Study design, setting, and participant eligibility

This cross-sectional study evaluates the feasibility of implementing a large-scale cervical precancer screening program based on concurrent hr-HPV testing and direct visual inspection in Ghana. The data were collected during the operational period of the initiative, which spanned from January 15 to February 22, 2025. The reporting of this study adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for cross-sectional studies. Facility-based opportunistic screening and treatment activities were conducted at 29 designated health facilities, encompassing a variety of governmental and private healthcare facilities, operating at different levels of service delivery (S1 Fig). This diverse range of sites was chosen to reflect the varied healthcare delivery landscape within Ghana and enhance the generalizability of our findings to other contexts.

Eligible participants were asymptomatic women 25 years and older invited to attend the designated screening sites during the project period. The exclusion criteria included current pregnancy, a history of total hysterectomy, and current treatment for CC. Women presenting with symptoms suggestive of invasive CC (e.g., unexplained vaginal bleeding, pelvic pain) were not enrolled in this screening pathway but were instead directed to appropriate diagnostic and clinical management protocols for symptomatic individuals.

Facility selection, project organisation, implementation, and oversight

The 29 facilities were purposively selected on the basis of (i) presence of at least one CCPTC-trained alumnus capable of unsupervised screening and (ii) geographic representation across all 16 regions and levels of care (CHPS compounds to regional hospitals), mirroring the expanded hub-and-spoke model previously described [12].

The initiative was conceived to screen about 1,600 women for cervical precancer using HPV DNA testing and VIA, provide thermal ablation for eligible lesions, raise public awareness about CC prevention, and promote early detection strategies [10]. The project was a multi-stakeholder partnership, with key collaborators being the Rotary Club of Accra-East (Ghana) and the Rotary Club of Eau Claire (Wisconsin, USA), who were the primary initiators and secured funding through The Rotary Foundation; the CCPTC at Catholic Hospital, Battor, which served as the lead technical partner for HW training, quality assurance, and logistical coordination of equipment distribution; Sansure Biotech Inc., who provided reagents for HPV testing and Omni Diagnostics, a local partner involved in the diagnostics supply chain. The Screen-and-Treat Research Group, Centre for Research in Applied Biology, Sunyani, provided research support. The initiative’s mobilization and awareness efforts were also supported by an extensive network of 53 Rotary Clubs, 3 Rotaract Clubs, and one Interact Club across Ghana [10].

A central coordinating committee, comprising representatives from the lead Rotary Clubs and CCPTC, was established to oversee the strategic planning, overall management, and execution of the initiative. The CCPTC played a pivotal role in guiding the technical aspects, including the standardized training of all participating HWs and the strategic distribution of thermal coagulator devices to the 29 selected screening sites across all 16 regions of Ghana. The screening components of the project were based on an expanded hub-and-spoke model for operational coordination and technical support, with the CCPTC serving as the central ‘hub,’ providing expertise, training, and ongoing mentorship, while the 29 screening sites functioned as the ‘spokes,’ delivering services within their communities [12]. This approach leveraged the existing strengths and reputation of CCPTC, facilitating a more sustainable and efficient scale-up of services compared to creating entirely new parallel systems. The central coordinating team regularly monitored key process indicators from the field data, such as the number of women screened per site, HPV and VIA ‘positivity’ rates, and treatment rates.

Health worker training, recruitment, and ‘telementoring’ as a means of quality assurance

The HWs involved in the PYPI were primarily graduates of the comprehensive double-module training program developed, hosted, and delivered by the CCPTC. Components of this training program (which last four weeks in total) have been described in details elsewhere [13,14]. In brief, Module 1 focuses on foundational knowledge and skills essential for establishing and running a cervical screening service, with emphasis on the administrative and logistical aspects of screening programs. Module 2 builds on the competencies acquired in Module 1, providing advanced training in VIA and mobile colposcopy. A significant part of Module 2 is equipping trainees with the knowledge and practical skills to treat cervical precancerous lesions using ablative methods, primarily thermal ablation, which was the method employed in the PYPI due to the distributed equipment. While graduates of the training program have been involved in screening in other nationwide programs of this nature [15], this was the first time in which an extended range of cervical precancer screening services were available to women, since all HWs involved were equipped with the skill and equipment to treat eligible cervical precancers.

