Genotypic distribution of human oncogenic papillomaviruses in sexually active women in Burkina Faso: Central, Central-Eastern and Hauts-Bassins regions.

OBJECTIVE
this study was conducted to determine the distribution of high-risk human papillomavirus (HR-HPV) genotypes in women in the general population of three regions of Burkina Faso.


METHOD
This multicenter, descriptive cross-sectional study involved 1321 sexually active women in five cities in three regions of Burkina Faso: Central, Central-Eastern and Hauts-Bassins regions. After collection of endocervical specimens, pre-cervical lesions were screened by visual inspection with acetic acid and lugol (VIA / VILI). HR-HPV genotypes were characterized by multiplex real-time PCR after extraction of viral DNA.


RESULTS
The mean age of women was 31.98 ± 10.09 years. The HR-HPV infection in the three regions ranged from 26.16% to 43.26% with 35.42% as overall prevalence in women. The most common HR-HPV genotypes in descending order were: HPV 56, 52, 66, 59, 39, 51, 18, 35. The prevalence of bivalent vaccine genotypes (HPV16 / 18) was 7.83% against 63.78% of genotypes not covered by HPV vaccine; 36.32% (170/468) of women had multiple concomitant HR-HPV infections.


CONCLUSION
this study showed significant regional variation and high prevalence of HR-HPV infection in women. The predominant genotypes differ from those covered by available vaccines in Burkina Faso. These results will help guide our health policies towards better prevention of cervical cancer. The diversity of oncogenic genotypes is sparking a large-scale study in the West African sub-region, particularly in cases of cancer and the introduction of the nonavalent vaccine which includes HPV 52 found among the predominant genotypes in this study.


Introduction
Persistent genital infection with high-risk human papillomavirus (HR-HPV) has been identified as the major etiologic agent associated with the development of invasive cervical cancer [1], a leading cause of morbidity and mortality in women in the world [2]. Affecting mainly the economically productive age group, cancer has a negative impact on the general health of the family.
In sub-Saharan Africa, invasive cancer of the cervix is the most common cancer in women [3] and the prevalence of HPV infection is 21.3% with significant regional variations, including 21.5% in West Africa [3]. In Burkina Faso, the annual number of cases of cervical cancer is estimated at 2517 with 2081 cases of annual deaths [4]. HR-HPV infection and induced cancers are a real public health problem, especially in Africa [5,6]. HPV vaccine and an organized approach to cervical cancer screening offer opportunities for preventing and controlling cervical cancer. For this purpose, the introduction of anti-HPV vaccination in developing countries is a recommendation of the World Health Organization (WHO). In order to broaden the scope of vaccination for girls in some African countries, some pharmaceutical companies have reduced the cost of the vaccine. However, the HPV vaccines available on the market only cover a few oncogenic genotypes including HPV 16 and 18, the most common types of HR-HPV, accounting for 70% of cervical cancer cases [7,8]. However, studies in Burkina Faso have reported the prevalence of other HR-HPV [9][10][11] genotypes found in cancer and precancerous lesions at different stages [12,13]. Moreover, the impact of vaccination can only be assessed with a better understanding of the epidemiological profile of HR-HPV in order to take into account circulating oncogenic genotypes in the development of HPV vaccines intended for populations. It therefore seems crucial to search in larger samples, HR-HPV genotypes circulating in the general population and in cases of cervical cancer in particular. In order to effectively prevent cervical cancer through prophylactic vaccination our study aims to describe the molecular epidemiology of HR-HPV genotypes in sexually active women without cervical lesions in three regions at risk of STIs in Burkina Faso [14].

Type and population of study
This cross-sectional, and multi-center descriptive epidemiological study collected 1321 cervical endocervical specimens from women in three STI-risk regions in Burkina Faso: Central, Central-Eastern and Hauts-Bassins regions. The target population was sexually active women (women who had been sexually active during their lives) in the cities of Ouagadougou, Bobo-Dioulasso, Orodara, Tenkodogo and Garango. Included in this study were all consenting sexually active women, regardless of age, who were not pregnant, menstruating no had undergone a total hysterectomy. Collection of endocervical specimens and screening for precancerous lesions of the cervix.
From December 2015 to March 2017, following an awareness campaign on the prevention of HPV infection and the risk of developing cervical cancer, we collected 1321 endocervical samples from sexually active women (15 to 76 years old) including 520 for the Center region, 535 for the Hauts-Bassins region and 266 for the Center-Est region. Information on socio-demographic, behavioral, sexual characteristics, HIV serology and level of knowledge on HPV and cervical cancer were collected using a standardized questionnaire [9,10,13,[15][16][17].
Using a single-use speculum, endocervical specimens were collected at the squamocolumnar junction using a sterile swab. The samples thus collected were frozen in a transport medium (medium transport) at -20°C and sent to the laboratory of molecular biology and genetics CERBA / LABIOGENE, University Joseph Ki-Zerbo, Burkina Faso for molecular biology analysis. Following sampling, screening for precancerous lesions was performed by Visual Inspection after application of Acetic Acid and Lugol (VIA/VILI).
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee for Health Research of Burkina Faso (CERS) (Deliberation N°2016-02-0012 of 03/02/2016) as well as those of the Regional Health Departments (DRS) targeted regions.
Informed consent: Informed consent has been obtained from all individuals included in this study

