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Design and feasibility of a novel program of cervical screening in Nigeria: self-sampled HPV testing paired with visual triage

Design and feasibility of a novel program of cervical screening in Nigeria: self-sampled HPV... Background: Accelerated global control of cervical cancer would require primary prevention with human papillomavirus (HPV) vaccination in addition to novel screening program strategies that are simple, inexpensive, and effective. We present the feasibility and outcome of a community-based HPV self-sampled screening program. Methods: In Ile Ife, Nigeria, 9406 women aged 30–49 years collected vaginal self-samples, which were tested for HPV in the local study laboratory using Hybrid Capture-2 (HC2) (Qiagen). HPV-positive women were referred to the colposcopy clinic. Gynecologist colposcopic impression dictated immediate management; biopsies were taken when definite acetowhitening was present to produce a histopathologic reference standard of precancer (and to determine final clinical management). Retrospective linkage to the medical records identified 442 of 9406 women living with HIV (WLWH). Results: With self-sampling, it was possible to screen more than 100 women per day per clinic. Following an audio- visual presentation and in-person instructions, overall acceptability of self-sampling was very high (81.2% women preferring self-sampling over clinician collection). HPV positivity was found in 17.3% of women. Intensive follow-up contributed to 85.9% attendance at the colposcopy clinic. Of those referred, 8.2% were initially treated with thermal ablation and 5.6% with large loop excision of transformation zone (LLETZ). Full visibility of the squamocolumnar junction, necessary for optimal visual triage and ablation, declined from 68.5% at age 30 to 35.4% at age 49. CIN2+ and CIN3+ (CIN- Cervical intraepithelial neoplasia), including five cancers, were identified by histology in 5.9 and 3.2% of the HPV-positive women, respectively (0.9 and 0.5% of the total screening population), leading to additional treatment as indicated. The prevalences of HPV infection and CIN2+ were substantially higher (40.5 and 2.5%, respectively) among WLWH. Colposcopic impression led to over- and under-treatment compared to the histopathology reference standard. (Continued on next page) * Correspondence: ajenifujako@yahoo.com Kanan T. Desai and Kayode O. Ajenifuja contributed equally to this work. Department of Obstetrics and Gynecology, Obafemi Awolowo University Teaching Hospital, Ile Ife, Nigeria Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 2 of 13 (Continued from previous page) Conclusion: A cervical cancer screening program using self-sampled HPV testing, with colposcopic immediate management of women positive for HPV, proved feasible in Nigeria. Based on the collected specimens and images, we are now evaluating the use of a combination of partial HPV typing and automated visual evaluation (AVE) of cervical images to improve the accuracy of the screening program. Keywords: Cervical screening, HPV, Self-sampling, Triage, Automated visual evaluation Introduction cervical intraepithelial neoplasia)] tend to look less nor- Nearly 85% of the annual 570,000 cervical cancer cases mal and are consequently harder to differentiate from and almost 90% of the 311,000 related deaths occur in precancer cases than HPV-negative controls. lower-resource countries [1, 2] due to the lack of effective This paper describes the field methods, feasibility, and cervical cancer prevention programs [3]. The COVID-19 preliminary descriptive results of Project Itoju in Ile-Ife, (coronavirus disease of 2019) pandemic threatens to re- Nigeria, designed to evaluate ultimately the combined duce elective procedures even further, including cervical strategy of self-sampled HPV typing combined with screening and related diagnostic procedures, especially in AVE triage on three different image capture devices. lower-resource settings, and will likely worsen cervical cancer health disparities. As a major advantage compared Materials and methods with cytology, the specimen for HPV (human papillomavi- Study design and overview rus) testing can be collected by the woman herself using a Women aged 30–49 years (comprising ~ 8% of the total vaginal self-collection device, yielding sensitivity for HPV population [10]) residing in the catchment area of Oba- infection that is similar to clinician-collected specimens femi Awolowo University Teaching Hospitals Complex when target-amplification methods like PCR (polymerase (OAUTHC) in Ile-Ife, Nigeria were invited by a public chain reaction) are used [4, 5]. Use of HPV testing will re- message campaign for self-sampled HPV testing. quire a second diagnostic modality for positives, because Women who screened HPV-positive were referred to majority of HPV infections are cleared within 1–2years of the colposcopy clinic and invited to participate in a re- initial detection. Only HPV infections that persist can search study (examining triage methods) under informed cause precancer and invasive cancer [6]. Thus, an im- consent. Standard colposcopic examination, including proved screening program must include triage methods to colposcopic images and treatment, if indicated, was of- focus treatment safely on the small fraction of HPV- fered to all HPV-positive women regardless of participa- positive women with precancer, the general term we use tion in the triage methods research. In addition, cervical to refer to lesions at substantial risk of invasion without images were collected with a cellphone and the EVA treatment [7]. Ideal characteristics of a triage test for (enhanced visual assessment) system (MobileODT, HPV-positive women, for use in low-resource regions, Israel), and a cervical sample was collected for subse- would include excellent risk discrimination (high precan- quent HPV typing, from all the participants in the study. cer risk in positives, low risk in negatives), low-cost, sim- The study was approved by National Cancer Institute plicity, and point-of-care use. (NCI) and OAUTHC ethical Institutional Review A new candidate for triage of HPV-positive women is Boards. automated visual evaluation (AVE) of cervical images using a deep-learning algorithm. A proof-of-principle Study timeline evaluation of AVE of Cervigrams (NTL Worldwide, Fen- The screening period was from November 2018 to De- ton, Missouri) for the diagnosis of cervical precancer, cember 2019. The colposcopy period was from Decem- demonstrated higher accuracy of AVE than expert ber 2018 to March 2020. gynecologist visual assessment or cytology [8]. Although these are promising results, the Cervigram Screening visit cervical images were based on film and a discontinued The screening visit started when a woman attended one expensive custom camera, and the technology is obso- of the three screening clinics (no appointment needed). lete. Thus, it is essential to advance the transfer of the A nurse-administered short screening questionnaire de- method to modern image acquisition devices (e.g., termined eligibility [age 30–49 years and not pregnant smartphones) [9]. There is also a need to evaluate the (self-reported)] for screening. If a woman was menstru- performance of AVE specifically for the triage of HPV- ating and was uncomfortable undergoing screening, she positive women, as HPV-positive controls [<CIN2 (CIN- was advised to return later. Known pregnant women Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 3 of 13 were excluded due to an “abundance of caution”, mainly of five contact attempts were made to approach and ad- to prevent any possibility that an unrelated adverse preg- vise a woman to attend the colposcopy clinic before a nancy outcome could be due, or even perceived to be woman was declared lost to follow-up. due, to the self-sampling for screening. The lower limit of age for screening was set at 30 years since younger Colposcopy clinic visit women have a high prevalence of HPV but a very low At the clinic, a nurse communicated the positive HPV risk of cancer [11]. The upper age limit was restricted to test result and its clinical meaning to the woman, ensur- 49 years due to age-related repositioning of the squamo- ing privacy. The woman was counseled to undergo a col- columnar junction (SCJ), where cancers arise, into the poscopy examination, preferably during that same visit endocervical canal, limiting the ability of any existing or later. visual screening or triage method to diagnose precancer Each woman was registered and interviewed to deter- accurately [12]. An informed consent at screening was mine eligibility for the triage methods research study obtained from eligible women seeking permission to (analyses in progress to be reported separately). Anyone