Trainees and alumni of CCPTC are typically integrated into professional WhatsApp groups moderated by CCPTC trainers, in order to facilitate peer-to-peer learning, rapid consultation on clinical queries, dissemination of updated information, and continuous professional development. To ensure sustained quality of screening and treatment services across the 29 geographically dispersed sites, this system of remote mentoring (termed ‘telementoring’) was used for ongoing QA in the PYPI, leveraging the CCPTC’s established ‘hub-and-spoke’ model of support. HWs were encouraged to share anonymized images from mobile colposcopes or mobile phones (if used for VIA documentation or difficult cases) and discuss challenging clinical scenarios with a gynecologist and experienced trainers at CCPTC via secure channels.

Cervical specimen collection and VIA

During cervical screening sessions, women first received comprehensive counseling about the procedure’s benefits, potential risks, and possible results. In the dorsal lithotomy position, a speculum was used to visualize the cervix, and a cervical sample was collected for HPV DNA testing using a sterile swab stick (Bioline Diagnostics LLP, New Delhi, India). This involved rubbing the swab stick in a circular motion on the ectocervix and inserting the brush or swab into the cervical os and rotating it 360 degrees to collect cells from the transformation zone (TZ). The dry swab samples were sealed in a plain tube. These samples were typically sent to the central laboratory of the CCPTC within 1 week. If delayed, they were stored refrigerated (0–4°C) or frozen (−16°C) at the collection site and submitted within two weeks. Before testing, the samples were kept frozen (−16°C).

Following HPV DNA sample collection, VIA was performed as per standard procedure [16]. In brief, 5% acetic acid was applied to the cervix, and examined after 120 seconds under good lighting for the presence of acetowhite lesions in the TZ (which would indicate a ‘positive’ result).

Transformation zone classification

During the VIA procedure, HWs assessed and classified the cervical TZ according to the 2011 International Federation for Cervical Pathology and Colposcopy (IFCPC) nomenclature [17,18], as a key determinant for eligibility for thermal ablation. The TZ types were recorded as:

Type 1: The entire TZ, including the entire squamocolumnar junction (SCJ), is ectocervical and therefore fully visible.

Type 2: The TZ has an endocervical component, but the entire SCJ is still fully visible, either directly or with gentle manipulation using a cotton-tipped applicator or an endocervical speculum if available.

Type 3: The TZ extends into the endocervical canal such that the upper limit of the SCJ is not fully visible, even with manipulation.

Laboratory processing of submitted cervical specimens and HPV DNA testing with the MA-6000 platform

At the central laboratory, the cervical specimens were preprocessed and tested for HPV DNA using HPV 13 + 2 DNA diagnostic kits (S3057E; Sansure Biotech Inc., Hunan, China) on an MA-6000 real-time PCR device (Sansure Biotech Inc.). All tests were run in strict accordance with the manufacturer’s instructions [19], and as described elsewhere [20]. For full genotyping, samples were processed in batches of up to 24, with the platform operating in full quantitative mode. This allowed for the detection and differentiation of the following high-risk HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68. Test results were typically available within approximately two hours.

General strategy for management of screen positives, follow-up, and referral pathways

Women were deemed eligible for immediate on-site treatment with thermal ablation if they were VIA positive and the entire TZ was fully visible (IFCPC TZ type 1 or 2), ensuring the full extent of the SCJ and any lesion could be assessed and reached by the ablation probe; the lesion was confined to the ectocervix, did not extend into the endocervical canal beyond the reach of the probe, did not extend onto the vaginal walls, and covered less than 75% of the cervical surface; and there was no clinical suspicion of invasive CC or glandular disease (e.g., adenocarcinoma in situ) [21]. The thermal ablation procedure involved the application of a pre-heated metallic probe (probe types such as 16 mm or 19 mm flat or nipple probes were available, selected based on cervical anatomy and lesion characteristics directly) to the abnormal cervical tissue within the TZ. Treatment was typically administered at a probe temperature of 100°C for 40 seconds per application, as recommended by WHO guidelines [21]. Multiple, slightly overlapping applications were performed as necessary to ensure complete ablation of the entire TZ, including the lesion and a small margin of surrounding normal tissue. The procedure was generally performed without anesthesia, though local anesthetic could be considered if available and deemed necessary by the provider.

