,2006) among hospital patients 65.4%, the study done in South Africa 65% on women attending health facility (David et al.,2008), study in Cameroon on hospital attendants 67.2% (Andrew et al.,2009) and a study conducted in Burkina Faso in female sex workers 66.1% (DidelotRousseau et al.,2006). High Risk HPV infection was found to be 78% and the LR HPV was 22% where the HR HPV was higher in both women with and without cervical cytology abnormality and in those with and without HIV infection, which is higher prevalence when compared with the study conducted in Zambia 69% on hospital patients (Christopher et al., 2006). In a study conducted in Kenya among Female sex workers, HR HPV prevalence was 55.6% (Stanley et al., 2010). In our study HPV 16 accounted for 46% of all HR HPV infections being the most frequent HPV, which is about 2.5 fold greater prevalence of HPV 16 compared with worldwide prevalence of 14% in the general population (Clifford et al., 2005). In this study, the second most prevalent HR HPV was HPV 35 (8.2%); meta-analysis from Africa showed HPV 35 to be most common in sub-Saharan African women (Lynette.2009), a study by Mihret in Addis Ababa indicated HPV 35 to be the second prevalent HPV type equivalent to HPV 18 (5.7%) (Mihret et al.,2005),a study on rural Gambia community 8% prevalence was noted ( Wal et al.,2005) and a study in Burkina Faso 9.4% (Didelot-Rousseau et al., 2006) which showed HPV 35 to be the second most common HR HPV type. HPV 35 was followed by HPV 56, HPV 45 and HPV 18 consecutively, which was not similar with studies done in Ethiopia on biopsy samples with suspected cervical cancer where the second most prevalent HPV was HPV 18 accounting 13.7%, 8.2% and 5.7% ( Fanta. 2005; Bekele et al., 2010; Mihret et al.,2005) respectively. In this study, the prevalence of HPV 18 was 4.7% which is similar in prevalence with the aggregate worldwide rates accounting 5% (Clifford, et al., 2005). In both HIV negative and positive groups the effect of age on the prevalence of HPV infection was not noticed, this is similar with the Kenya research on female sex workers (Stanley et al., 2010). The prevalence almost remained constant specifically in HIV negative women in all age categories while in HIV positive women the prevalence of HPV infection was increasing with increasing age, this is not consistent with studies done worldwide (Carolyn.2007), where peak of HPV prevalence was seen before the age of 25 – 35 and decreased with increasing age. But our study was similar with the studies conducted in Africa countries such as Nigeria (Thomas et al., 2004) and Gambia (Wall et al., 2005). The increasing rate of HPV infection in older women among HIV-positive subjects in our study can be explained by a decreased ability to clear HPV infection, reactivation of HPV or re-infection with HPV types previously cleared, all of which more likely to occur more commonly in HIV-infected women (Cynthia et al., 2009). The prevalence of cervical cytology abnormality in the overall study participants was low 26.1%, an explanation could be the very low sensitivity of conventional cytology technique and the sample collection technique used compared to other more sensitive techniques for Pap smear (Cynthia et al., 2009; Carolyn.2007). The association between HPV infection and cervical cytology abnormality found in our study accords with previous studies conducted worldwide, where HR HPV was detected in 93.6% of women with cervical cytology abnormality and in 100% of the Squamous cell carcinomas. This is in agreement with other studies done in Ethiopia (Fanta. 2005; Bekele et al., 2010; Mihiret et al., 2005) and worldwide meta- analysis (Clifford et al., 2003; Lynette.2009). Over all prevalence of HPV infection specifically HR HPV infection in women with normal cervical cytology result is high (68.1%), compared with study conducted by Fanta 50% (Fanta. 