store the left-over sample after HPV testing for future with a history of cervical cancer, hysterectomy, or who research and to be contacted in future for a follow-up was pregnant at the time of enrollment (confirmed with study. However, the screening effort was a public health rapid pregnancy test) was excluded from the study. A fe- intervention and not an experimental study. male nurse took informed consent from all the eligible After enrollment, eligible women were provided with women for participation in a research study (examining an HPV self-sample collection kit containing a cervical triage methods). An anonymized picture of the cervix brush and a Specimen Transport Medium (STM) tube was shown to the woman at the time of consent in order (Qiagen, USA) [13]. Women were shown a 5-min ani- to reassure her about confidentiality and privacy of mated video on how to collect a vaginal self-sample image collection and to minimize refusals. [https://www.youtube.com/watch?v=JiNqrDntbTc][14] Following the interview, a colposcopy examination was while waiting. Women went into a private self-sample performed by one of the study gynecologists (KOA, collection area one at a time to self-sample. After col- CAA). Cervical images for research were collected one lecting the sample, each woman left the brush in the minute after applying 5% acetic acid for each device, se- STM vial in a rack. A nurse helped to break the stem of quentially with three different devices: 1) a Samsung the brush, closed and labelled the vial, and cleaned the Galaxy S8 [16] smartphone; 2) a MobileODT EVA de- outside of the vial and rack with an alcohol wipe. The vice that provided lighting and magnification for a Sam- nurse was available to assist women in specimen collec- sung Galaxy J5 smartphone [17]; and 3) a Zeiss FC150 tion upon request. The collection room was cleaned be- colposcopic image captured via a beam splitter by a tween collections. Before leaving the clinic, participants DSLR (digital single-lens reflex) camera [18, 19]. After completed an anonymous feedback form regarding their image collection, a cervical specimen was collected using experience with self-sampling. The specimens were a cervical sample collection kit containing a cervical stored at room temperature and transferred to the HPV brush and a STM tube (Qiagen, USA) [13] and stored at laboratory at the end of the day, to be stored at 2–8 C 2-8 °C. We plan to test this sample along with the re- until testing. sidual sample from screening for HPV typing using the TypeSeq HPV test [20] at the NCI and report the results HPV testing in future publications. The primary HPV test used in the study was the Digene At the beginning of the study, a dry swab sample was Hybrid Capture-2 (HC2) HPV DNA (deoxyribose nu- collected at colposcopy, before collecting images and ap- cleic acid) Test (Qiagen, USA), which is a US Food and plying acetic acid, for the two-type OncoE6 test (Arbor Drug Administration approved nucleic acid Vita, USA). The test is known to have high positive pre- hybridization assay with signal amplification using mi- dictive value; in fact, three of the five positives (all for croplate chemiluminescence targeting 13 high-risk types HPV 16) were diagnosed with CIN2+. However, the test of HPV DNA in cervical and vaginal specimens, without was dropped after testing 373 samples due to rare posi- distinguishing between them [15]. tivity and low yield. Trained nurses called participants by phone, when Finally, a standard colposcopy examination was per- their HPV test results were available (within two weeks formed to assess the presence and possible nature of cer- of collection for most of the study period). HPV- vical lesions and to take biopsies of acetowhite lesions, negative women were informed and educated about the up to a maximum of four biopsies. In addition, endocer- test result over the phone, and their questions were an- vical curettage (ECC) was performed in cases where the swered. HPV-positive women were asked to visit the col- SCJ was not fully visible, even in the absence of aceto- poscopy clinic to receive their test results. A minimum whitening. All women with acetowhite lesions were Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 4 of 13 offered immediate treatment without waiting for histo- servers and portal for analysis and remote quality pathology results, following the American Society of assurance. Colposcopy and Cervical Pathology 2012 Consensus guidelines [21], leaning towards the more clinically- Data analysis aggressive options for women at risk of being lost to The preliminary data were analyzed using SPSS 20 (Stat- follow-up. Either thermal ablation or large loop excision istical Package for the Social Sciences) [24] and Epi Info of the transformation zone (LLETZ) was performed, de- [25]. Descriptive results were presented as frequencies pending on the colposcopy examination findings. Abla- and percentages. Chi-square tests were used to compare tion was performed only if the SCJ was fully visible, the the yield of disease between different subgroups. In fu- entire lesion was visible, the lesion did not cover > 75% ture analyses to evaluate the triage tests, the area under of the ectocervix, and cervix architecture was appropri- the curve (AUC) on a ROC (Receiver Operating Charac- ate for the ablation probe [22]. teristic) curve will be used. Additional details on study methods (i.e. study site, organization of the clinics, training of staff, and image Histopathology and final diagnosis collection protocol) are provided in the Additional file 1. Histopathological confirmation of CIN2 or CIN3 was used as the reference standard for the presence of pre- Results cancer, against which other experimental tests were eval- A total of 9625 women came for screening, of which uated and final clinical review decisions were made. 9406 (97.7%) eligible women aged 30–49 years were Even though from the clinical management purposes, all screened; and 219 (2.3%) ineligible women (1.7% due to high-grade (CIN2+) lesions were treated equivalently; age restrictions) were not screened (Details on enroll- for the true yield of the screening effort, we reported the ment and exclusions are provided in the Additional file 1). prevalence of CIN2+ and CIN3+ lesions separately to A total of 442 (4.7%) of 9406 women living with HIV avoid the ambiguity of equivocal CIN2 lesions (a mixture (WLWH) were noted when cross referenced with the of HPV infections, true precancers, and an error in his- HIV clinic of OAUTHC, whereas HIV status was un- topathologic diagnosis) [23]. The study pathologist (AB) known for the remaining 8964 (95.3%) women. at the University of Lagos performed all pathology With self-sampling, we were able to screen an average diagnoses. of 20 women per working day (with a peak of up to 100 women in a single day per clinic with two self-sample Quality assurance review and treatment recalls collection areas). Roughly 9065 participants provided All cases were reviewed for adequate clinical manage- written feedback. Of those, 80.8% women said that they ment by a US gynecologic oncologist (AN). The more were able to collect the self-sample without any help complex cases were discussed in a case conference call. from a nurse and 81.2% women said that they would Recall was recommended for women needing further prefer self-sampling to provider’s sampling in the future. management and such cases were re-reviewed once the Asked to score their impressions, 78.5% women were recall was completed until the case was determined to very confident in their ability to collect the self- be adequately treated. We planned to reach all women sampling, 91% found it very easy, 88.5% found it very needing recall for additional management, a minimum comfortable (not painful at all), 95.1% found the video to of seven times. However, we restricted our attempts, and be very helpful in guiding how to collect the sample, and recalled only those women at the highest immediate risk 97.8% said they are very likely to recommend self- of invasive cancer because of the spread of the COVID- sampling to others. The difficult components of self- 19 pandemic in March 2020. sampling reported (one or more issues for 2322 respon- dents) were: the decision on how deep to insert the Screening and management project software brush into the vagina for 52% of 2322, how to insert the All data and images were collected with a HIPAA brush into the vagina for 49.1%, identifying the vaginal (Health Insurance Portability and Accountability Act) opening for 39.6%, rotating the brush inside the vagina compliant smartphone application ‘EVA for research’ for 23.3%, and proper handling to put the brush into the (MobileODT, Israel). The data platform was custom de- tube after collection for 