HPV results were communicated to participants and providers within two weeks via telephone or in-person return visits arranged by the screening site. HPV-positive,VIA-negative women were advised to undergo HPV DNA testing again after 1 year; they were also given the choice of retesting at a nearby facility or at the ‘spoke’ where they were initially screened. HPV-negative women were reassured and advised to return for routine screening in 5 years (if VIA negative) or in 6 months to 1 year (if minor changes were found at VIA). HPV negative, VIA negative women who were HIV positive were counselled to be rescreened in 3 years. Women with major cervical lesions who were ineligible for on-site thermal ablation were counseled and referred for further diagnostic evaluation and appropriate management (e.g., LEEP) at a higher-level facility.

Ethical considerations

The study complied with the Declaration of Helsinki (1964) and its later amendments. Written informed consent was sought from all women prior to registration, interviews, sample collection, and cervical screening. The study was approved by the Committee for Human Research and Ethics, University of Energy and Natural Resources, Sunyani, Ghana (ref. CHRE/CA/363/025).

Sample size

This study comprised all women screened by CCPTC graduates as part of the Rotary PYPI. We did not perform an a priori sample size estimation because the study was embedded within a service-delivery initiative funded by a fixed Rotary Foundation grant. Thus, realised sample of 1,636 women was determined by the availability of funds/resources in the initiative instead of research-driven statistical requirements.

Data collection and transmission

Standardized paper-based case report forms (CRFs) in routine use at the CCPTC for clinical data collection were used for the PYPI consistently across all 29 screening sites. These CRFs were developed to capture all essential demographic, clinical, screening, and treatment variables. After providing education and counseling and obtaining written consent, data were collected by HWs and subsequently sent by courier to the CCPTC, where the data were transcribed into REDCap version 11·0·3 (Vanderbilt University, Nashville, TN, USA) [22] and stored securely within databases hosted and managed by the CCPTC. Prior to the analyses reported here, the databases were queried, and data were extracted and converted into an Excel spreadsheet (S2 File), followed by manual cross-checking for accuracy.

Study variables and outcomes

Data captured in the CRFs included sociodemographic information, behavioral and lifestyle factors, clinical history, HPV specimen information, VIA findings, adequacy of examination, and treatment information (if applicable). HPV test results, including the overall hr-HPV status and specific genotype(s) identified, were recorded once testing was completed at the CCPTC. The primary screening outcomes were rates of hr-HPV infection and of cervical lesions observed at VIA. Secondary outcomes specific to the PYPI were referral efficiency, defined as the effectiveness of the referral system between satellite centers and the central laboratory for cases requiring advanced diagnostic testing or treatment and treatment rates, defined as the proportion of women who received follow-up care, including those treated with procedures like thermal ablation or loop electrosurgical excision procedure.

Statistical methods

Continuous variables were inspected for distributions using histograms. Normally distributed continuous variables are summarized descriptively as means with standard deviations (SDs), while skewed variables are summarized as medians with interquartile ranges (IQRs). Categorical data are reported as frequencies and percentages. The overall prevalence of hr-HPV and cervical lesions (VIA ‘positivity’) are expressed as proportions, accompanied by their 95% Clopper–Pearson confidence intervals (CIs). The prevalence of hr-HPV was further disaggregated by region, specific hr-HPV genotype (and grouping based on the IARC’s classification [23]), and whether infections involved single or multiple genotypes. We fit separate univariable and multivariable logistic regression models to identify factors potentially associated with hr-HPV infection and VIA ‘positivity’. Both multivariable models were fitted using the backward stepwise elimination procedure with a liberal probability cut-off of 0·25 for variable selection. Given the minimal amount of missing data across the assessed variables, participants with missing values were listwise-deleted from the regression analyses. Effect estimates and directionality of the observed associations are reported as odds ratios and adjusted odds ratios (aORs), each with its corresponding 95% CI. All statistical analyses were performed using Stata release 17·0 (StataCorp LLC, College Station, TX, USA). Hypothesis tests were considered statistically significant at a two-tailed alpha level of 5%.

