The “COMPASS-DUST” is a two-phase study that will be conducted in two accredited adult HIV treatment clinics in Lagos, Nigeria–The Lagos University Teaching Hospital (LUTH) in Idi-Araba and the Nigerian Institute of Medical Research (NIMR) in Yaba, Lagos, Nigeria. The two clinics provide comprehensive care for people living with HIV (PLHIV) in Lagos and its surrounding states in Southwest Nigeria. Together, these clinics attend to ~19,000 PLHIV each month and offer integrated care to sexually active women with HIV infection, including reproductive health services such as routine cervical cancer screening with either Pap smear, HPV DNA testing or VIA depending on availability and affordability.

In Phase 1, we will conduct an intra-participant comparative cross-sectional study involving sexually active women with well-controlled HIV who are otherwise healthy and aged 25–65 years [28] at the participating sites. Women with suspicious cervical lesions, those with ongoing pregnancy or within 6 weeks of childbirth, those with a previous history of hysterectomy, HPV DNA testing or Pap smear within the past year, previous HPV vaccination, or history of ICC or therapy for benign or malignant cervical lesions will be excluded from enrollment. A total of 1,123 WLHIV will be required to achieve 80% power at a target significance level of 5%. This is based on a comparative specificity of 96.5% for DUST and 93.2% for Pap smear [29] respectively for CIN2+ detection in the primary and HPV-triage of WLHIV with adjustment made for an estimated non-response or data recording error rate of 10%.

In Phase 2, a prospective cohort study will be conducted to involve WLHIV and diagnosed with CIN1 in Phase 1. Women with the diagnosis of CIN2+ at baseline assessment, lack of willingness to continue in the study, and withdrawal of consent at any time during the follow-up period will be excluded. We will enrol all WLHIV who are diagnosed with CIN1 for a 6-monthly follow-up for 2 years. As there are no previous studies that assessed the risk of CIN progression based on DUST status, the data generated will enable us to formulate hypotheses for testing in future studies.

In Phase 1, potentially eligible participants will be enrolled by members of the study team using the consecutive sampling technique [Fig 1]. Study intent and procedures will be introduced, and informed consent is obtained before any study procedure. An electronic data collection form designed on the REDCap database will collect information on socio-demographic variables, HIV history (duration of diagnosis), ART history (types and duration of use), immune status (latest CD4+ cells count and viral load), and other relevant information aimed at evaluating the risk factors for cervical cancer [30]. Information on participants’ HIV history, ART use, and immune status will be extracted from the patient’s electronic database at the adult HIV clinics. The women will then undergo pelvic examination and cervical liquid-based cytology (LBC) sample collection using a Cytobrush that is broken off and placed into a ThinPrep vial containing PreservCyt Solution (Hologic Inc, Marlborough, MA 01752, USA) for sequential analysis by the study pathologists for human papillomavirus (HPV) DNA testing, Papanicolaou (Pap) smear, and DUST. All the participants will subsequently undergo visual inspection with acetic acid (VIA) using 3–5% acetic acid by a study nurse as recommended by the WHO [31] and interpreted according to IARC (International Agency for Research on Cancer) criteria [32]. Any dense opaque aceto-white lesion observed near the squamo-columnar junction will be reported as VIA positive (VIA+). Thereafter, a colposcopic evaluation (reference gold standard) will be performed during which 3–5 different images will be taken for documentation and subsequent post-evaluation review by the study clinical team for quality control. The clinical team members will take a turn on each of the three selected days of the week to perform the colposcopic examination and the photographic images of the findings will be jointly reviewed at the end of each week for concurrence and quality control. Lesions suspicious for CIN, as per usual care, will be biopsied and collected in a 10% buffered formalin-containing recipient for transportation and confirmation by histopathologic analysis at the Anatomic and Molecular Pathology (AMPATH) laboratory. The histology results will be interpreted as normal, CIN1, or CIN2+.

In Phase 2, all consecutively consenting WLHIV with the diagnosis of CIN 1 in Phase 1, will be identified and invited by the study investigators to participate. The enrolled women will undergo repeat Pap smear and hr-HPV DNA co-testing as per standard of care [33] at the 6th, 12th, 18th, and 24th months for a cumulative 2-year period. This is the minimum period required to see a difference in CIN progression between the screening methods [34]. At enrolment, a participant’s locator form will be completed and then reviewed at each follow-up visit to ensure that the participant’s contact information is up to date. Women with ≥ASCUS or HPV+ or both will be recalled for further evaluation using colposcopy with(out) biopsy and histopathologic analysis. The study pathologists will be blinded to the baseline results or other follow-up data of the participants. Women with normal colposcopic (and/or histologic) findings will continue in the study while those with a positive test defined as CIN1 persisting till the last follow-up at the 24th month (persistent CIN1) or CIN2+ at any time will be offered standard treatment.

