High human papillomavirus prevalence among females attending high school in the Eastern Cape Province of South Africa

As part of the human papillomavirus (HPV) vaccination strategy in South Africa, it is essential to have information on HPV prevalence, and HPV types distribution among the unvaccinated population. Information on the prevalence of HPV and the distribution of HPV types in adolescents and young women in South Africa’s Eastern Cape Province is minimal. Therefore, this study investigates the prevalence, distribution of HPV types, and factors associated with HPV infection amongst unvaccinated female learners. A sample composed of 213 sexually active female learners attending high schools in the Eastern Cape Province of South Africa; median age 18 years, who provided self-collected vaginal specimens. Roche Linear Array HPV genotyping assay that detects 37 HPV genotypes was used to detect HPV infection. HPV infection was detected in 76.06% (162/213) of participants. Of these 14.55% (31/213) were positive for HPV types targeted by the Cervarix® HPV vaccine (HPV-16 and/or 18), 20.66% (44/213) by Gardasil®4 (HPV-6, -11, -16 and/or -18) and 37.09% (79/213) by Gardasil®9 (HPV-6, -11, -16, -18, -31, -33, -45, -52 and/or -58). HPV-35, commonly detected in cervical cancer cases among women of African ancestry, was frequently detected (9.40%). Participants who reported to have ever consumed alcohol had a significantly higher risk of HPV infection (OR: 2.91, 95% CI: 1.38–6.11, p = 0.005). High HPV prevalence was observed among participants. The high prevalence of HPV types targeted by the Gardasil®9 vaccine encourages the introduction of the Gardasil®9 vaccine. Data from this study will inform both vaccination campaigns and monitor the impact on HPV types after vaccination.

Introduction prevalence and distribution of HPV genotypes; and factors associated with HPV amongst high school female learners in Eastern Cape, South Africa.

Ethics statement
This study was approved by the University of Cape Town Human Research Ethics Committee (HREC: 369/2015). Permission to conduct research in the Eastern Cape was granted by the Eastern Cape Provincial Health Research Committee (EC_2016RP29_562). Both the Provincial Department of Health and Education in the Eastern Cape granted permission to investigators to conduct this study. Participation in the study was voluntary, with written informed consent and parental assent obtained for participants younger than 18 years.

Study population and specimen collection
Participants of this study were recruited from grade 8 to 12 learners who participated in the HPV education intervention study conducted between April and May 2019 in two high schools situated in Chris Hani District Municipality of the Eastern Cape Province, South Africa. The participating schools belong to quintile one (no-fee paying schools) South African Department of education quintile ranking. The high schools were randomly selected. After HPV education intervention, females (�15 years, regardless of sexual history status) were invited to the nearest primary care facilities where the study was conducted. Participants received information on study procedures, objectives, and other important information. After which, they responded to closed-ended questionnaires enquiring about their demographics, sexual practices, contraceptive use, smoking habits, and alcohol consumption. Questionnaires were self-administered while the researcher was reading out questions and explaining where necessary. Pre and post-HIV counseling, rapid HIV tests were conducted by a qualified clinic staff member or HIV lay counselor. All participants with a positive HIV test had necessary follow-up according to the Health Department's protocol guidelines.
The study reports on results only for those participants who were sexually experienced. A total of 257 female learners responded to the invitation, of whom 221 were sexually experienced, while 36 were not (Table 1). Self-collected vaginal specimens were only obtained from sexually experienced females who were not menstruating on the day of the visit (as per selfreport). Only one participant noticed menstruation before collecting the specimen and was, therefore, excluded. A total of 220 sexually experienced participants provided self-collected vaginal specimens. A health professional demonstrated to study participants on how to collect specimen using the Evalyn1 Brush (Rovers1 Medical Devices B.V. Oss, Netherlands). Instruction leaflets that Rovers1 Medical Devices provided were also used as a reference. In order to collect the self-collected vaginal specimen, participants were requested to gently insert the Evalyn1 Brush as far as possible into the vagina while in a standing or squatting position, rotate the brush five times in the same direction, remove the brush, pull back the plunger till the brush enters the casing and place the cap back on the Evalyn1 Brush. Participants were assured that the demonstration brush used for specimen collection was soft and allowed to touch it. The Evalyn1 Brushes with vaginal specimens were stored at room temperature and transported to the University of Cape Town HPV laboratory within 30 days.

Nucleic acid extraction
The white brush part was detached from the Evalyn1 Brush device and placed in sterile 2ml cryo-tubes. One milliliter of Digene specimen transport medium (Qiagen, Hilden, Germany) was added and vortexed three times, 10 minutes apart, to detach the cells from the white brush. A total of 400μl were used for nucleic acid extraction that was conducted using an automated procedure of MagNA Pure Compact (Roche Molecular Systems, Inc., Branchburg, NJ, USA) and MagNA Pure Compact Nucleic Acid Isolation Kit (Roche Molecular Systems, Inc., Branchburg, NJ, USA).