Participant selection and dispositions

A total of 1,685 women presented for cervical precancer screening (Fig 1), among whom 1,636 underwent and had valid results for both HPV DNA testing and VIA, and were included in the present analyses. 435 women (26·6%) tested positive for hr-HPV; among these, 416 (95·6% of hr-HPV positives; 25·4% of the total screened) were hr-HPV positive and VIA negative. Nineteen women (4·4% of hr-HPV positives; 1·2% of the total screened) were positive for both hr-HPV and VIA. Among hr-HPV + /VIA+ women, 6 (31·6%) were treated (all via thermal coagulation) and 13 (68·4%) were managed conservatively.

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Fig 1. Flow diagram illustrating participant enrollment, screening with HPV DNA testing and VIA, and subsequent management in the Rotary ‘Protect Your Pearl’ Initiative.

https://doi.org/10.1371/journal.pone.0350573.g001

The sociodemographic and clinical details of women screened in the PYPI are shown in Table 1. The mean age was 38·5 (SD, 10·4) years, a greater proportion were married or cohabiting (n = 1,089, 66·6%), and the median parity was 2 (IQR: 1, 3). Regarding clinical history, 1·2% (n = 20) reported being HIV positive, while the HIV status was unknown or missing for 595 (36·4%). Prior precancer screening was reported among 322 (19·7%) women.

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Table 1. Sociodemographic and clinical details of the 1,636 women screened using HPV DNA testing and VIA under the Rotary ‘Protect Your Pearl’ Initiative.

https://doi.org/10.1371/journal.pone.0350573.t001

Screening characteristics and outcomes among the study participants

Gross cervical inspection revealed abnormality in 19 (1·2%) participants. The commonest TZ type was type 3 (68·9%), followed by type 2 (22·2%) and type 1 (8·4%). The VIA ‘positivity’ rate was 4·0% (95% CI, 3·1–5·0) (Table 2). The overall hr-HPV prevalence was 26·6% (95% CI, 24·5–28·8). HPV52 was the most prevalent at 5·3% (95% CI, 4·3–6·5), followed by HPV58 (4·4%, 95% CI, 3·5–5·5) and HPV51 (3·6%, 95% CI, 2·8–4·6). HPV35 was detected in 2·7% (95% CI, 2·0–3·6), HPV18 in 2·4% (95% CI, 1·8–3·3), HPV31 in 2·5% (95% CI, 1·9–3·4), HPV56 in 2·4% (95% CI, 1·8–3·3), and HPV16 in 1·8% (95% CI, 1·3–2·6). Co-infection with high-risk and probable high-risk HPV types was observed in 2·0% (95% CI, 1·4–2·8) of women (Table 2).

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Table 2. Gross clinical characteristics and laboratory outcomes (including HPV genotype distributions) recorded among the 1,636 women screened using HPV DNA testing and VIA under the Rotary ‘Protect Your Pearl’ Initiative.

https://doi.org/10.1371/journal.pone.0350573.t002

Factors associated with hr-HPV infection and VIA ‘positivity’

In the multivariable model, although based on a small number of HIV-positive women (n = 20), HIV infection was associated with substantially higher odds of hr-HPV infection (aOR=5·77; 95% CI, 2·07–16·13, P = 0.001). Compared to single women, those with a steady partner had more than twice the odds of infection (aOR=2·02; 95% CI, 1·22–3·36). In contrast, women who were married/cohabiting (aOR=0·51; 95% CI, 0·38–0·69) or widowed (aOR=0·43; 95% CI, 0·23–0·82) were independently associated with lower odds of hr-HPV infection (aOR 0.51 [95% CI 0.38–0.69], P < 0.001; aOR 0.43 [0.23–0.82], P = 0.011; and aOR 0.67 [0.48–0.92], P = 0.014) respectively. Further, women who had prior cancer screening showed significantly lower odds of infection compared to those who had never been screened (aOR=0·67; 95% CI, 0·48–0·92). Other variables, including religion, NHIS coverage, TZ type, and abnormal findings on gross vulval inspection, did not show independent associations with hr-HPV infection (Table 3).

