Study design and overview
Women aged 30–49 years (comprising ~ 8% of the total population ) residing in the catchment area of Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC) in Ile-Ife, Nigeria were invited by a public message campaign for self-sampled HPV testing. Women who screened HPV-positive were referred to the colposcopy clinic and invited to participate in a research study (examining triage methods) under informed consent. Standard colposcopic examination, including colposcopic images and treatment, if indicated, was offered to all HPV-positive women regardless of participation in the triage methods research. In addition, cervical images were collected with a cellphone and the EVA (enhanced visual assessment) system (MobileODT, Israel), and a cervical sample was collected for subsequent HPV typing, from all the participants in the study.
The study was approved by National Cancer Institute (NCI) and OAUTHC ethical Institutional Review Boards.
The screening period was from November 2018 to December 2019. The colposcopy period was from December 2018 to March 2020.
The screening visit started when a woman attended one of the three screening clinics (no appointment needed). A nurse-administered short screening questionnaire determined eligibility [age 30–49 years and not pregnant (self-reported)] for screening. If a woman was menstruating and was uncomfortable undergoing screening, she was advised to return later. Known pregnant women were excluded due to an “abundance of caution”, mainly to prevent any possibility that an unrelated adverse pregnancy outcome could be due, or even perceived to be due, to the self-sampling for screening. The lower limit of age for screening was set at 30 years since younger women have a high prevalence of HPV but a very low risk of cancer . The upper age limit was restricted to 49 years due to age-related repositioning of the squamocolumnar junction (SCJ), where cancers arise, into the endocervical canal, limiting the ability of any existing visual screening or triage method to diagnose precancer accurately . An informed consent at screening was obtained from eligible women seeking permission to store the left-over sample after HPV testing for future research and to be contacted in future for a follow-up study. However, the screening effort was a public health intervention and not an experimental study.
After enrollment, eligible women were provided with an HPV self-sample collection kit containing a cervical brush and a Specimen Transport Medium (STM) tube (Qiagen, USA) . Women were shown a 5-min animated video on how to collect a vaginal self-sample [https://www.youtube.com/watch?v=JiNqrDntbTc]  while waiting. Women went into a private self-sample collection area one at a time to self-sample. After collecting the sample, each woman left the brush in the STM vial in a rack. A nurse helped to break the stem of the brush, closed and labelled the vial, and cleaned the outside of the vial and rack with an alcohol wipe. The nurse was available to assist women in specimen collection upon request. The collection room was cleaned between collections. Before leaving the clinic, participants completed an anonymous feedback form regarding their experience with self-sampling. The specimens were stored at room temperature and transferred to the HPV laboratory at the end of the day, to be stored at 2–80 C until testing.
The primary HPV test used in the study was the Digene Hybrid Capture-2 (HC2) HPV DNA (deoxyribose nucleic acid) Test (Qiagen, USA), which is a US Food and Drug Administration approved nucleic acid hybridization assay with signal amplification using microplate chemiluminescence targeting 13 high-risk types of HPV DNA in cervical and vaginal specimens, without distinguishing between them .
Trained nurses called participants by phone, when their HPV test results were available (within two weeks of collection for most of the study period). HPV-negative women were informed and educated about the test result over the phone, and their questions were answered. HPV-positive women were asked to visit the colposcopy clinic to receive their test results. A minimum of five contact attempts were made to approach and advise a woman to attend the colposcopy clinic before a woman was declared lost to follow-up.
Colposcopy clinic visit
At the clinic, a nurse communicated the positive HPV test result and its clinical meaning to the woman, ensuring privacy. The woman was counseled to undergo a colposcopy examination, preferably during that same visit or later.
Each woman was registered and interviewed to determine eligibility for the triage methods research study (analyses in progress to be reported separately). Anyone with a history of cervical cancer, hysterectomy, or who was pregnant at the time of enrollment (confirmed with rapid pregnancy test) was excluded from the study. A female nurse took informed consent from all the eligible women for participation in a research study (examining triage methods). An anonymized picture of the cervix was shown to the woman at the time of consent in order to reassure her about confidentiality and privacy of image collection and to minimize refusals.