18.9% women. signed for the project (led by CS) using an advanced bar- A total of 1630 (17.3%) [95% confidence interval (CI): code scanning system to limit human error from manual 16.6–18.1%] of 9406 screened women were HC2- key-in. The data and images from different sources on positive. The rate of HC2 positivity was 16.2% among study assigned smartphones were held locally until inter- the 8964 women with unknown HIV status and 40.5% net connectivity was available, at which point all data among the 442 women living with HIV (P < 0.001, inde- automatically transferred and aggregated to cloud pendent chi-square) (Table 1, Table 2). The strong Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 5 of 13 Table 1 Age-specific prevalence of high-risk HPV and precancer among women with unknown HIV status b c b Age at No. of women No. of HPV-positive (% Colposcopy Histopathology results (% of women attending colposcopy ) Yield of the screening effort (% of total screening screened in the of women screened) attendance (95% CI) women screened) (years) age group (95% CI) among CIN2+ CIN3+ CIN2+ CIN3+ HPV- positive 30–34 2553 476 (18.6%) (17.2–20.2%) 393 (82.6%) 26 (6.6%) (4.4–9.6%) 14 (3.6%) (2.0–5.9%) 1.0% 0.5% 35–39 2646 411 (15.5%) (14.2–17.0%) 354 (86.1%) 18 (5.1%) (3.0–7.9%) 10 (2.8%) (1.4–5.1%) 0.7% 0.4% 40–44 2282 351 (15.4%) (13.9–16.9%) 309 (88.0%) 12 (3.9%) (2.0–6.7%) 5 (1.6%) (0.5–3.7%) 0.5% 0.2% 45–49 1479 213 (14.4%) (12.7–16.3%) 194 (91.1%) 15 (7.7%) (4.4–12.4%) 7 (3.6%) (1.5–7.3%) 1.0% 0.5% Total 8960 1451 (16.2%) (15.4–17.0%) 1250 (86.1%) 71 (5.7%) (4.5–7.1%) 36 (2.9%) (2.0–4.0%) 0.8% 0.4% Not including four women with missing data on age CIN2+ and CIN3+ include a total of four cases of squamous cell carcinoma (two of them were in 49-year old women, one in a 47-year old woman, and one in a 33-year old woman; all four cases were diagnosed on biopsy). We also presumptively included five women with OncoE6 HPV 16 positivity because of the known high positive predictive value of the biomarker (Three of the five had confirmed CIN2+; in two LLETZ was recommended and is still incomplete) Histopathology report was not completed for four participants due to COVID-19 pandemic spread and lockdown. These four participants were excluded from the denominator Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 6 of 13 Table 2 Age-specific prevalence of high-risk HPV and precancer among women living with HIV (WLWH) Age at No. of No. of HPV-positive Colposcopy attendance Histopathology results (% of women Yield of the screening b b screening women (% of women among HPV-positive attending colposcopy ) (95% CI) effort (% of total (years) screened screened) (95% CI) women screened) in the CIN2+ CIN3+ CIN2+ CIN3+ age group 30–34 77 35 (45.5%) (34.1–57.2%) 27 (77.1%) 1 (3.8%) (0.1–19.6%) 1 (3.8%) (0.1–19.6%) 1.3% 1.3% 35–39 160 57 (35.6%) (28.2–43.6%) 41 (71.9%) 2 (5.0%) (0.6–16.9%) 2 (5.0%) (0.6–16.9%) 1.3% 1.3% 40–44 134 46 (34.3%) (26.3–43.0%) 40 (87.0%) 4 (10.0%) (2.8–23.7%) 3 (7.5%) (1.6–20.4%) 3.0% 2.2% 45–49 71 41 (57.7%) (45.4–69.4%) 35 (85.4%) 4 (11.4%) (3.2–26.7%) 3 (8.6%) (1.8–23.1%) 5.6% 4.2% Total 442 179 (40.5%) (35.9–45.2%) 143 (79.9%) 11 (7.8%) (4.0–13.5%) 9 (6.4%) (3.0–11.8%) 2.5% 2.0% CIN2+ and CIN3+ includes one case of squamous cell carcinoma in a 49-year old woman Histopathology report was not completed for two participants due to COVID-19 pandemic spread. These two participants were excluded from the denominator differences in HPV positivity between the two groups only one scheduling contact. An additional 299 (21.4%), persisted in all age groups. 166 (11.9%), 85 (6.1%), 77 (5.5%) and 64 (4.6%) came Out of 1630 HPV-positive women, 1400 (85.9%) en- after two, three, four, five, and more than five contact rolled for the study at colposcopy, of which seven cases attempts. were excluded due to unsatisfactory colposcopy and dif- Following the protocol of immediate management by ficulty in image collection due to various reasons out- colposcopic impression, without awaiting histopathology lined in the Additional file 1. Out of the 1400 who diagnosis of the biopsies taken, 114 (8.2%) women were enrolled, 709 (50.6%) women came for colposcopy after treated with thermal ablation and 78 (5.6%) with LLETZ. Fig. 1 Squamocolumnar junction visibility by age (n = 1393) Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 7 of 13 Fig. 2 Squamocolumnar junction (SCJ) visibility, by age and parity (n = 1380) Overall, CIN2+ and CIN3+ (including five cancers), and 6.4%, respectively (P = 0.31 for CIN2+ and P = 0.03 were detected in 5.9% (95% CI:4.7–7.3%) and 3.2% (95% for CIN3+, independent chi-square test in comparison CI:2.4–4.3%) of the HPV-positive women attending col- to women with unknown HIV status) (Table 2). The poscopy (85% attendance among the 1630), respectively. yield of the screening effort among women living with Thus, overall yield of the screening effort was 0.9% of HIV was 2.5% for CIN2+, of which all except two were 9406 women for CIN2+ and 0.5% for CIN3 + . actually CIN3+ (P < 0.001 for CIN2+ and CIN3+, inde- The prevalences of CIN2+ and CIN3+ among the pendent chi-square in comparison to women with un- HPV-positive women with unknown HIV status attend- known HIV status) (Table 2). Relatively small age- ing colposcopy were 5.7 and 2.9%, respectively, whereas specific numbers preclude any conclusions regarding the overall yields of the screening effort in these groups a trend in the prevalence of HPV or precancer by were 0.8 and 0.4%, respectively (Table 1). The age- age. The proportion of HPV-positive women in- specific prevalences of high-risk HPV positivity and creased with decrease in CD4 (cluster of differenti- precancer among women with unknown HIV status ation 4) count (64% in CD4 < 200/mm vs 31.1% in are shown in Table 1. The prevalence of HPV de- CD4 > 500/ mm3, P = 0.001, chi-square for trend) and creased from 18.6% at age 30–34 years to 14.4% at increase in HIV viral load (36.3% in <=20 copies/ml age 45–49 years (P = 0.0003, chi-square for trend). No vs 50% in > 20 copies/ml, P = 0.02, independent chi- meaningful trend was observed in the prevalence of square), however small numbers precluded trend ana- precancer by age (P = 0.87, chi-square for trend for lysis of precancer/cancer. CIN2+), except for a very high 24.1% (seven CIN2+ Out of a total of 75 histopathologically confirmed including three cancers) overall prevalence at self- cases, 59 (78.7%) were diagnosed through biopsy or reported age of 49 years. LLETZ of acetowhite lesions at colposcopy visit, 35 of The prevalences of CIN2+ and CIN3+ among the which (59.3% of 59) had a high-grade colposcopic im- HPV-positive WLWH attending colposcopy were 7.8 pression. QA review of colposcopic images revealed Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 8 of 13 Table 3 Management of women by histopathologic diagnosis Histopathologic diagnosis Management Row total No treatment Immediate management based on colposcopic Delayed excisional treatment on a recall visit indicated (row%) impression (row%) (column%) (row%) (column%) (column%) With ablation With LLETZ Completed Pending <CIN2 1158 (88.7%) (100.0%) 90 (6.9%) (78.9%) 54 (4.1%) (69.2%) 3 (0.2%) (12.0%) 0 1305 (100%) CIN2 Diagnosed on biopsy/LLETZ 0 10 (40.0%) (8.8%) 11 (44.0%) (14.1%) 2 (8.0%) (8.0%) 2 (8.0%) (16.7%) 25 (100%) Diagnosed on ECC 0 0 0 8 (66.7%) (32.0%) 4 (33.3%) (33.3%) 12 (100%) CIN3 Diagnosed on biopsy/LLETZ 0 13 (38.2%) (11.4%) 12 (35.3%) (15.4%) 5 (14.7%) (20.0%) 4 (11.8%) (33.3%) 34 (100%) Diagnosed on ECC 0 1 (16.7%) (0.9%) 0 3 (50.0%) (12.0%) 2 (33.3%) (16.7%) 6 (100%) Cancer Diagnosed on biopsy/LLETZ00 1 (20.0%) (1.3%) 4 (80.0%) (16.0%) 0 5 (100%) Column total 1158 (100%) 114 (100%) 78 (100%) 25 (100%) 12 (100%) Grand total = 1387 Recall attempts are temporarily paused due to COVID-19 pandemic spread Includes two women with OncoE6 HPV 16 positive, in whom LLETZ was recommended on recall and is still incomplete Includes one case with colposcopic impression of cancer treated with hysterectomy of what eventually turned out to be <CIN2; other two cases were recalled for LLETZ due to reporting error Invasive squamous cell carcinoma with CIN3 at margins was diagnosed on on-site LLETZ leading to a recall for a repeat LLETZ with CIN3 diagnosis leading to a 2nd recall for a hysterectomy Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 9 of 13 potential under-biopsying of more subtle acetowhite le- to be explained more clearly in future self-sampling pro- sions. 