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Table 3. Exploratory logistic regression analysis of selected sociodemographic and clinical variables associated with high-risk HPV infection among the 1,636 women screened using HPV DNA testing and VIA under the Rotary ‘Protect Your Pearl’ Initiative.

https://doi.org/10.1371/journal.pone.0350573.t003

In the multivariable logistic regression (Table 4), HIV-positive status was significantly associated with higher odds of VIA ‘positivity’ (aOR=7.49; 95% CI, 1.99–28.19, P = 0.003). The wide confidence interval signals limited statistical precision and warrants cautious interpretation pending larger studies. In addition, women with a type 2 TZ had significantly increased odds of VIA positivity compared to those with a type 3 TZ (aOR=2.48; 95% CI, 1.43–4.31).

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Table 4. Exploratory logistic regression analysis of selected sociodemographic and clinical variables associated with VIA ‘positivity’ among the 1,636 women screened using HPV DNA testing and VIA under the Rotary ‘Protect Your Pearl’ Initiative.

https://doi.org/10.1371/journal.pone.0350573.t004

Regional distributions of hr-HPV prevalence

We observed substantial variability in prevalence across the 16 regions of Ghana (Fig 2). Bono East region showed the highest prevalence at 39·2% (95% CI, 25·8–53·9), followed by Eastern region (34·0%; 95% CI, 24·9–44·0) and Western North region (33·8%; 95% CI, 23·0–46·0). Western region showed the lowest prevalence at 10·0% (95% CI, 3·8–20·5), with Savanna region and Ahafo region following closely with relatively low prevalence estimates of 17·4% (95% CI, 9·3–28·4) and 19·4% (95% CI, 12·1–28·6), respectively.

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Fig 2. Forest plot showing the distribution of hr-HPV prevalence stratified by region among women screened under the Rotary ‘Protect Your Pearl’ Initiative. n, number of women screened; CI, confidence interval.

https://doi.org/10.1371/journal.pone.0350573.g002

Strengths and limitations

To our best knowledge, this is the first time a non-targeted nationwide cervical screening intervention in Ghana has been systematically evaluated and documented. This study has several notable strengths. Its large-scale and nationwide reach, encompassing all 16 regions of Ghana, provides valuable data on the implementation of cervical screening across diverse settings within the country. The use of hr-HPV DNA testing aligns with current international best practices and WHO recommendations, and relying on HWs trained through the standardized, comprehensive CCPTC curriculum likely enhanced the consistency of screening procedures compared to programs with less structured training. Although the model required central laboratory HPV testing and thermal-ablation equipment, it leveraged existing CCPTC alumni infrastructure and ubiquitous mobile technology for telementoring, rendering it more feasible for scale-up than parallel vertical programmes. Ghana’s current opportunistic VIA/Pap strategy can be incrementally strengthened by embedding this alumni-driven concurrent-testing approach within routine services, thereby addressing the critical human-resource and equipment gaps identified in the 2014 policy [4].

Together with prior small-scale studies, our findings support a shift toward HPV screening complemented by VIA triage in resource-limited settings. The high prevalence of diverse hr-HPV types highlights the need for broad-coverage HPV tests and eventual adoption of vaccines covering non-16/18 genotypes. The demonstrated feasibility of leveraging existing postal networks for specimen transport and mobile telementoring for quality assurance provides a pragmatic blueprint for national programs aiming to meet WHO cervical cancer elimination targets. Policymakers should consider integrating this hub-and-spoke approach into routine health services, investing in decentralized laboratory capacity or sustained postal partnerships, and scaling up workforce training and digital mentorship to close the cervical cancer equity gap across sub-Saharan Africa.

However, the study also has several limitations. A major limitation is the lack of histological confirmation for the vast majority of participants; VIA ‘positivity’ was used as a subjective surrogate endpoint together with HPV DNA testing. Second, there is the potential for selection bias, as the participants were women who voluntarily presented for screening, who may differ from the general eligible population in Ghana, potentially being more health-conscious or educated, which could affect the generalizability of findings like hr-HPV prevalence. Third, we relied on self-reported data for variables such as HIV status, smoking history, and prior clinical history (which introduces potential recall or social desirability bias) and a high proportion of women had unknown HIV status. Fourth, despite standardized training, the inherent subjectivity of VIA interpretation and TZ classification could lead to inter-observer variability among HWs across the 29 sites [37], although telementoring aimed to mitigate this. Finally, the acknowledged absence of an a priori sample size calculation means that the statistical power for certain secondary analyses, such as subgroup comparisons or multivariable modeling, might be limited, although the overall sample size is substantial for a descriptive report of the screening program.