2005) and two studies in Kenya showed, a prevalence of 50.5% and 61.5% on female sex workers and in women attending family planning service respectively (Stanley et al., 2010; DeVuyst et al., 2003). Worldwide meta-analysis showed the prevalence of HPV infection in the general population with normal cervical cytology finding ranging from 1.4% – 25.6% (Clifford et al., 2006). In another meta analysis global prevalence was estimated to be 10.4% (Lynette.2009). Also prevalence of HPV among women with normal cytology ranges 11.4% worldwide and 33.6 in eastern Africa (WHO. 2010). A world wide metanalysis on HPV genotypes on women with cervical cancer showed the most common HPV types to be, in order of decreasing prevalence, HPV16, 18, 45, 31, 33, 58, 52, 35, 59, 56, 6, 51, 68, 39, 82, 73, 66 and 70. Women with cervical cancer in Africa usually have the following HPV types, in descending order, 16, 18, 33, 45, 35, 31, 58, and 52. On the other hand, HPVs 16, 33, 31, 18, 52, 58, 35 and 56 were those most frequently present in women with CIN II and CINIII (Clifford et al., 2006). Studies conducted in Ethiopia showed the prevalence of HPV genotypes in cervical cancer cases in descending order, HPV 16, 18, 45, 58 (Fanta. 2005) and HPV16, 18, 56, 45, 39, 52, 31, 35, 58, 33 and 59 (Bekele et al., 2010). In this study Women with cervical cytology abnormality had the following HPV types, in descending order, 16, 45, 35, 18, 31, 56, 59, 52, 58, 66, 70, 73, 82 and women with SCC had HPV types 16, 45, 35, 56 in a prevalence order indicting presence of difference in genotype distribution. In general, from this study we can see that with increasing severity of cervical cytology abnormality, there was an increasing prevalence of HR-HPV infection, specifically HPV 16 was the most frequent and strongly associated HR HPV genotype among women with abnormal cervical cytology in all disease stages. This is in line with studies conducted in Ethiopia, (Fanta. 2005; Bekele et al., 2010; Mihiret et al., 2005) and worldwide meta analysis (Clifford et al., 2005; WHO. 2010) underlining its carcinogenic importance from all continents studied. Unlike other Ethiopian studies and the world wide meta analysis (Lynette. 2009) HPV 45 was the 2nd most prevalent HPV genotype involved in abnormal cervical cytology findings in our study while HPV 18 had low association with abnormal cervical cytology findings where it was not detected in women with SCC. Age has statistically significant association with cervical cytology abnormality where women above the age of 30 had a greater risk of developing cervical abnormality than women with younger age (<30 years of age). This is consistent with other studies (Wall et al., 2005; DeVuyst et al., 2003) conducted in Africa and worldwide that stated older age as a risk factor for cervical cytology abnormality development (Ian et al., 2007;Hugo et al., 2006; Noor et al., 2006). In addition women living out of Addis Ababa had a significant association with cervical cytology abnormality indicating absence of medical care and screening program in areas outside Addis Ababa where most cases often presented at advanced stages of disease in this hospital. HPV prevalence was similarly very high in both HIV negative and HIV positive women 68.7% and 62.4% respectively; which didn’t indicate HIV infection as a risk factor for increased prevalence of HPV infection. This is not similar with the Burkina Faso (Didelot-Rousseau et al 2006), Kenya (Stanley et al., 2010) and Zambia (Christopher et al., 2006) studies which showed HPV prevalence to be high among HIV positive individuals. A study conducted in Tanzania among antenatal women showed absence of significant statistical association of HPV and HIV infection which is similar to our study (Philippe et al., 2001). The difference among the studies could be attributed to absence of matching of the two groups which is important to eliminate confounding risk factors such as age and other reproductive health behaviors that obscure the