16 (21.3%) of 75 histopathologic precancer cases grams. Compliance with follow-up for colposcopy was were identified, in the absence of visible acetowhitening, also very high. only on ECC (1.2% of colposcopy examinations and 0.2% The two previous phases of Project Itoju established of the screening population). This is important in light the epidemiology of HPV infection in rural Nigeria [26], of the fact that even amongst women as young as age validated a low-cost HPV test (careHPV, Qiagen) [27] 30, the SCJ was only partially visible in 8.3% and not vis- (phase 1) and attempted (ultimately unsuccessfully) to ible in 23.1% of women (31.5% total), rising to 12.5 and determine whether immunosuppression due to soil- 52.1% (64.6% total) by age 49 years (P < 0.001, chi-square borne helminth infections or other parasitoses was the for trend) (Fig. 1). Interestingly, multiple vaginal deliver- cause of high HPV prevalence among older women in ies were found to increase full SCJ visibility (Fig. 2). the region (phase 2). In the current phase of Project Among 192 women treated on-site based on colpo- Itoju, we reconfirmed the overall high prevalence of scopic impression, without awaiting histopathology re- HPV infections even after restricting the age of screen- sults, only 48 (25%) were eventually diagnosed with ing to 30–49 years of age [28]. Although the prevalence CIN2+ on histopathology (Table 3). Viewing overtreat- of HPV decreased to 16% at age 45–49 years from 19% ment from another perspective, 11% of women with his- at age 30–34 years, it was still much higher than the glo- topathologic <CIN2 were treated based on colposcopic bal average of around 5% for women age 45–54 years impression. On the other hand, 43.2% of CIN2 (16 out and consistent with what is observed for the women in of 37) and 35% of CIN3 (14 out of 40) were not treated sub-Saharan Africa [29]. Despite the high prevalence of immediately; either because of underdiagnosis [diag- HPV infection, the prevalence of precancer was less than nosed later on ECC] or a variety of programmatic issues 1% in the general population of mostly unscreened such as equipment failure. A total of 12 (1.1% of colpos- women 30–49 years old. One reason for the low preva- copy population) precancer cases needed more than one lence of precancer could be under biopsy of more subtle treatment visit in order to obtain clear margins free of acetowhite lesions [30]. It also underscores the need to high-grade findings. further study the type-specific natural history in the re- Out of 1138 (82%) patients with full clinical quality as- gion of HPV acquisition, clearance, persistence, and pro- surance review completed, 143 (12.6%) cases were rec- gression, particularly in the setting of HIV infection. In ommended for recall; mainly (113, or 9.9% of total) for this current study, we obtained samples for HPV geno- repeat ECC; commonly because the tissue was insuffi- typing from women at screening and again at colposcopy cient for diagnosis on the prior ECC (100, or 8.8% of visit. We will be testing and reporting the results of total) or because the ECC required dilatation/sedation these tests elaborating the prevalence of various HPV ge- due to stenotic os or tethered cervix (13, or 1.1% of notypes and short-term clearance of infection in subse- total). However, the overall yield of precancer from re- quent papers. We also hope to retest the women in the call for ECC was only 0.2% for the colposcopy popula- future to explore the long-term persistence of HPV tion and 0.02% for the general population of women infection. aged 30–49 years. The prevalence of HPV and precancer (particularly CIN3+) was markedly high among WLWH as also re- Discussion ported by others [28]. This was observed despite the Our results showed the feasibility and acceptability of suppressed viral load with a documented average of < 20 self-sampled HPV testing and smartphone-based cervical copies/ml. This finding was expected since HPV and image collection in Nigeria. Nevertheless, the combin- HIV can be co-transmitted and women with HIV tend ation of higher HPV prevalence (17.4%) with a much to have lower clearance of acquired HPV infections. The lower risk of precancer (0.9%) suggests the need for tri- management of HPV in WLWH is an important topic age to improve the accuracy of the screening program. beyond the scope of this article. In addition, any visual triage method would require According to the World Health Organization (WHO), restricting the upper age limit of inclusion to increase Nigeria has only four physicians per 10,000 population program effectiveness. [31]. At present, only < 10% of women > 15 years have There was a very positive response and clear accept- ever been screened for cervical cancer in Nigeria [32]. In a ability for vaginal self-sampling for HPV testing. The dif- resource-constrained setting with a shortage of expert gy- ficult aspects of self-sampling reported by some necologic providers and infrastructure, avoidable referral participants included identifying the vaginal opening, de- to colposcopy and substantial overtreatment are not sus- termining depth of the insertion of the brush into the tainable. On the contrary, in settings with once in a life- vagina, rotating the brush inside the vagina, and putting time screening opportunity for a majority of women, the brush back into the tube after collection; these need substantial missed diagnosis and undertreatment is not Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 10 of 13 Fig. 3 Effect of image capture method on cervical appearance and limitation of visual triage method. a. Cervical images of the same cervix showing histopathologic <CIN2 (left trio) and CIN2+ (right trio), captured with (clockwise starting from upper left) a Samsung S8 smartphone camera and its flashlight, a MobileODT EVA device (Samsung J5 phone with an extra light source and a zoom lens), and a Zeiss FC150 colposcope with a beam splitter and DSLR camera (see supplement). b. Cervical images of histopathologic CIN2+ cases with squamocolumnar junction (clockwise from upper left) fully visible, partially visible, and not visible (diagnosed on ECC); captured with a Zeiss FC150 colposcope with a beam splitter and DSLR camera acceptable either. It is therefore essential that simple yet images from three different devices (Fig. 3a) has raised an accurate triage tests, separately or in combination, are important research question regarding the device portabil- available to stratify risk of precancer/cancer among HPV- ity of deep-learning based AVE algorithms due to vari- positive women such that treatment intensity can be tai- ation in color, brightness, reflection, glare imparted by lored to risk of cancer and sustained with local resources. each device camera, light source and image processing ap- In the future, we will be assessing the machine-learning plication. Training or automation to capture an in-focus, based AVE of cervical images using three different image non-blurry good quality cervical image, capturing entire collection methods. We will be reporting the results of the SCJ, for evaluation by AVE, is yet another challenge. assessment of the combination of AVE with HPV geno- It is important to note that AVE, like other visual as- typing for triage of HPV-positive women in subsequent sessment methods, requires that the cervical SCJ be vis- papers. However, preliminary analysis of the cervical ible. In the current study population, we found that the Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 11 of 13 SCJ was not fully visible for almost 64.6% of women by high risk of progression to invasion. Even though in the age 49 years. We also corroborated an earlier poorly study we managed to achieve a relatively higher rate of understood observation that women with multiple vagi- compliance with follow-up, the compliance in the real- nal deliveries were more likely to have fully visible SCJs world setting needs to be monitored and factored in the [33]. No visual screening methods or ablative treatment effectiveness of the screening programs. methods work when the transformation zone, where cer- We had to halt the recall activities under the screening vical cancer typically arises, is not visible (Fig. 3b) [12, program as the global spread of the COVID-19 pan- 34]. This could partially explain the decrease in the demic led to pausing of field efforts in Nigeria, including prevalence of precancer with age despite the high HPV a very few treatment visits still pending, and changed the prevalence, found in this study. It is, therefore, particu- risk-benefit ratio for cervical cancer screening. When we larly crucial to emphasize restricting visual approaches reopen, the first step will be to complete these treatment for screening or triage of older women to avoid giving visits. Then, moving forward in the COVID-19 era, we false reassurance to women in these age groups and to are considering the importance of self-sampled HPV avoid identifying high-risk HPV-positive women, par- testing with a sterile kit at a household level, avoiding ticularly with HPV 16, 