Extraction of HR-HPV viral DNA and molecular characterization of HR-HPV genotypes by real-time multiplex PCR
HR-HPV viral DNA was extracted using the DNA-Sorb-A kit (Sacace Biotechnologies, Como, Italy) from the endocervical samples following the protocol provided by the manufacturer. The extracted viral DNA was amplified using the "HPV Genotypes 14 Real

Statistical analysis
The data was processed and analyzed on the microcomputer using IBM SPSS 21 and Epi Info v7.0. The chi-square test was used for comparisons with a significant difference for p < 0.05.

Results
Sociodemographic, sexual and behavioral characteristics of women in our study.
The mean age of women in this study was 31.98 ± 10.09 years (range 15-76 years, median = 31 years), of which 37.37 ± 9.9 years (range 20-60 years) for Central-East region, 28.02 ± 10.14 years (15-57 years) for the Central region and 33.14 ± 8.49 years (16-76 years) for the Hauts-Bassins region. Women under 35 years of age accounted for a majority of 61.09% (807/1321) of the study population. In this study, 71.15% (940/1321) of women were married and 37.47% (495/1321) had a secondary education. The mean age at first intercourse was 18.45 years ± 2.51 years (7 years to 33 years); 90.54% (1196/1321) of women reported having only one sexual partner at the time of the study. Of the women in the study, 47.92% (633/1321) had never used a condom; 40.65% (537/1321) had more than two sexual intercourse a week and 54.20% (716/1321) did not use a contraceptive method. Almost half of women (45.80%) had a history of sexually transmitted infection (STI) and 0.91% (12/1321) were HIV-positive. Screening of precancerous cervical lesions by VIA / VILI in the women in the study showed a prevalence of 4.39% (58/1319) of cervical dysplasia. In addition, of the 1321 women recruited, two of them were not screened by VIA / VILI. Table I summarizes the characteristics of the study population.

Portage of HR-HPV infection in women in the three regions of Burkina Faso.
All samples tested were positive for the β-globin gene used as an internal control.  (Figure 1). But these differences were not statistically significant.
In this table, the HR-HPV genotypes are presented in descending order according to their prevalence. In fact, the genotypes mentioned in red indicate the most common genotypes at the top of the list in each population of women. HPV 16 covered by HPV vaccines is absent in women in the Hauts-Bassins region. The prevalence of 35.42% in bold indicates the overall prevalence of highrisk HPV infection among women in Burkina Faso through this study.

Genotypic distribution and prevalence of HR-HPV identified by HPV vaccines available on the market.
Despite the high proportion of subjects infected with HR-HPV, the proportion of women infected with genotypes covered by the bivalent / quadrivalent vaccine (HPV16 and / or HPV18) was modest, at 7.83% against 36.22% of genotypes covered by the nonavalent vaccine (HPV 6/11/16/18/31/33/45/52/58). In addition, 63.78% of HR-HPV genotypes identified in women were not covered by any currently available HPV vaccine on the market (Figure 3).

Risk factors associated with HR-HPV carriage in women in our study.
The co-variables for which we collected data (age, age of first sexual intercourse, condom use during sex, contraceptive use, history of STIs, HIV serology, VIA / VILI results) were not significantly associated with the risk of HR-HPV genotype infection. However, risk factors such as educational level, marital status, occupation, number of sexual partners and frequency of sexual intercourse were significantly associated with HR-HPV infection (p < 0.005).    Figure 3: Frequency of HR-HPV identified in the women in our study according to their coverage by the HPV vaccines available on the market.  Considering the use of condoms as a risk factor associated with HR-HPV infection, 124 women always used condoms during sexual intercourse in the present study. Among them, 47 were infected with at least one genotype of HR-HPV. Out of these 47 HPV + women, 15 multiple infections and 32 isolated infections were observed. Taking multiple infections into account, the cumulative genotype was 68 and the predominant genotype was HPV 59 followed by HPV 56 and 52 as shown in the table VI.