effect of HIV on prevalence of HPV infection. Prevalence of HR HPV is high in both HIV negative (78.4%) and HIV positive (75.9%) women this is not consistent with studies conducted in Kenya (Stanley et al., 2010) and Zambia (Christopher et al., 2006) which have indicated HR HPV to be more prevalent in HIV positive women. High prevalence of HR HPV in HIV negative women in this study might be associated with presence of high rate of abnormal cervical cytology finding in HIV negative women. HPV 16 was the most prevalent genotype in those with and without abnormal cervical cytology findings in both groups. HIV-infected women harbor a distribution of HR HPV that does not differ much from that of HIV negative women, where among HIV positive the HR HPVs in descending order were HPV 16, 18, 56, 35, 45, 58, 70, 31, 39 while among HIV negatives were 16, 35, 56, 45, 18, 31, 58, 59, 70, 39 and this is similar with the study conducted in Kenya where there was no difference in genotype distribution in the two groups (Stanley et al., 2010). Cervical cytology abnormality is more prevalent in HIV negative women (30.6%) compared with HIV positives (16.5%), which was inconsistent with other studies done in sub Saharan African countries. A study in Uganda (Blossom et al.,2007) showed 73% of HIV-positive women versus 16% of HIV-negative women to have abnormal Pap smears and a study conducted in Kenya (Stanley et al., 2010) on female sex workers showed 27% versus 8% cervical abnormality in HIV positive and HIV negative women respectively. The difference could be attributed to the selection bias of study participants and the study site; where most of HIV negative women were suspected of cervical cancer referred from outside Addis Ababa to Tikur Anbessa Teaching Hospital, which is the only referral hospital for radiotherapy. Prospective studies demonstrated that women with HIV infection are much more likely than HIV negative women to have persistent HPV infection and the effect is stronger in women of older ages and with lower CD4 counts (<200 cells/µl) ( Miotti et al., 1996; Sun X-W et al., 1997; Alex et al., 2003; Ferenczy et al., 2003; Cynthia et al., 2009). In our study 68.7% of women had a CD4 count above 200 cells/µl and the mean CD4 count (530cells/µl) of women with HPV infection and with normal cervical cytology finding and being young age (mean age 33.5) can be justifications for low prevalence of cervical cytology abnormality in HIV positive women. Even if the prevalence of cervical cytology abnormality was expected to be higher in HIV positive women than the HIV negatives the rate is within the rage compared to the review which has reported the overall prevalence to be ranging from 6% to 32%, with that of low-grade lesions ranging from 11% to 19% and high-grade lesions from 7% to 15% (Stany et al., 2005). In HIV positive women presence of CIN I (LSIL) was high compared to (CIN II and CINIII) high grade lesions. In HIV negative women prevalence of CIN III was high which was associated with old age and high parity. Late stage of cervical cytology abnormality is less prevalent in HIV positive women where no woman was detected to have SCC. Since our study participants were ART naïve; this finding is in agreement with other studies done in developing countries indicating short life expectancy in untreated HIV positive women in relation to the 10 years on average required for CIN to progress to SCC (Gichangi et al., 2002; Mandelblatt et al., 1999; Palefsky et al., 2003). Among women with cervical cytology abnormality, HIV positives were much younger than those that were HIV negative (mean age 34 years and 52 years for HIV positives and HIV negatives respectively). This indicates that development of abnormal cervical cytology at early age in HIV positive women, which is consistent with other studies done worldwide (Clarke et al., 2002; Cynthia .2009). CD4 count level in HIV positive women was not associated with HPV infection which is inconsistent with studies done in other African countries (Vuyst et al.,2003; Cynthia et al., 2010) which has indicated increment of HPV infection in accordance with lower CD4 counts. The CD4 count was statistically associated with cervical cytology abnormality development which agrees with studies done in other sub Saharan African countries or worldwide (Vuyst et al.,2003; Groesbeck et al., 2006; Stephen et al., 2003) indicating Immunosuppression as a result of HIV infection to be important for malignant progression in the cervix. 