18/45, with no available means of any mass gathering and minimizing the need for further triage. It is worth noting however that even at speculum examination. A small percentage of HPV- age 30, the SCJ was not fully visible in up to 30% of positive women with high-risk types could be triaged women, thus age-restriction to women <=49 years does and treated, spacing community clinic appointments in a not eliminate inadequacies in visual triage. The current COVID conscious manner [36]. standard practice is to collect an ECC sample whenever The limitations of this study should be noted. The the SCJ is not fully visible, to rule out cancer within the population selected for the study was a volunteer popu- endocervical canal. But an ECC needs an interpretation lation of women residing in the university town of Ile- by an expert histopathologist, which is challenging in Ife, which may have a lower HPV prevalence than the low resource settings. Difficulty in collecting ECC in general population in Nigeria. Also, HC2 is not generally women with a stenotic cervical os and insufficient tissue used for self-sampling, as it is slightly less sensitive for in the ECC sample to rule out carcinoma are other chal- detection of precancer than PCR-based HPV test lenges with ECC. Thus, the development of low-cost methods [37]. A moderate amount of cross-reactivity simple triage alternatives for ECC remains an unsolved against other genetically related but less oncogenic HPV challenge for improving cervical cancer screening pro- types with HC2 is well-documented. This may partly ex- grams. Research is also needed on simpler alternatives plain the low precancer to HPV ratio found in this study. for LLETZ, that could be performed easily by a general Despite the known limitations of HC2, its operational physician or a nurse without the need of an expert simplicity, and easy trainability, as supported by virtually gynecologist, particularly in low-resource settings where trouble-free operation of HC2 throughout the study sup- the needed resources are in short supply especially in ported its use. rural underserved areas. In future work we will assess misclassification by It is worth noting that even though the yield of pre- retesting residual screening samples with a more sensi- cancer from ECC at colposcopy visit was 3.2% (21% of tive whole-genome sequencing method [20]; review and total precancers diagnosed on ECC, possibly due to not recall women with highest risk HPV types and potential taking enough biopsies of subtle acetowhite lesions), the missed biopsies of subtle acetowhite lesions; and additional yield of precancer from the women recalled digitalize the histopathology slides for a second review, for insufficient ECC or difficult ECC collection was only particularly for borderline cases. 0.02% for the overall screened population. Another study has also noted overall low yield of ECC with an increase Conclusion in proportionate additional yield of ECC when fewer bi- A cervical cancer screening program using self-sampled opsies are taken [35]. It is worth exploring the cost- HPV testing, with colposcopic immediate management effectiveness of recalls for ECC to avert a very low risk of women positive for HPV, is feasible in Nigeria but re- of adenocarcinoma in low-resource settings where no sults in both over-and under-treatment. There are newer organized follow-up is available. Any recommendation HPV tests entering the marketplace that cost less than for follow-up outside the study in these settings is likely ten dollars per test, take less than an hour to perform, to remain a theoretical reassurance. In this regard, there and provide genotyping, suggesting that a self-sampled were a total of 12 women (1.1% of colposcopy popula- HPV based screening program would be feasible in this tion) who needed multiple rounds of treatment in order population in the future [38]. Having proven feasibility, to obtain clear margins on excision. These are women we are now evaluating the accuracy and efficacy in diag- with confirmed high-grade lesions and hence also at nosing CIN2+ of smartphone-based automated visual Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 12 of 13 evaluation of cervical images combined with HPV geno- Author details Division of Cancer Epidemiology and Genetics, National Cancer Institute, typing as an assistive strategy to improve visual triage. NIH, Rockville, USA. Oak Ridge Institute of Science and Education, Oak Ridge, USA. Department of Obstetrics and Gynecology, Obafemi Awolowo University Teaching Hospital, Ile Ife, Nigeria. Lagos University Teaching Supplementary information Hospital, Lagos, Nigeria. Rutgers New Jersey Medical School and Cancer Supplementary information accompanies this paper at https://doi.org/10. Institute of New Jersey (CINJ), Newark, USA. Hela Health, Tel Aviv, Israel. 1186/s13027-020-00324-5. 7 8 Global Health Labs, Bellevue, USA. Albert Einstein College of Medicine, New York City, USA. National Library of Medicine, NIH, Bethesda, USA. Additional file 1. Received: 28 July 2020 Accepted: 22 September 2020 Abbreviations AUC: Area under the curve; AVE: Automated visual evaluation; CD4: Cluster of differentiation 4; CI: Confidence interval; CIN: Cervical intraepithelial References neoplasia; COVID-19: Coronavirus disease of 2019; DNA: Deoxyribose nucleic 1. Arbyn M, Weiderpass E, Bruni L, de Sanjosé S, Saraiya M, Ferlay J, et al. acid; DSLR: Digital single lens reflex; ECC: Endocervical curettage; Estimates of incidence and mortality of cervical cancer in 2018: a worldwide EVA: Enhanced visual assessment; HC2: Hybrid capture-2; HIPAA: Health analysis. Lancet Glob Heal. 2020;8(2):e191–203. Insurance portability & accountability act; HPV: Human papillomavirus; 2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. HIV: Human immunodeficiency virus; LLETZ: Large loop excision of the Cancer incidence and mortality worldwide: sources, methods and major transformation zone; NCI: National Cancer Institute; OAUTHC: Obafemi patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–86. Awolowo University Teaching Hospitals Complex; OAU: Obafemi Awolowo 3. Vaccarella S, Lortet-Tieulent J, Plummer M, Franceschi S, Bray F. Worldwide University; PCR: Polymerase chain reaction; RLU/CO: Relative light unit/cut-off; trends in cervical cancer incidence: impact of screening against changes in RNJMS: Rutgers New Jersey Medical school; ROC: Receiver operating disease risk factors. Eur J Cancer. 2013;49(15):3262–73. characteristic; SCJ: Squamocolumnar junction; SPSS: Statistical package for 4. Polman NJ, Ebisch RMF, Heideman DAM, Melchers WJG, Bekkers RLM, Molijn social studies; STM: Specimen transport media; US: United States; AC, et al. Performance of human papillomavirus testing on self-collected WLWH: Women living with HIV versus clinician-collected samples for the detection of cervical intraepithelial neoplasia of grade 2 or worse: a randomised, paired screen-positive, non- Acknowledgements inferiority trial. Lancet Oncol. 2019;2045(18):1–10. This research was supported in part by an appointment to the National 5. Porras C, Hildesheim A, González P, Schiffman M, Rodríguez AC, Wacholder Cancer Institute Research Participation Program. This program is S, et al. Performance of Self-collected Cervical Samples in Screening for administered by the Oak Ridge Institute for Science and Education through Future Precancer using Human Papillomavirus DNA Testing. J Natl Cancer an interagency agreement between the U.S. Department of Energy and the Inst. 2015;107(1):1–9 Available from: http://www.ncbi.nlm.nih.gov/ National Institutes of Health. The video on self-sampling was designed by pubmed/25479804. [cited 2019 Jun 13]. Livinghealth International, Nigeria. 6. Rodríguez AC, Schiffman M, Herrero R, Hildesheim A, Bratti C, Sherman ME, et al. Longitudinal Study of Human Papillomavirus Persistence and Cervical Intraepithelial Neoplasia Grade 2/3: Critical Role of Duration of Infection. J Authors’ contributions Natl Cancer Inst. 2010;102(5):315–24 Available from: https://www.ncbi.nlm. MS, KOA, KTD, CAA, AN, MHE, PC contributed substantially to the conception nih.gov/pmc/articles/PMC2831050/. [cited 2020 Mar 17]. and design of the study. KOA, AB, CAA, TO, TOO, MMO, KTD, AN, MHE, and 7. Wentzensen N, Schiffman M, Palmer T, Arbyn M. Triage of HPV MS contributed to acquisition of data or the analysis and interpretation. MS, positive women in cervical cancer screening. J Clin Virol. 2016;76: KOA, and KTD drafted the manuscript. All authors provided critical revision of S49–55. the article and provided final approval of the version to publish. 8. Hu L, Bell D, Antani S, Xue Z, Yu K, Horning MP, et al. An observational study of deep learning and automated evaluation of cervical images for Funding Cancer screening. J Natl Cancer Inst. 2019;11(9):923–32. The study was funded by the NCI intramural research program and Global 9. Xue Z, Novetsky AP, Einstein MH, Marcus JZ, Befano B, Guo P, et al. A Good (Now, Global Health Labs) (Bellevue, USA). demonstration of automated visual evaluation (AVE) of cervical images taken with a smartphone camera. Int J Cancer. 2020:ijc.33029 Available Availability of data and materials from: https://onlinelibrary.wiley.com/doi/abs/10.1002/ijc.33029.[cited The datasets used and/or analysed during the current study are available 2020 May 14]. from the corresponding author on reasonable request. 10. Nigeria Data Portal. Population Distribution by Age 2006-Osun Demographic pyramid. National Bureau of Statistics. 2006. 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BMJ. 2018;363:k4823. 38. Zhang W, Du H, Huang X, Wang C, Duan X, Liu Y, et al. Evaluation of an isothermal amplification HPV detection assay for primary cervical cancer screening. Infect Agents Cancer. 2020. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Design and feasibility of a novel program of cervical screening in Nigeria: self-sampled HPV testing paired with visual triage

