PREVALENCE AND MOLECULAR CHARACTERISATION OF HUMAN PAPILLOMA VIRUS AMONG WOMEN ATTENDING SELECTED HOSPITALS IN FEDERAL CAPITAL TERRITORY, ABUJA, NIGERIA

ABSTRACT

Cervical Cancer is the second most common cancer caused by Human Papilloma Virus (HPV). Human papillomavirus infection is the most common sexually transmitted viral infection globally, with approximately 75% of sexually active women and men contracting an infection at some point during their lives. HPV infection and cervical cancer have been associated for a long time. HPV is discovered in 99.7% of cervical cancer specimens. This study determined the prevalence and molecular characterisation of HPV infection among women attending selected hospitals in Federal Capital Territory, Nigeria. A total of five hundred and one (501) women volunteers were screened using Enzyme Linked Immunosorbent Assay (ELISA). HPV DNA was obtained by extraction and HPV types identified by PCR method using consensus primer sets MY09/MY11 and GP5+/GP6+. Phylogenetic analysis was  determined  using  CLUSTALW  on  UniproUgene  software.  Data  from  administered questionnaires was analysed using Chi square (ᵡ2) at 95% confidence interval and 0.05 significance level. The analysis was performed using IBM Statistical Package for Social Sciences version 23 statistical software package. The overall prevalence of HPV infection in this study was 10.98%. Women who participated were within the ages of 15 to 64 years. The results obtained showed high prevalence of HPV among women within the age range of 35 – 44 years (4.80%), while a low prevalence was observed among women between the age range of 15 – 24 years (0.20%).Married women were found to be more infected (7.98%) when compared to the women that were single, divorced or widowed. In consideration of occupation, there was a high prevalence among civil servants (5.60%) compared to farmers and other occupations. Women with tertiary education qualification recorded more rate of infection (7.90%) when compared to those with lower level of education. Demographic data and risk factors such as Age group (ᵡ2= 9.508, P = 0.050), Educational Status (ᵡ2 = 55.909, P = 0.000), Marital Status (ᵡ2  = 15.390, P = 0.000), HIV Status (ᵡ2  = 11.871, P = 0.001) and Number of sexual Partners (ᵡ2 = 6.252, P = 0.012) were found to have significant association with HPV infection. Nine HPV types (HPV- 6, 16, 18, 31, 58, 66, 70, 72 and 81) were detected in this study with HPV- 70 being the most predominant (26.67%).The phylogenetic tree was constructed using 15 isolates from this study against 45 reference strains selected from NCBI data based on percentage similarity. HPV-6 isolated in this study clustered with isolate from Iran while HPV-18 clustered with isolate from Bayelsa, Nigeria. Mutation was also detected in HPV 6 and HPV 18 strain isolated in the study. There is a need to increase the level of surveillance on females at risk of cervical cancer in the study area, since significant proportion of highly oncogenic strains with a high tendency to transform into malignancy were observed in this study. There is the need for sexual behaviour education and awareness about HPV to be intensified in order to reduce the spread of the infection in the study area.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the study

Human Papilloma Virus (HPV) is an icosahedral virus, measuring 55nm in diameter. The viruses are non-envelope, double stranded DNA measuring about 8 kilo bases in length (Brooks et al., 2007). The virus belongs to the Papillomaviridae family and is capable of infecting humans. Like all Papilloma viruses, HPV establishes productive infections only at the stratified epithelium of the skin or mucous membrane leading to the development of cancer (Brooks et al., 2007). Human papillomavirus (HPV) infection is the most common sexually transmitted viral infection  globally, with approximately 75% of sexually active women and men contracting an infection at some point during their lives (zur Hausen, 2009). HPV infection and cervical cancer have been associated for a long time; HPV is discovered in 99.7% of cervical cancer specimens (Walboomers et al., 1999; Denny et al., 2014).

Approximately 200 species of HPV have been identified and their classification was dependent on the degree of homology between the viral genomes detected by DNA hybridization  (WHO/ICO,  2014).  About  40  HPV  species  have  been  identified  to  be associated with the genital mucosal, which are categorized according to their carcinogenic potential (WHO/ICO, 2014). The virus is mostly transmitted through sexual intercourse and it is not easily detected at the early stage of infection. Clinical manifestations of HPV infection include genital warts, recurrent respiratory papillomatosis, Cervical Intraepithelial Neoplasia (CIN), and cancers, including cervical, anal, vaginal, vulva, penile, head and neck cancer (Avci et al., 2013).

Cervical cancer is the second most common malignant cancer in women and is a major cause of cancer related death among women worldwide (Anorlu et al.,2007; Fadahunsi et al., 2013; Kolawole et al., 2015). Studies have shown that about 500,000 new cases are diagnosed every year with approximately 85% of deaths occurring in developing countries of the world (Ferlay et al., 2010; WHO, 2012). The incidence of cervical cancer is 14,550 per 100,000 and the mortality rate is 9,659 per 100,000 (WHO, globocan2012).

Epidemiological studies have shown that human papillomavirus (HPV) is the main cause of cervical cancer and precancerous lesions (Walboomers et al., 1999; WHO, 2012). The strains involved in the onset of cancers are known as High-risk HPV (HR-HPV) and they include: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 -66 and -68 (Gheit & Tommasino, 2011).