7. Limitations • The first limitation of this study was study site where most of the women were patients with any gynecological problems which doesn’t represent the general population. • The second limitation was study design which should have been a longitudinal casecontrol study that could enable us to see the HPV persistence and development of abnormal cervical cytology. Also it would be possible to see the effect of HIV infection on HPV prevalence and cervical cytology abnormality development in the study population. This would also decrease the effect of other confounding socio-demographic factors for HPV prevalence and cervical cytology abnormality development. • Simple cotton tipped wooden applicator stick was used for cervical swab collection which has decreased the quality of the sample on its sensitivity for Pap smear and HPV DNA detection. • Pap smear was done by conventional technique which was known to be less sensitive to detect presence of cervical cytology abnormality. • Inability to detect presence of multiple HPV infection, the laboratory where HPV genotyping was performed didn’t have the capacity to identify multiple infections. 8. Conclusion This study provides baseline information on the pattern of Human Papillomavirus infection and genotype distribution in both HIV positive and HIV negative women with normal and abnormal cervical cytology findings in Ethiopia. The overall HPV infection prevalence was 64.4%, and HR HPV genotypes were more frequently detected than LR HPV genotypes in both groups of women with and without cervical cytology abnormalities and in HIV positive and HIV negative women. The most frequent HR HPV genotype was HPV 16 followed by HPV 35, 56, 45 and 18 in decreasing order. HPV infection was high across all age groups in both HIV positive and HIV negative women which has indicated the presence of high persistence of HPV infection. Presence of abnormal cervical cytology was significantly associated with HR HPV infection (P=0.000). HPV 16 infection was highly associated with abnormal cervical cytology findings in HIV positive and negative women, which is the most frequently detected genotype in each stage of cervical cytological abnormalities. The mean age of women with abnormal cervical cytology result was 48yrs indicating presence of cervical cytology abnormality at older age compared to women with normal cervical cytology finding, which was 38yrs. The prevalence of abnormal cervical cytology was high in HIV negative women compared to HIV positive women, specifically in women above 50 years of age. Among HIV positive women, the finding of abnormal cervical cytology was more prevalent at early age where most low grade lesions were detected in women aged <30 years. SCC was detected only in HIV negative women, which could be associated with the older age as compared to HIV positives. CD4 count level in HIV positive women was associated with development of abnormal cervical cytology. CD4 counts of less than 50 cells/µl were associated with high prevalence of abnormal cervical cytology. In general risk factors for HPV infections such as age, HIV infection, presence of multiple sexual partners, presence of history of other STIs, use of condom were not statistically associated with HPV status which were obscured with study site and study participant selection bias. Also risk factors, such as presence of other STIs including HIV, oral