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10.1186/s13027-020-00324-5
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Abstract

Background: Accelerated global control of cervical cancer would require primary prevention with human papillomavirus (HPV) vaccination in addition to novel screening program strategies that are simple, inexpensive, and effective. We present the feasibility and outcome of a community-based HPV self-sampled screening program. Methods: In Ile Ife, Nigeria, 9406 women aged 30–49 years collected vaginal self-samples, which were tested for HPV in the local study laboratory using Hybrid Capture-2 (HC2) (Qiagen). HPV-positive women were referred to the colposcopy clinic. Gynecologist colposcopic impression dictated immediate management; biopsies were taken when definite acetowhitening was present to produce a histopathologic reference standard of precancer (and to determine final clinical management). Retrospective linkage to the medical records identified 442 of 9406 women living with HIV (WLWH). Results: With self-sampling, it was possible to screen more than 100 women per day per clinic. Following an audio- visual presentation and in-person instructions, overall acceptability of self-sampling was very high (81.2% women preferring self-sampling over clinician collection). HPV positivity was found in 17.3% of women. Intensive follow-up contributed to 85.9% attendance at the colposcopy clinic. Of those referred, 8.2% were initially treated with thermal ablation and 5.6% with large loop excision of transformation zone (LLETZ). Full visibility of the squamocolumnar junction, necessary for optimal visual triage and ablation, declined from 68.5% at age 30 to 35.4% at age 49. CIN2+ and CIN3+ (CIN- Cervical intraepithelial neoplasia), including five cancers, were identified by histology in 5.9 and 3.2% of the HPV-positive women, respectively (0.9 and 0.5% of the total screening population), leading to additional treatment as indicated. The prevalences of HPV infection and CIN2+ were substantially higher (40.5 and 2.5%, respectively) among WLWH. Colposcopic impression led to over- and under-treatment compared to the histopathology reference standard. (Continued on next page) * Correspondence: ajenifujako@yahoo.com Kanan T. Desai and Kayode O. Ajenifuja contributed equally to this work. Department of Obstetrics and Gynecology, Obafemi Awolowo University Teaching Hospital, Ile Ife, Nigeria Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 2 of 13 (Continued from previous page) Conclusion: A cervical cancer screening program using self-sampled HPV testing, with colposcopic immediate management of women positive for HPV, proved feasible in Nigeria. Based on the collected specimens and images, we are now evaluating the use of a combination of partial HPV typing and automated visual evaluation (AVE) of cervical images to improve the accuracy of the screening program. Keywords: Cervical screening, HPV, Self-sampling, Triage, Automated visual evaluation Introduction cervical intraepithelial neoplasia)] tend to look less nor- Nearly 85% of the annual 570,000 cervical cancer cases mal and are consequently harder to differentiate from and almost 90% of the 311,000 related deaths occur in precancer cases than HPV-negative controls. lower-resource countries [1, 2] due to the lack of effective This paper describes the field methods, feasibility, and cervical cancer prevention programs [3]. The COVID-19 preliminary descriptive results of Project Itoju in Ile-Ife, (coronavirus disease of 2019) pandemic threatens to re- Nigeria, designed to evaluate ultimately the combined duce elective procedures even further, including cervical strategy of self-sampled HPV typing combined with screening and related diagnostic procedures, especially in AVE triage on three different image capture devices. lower-resource settings, and will likely worsen cervical cancer health disparities. As a major advantage compared Materials and methods with cytology, the specimen for HPV (human papillomavi- Study design and overview rus) testing can be collected by the woman herself using a Women aged 30–49 years (comprising ~ 8% of the total vaginal self-collection device, yielding sensitivity for HPV population [10]) residing in the catchment area of Oba- infection that is similar to clinician-collected specimens femi Awolowo University Teaching Hospitals Complex when target-amplification methods like PCR (polymerase (OAUTHC) in Ile-Ife, Nigeria were invited by a public chain reaction) are used [4, 5]. Use of HPV testing will re- message campaign for self-sampled HPV testing. quire a second diagnostic modality for positives, because Women who screened HPV-positive were referred to majority of HPV infections are cleared within 1–2years of the colposcopy clinic and invited to participate in a re- initial detection. Only HPV infections that persist can search study (examining triage methods) under informed cause precancer and invasive cancer [6]. Thus, an im- consent. Standard colposcopic examination, including proved screening program must include triage methods to colposcopic images and treatment, if indicated, was of- focus treatment safely on the small fraction of HPV- fered to all HPV-positive women regardless of participa- positive women with precancer, the general term we use tion in the triage methods research. In addition, cervical to refer to lesions at substantial risk of invasion without images were collected with a cellphone and the EVA treatment [7]. Ideal characteristics of a triage test for (enhanced visual assessment) system (MobileODT, HPV-positive women, for use in low-resource regions, Israel), and a cervical sample was collected for subse- would include excellent risk discrimination (high precan- quent HPV typing, from all the participants in the study. cer risk in positives, low risk in negatives), low-cost, sim- The study was approved by National Cancer Institute plicity, and point-of-care use. (NCI) and OAUTHC ethical Institutional Review A new candidate for triage of HPV-positive women is Boards. automated visual evaluation (AVE) of cervical images using a deep-learning algorithm. A proof-of-principle Study timeline evaluation of AVE of Cervigrams (NTL Worldwide, Fen- The screening period was from November 2018 to De- ton, Missouri) for the diagnosis of cervical precancer, cember 2019. The colposcopy period was from Decem- demonstrated higher accuracy of AVE than expert ber 2018 to March 2020. gynecologist visual assessment or cytology [8]. Although these are promising results, the Cervigram Screening visit cervical images were based on film and a discontinued The screening visit started when a woman attended one expensive custom camera, and the technology is obso- of the three screening clinics (no appointment needed). lete. Thus, it is essential to advance the transfer of the A nurse-administered short screening questionnaire de- method to modern image acquisition devices (e.g., termined eligibility [age 30–49 years and not pregnant smartphones) [9]. There is also a need to evaluate the (self-reported)] for screening. If a woman was menstru- performance of AVE specifically for the triage of HPV- ating and was uncomfortable undergoing screening, she positive women, as HPV-positive controls [<CIN2 (CIN- was advised to return later. Known pregnant women Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 3 of 13 were excluded due to an “abundance of caution”, mainly of five contact attempts were made to approach and ad- to prevent any possibility that an unrelated adverse preg- vise a woman to attend the colposcopy clinic before a nancy outcome