Using the collected LBC sample, a study pathologist will perform the HPV testing using HPV DNA target amplification by Polymerase Chain Reaction (PCR) followed by nucleic acid hybridization for the detection of 14 high-risk HPV genotypes–HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Two other study pathologists will subsequently read the other prepared slides from the PreservCyt residual LBC sample separately for Pap smear and DUST. The results of Pap smear cytology. will be classified according to the revised Bethesda’s classification [35] as negative for intraepithelial lesion and malignancy (NILM), Atypical squamous cells of undetermined significance (ASCUS), Atypical squamous cells cannot exclude HSIL (ASC-H), Low grade squamous intraepithelial lesion (LSIL) and High grade squamous intraepithelial lesion (HSIL). The slide for p16/Ki-67 dual staining (DUST) is prepared from the residual material using the ThinPrep 2000 Processor (Hologic) and will then be tested with the CINtec PLUS Cytology kit (manufactured by Roche mtm Laboratories AG, Mannheim, Germany). For the study, DUST tested sample is interpreted as either positive (DUST+) or negative (DUST-). Per the manufacturer’s instructions, CINtec PLUS detects a positive sample as the simultaneous appearance of brownish cytoplasmic staining (p16) and red nuclear staining (Ki-67) in one or more cells (DUST+), independent of cellular morphology. Slides without any double-stained cells will be labelled as negative for p16/Ki-67 dual staining (DUST-). A case will be scored as inadequate if adequate evaluation is impossible, no p16 and/or Ki-67 staining is visible as internal control, or if slides do not meet the squamous cellularity criteria as specified in the Hologic criteria (≥5000 cells per slide). Inadequate cases will be classified as negative (DUST-). The pathologist reading any slide will be blinded to the other screening tests or histology results, or other participants’ data.

Data will be entered into the REDCap database and then exported to R Statistical Computing Software (R-4.1.2) for Windows for statistical analysis. Missing or incomplete data will be handled by running a missing values analysis using pairwise or listwise deletion if no pattern is detected or the multiple imputation (MI) method if a pattern is detected [36]. Descriptive statistics will be used to describe the characteristics of the participating HIV-infected women and quantitative data will then be tested for normality with the Kolmogorov–Smirnov test with Lilliefors significance correction. Odds ratios and 95% confidence interval by 2×2 tables will be used to assess the association between each of the screening tests (DUST, Pap smear and HPV16/18) and histological outcomes. The sensitivity, specificity, positive predictive value, and negative predictive value of abnormal Pap smear (≥ASCUS), hr-HPV+, HPV16/18, or VIA+ as primary or HPV-triage tests will be compared to DUST. Multivariate analysis using a binary logistic regression model with the forward stepwise selection technique will be used to assess the associations between DUST+ and severity of cervical cytologic abnormalities and grade of CIN (CIN 1–3) while adjusting for other important covariates such as Age, ART history (type and duration), immune status (CD4+ cells count and viral load, etc.). Risks of persistence and progression of CIN1 will be compared with clinical management thresholds for colposcopy referral (and positive histologic diagnosis) and a 6-month return interval. Relative risks (RR) will be used to determine the strength of associations between participants’ baseline data and CIN1 progression (and persistence at 1- and 2 years). Women with <CIN1 at 2-year follow-up assessment will be censored. Longitudinal data will be evaluated using the multivariate Cox regression model with forward stepwise selection to test the association between baseline DUST status and progression of CIN1 to CIN2+ (and persistence of CIN1) at 1-year and 2-year. Adjustments will be made in the final multivariate models with the inclusion of other baseline variables with P<0.20 in the univariate analysis. Statistical significance for a two-tailed test will be reported at P<0.05.

This study protocol was reviewed and approved by the Health Research Ethics Committees of the College Medicine University of Lagos (CMUL/HREC/07/22/1075), Lagos University Teaching Hospital (ADM/DSCST/HREC/APP/5204) and Nigerian Institute of Medical Research (IRB/22/043). The research will be conducted ethically according to the World Medical Association Declaration of Helsinki. All potential study participants will give their written informed consent before enrolment and strict adherence to the privacy and confidentiality of participant’s information will be ensured during and after the conduct of the study.