contraceptive use for more than five years were not statistically associated with abnormal cervical cytology finding which has resulted from the study design bias. 9. Recommendation Based on the findings of this study, it is recommend that; • The currently available quadrivalent vaccine can prevent more than sixty five percent of cervical cytology abnormality caused by HPV 16 and HPV 18 in Ethiopia. • HPV infection is equally an important STI in both HIV negative and HIV positive women in Ethiopia, which confirm a need for further population-based study design, to determine which HR HPV genotypes are the most frequent ones. This is important, the efficiency of the available vaccine decreases as the prevalence of HR HPV genotypes that are not preventable by the currently available vaccine increases. • Even though much is known about cervical diseases and their optimal control, current measures to prevent HPV and cervical problem in both HIV positive and negative women are clearly inadequate. Ethiopia needs to develop a new and expanded cervical cancer prevention programs to all strata of the population. The first age group to start should be before the age of the start of sexual activity. • Pap smear screening programme has reduced prevalence of cervical problems by 90% in developed countries. Ethiopia needs to develop a new strategy to have a population based screening programme, emphasizing among women aged 35 to 50. This age range is a reasonable target group for a new cervical cancer prevention program with limited resources. • Low CD4 count is found to be associated with cervical cytology abnormality. Therefore, early commencement of ART for HIV positive women is highly recommended to prevent further progression to cervical cancer. Screening for cervical cytology abnormality should be part of the routine care and support package for HIV positive women under regular follow up. • Increasing awareness to cervical cancer and preventive methods should become a high priority on the public health agenda. 10. 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G, Robert T , Mark S (2003) Determinants of Human Papillomavirus Load among Women with Histological Cervical Intraepithelial Neoplasia 3: Dominant Impact of Surrounding Low-Grade Lesions. CEBP 12: 1038–1044 . Stanley M L,Davy V B,Matthew F C, Annalene N, Wim D, Kishor M, Christophe E D, Patricia C, John-Paul B, Marleen T (2010) Association of HIV infection with distribution and viral load of HPV types in Kenya: a survey with 820 female sex workers. BMC Infectious Diseases 10:1-10. Stany M, Rose G, Zahn C (2005) Special situations: abnormal cervical cytology in immunocompromised patients. Clin Obstet Gynecol 48:186–92. Stefan D and Karl M (2002) The Human Papillomavirus Type 16 E6 and E7 Oncoproteins Independently Induce Numerical and Structural Chromosome Instability. JCR 62: 7075-82. Stephen E. H, Cathy W. C, Mame A. F N, Mame B. D, Aissatou D, Papa T,Abdoul Aziz K, Birama D, Papa Salif S, Awa M. C-S, Jane M. K, Nancy B. K(2003) Increased Risk of HighGrade Cervical Squamous Intraepithelial Lesions and Invasive Cervical Cancer among African Women with Human Immunodeficiency Virus Type 1 and 2 Infections. The Journal of Infectious Diseases 188:555–63. Stephanie T, Andreas M, Konstantin M, Martin S ,Gertrud S (2007) Protein tyrosine phosphatase H1 is a target of the E6 oncoprotein of high-risk genital human papillomaviruses .JGV 88:2956–2965. Sun X-W, Kuhn L, Ellerbrock TV, et al.