could be due, or even perceived to be woman was declared lost to follow-up. due, to the self-sampling for screening. The lower limit of age for screening was set at 30 years since younger Colposcopy clinic visit women have a high prevalence of HPV but a very low At the clinic, a nurse communicated the positive HPV risk of cancer [11]. The upper age limit was restricted to test result and its clinical meaning to the woman, ensur- 49 years due to age-related repositioning of the squamo- ing privacy. The woman was counseled to undergo a col- columnar junction (SCJ), where cancers arise, into the poscopy examination, preferably during that same visit endocervical canal, limiting the ability of any existing or later. visual screening or triage method to diagnose precancer Each woman was registered and interviewed to deter- accurately [12]. An informed consent at screening was mine eligibility for the triage methods research study obtained from eligible women seeking permission to (analyses in progress to be reported separately). Anyone store the left-over sample after HPV testing for future with a history of cervical cancer, hysterectomy, or who research and to be contacted in future for a follow-up was pregnant at the time of enrollment (confirmed with study. However, the screening effort was a public health rapid pregnancy test) was excluded from the study. A fe- intervention and not an experimental study. male nurse took informed consent from all the eligible After enrollment, eligible women were provided with women for participation in a research study (examining an HPV self-sample collection kit containing a cervical triage methods). An anonymized picture of the cervix brush and a Specimen Transport Medium (STM) tube was shown to the woman at the time of consent in order (Qiagen, USA) [13]. Women were shown a 5-min ani- to reassure her about confidentiality and privacy of mated video on how to collect a vaginal self-sample image collection and to minimize refusals. [https://www.youtube.com/watch?v=JiNqrDntbTc][14] Following the interview, a colposcopy examination was while waiting. Women went into a private self-sample performed by one of the study gynecologists (KOA, collection area one at a time to self-sample. After col- CAA). Cervical images for research were collected one lecting the sample, each woman left the brush in the minute after applying 5% acetic acid for each device, se- STM vial in a rack. A nurse helped to break the stem of quentially with three different devices: 1) a Samsung the brush, closed and labelled the vial, and cleaned the Galaxy S8 [16] smartphone; 2) a MobileODT EVA de- outside of the vial and rack with an alcohol wipe. The vice that provided lighting and magnification for a Sam- nurse was available to assist women in specimen collec- sung Galaxy J5 smartphone [17]; and 3) a Zeiss FC150 tion upon request. The collection room was cleaned be- colposcopic image captured via a beam splitter by a tween collections. Before leaving the clinic, participants DSLR (digital single-lens reflex) camera [18, 19]. After completed an anonymous feedback form regarding their image collection, a cervical specimen was collected using experience with self-sampling. The specimens were a cervical sample collection kit containing a cervical stored at room temperature and transferred to the HPV brush and a STM tube (Qiagen, USA) [13] and stored at laboratory at the end of the day, to be stored at 2–8 C 2-8 °C. We plan to test this sample along with the re- until testing. sidual sample from screening for HPV typing using the TypeSeq HPV test [20] at the NCI and report the results HPV testing in future publications. The primary HPV test used in the study was the Digene At the beginning of the study, a dry swab sample was Hybrid Capture-2 (HC2) HPV DNA (deoxyribose nu- collected at colposcopy, before collecting images and ap- cleic acid) Test (Qiagen, USA), which is a US Food and plying acetic acid, for the two-type OncoE6 test (Arbor Drug Administration approved nucleic acid Vita, USA). The test is known to have high positive pre- hybridization assay with signal amplification using mi- dictive value; in fact, three of the five positives (all for croplate chemiluminescence targeting 13 high-risk types HPV 16) were diagnosed with CIN2+. However, the test of HPV DNA in cervical and vaginal specimens, without was dropped after testing 373 samples due to rare posi- distinguishing between them [15]. tivity and low yield. Trained nurses called participants by phone, when Finally, a standard colposcopy examination was per- their HPV test results were available (within two weeks formed to assess the presence and possible nature of cer- of collection for most of the study period). HPV- vical lesions and to take biopsies of acetowhite lesions, negative women were informed and educated about the up to a maximum of four biopsies. In addition, endocer- test result over the phone, and their questions were an- vical curettage (ECC) was performed in cases where the swered. HPV-positive women were asked to visit the col- SCJ was not fully visible, even in the absence of aceto- poscopy clinic to receive their test results. A minimum whitening. All women with acetowhite lesions were Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 4 of 13 offered immediate treatment without waiting for histo- servers and portal for analysis and remote quality pathology results, following the American Society of assurance. Colposcopy and Cervical Pathology 2012 Consensus guidelines [21], leaning towards the more clinically- Data analysis aggressive options for women at risk of being lost to The preliminary data were analyzed using SPSS 20 (Stat- follow-up. Either thermal ablation or large loop excision istical Package for the Social Sciences) [24] and Epi Info of the transformation zone (LLETZ) was performed, de- [25]. Descriptive results were presented as frequencies pending on the colposcopy examination findings. Abla- and percentages. Chi-square tests were used to compare tion was performed only if the SCJ was fully visible, the the yield of disease between different subgroups. In fu- entire lesion was visible, the lesion did not cover > 75% ture analyses to evaluate the triage tests, the area under of the ectocervix, and cervix architecture was appropri- the curve (AUC) on a ROC (Receiver Operating Charac- ate for the ablation probe [22]. teristic) curve will be used. Additional details on study methods (i.e. study site, organization of the clinics, training of staff, and image Histopathology and final diagnosis collection protocol) are provided in the Additional file 1. Histopathological confirmation of CIN2 or CIN3 was used as the reference standard for the presence of pre- Results cancer, against which other experimental tests were eval- A total of 9625 women came for screening, of which uated and final clinical review decisions were made. 9406 (97.7%) eligible women aged 30–49 years were Even though from the clinical management purposes, all screened; and 219 (2.3%) ineligible women (1.7% due to high-grade (CIN2+) lesions were treated equivalently; age restrictions) were not screened (Details on enroll- for the true yield of the screening effort, we reported the ment and exclusions are provided in the Additional file 1). prevalence of CIN2+ and CIN3+ lesions separately to A total of 442 (4.7%) of 9406 women living with HIV avoid the ambiguity of equivocal CIN2 lesions (a mixture (WLWH) were noted when cross referenced with the of HPV infections, true precancers, and an error in his- HIV clinic of OAUTHC, whereas HIV status was un- topathologic diagnosis) [23]. The study pathologist (AB) known for the remaining 8964 (95.3%) women. at the University of Lagos performed all pathology With self-sampling, we were able to screen an average diagnoses. of 20 women per working day (with a peak of up to 100 women in a single day per clinic with two self-sample Quality assurance review and treatment recalls collection areas). Roughly 9065 participants provided All cases were reviewed for adequate clinical manage- written feedback. Of those, 80.8% women said that they ment by a US gynecologic oncologist (AN). The more were able to collect the self-sample without any help complex cases were discussed in a case conference call. from a nurse and 81.2% women said that they would Recall was recommended for women needing further prefer self-sampling to provider’s sampling in the future. management and such cases were re-reviewed once the Asked to score their impressions, 78.5% women were recall was completed until the case was determined to very confident in their ability to collect the self- be adequately treated. We planned to reach all women sampling, 91% found it very easy, 88.5% found it very needing recall for additional management, a