According to global data on HPV infection, Africa has the greatest prevalence (22.1%) (de Sanjose et al., 2007). The prevalence of HPV infection has been reported in Sub-Saharan Africa and it is dependent on the age of the patient and the presence of cytological abnormalities. In some populations, cross-sectional studies have revealed high prevalence rate of infection to be between 20% – 40% among sexually active young women and the prevalence decrease as they age (Brooks et al., 2007).Young age, early age at first sexual contact, sexual promiscuity, and immunosuppression are some risk factors that have all been linked to HPV infection in women. The risk rises as the number of recent and lifetime sexual partners increases. Other factors, such as long-term hormonal contraception use, tobacco smoking, low socioeconomic position, and poor diet, have been linked to HPV infection less consistently (Das et al., 2000; CDC, 2013).

Many  molecular  methods  for  HPV  testing  and  typing  are  currently  available  but  PCR amplification of HPV genomes is the most sensitive technique and can detect between 5 and 100 DNA molecules in a specimen (van den Brule et al., 1993). PCR utilizing consensus primers directed at relatively conserved regions of the HPV genome allows for the amplification of a broad spectrum of HPV genotypes in a single reaction. A number of different  primer  combinations  amplifying  fragments  from  various  regions  of  the  HPV genome have been developed (van den Brule et al., 1993).

The most frequently used amplification systems for the detection of HPV DNA in clinical samples are based on MY09/MY11 and GP5+/GP6+ consensus primer sets mediated PCR which amplifies DNA fragments in the conserved L1 region of approximately 450bp and 150bp respectively (Shikova et al., 2009).

Several studies have point to the fact that any single method or technique for the detecting of HPV  may  underestimate  the  true  prevalence  of  HPV  in  cervical  samples  and  so  more techniques should be employed to allow for comparison (Karlsen et al., 1996). Based on these, detection of HR-HPV infections which is crucial for identifying women at increased risk of developing cervical lesion involve the optimization of HPV testing methods. In order to optimize the testing methods, for HPV identification, different primer sets are compared to determine their potency in detecting and flanking the gene of interest (Shikova et al., 2009). Cervarix and Gardasil are the only two prophylactic HPV vaccines that have been approved by the US Food and Drug Administration (FDA) so far. These vaccinations are available for early prevention against infection with HPV strains that cause cancer. All three HPV vaccines have been shown to protect against HPV types 16 and 18. Furthermore, Gardasil 9 can protect against cervical cancer in up to 90% of cases(Zhai & Tumban, 2016).HPV vaccines have been shown to be both safe and effective, providing long-term protection from HPV infections (Mejilla et al., 2017). To the best of our knowledge, the majority of Nigerian healthcare facilities have yet to implement in their national vaccination program, the HPV vaccine. As a result, statistics on HPV genotypes, regional distribution, and risk factors among childbearing women are needed. The best HPV vaccines are necessary for the prevention of cervical cancer

1.2 Statement of the Research Problem

Cervical  Intraepithelial  Neoplasia  (CIN);  and  cancers,  including  cervical,  anal,  vaginal, vulva, penile, head and neck cancer are caused by HPV (Malloy et al., 2000). Cervical cancer is the third most common cancer among women and the seventh most common cancer in the world with 250,000 deaths and 500,000 new patients worldwide, annually (WHO, 2012). In Nigeria, the number of women at risk of cervical cancer is estimated to be 50.3million and the number of deaths attributed to cervical cancer is 8,240 (Bruni et al., 2010).

The use of chemotherapy for the treatment of cancer related issues has failed. One option that appears to  be promising is  the prophylactic measure  (use of  vaccine)  towards effective prevention and control of cancers in Nigeria. This study intends to identify the strains of Human Papilloma Virus (HPV) in circulation within the Federal Capital Territory Abuja, Nigeria in order to enrich the data bank for the formulation of indigenous vaccine for HPV in future.

1.3 Justification for the Study

Previous studies on Human papilloma virus among Nigerian women have been conducted but limited (Thomas et al.,2004; Okolo et al.,2010; Nnodu et al.,2010; Akarolo- Anthony et al.,2014; Kolawole et al.,2015; Okwuraiwe et al.,2015 and Nyengidiki et al.,2016). Despite the high rate of cervical cancer morbidity and death in Nigeria, no reliable nationwide data on HPV genotypes in Nigerian women exists. A study by Akarolo- Anthony et al. (2014) in Abuja was restricted to just National hospital which is located in one municipal area council as against the six municipal area councils. This study intends to capture all the six municipal area councils and determine the HPV genotypes in circulation within the Federal Capital

Territory, Abuja.  This study differs from the previous studies in the sense that aside DNA sequencing of the HPV isolated was performed, phylogenetic analysis was also conducted to know the evolutionary history of the HPV in circulation in Abuja. This study has provided baseline information on the circulating genotypes/subtypes of the HPV in the study area for the development of indigenous vaccine in future.

1.4 Aim and Objectives

Aim

This study determined the prevalence and molecular characterization of HPV among women attending six selected hospitals in the Federal Capital Territory, Abuja, Nigeria.

Objectives

i.      To detect HPV from samples obtained from women attending six selected hospitals in the Federal Capital Territory, Abuja, using ELISA technique.

ii.      To identify socio demographic and risk factors associated with HPV infection in the study area.

iii.     To extract the DNA of HPV from the samples collected and amplify, using PCR technique.

iv.      To sequence the amplicons.

v.         To construct a phylogenetic tree that will determine the evolutionary relationship of the different strains identified.

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