(1997) Human papillomavirus infection in women infected with the human immunodeficiency virus. N Engl J Med 337:1343–9. Suzanne D V, Elizabeth R U, Margaret A P, Sibailly T S,Stefan Z W, Peter D G, Donna L M, Ira R H, Alan E ,William C R (1999) HIV and human papillomavirus as independent risk factors for cervical neoplasia in women with high or low numbers of sex partners. Sex Transm Inf 75:258–260. Svetlana V, Nicolas W, Jens E, Ruediger K, Corina Z, Peter M, Heike S, Lars-Christian H, Michael H, Magnus von K Dz (2005) Clonal History of Papillomavirus-Induced Dysplasia in the Female Lower Genital Tract. J Natl Cancer Inst.97: 1816 – 21. Tedd V. E, Mary A C, Timothy J. B, Xiao-Wei S, Dorothy S, Karen B,Thomas C. W(2000) Incidence of Cervical Squamous Intraepithelial Lesions in HIV-Infected women.JAMA.283:1031-1037. Thomas JO, Herrero R, Omigbodun AA, Ojemakinde K, Ajayi IO, Fawole A, Oladepo O, Smith JS, Arslan A, Mun˜oz N, Snijders PJF, Meijer CJLM, S Franceschi S (2004) Prevalence of papillomavirus infection in women in Ibadan,Nigeria: a population-based study. British Journal of Cancer 90: 638 – 45. Thomas I, Luisa Lina V (2003) Chapter 12: Human Papillomavirus Technologies. JNCI 31:80– 8. Tomomi N, Woei L P, John D, Denis L, Paul F. L (2005) Human Papillomavirus Type 16 E1E4 Contributes to Multiple Facets of the Papillomavirus Life Cycle. JV 79:13150-165. Wall SR, Scherf CF, L Morison L, Hart KW, West B, Ekpo G, Fiander AN, S Man1, Gelder CM, Walraven G, LK Borysiewicz LK (2005) Cervical human papillomavirus infection and squamous intraepithelial lesions in rural Gambia, West Africa: viral sequence analysis and epidemiology. BJC 93: 1068-76. WHO/ICO HPV Information Centre. Summary Report 2010.www. who. int/ hpv centre Xavier C, Nubia M (2003) Cofactors in Human Papillomavirus Carcinogenesis—Role of Parity, Oral Contraceptives, and Tobacco Smoking. JNC 31:20–28. Xavier C , Mireia D , Silvia de S (2006)Worldwide Human Papillomavirus Etiology of Cervical Adenocarcinoma and Its Cofactors: Implications for Screening and Prevention. J Natl Cancer Inst 98:303 – 15. Annexes I: Information Sheet Human papillomavirus infection and genotype distribution in relation to cervical cytology abnormalities and HIV-1 infection in Ethiopia. Introduction and Purpose You are being invited to participate in a research study entitled “Human papillomavirus infection and genotype distribution in relation to cervical cytology abnormalities and HIV1 infection in Ethiopia”. Your doctor will explain the details of the study to you. You should feel free to ask any questions about the study, your participation in the study, or procedures required for participating. You must be at least 18 years old to participate. Cervical cancer is the second most common cancer among women in the developing countries. The majority of cervical cancers are nowadays attributed to infection with human papilloma viruses (HPV). Sexualy transmitted infections (STIs), including HPV, are major public health problems in Ethiopia. Cervical cancer is also significantly associated with HIV co-infection. Very little is known, however, about the association of cervical abnormal cytology and HPV and HIV infection in our country. This study is designed to find out how many women are infected with HPV and HIV as well as how many women have abnormalities in their cervix. Moreover, this study will find out on the interaction among the above-noted diseases/infections. Early cervical abnormalities that may lead to cancer can be detected using so called Pap screening.There is a laboratory test available to determine whether or not HPV is present in your genital organs. We do hope too that you are aware about the availability of an HIV laboratory test in the blood. Study Design We will offer and perform HIV testing on up to 1300 eligible women presenting for pap screening at Gyn/Obs Clinic of Tikur Anbessa Hospital, Faculty of Medicine, AAU. The study will run for a period of 1 year in Addis Ababa starting________ 2008. Study Procedure If you decide to participate in the study, you will be asked to provide a medical history and give about 10 milliliters of blood for two laboratory tests: HIV 1/2 antibody. If your HIV test result is positive, we will also do an additional test for CD4 cell count to determine your immune status. In addition, the study staff may take samples from genital secretions, for pap screen. Possible Risks The biggest