HPV detection
Roche Linear Array HPV Genotyping Test (Roche Molecular Systems, Inc., Branchburg, NJ, USA) was used to detect HPV genotypes in extracted nucleic acid from vaginal specimens and manufacturer instructions were followed. The Linear Array HPV Genotyping Test amplifies the target HPV DNA for 37 anogenital HPV genotypes and include 24 low risk (LR) HPV types (HPV-

Data analysis
All variables were captured and coded in Microsoft excel 2013 and exported to Stata 14.1 for analysis. Participants were counted more than once when determining the prevalence of LR-HPV, HR-HPV, and probable HR-HPV if they have types that belong to more than one category. Multiple HPV infections were defined as the detection of two or more HPV types in the same sample. Numerical variables were explored using the Shapiro Wilk test, histogram, and/or box-and-whisker plot. The median and interquartile range (IQR) are used to summarise age in years since they were not normally distributed. Age was later categorised into three categories. The Wilcoxon rank-sum test was used to compare the median ages of males and females. Categorical variables are presented using frequency tables, percentages, and graphs.
The two-sample test of proportions was performed to compare demographic characteristics of males and females, HPV status by the study site, and the age gap in years of current and/or previous sexual partner(s). Logistics regression was used to determine the bivariate association of HPV and the other variables to determine the Odds Ratio (OR). The unadjusted model (bivariate associations) is presented. The 95% Confidence Interval (CI) was used to estimate the precision of estimates. The level of significance was set at 5% (p-value � 0.05) for statistical significance.

HPV type distribution and prevalence of HPV types targeted by HPV vaccines
In Fig 2,    targeted by current commercial HPV vaccines but commonly detected in cervical cancer cases among women of African ancestry origin, HPV-35 was detected in 9.39% female learners (Fig 2).  (Table 4).

Discussion
According to our knowledge, this is the first peer-reviewed report on HPV prevalence and genotype distribution among female learners attending high schools in the rural areas of Eastern Cape Province of South Africa. High overall HPV prevalence (76.06%) and infection with 3-10 different HPV types (42.72%) are of public concern in this population. Among those that tested for HIV infection, the HPV prevalence was still found to be high among the HIV-negative  [17]. The observed HPV prevalence was higher than the one previously reported among HIV-negative adolescents and young adults in the Western Cape (68.2%) and Gauteng (65.0%) provinces of South Africa [19]. However, it was in the range of the HPV prevalence previously reported in literature among South African adolescents and young women [14][15][16][17][18][19][20]. Compared with the population outside South Africa, the observed HPV prevalence was lower (60.3%) among HPV unvaccinated Colombian women between the ages of 18-25 years. The detection of multiple HPV infections was also common than a single HPV infection. It is important to note that the Roche Linear Array HPV Genotyping Test was also used in a Colombian study [39]. Girls who were vaccinated in 2014 when South Africa initially introduced the HPV schoolbased vaccination program were 14 years of age in 2019 when this current study was conducted. Therefore, the probability that some of the study participants would have received the HPV vaccination a few years before is low as the current study participants were between the age of 15 and 22 years. None of the study participants reported having received HPV vaccination at primary school. The high prevalence of HPV types targeted by Cervarix1 HPV vaccine (HPV-16 and/or 18; 14.55%) and Gardasil19 (HPV-6, -11, -16, -18, -31, -33, -45, -52 and/or -58; 37.09%) was similar to the one previously reported among HIV-negative Western Cape (Cape Town) and Gauteng (Soweto) adolescents and young women (18.6% and 38.5% respectively) [19]. The high prevalence of HPV types targeted by Gardasil19 promotes the introduction of Gardasil19 HPV vaccine as it will offer protection to more HPV types associated with cancers [22]. HPV-35, known to be common in African ancestry women with cervical cancer [33,34], was common (9.4%) in this study.
It is not clear why the overall HPV prevalence was found to differ between school-clinic-1 and school-clinic-2 as the participants' demographic data was not found to differ; both the school-clinic sites are located in rural areas far from town. Participants who reported having ever consumed alcohol had a higher risk of HPV infection, which could indicate high-risk sexual behaviour [40]. However, few participants reported being drunk during the last sexual intercourse. Only 38.19% of learners reported having used condoms during their last sexual encounter, and 32.32% were not using any contraceptives. The South African National Youth Risk Behaviour Survey (SANYRBS) 2011 among learners attending high schools in Eastern Cape Province, reported that 37.1% had had sex in their lifetime, 43.0% had at least 2 sexual partners in their lifetime, and 28.7% mostly used condoms during sexual intercourse [41]. The low condom usage among learners in the SANYRBS and the current study suggest that learners are highly exposed to sexually transmitted diseases (STDs) and adolescent pregnancy. The observed high burden of HPV infection among female high school learners emphasizes the importance and need for HPV awareness programs in schools and communities. In view of the majority of learners reported to be sexually active, knowledge about different STDs, risk factors of acquiring STDs, and disease associated with sex is a fundamental need for this population [42]. HPV awareness programs may also support vaccination and cervical cancer screening programs.
It is acknowledged that the study population was from two communities and does not represent the population of Eastern Cape Province, and cannot be generalised. Despite these limitations, the information reported remains essential for this province and South Africa as there is currently limited HPV information on this population. There are few chances that the participants could have received the HPV vaccine during the national school HPV vaccination program or elsewhere. However, the possibility of receiving the HPV vaccine is not completely ruled out because the vaccines were available in the country.

Conclusion
High HPV prevalence was observed among high school female learners. The high prevalence of HPV types targeted by the Gardasil19 vaccine encourages the introduction of the Gardasil19 vaccine. It is envisaged that these results will contribute to HPV baseline data among adolescents and young women of Eastern Cape Province and will be utilized to evaluate the impact of HPV vaccination. Research or surveillance projects to monitor HPV prevalence and distribution among HPV vaccinated and unvaccinated adolescents and young women are necessary to monitor the impact of HPV vaccination in South Africa.