Discussion
This epidemiological study included 1321 sexually active women, regardless of age and has shown that the carrying of HR-HPV is quite considerable in the female population. In fact, the overall prevalence of 35.42% (468/1321) of HR-HPV infection was close to that of 33.2% in Benin [18] and 35% in England [19]. On the other hand, it was lower than those of other countries, particularly in Africa: 41.5% in Burkina Faso [10], 42.6% in Ghana [20] , 44.61% in France [21] , 54% in Africa South [2], 74% in Tanzania [22] and 83.2% in Ethiopia [23]. However, it was higher than those reported by other authors for a population similar to ours: 18.2% in Egypt [24] , 15.6% in Nigeria [25] , 14.5% in China [26] , 15.9% in Great Britain [27] and 7.8% in Italy [28].
Our results showed significant regional variation in HR-HPV infection, which was 38.72% for Central East, 43.26% for Central and 26.16% for Hauts-Bassins regions. Our results corroborate those of other authors who have shown that the prevalence of genital HPV infection among women differs considerably between countries and regions as well as between different risk groups [29][30][31][32].
In our study, women aged less than 35 years were predominantly represented (61.09%) and were the most affected group for HR-HPV infection (36.68%). This corroborates those of Monsonego, 2007;Louie et al., 2008 [33, 34] who had shown that this infection had a high peak at the beginning of sexual activity and then this peak decreased with age to reach a prevalence of less than 10% beyond the group of women aged 30 to 35 in resourcelimited countries. This could be explained by both the clearance of the virus and the acquisition of immunity, reflecting the transient nature of HPV infection [34,35]. In addition, the high prevalence of HR-HPV in the Central Region could be explained not only by the average age of this particularly young female population but also by the fact that almost half of the women in this region (47.69% or 248/520) never used a condom during sex. According to Winer et al., 2006 [36], consistent condom use appears to reduce the risk of cervical and vulvovaginal HPV infection.
In the general population of women, the predominant genotypes were respectively HPV 56/52/66/59/39/51 topping the list and total absence of HPV16 in the region of Hauts-Bassins. Our results differ from those in the literature according to which HPV 16/18 is the most common in the world, particularly in cases of cervical cancer [37][38][39][40].
In addition, it is in the less common HPV series that we find the two HPV-16 and 18 genotypes that are covered by the available HPV vaccines. The absence of HPV16 in the Hauts-Bassins area, the low prevalence of HPV 16/18 and the variation in genotype distribution of HR-HPV could be explained by genetic polymorphisms. Indeed, according to Stanczuk et al., 2003 andYang et al., 2013 [41, 42] HPV infection is thought to be associated with the polymorphism of genes coding for pro-inflammatory cytokines.
In addition, the predominant genotypes identified in precancerous lesions in the women in our study were HPV 52-59 / 56-66-51-45-39 / 68-31 / 18-33. However, according to the literature, after HPV16 / 18, the most common genotypes are HPV 31/33/35/45/52/58, involved in 20% of cases of cervical cancer worldwide [43]. Also, some no-vaccine genotypes identified in our study had already been found in cases of cervical cancer and high-grade precancerous lesions. [12,13,44,45] The presence of HPV 52 and these genotypes not covered by the vaccine would seem to imply a risk of subsequent progression of precancerous lesions to cervical cancer [46,47] because even though HPV16 and 18 worldwide (both types preventable by vaccination) account for more than 70% of cervical cancers [4,48], studies have already shown the existence of other predominant genotypes also having a considerable responsibility for the occurrence of these cancers, high grade cervical intraepithelial cancers and neoplasia (CIN 1 and 2) [12,13,44,45].
However, HPV 16/18 found in a small proportion among the women in our study are the only HR-HPV covered by the vaccines available in Burkina Faso. The distribution of HR-HPV therefore varies by geographic and demographic factors and may influence the effectiveness of vaccination [43].
According to HIV serology, nearly half (41.  [17,20,49], who showed that HIV is an additional risk factor for the maintenance of viral persistence..
The risk factors for HPV infection among women in our study for whom a statistically significant difference was observed were education level, marital status, occupation, number of sexual partners and frequency of sexual intercourse. Other studies have also noted that these risk factors would influence or increase the risk of HPV infection [16,39,50].
Furthermore, the distribution of HR-HPV genotypes among women who always used condoms during sexual intercourse suggest that in addition to sexual transmission a direct skin-to-skin infection of portions not covered by condom during microtrauma could be considered.
This mapping of genotypes of HR-HPV in women in the general population shows a difference in the distribution of HR-HPV genotypes and raises questions about effective prophylactic actions to control HPV infection. It is clear from this study that women in Burkina Faso would benefit from being treated with the nonavalent vaccine that includes the predominant HPV 52 in the population. In addition, mapping of HR-HPV genotypes in cases of invasive cervical cancer in West Africa would reinforce this control.

Conclusion
This study contributes to the development of a mapping of high-risk HPV genotypes in sexually active women in Burkina Faso. The prevalence of HR-HPV infection was high among these women and showed significant regional variation. In addition, the predominant genotypes (HPV 56, 52, 66, 59, 39, 51) differ from those covered by available vaccines in Burkina Faso. It would be necessary to conduct a large-scale study in the West African subregion, particularly in cases of cervical cancer. Nevertheless, prophylactic vaccination against broad-spectrum HPV would be of paramount importance but should be adapted to the realities of our populations. However, the protection of women could be extended with the introduction of the nonavalent vaccine which includes HPV52 found among the predominant genotypes of this study.