Participant enrolment and sample collection
We randomly selected 15 out of 300 registered high schools in Jos, North Central Nigeria for this study and received permission to conduct the study in 10 (66.7%) of the schools. Between May and November 2016, we contacted 700 female students and their parents, of whom 232 girls and their parents agreed to participate in the study giving a 33.1% response rate. We obtained consent from the parents of girls younger than 18 years of age and assent from the girls. Older girls gave consent in their self-cognizance.
We conducted face-to-face interview with the girls in company of female nurses or researchers using structured questionnaires that covered socioeconomic factors, family characteristics, gynecologic history, sexual hygiene and practices. Questionnaire designs were guided by the research questions and literature review. The questionnaires were pre-tested on 10 individuals to evaluate their performance and corrected as required.
Prior to sample collection, we trained study participants on how to self-obtain vaginal samples using sterile swab sticks - a small cotton swab on a wooden handle packaged in an individual reclosable plastic pack. After the training, each girl was given 2 swab sticks and instructed to:
- Remove the swab from the reclosable pack.
- Gently insert the cotton swab into the vagina while squatting or standing with legs apart.
- Gently turn the swab three times and remove.
- Replace the swab in the plastic pack.
- Repeat this procedure with the second swab stick.
- Swabs were inserted for about 3 cm and NC was always present to guide the girls
The first collected vulvo-vaginal swab was immediately stored on ice pack in a cooler until transported to the Plateau State Human Virology Research Centre (PLASVIREC) sample repository for storage at -80oC, usually within three hours of collection. The second swab was immediately applied on Hydrion pH test paper (pH 2.8-4.6, 4.5-6.1 & 5.5-8.0) (Micro Essential Lab. Inc. New York) the colour change was compared with the provided standards.
HPV detection and genotyping
Collected samples were transported to the African Collaborative Centre for Microbiome and Genomics Research (ACCME), Institute of Human Virology, Abuja, Nigeria for HPV detection and genotyping. To prepare the vulvo-vaginal swab for DNA extraction, 1 millilitre of PBS was added to the dry swabs inside the cryovials and vortexed to dislodge the cells from the cotton wool.
DNA extraction was carried out using Cador® Pathogen 96 QIAcube® HT Kit (Qiagen, Germany) on QIAcube HT (QIAGEN, Germany). The DNA was amplified and HPV detected using SPF10 (DDL Diagnostic Laboratories, The Netherlands) which simultaneously detects HPV types 2, 3, 4, 5, 6, 7, 8, 11, 13, 14, 16, 18, 20, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55 (re-classified as a subtype of HPV44), 56, 57, 58, 59, 61, 62, 64 (re-classified as a subtype of HPV34), 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 95, 97, 102, 106, 114 and 115. Positive samples were tested with reverse hybridization line probe assay (LiPA25) which can simultaneously identify 25 HPV types (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68–73, 70, and 74) according to the manufacturer’s (DDL Laboratories, Netherlands) instructions. This is a highly sensitive broad-spectrum PCR-based assay for qualitative detection of HPV and suitable for epidemiological studies.
Ethical approval and informed consent
The study protocol was approved by the institutional review board of the Jos University Teaching Hospital (JUTH).
Data management and Statistical analysis
We entered data into REDCap electronic database and analyzed using Stata version 14 (StataCorp, College Station, Texas USA). High-risk HPV types were defined as types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 88, 73 and 82, including the probable hrHPV types and low-risk types were defined as types 6, 11, 40, 42, 43, 44, 54, 61, 68/73, 70, 72, 74, 81 and CP6108[18]. Univariate analysis was done using frequency distribution and proportion for categorical variables and descriptive statistics for continuous variables. Bivariate analysis to test for association was done using chi square or fisher’s exact test for categorical variables and t-test for continuous variables. A p-value < 0.20 was used as a criterion for the inclusion of variables in the multivariable analysis. We set a p-value <0.05 as statistically significant