risk identified in this study is your identification of HIV status. If you have a partner, we encourage you to undertake the counseling session along with your partner. Our counselors will educate you and your partner with regards to HIV transmission in the enrollment process and reinforced throughout the counseling. There are no risks, except for the usual risks associated with blood drawing or cervical sampling such as bruising, bleeding, and minor discomfort. In most situations the additional cervical sample will be taken during a routine Pap smear screen as part of your normal diagnostic workup. Benefits If you participate in this study, you will know your HIV status to help you adjust your behaviour, protect yourself or those you love and the community at large. Your participation will contribute to the understanding of the associations between HIV/HPV/ Cervical abnormalities in our country. It is hoped that what we learn from this study will help to prevent the development of HPV infection that causes cervical cancer in the future. However, you may not directly benefit from participating in this study. There is no payment for your participation in this study. All extra laboratory tests performed for the study are for free. However, you will be re-imbursed to cover your transport costs as well as compensation for the time lost in the clinic. Confidentiality Your name and other information that can be used to identify you will be kept private and confidential. The blood and cervical samples will be sent to a laboratory for analysis. The researchers conducting this study will review your records and follow the progress of the research, but nothing that can be used to identify you will be used in reports of this study. Authorized representatives from the sponsor, any relevant governmental agency and the Institutional Review Board of your clinic (provided that such inspectors are legally obligated to protect any identifiable information from public disclosure, except where disclosure is otherwise required) may inspect your records. This proposal has been reviewed and approved by appropriate Ethics Review Committee whose task is to make sure that research participants are protected from harm. If you wish to find about more about the Review Committee, contact at telephone no. (011) 553 4945 or if you have any question about the study, contact the principal investigator, at telephone no. (0911)-661742 or send e-mail at [email protected]. Consent Participation in research is voluntary. You have the right to not participate. Your choice will not affect your relationship with your doctor or your access to medical care. By signing this document, you do not give up any of your legal rights. A copy of this form will be kept in your medical records. I have read this Patient Information and Consent Form, or this form has been read to me. I have had the opportunity to ask, and I have received answers to, any questions I had regarding the study. I understand that if I have any additional questions, I may contact my treating physician. I agree to participate in this study and I have received a copy of this Patient Information and Consent Form. Signature participant:__________________________________ Name participant:_____________________________________ Date:______________________________________ Signature physician:___________________________________ Name physician:______________________________________ Date:______________________________________________ (For those who can not write) Signature witness:____________________________________ Name physician:______________________________________ Date:______________________________________________ Annex IA: Patient Information sheet and Informed Consent Form (Amharic Version) ¾G<T” ûúKAT zÃ[e ui “ ²[²[-SM e`߃ ŸTIì” ›õ "”W` i ‹“ Ÿ›?‹.›Ã .y= Ò` ÁK¨< Ó”– Ó”–<’ƒ KT¾ƒ ¾T>"H@É Ø“ƒ SÓu=Á“ ¾Ø“~ ª“ ¯LT' ›G<” ¾U”ÖÃp ¾G<T” ûúKAT zÃ[e ŸTIì” ›õ "”W`“ uiታታ‹“ ታ”Ç=G<U ŸK?KA‹ ¾›vL²` uiታታ‹ ›?