Following the interview, a colposcopy examination was performed by one of the study gynecologists (KOA, CAA). Cervical images for research were collected one minute after applying 5% acetic acid for each device, sequentially with three different devices: 1) a Samsung Galaxy S8  smartphone; 2) a MobileODT EVA device that provided lighting and magnification for a Samsung Galaxy J5 smartphone ; and 3) a Zeiss FC150 colposcopic image captured via a beam splitter by a DSLR (digital single-lens reflex) camera [18, 19]. After image collection, a cervical specimen was collected using a cervical sample collection kit containing a cervical brush and a STM tube (Qiagen, USA)  and stored at 2-8 °C. We plan to test this sample along with the residual sample from screening for HPV typing using the TypeSeq HPV test  at the NCI and report the results in future publications.
At the beginning of the study, a dry swab sample was collected at colposcopy, before collecting images and applying acetic acid, for the two-type OncoE6 test (Arbor Vita, USA). The test is known to have high positive predictive value; in fact, three of the five positives (all for HPV 16) were diagnosed with CIN2+. However, the test was dropped after testing 373 samples due to rare positivity and low yield.
Finally, a standard colposcopy examination was performed to assess the presence and possible nature of cervical lesions and to take biopsies of acetowhite lesions, up to a maximum of four biopsies. In addition, endocervical curettage (ECC) was performed in cases where the SCJ was not fully visible, even in the absence of acetowhitening. All women with acetowhite lesions were offered immediate treatment without waiting for histopathology results, following the American Society of Colposcopy and Cervical Pathology 2012 Consensus guidelines , leaning towards the more clinically-aggressive options for women at risk of being lost to follow-up. Either thermal ablation or large loop excision of the transformation zone (LLETZ) was performed, depending on the colposcopy examination findings. Ablation was performed only if the SCJ was fully visible, the entire lesion was visible, the lesion did not cover > 75% of the ectocervix, and cervix architecture was appropriate for the ablation probe .
Histopathology and final diagnosis
Histopathological confirmation of CIN2 or CIN3 was used as the reference standard for the presence of precancer, against which other experimental tests were evaluated and final clinical review decisions were made. Even though from the clinical management purposes, all high-grade (CIN2+) lesions were treated equivalently; for the true yield of the screening effort, we reported the prevalence of CIN2+ and CIN3+ lesions separately to avoid the ambiguity of equivocal CIN2 lesions (a mixture of HPV infections, true precancers, and an error in histopathologic diagnosis) . The study pathologist (AB) at the University of Lagos performed all pathology diagnoses.
Quality assurance review and treatment recalls
All cases were reviewed for adequate clinical management by a US gynecologic oncologist (AN). The more complex cases were discussed in a case conference call. Recall was recommended for women needing further management and such cases were re-reviewed once the recall was completed until the case was determined to be adequately treated. We planned to reach all women needing recall for additional management, a minimum of seven times. However, we restricted our attempts, and recalled only those women at the highest immediate risk of invasive cancer because of the spread of the COVID-19 pandemic in March 2020.
Screening and management project software
All data and images were collected with a HIPAA (Health Insurance Portability and Accountability Act) compliant smartphone application ‘EVA for research’ (MobileODT, Israel). The data platform was custom designed for the project (led by CS) using an advanced barcode scanning system to limit human error from manual key-in. The data and images from different sources on study assigned smartphones were held locally until internet connectivity was available, at which point all data automatically transferred and aggregated to cloud servers and portal for analysis and remote quality assurance.
The preliminary data were analyzed using SPSS 20 (Statistical Package for the Social Sciences)  and Epi Info . Descriptive results were presented as frequencies and percentages. Chi-square tests were used to compare the yield of disease between different subgroups. In future analyses to evaluate the triage tests, the area under the curve (AUC) on a ROC (Receiver Operating Characteristic) curve will be used.
Additional details on study methods (i.e. study site, organization of the clinics, training of staff, and image collection protocol) are provided in the Additional file 1.