‹.›Ã.y=. zÃ[eU ÚUa ÁK¨<” l`˜’ƒ KT¾ƒ uT>Å[Ó Ø“ƒ ¨<eØ ታ”Ç=d}ó ’¨<:: ¾c?„‹ ¾TIì” ›õ "”c` uታÇÑ> ›Ña‹ ¾G<K}— Å[Í Là ÃÑ—M:: u}KÃU ¾c?„‹ ¾TIì” ›õ "”c` uG<T” ûúKAT zÃ[e (HPV) U¡”Áƒ ታ”ÅT>Ÿcƒ ¾}KÁ¿ Ø“„‹ ÁSK¡ታK<:: u}ÚT]U ¾}KÁ¿ ¾›vK²` uiታታ‹ u›Ñ^‹” u›=ƒታåÁ uw³ƒ }c^ß}¨< ÃÑ—K<:: ¾c?„‹ ¾TIì” ›õ "”c` Ÿ›?‹.›Ã.y=. zÃ[e Ò` ታታታታታ ታ”ÇK¨< ›”Ç”É S[Í‹ ÃÖlTK<:: J•U u›Ñ^‹” ¾c?„‹ ¾TIì” ›õ "”c` Ÿ›?‹.›Ã.y= ታ“ G<T” ûúKAT zÃ[e ÁK¨< Ó”–<’ƒ S[Í u×U ¨<e” ’ታ:: ¾²=G< Ø“ƒ ª“ ¯LT u›G<’< Ñ>²? uG<T” ûúKAT zÃ[e ›”Ç=G<U uK?KA‹ ¾›vK²` uiታታ‹“ ›?‹.›Ã.y=” ÚUa Ÿc?„‹ ¾TIì” ›õ "”W` Ò` ÁK¨<” Ó”–<ƒ uØMkƒ KT¾ƒ ’¨<:: ¾TIì” u` "”W` Ñ“ ŸSŸc~ uòƒ KT¨p ¾T>Å[Ó U`S^ uታ”ÓK=´— “PaP screening” }wKA Ãታ¨nM:: u}ÚT]U uTIì” ›Ÿvu= ¾G<T” ûúKAT zÃ[e ታ“ K?KA‹U ¾›vL²` uiታታ‹” ¾T>ÁSÖ< Iªeƒ” KT¨p ¾T>[Ç ¾Lw^„] U`S^‹ ›K<:: ታ”Ç=G<U ¾›?‹.›Ã.y= uÅU U`S^ KT¨p ታ”ÅT>‰M ታ”ÅT>Á¨<l }có ታ“Å`ÒK”:: ¾Ø“~ ”Éõ' uØl` ›”ud JeúታM ¾TIì” ¡K=’>¡ ŸTIì” U`S^“ ታ”Ç=G<U K’pap screening’ KT>ðMÑ< ታeŸ 1300 KT>J’< c?„‹ ¾›?‹.›Ã.y= U¡`“ U`S^ ታ“Å`ÒK”:: ¾Ø“~ H>Ń' u²=G< Ø“ƒ KSd}õ õnÅ— ŸJ’< ' eK ታ`eታ Iw[}cv© ìvÓ ¾Ö?“ G<’@ታ ›”Ç”É ØÁo‹” ታ”ÖÃታK”:: ¾›?‹.›Ã.y= ታ“ ¾mؘ uiታ (syphilis) S•\”“ ›KS•\” KT¨p U`S^ ÃÅ[ÒM:: u}ÚT]U ŸTIì” ðdi "K' Ÿðdg< “S<“ }¨eÊ K}KÁ¿ ¾›vL²` U`S^‹ ÃÅ[ÒM:: ¾ÅU U`S^ ¨<Ö?ƒ ¾›?‹.›Ã.y= zÃ[e ÁK¨< SJ’< Ÿ}[ÒÑÖ u}ÚT] ¾CD4 U`S^ ÃÅ[ÓMታM:: ÃI”U ¾ታ`ታ” ¾›?‹.›Ã.y= Å[Í G<’@ታ KT¨p Ã[ÇM:: uØ“~ Ÿ}d}ñ ¾T>Å`e Ñ<ǃ' u²=I ýaË¡ƒ c=d}õ U”U ›Ã’ƒ Ñ<ǃ ›ÃÅ`ewƒU:: J•U “S<“¨< uT>¨cÉuƒ ¨pƒ ታ”Å T”–¨<U Ñ>²? ƒ”i ¾SleM“ ƒ”i ¾SÉTƒ G<’ታ K=ÁÒØU ËLM:: K²=G<U }Ñu=¨<” I¡U“ ÁÑ—K<:: u²=G< Ø“ƒ Ÿ}d}ó ƒMl ‹Ó` K=ðÖ` ¾T>‹M ’Ñ` u=•` ¾ታ`e ¾›?‹.›Ã.y= U`S^ ¨<Ö?ƒ G<’@ታ ’¨<:: u}KÃU ¾ƒÇ` ÕÅ— ¨Ã”U ¾õp` ÕÅ— "Mƒ' G<Kታ‹G<U U¡¡`“ U`S^ ታ”ታታÅ`Ñ< ታ“devK”:: u}ÚT]U KG<Kታ‹G< eK ›?‹.›Ã.y= S}LKòÁ S”ÑÊ‹“ K?KA‹ ›eðLÑ> ƒUI`ƒ ÃcׇHM:: ¾›vK²` SÉH’>„‹ ›”Ç”É Ønp” ¾TÃðKÑ< “side effects” K=•ታ†¨< ታ”ÅT>‹K< ታ“deታK”:: J•U Ÿõ}— ›ÅÒ ›ÃÅK<U:: uØ“~ Ÿ}d}ñ ¾T>ÁÑ–<ƒ ØpU' u²=I Ø“ƒ uSd}õ w‰ ¾T>ÁÑ–<ƒ kØ}— ØpU LÕ` ËLM:: u²=I ¡K=’>¡ ታታታታታታታ ¡w"u?“ ¾ì[-›?‹.›Ã.y>. I¡U“ uSÅu—¨< ¾wH@^© ýaÓ^U TÓ–~” ÃkØLK<:: Ÿ²=I Ø“ƒ ¾T>Ñ–¨< S[Í K`e“ KK?KA‹ ŸzÃ[c< Ò` KT>•\ ›=ƒÄåÁ¨<Á” ¾T>cÖ¨<” ¾I¡U“ ›ÑMÓKAƒ Ø^ƒ KThhM ÃÖpTM:: u}ÚT]U u²=G< Ø“ƒ uT>d}óuƒ ¨pƒ KT>Å[ÓMƒ ¾›?‹.›Ã.y>. ታ“ ¾›?‹.›Ã.y>. ¾CD4 U`S^ ¨<Ö?„‹ uGŸ=V uŸ<M ÃeታM:: K²=IU U`S^ ¾T>ŸðK<ƒ ’Ñ` ¾KU:: Ÿ²=I Ø“ƒ uSd}õ ¾T>Ÿðታታƒ ’Ñ` ¾KU:: ¾Ø“~ }dታò S[Í ›ÁÁ´' Ÿ²=I ¡K=’>¡ ¨<Ü uT>LŸ¨< ታ`ታ” ŸT>SKŸ}¨< S[Í Là eU”& ›É^h”“ K?KA‹ ታ`ታታ” KSK¾ƒ K=ÁÑKÓK< ¾T>‹K< ’Ña‹ uS<K< ታ“ÖóK”:: ታ`ታታ” ¾T>SKŸƒ S[Í uS<K< }qMö ¾T>kSØ c=J” u¢Uý¿}` ¾U”ò¨< S[ÍU ¾}Öuk ÃJ“M:: ¾ታ`ታ” S[Í ¾Á² Êc? ታ²=I ¡M’>¡ w‰ ÃkS×M:: ÃI”” S[Í ¾T¾ƒ Swƒ K¡K=’>Ÿ<“ u²=I Ø“ƒ }dታò ŸJ’< É`ÏŒ‹ KSÖ< vKVÁ‹ w‰ ¾}cÖ ÃJ“M: ÃIU ¾T>J’¨< ¾S[Í ›evew Ø^ƒ” KSŸታ}M c=vM w‰ ’¨<:: ÃI ¾Ø“ƒ“ ¾U`U\ c’É uÖ?“ U`U`“ ¾e’ UÓv` ›×] ¢T>ታ ታÄ ¾ìÅk SJ’<”“' T”—¨<”U Ø“~” u}SKŸ} ØÁo S[Í ŸðKÑ< ue.l. (011) 551 4945 ¨Ã”U ¾Ø“~ ª“ ›e}vv] ue.l. (0911) 661742 ታ”Ç=G<U ታE-mail [email protected] u=MŸ<M” ›eðLÑ>¨<” S[Í G<K< ታ”cታK”:: u²=I Ø“ƒ KSd}õ ¾T>Å[Ó eUU’ƒ' u²=I Ø“ƒ ¾T>d}óƒ uS<K< ðnÅ–’ƒ w‰ ’¨<:: ›Md}õU u=K<' KSd}õ Ÿ}eTS< uታLU u=J” KSkÖM "MðKÑ<' ¨Ã”U u²=G< Ø“ƒ Ÿ}d}ó uታL KSkÖM "MðKÑ< uT”—¨<U Ñ>²? ›MðMÓU ¾TKƒ Swƒ ¾}Öuk J•' ÁKU”U }êታ• ታ”Å uò~ I¡U“ታታ TÓ–ƒ ÃkØLK<:: ÃI ¾eUU’ƒ pê ’¨<:: eKØ“~ ¾}¨c’ S[Í Ãe؃“ KSd}õ ›MÁU LKSd}õ ታ”Ç=¨e’< Ã[ÇታM:: KSd}õ Ÿ}eTS< ò`T ›MÁU ¾×ƒ ›h^ ታ²=I pê Là Á•^K<:: J•U uSð[U ¾ታ`eታ Swƒ uT”—¨<”U Ñ>²? ›Ãh`U:: ¾eUU’~ pê ¢ú(pÍ=) ŸI¡U“ "`É Ò` ÃkS×M:: Ÿ²=I uLà ¾}ÑKì¨<” S[Í“ ¾eUU’ƒ pê ›”wu? ¨Ã”U }’xM˜ uÅ”w ¾}[ÇG< SJ’@” ታÑMéKG<˜:: eK Ø“~ ÁMÑv˜” ’Ñ` G<K< ¾SÖ¾p °ÉM ›Ó˜Š }Ñu= SMe G<K< ›Ó˜Š›KG<˜:: K¨ÅòƒU KSÖ¾p ŸðKÓŸ<˜ NŸ=VŠ” KSÖ¾p ታ”ÅU‹M }’Óa—M:: Ÿ²=I Ø“ƒ ¾T>Ñ–¨<” ¨<Ö?ƒ T¨p ŸðKÓŸ<˜ u¡K=’>Ÿ< vKVÁ uŸ<M TÓ–ƒ ታ”ÅU‹M }’Óa—M:: u²=I Ø“ƒ KSd}õ }eTU‰KG< ÃI”” uò`T ›[ÒÓ×KG<˜:: ¾²=I ›"M uSJ” Ÿ}eu¨< ¾ÅT@ “S<“ Là uÅT@ ¨<eØ ¾›?‹.›Ã.y>. zÃ[e ታ“ ¾mؘ ›UÜ v¡{]Á U`S^‹ ታ”Ç=e\M˜' u}ÚT]U ŸTIì’@ ¾TIì” ›õ ’pap screen’ ታ“ ¾TIì” ¾TIì” ›õ ŸT>¨×¨< ðdi ¾›vK²` U`S^‹ ›”Ç=Å[Ñ< ðpÍKG<˜:: uÅT@ ¨<eØ ¾›?‹.›Ã.y= zÃ[e Ÿ}Ñ– u} ÚT]U ¾CD4 U`S^ ታ”Ç=Å[ÓM˜ ðpΛKG<˜:: ¾Ø“~ }dታò eU: ò`T: k”: ¾›dታò eU: ò`T: k”: (¾eUU’ƒ pê” T”uw KTËK<): ¾Ue¡` eU: ò`T: k”:
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