minimum comfortable (not painful at all), 95.1% found the video to of seven times. However, we restricted our attempts, and be very helpful in guiding how to collect the sample, and recalled only those women at the highest immediate risk 97.8% said they are very likely to recommend self- of invasive cancer because of the spread of the COVID- sampling to others. The difficult components of self- 19 pandemic in March 2020. sampling reported (one or more issues for 2322 respon- dents) were: the decision on how deep to insert the Screening and management project software brush into the vagina for 52% of 2322, how to insert the All data and images were collected with a HIPAA brush into the vagina for 49.1%, identifying the vaginal (Health Insurance Portability and Accountability Act) opening for 39.6%, rotating the brush inside the vagina compliant smartphone application ‘EVA for research’ for 23.3%, and proper handling to put the brush into the (MobileODT, Israel). The data platform was custom de- tube after collection for 18.9% women. signed for the project (led by CS) using an advanced bar- A total of 1630 (17.3%) [95% confidence interval (CI): code scanning system to limit human error from manual 16.6–18.1%] of 9406 screened women were HC2- key-in. The data and images from different sources on positive. The rate of HC2 positivity was 16.2% among study assigned smartphones were held locally until inter- the 8964 women with unknown HIV status and 40.5% net connectivity was available, at which point all data among the 442 women living with HIV (P < 0.001, inde- automatically transferred and aggregated to cloud pendent chi-square) (Table 1, Table 2). The strong Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 5 of 13 Table 1 Age-specific prevalence of high-risk HPV and precancer among women with unknown HIV status b c b Age at No. of women No. of HPV-positive (% Colposcopy Histopathology results (% of women attending colposcopy ) Yield of the screening effort (% of total screening screened in the of women screened) attendance (95% CI) women screened) (years) age group (95% CI) among CIN2+ CIN3+ CIN2+ CIN3+ HPV- positive 30–34 2553 476 (18.6%) (17.2–20.2%) 393 (82.6%) 26 (6.6%) (4.4–9.6%) 14 (3.6%) (2.0–5.9%) 1.0% 0.5% 35–39 2646 411 (15.5%) (14.2–17.0%) 354 (86.1%) 18 (5.1%) (3.0–7.9%) 10 (2.8%) (1.4–5.1%) 0.7% 0.4% 40–44 2282 351 (15.4%) (13.9–16.9%) 309 (88.0%) 12 (3.9%) (2.0–6.7%) 5 (1.6%) (0.5–3.7%) 0.5% 0.2% 45–49 1479 213 (14.4%) (12.7–16.3%) 194 (91.1%) 15 (7.7%) (4.4–12.4%) 7 (3.6%) (1.5–7.3%) 1.0% 0.5% Total 8960 1451 (16.2%) (15.4–17.0%) 1250 (86.1%) 71 (5.7%) (4.5–7.1%) 36 (2.9%) (2.0–4.0%) 0.8% 0.4% Not including four women with missing data on age CIN2+ and CIN3+ include a total of four cases of squamous cell carcinoma (two of them were in 49-year old women, one in a 47-year old woman, and one in a 33-year old woman; all four cases were diagnosed on biopsy). We also presumptively included five women with OncoE6 HPV 16 positivity because of the known high positive predictive value of the biomarker (Three of the five had confirmed CIN2+; in two LLETZ was recommended and is still incomplete) Histopathology report was not completed for four participants due to COVID-19 pandemic spread and lockdown. These four participants were excluded from the denominator Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 6 of 13 Table 2 Age-specific prevalence of high-risk HPV and precancer among women living with HIV (WLWH) Age at No. of No. of HPV-positive Colposcopy attendance Histopathology results (% of women Yield of the screening b b screening women (% of women among HPV-positive attending colposcopy ) (95% CI) effort (% of total (years) screened screened) (95% CI) women screened) in the CIN2+ CIN3+ CIN2+ CIN3+ age group 30–34 77 35 (45.5%) (34.1–57.2%) 27 (77.1%) 1 (3.8%) (0.1–19.6%) 1 (3.8%) (0.1–19.6%) 1.3% 1.3% 35–39 160 57 (35.6%) (28.2–43.6%) 41 (71.9%) 2 (5.0%) (0.6–16.9%) 2 (5.0%) (0.6–16.9%) 1.3% 1.3% 40–44 134 46 (34.3%) (26.3–43.0%) 40 (87.0%) 4 (10.0%) (2.8–23.7%) 3 (7.5%) (1.6–20.4%) 3.0% 2.2% 45–49 71 41 (57.7%) (45.4–69.4%) 35 (85.4%) 4 (11.4%) (3.2–26.7%) 3 (8.6%) (1.8–23.1%) 5.6% 4.2% Total 442 179 (40.5%) (35.9–45.2%) 143 (79.9%) 11 (7.8%) (4.0–13.5%) 9 (6.4%) (3.0–11.8%) 2.5% 2.0% CIN2+ and CIN3+ includes one case of squamous cell carcinoma in a 49-year old woman Histopathology report was not completed for two participants due to COVID-19 pandemic spread. These two participants were excluded from the denominator differences in HPV positivity between the two groups only one scheduling contact. An additional 299 (21.4%), persisted in all age groups. 166 (11.9%), 85 (6.1%), 77 (5.5%) and 64 (4.6%) came Out of 1630 HPV-positive women, 1400 (85.9%) en- after two, three, four, five, and more than five contact rolled for the study at colposcopy, of which seven cases attempts. were excluded due to unsatisfactory colposcopy and dif- Following the protocol of immediate management by ficulty in image collection due to various reasons out- colposcopic impression, without awaiting histopathology lined in the Additional file 1. Out of the 1400 who diagnosis of the biopsies taken, 114 (8.2%) women were enrolled, 709 (50.6%) women came for colposcopy after treated with thermal ablation and 78 (5.6%) with LLETZ. Fig. 1 Squamocolumnar junction visibility by age (n = 1393) Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 7 of 13 Fig. 2 Squamocolumnar junction (SCJ) visibility, by age and parity (n = 1380) Overall, CIN2+ and CIN3+ (including five cancers), and 6.4%, respectively (P = 0.31 for CIN2+ and P = 0.03 were detected in 5.9% (95% CI:4.7–7.3%) and 3.2% (95% for CIN3+, independent chi-square test in comparison CI:2.4–4.3%) of the HPV-positive women attending col- to women with unknown HIV status) (Table 2). The poscopy (85% attendance among the 1630), respectively. yield of the screening effort among women living with Thus, overall yield of the screening effort was 0.9% of HIV was 2.5% for CIN2+, of which all except two were 9406 women for CIN2+ and 0.5% for CIN3 + . actually CIN3+ (P < 0.001 for CIN2+ and CIN3+, inde- The prevalences of CIN2+ and CIN3+ among the pendent chi-square in comparison to women with un- HPV-positive women with unknown HIV status attend- known HIV status) (Table 2). Relatively small age- ing colposcopy were 5.7 and 2.9%, respectively, whereas specific numbers preclude any conclusions regarding the overall yields of the screening effort in these groups a trend in the prevalence of HPV or precancer by were 0.8 and 0.4%, respectively (Table 1). The age- age. The proportion of HPV-positive women in- specific prevalences of high-risk HPV positivity and creased with decrease in CD4 (cluster of differenti- precancer among women with unknown HIV status ation 4) count (64% in CD4 < 200/mm vs 31.1% in are shown in Table 1. The prevalence of HPV de- CD4 > 500/ mm3, P = 0.001, chi-square for trend) and creased from 18.6% at age 30–34 years to 14.4% at increase in HIV viral load (36.3% in <=20 copies/ml age 45–49 years (P = 0.0003, chi-square for trend). No vs 50% in > 20 copies/ml, P = 0.02, independent chi- meaningful trend was observed in the prevalence of square), however small numbers precluded trend ana- precancer by age (P = 0.87, chi-square for trend for lysis of precancer/cancer. CIN2+), except for a very high 24.1% (seven CIN2+ Out of a total of 75 histopathologically confirmed including three cancers) overall prevalence at self- cases, 59 (78.7%) were diagnosed through biopsy or reported age of 49 years. LLETZ of acetowhite lesions at colposcopy visit, 35 of The prevalences of CIN2+ and CIN3+ among the which (59.3% of 59) had a high-grade colposcopic im- HPV-positive WLWH attending colposcopy were 7.8 pression. QA review of colposcopic images revealed Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 8 of 13 Table 3 Management of women by histopathologic diagnosis Histopathologic diagnosis Management Row total No treatment Immediate management based on colposcopic Delayed excisional treatment on a recall visit indicated (row%) impression (row%) (column%) (row%) (column%) (column%) With ablation With LLETZ Completed Pending <CIN2 1158 (88.7%) (100.0%) 90 (6.9%) (78.9%) 54 (4.1%) (69.2%) 3 (0.2%) (12.0%) 0 1305 (100%) CIN2 Diagnosed on biopsy/LLETZ 0 10 (40.0%) (8.8%) 11 (44.0%) (14.1%) 2 (8.0%) (8.0%) 2 (8.0%) (16.7%) 25 (100%) Diagnosed on ECC 0 0 0 8 (66.7%) (32.0%) 4 (33.3%) (33.3%) 12 (100%) CIN3 Diagnosed on biopsy/LLETZ 0 13 (38.2%) (11.4%) 12 (35.3%) (15.4%) 5 (14.7%) (20.0%) 4 (11.8%) (33.3%) 34 (100%) Diagnosed on ECC 0 1 (16.7%) (0.9%) 0 3 (50.0%) (12.0%) 2 (33.3%) (16.7%) 6 (100%) Cancer Diagnosed on biopsy/LLETZ00 1 (20.0%) (1.3%) 4 (80.0%) (16.0%) 0 5 (100%) Column total 1158 (100%) 114 (100%) 78 (100%) 25 (100%) 12 (100%) Grand total = 1387 Recall attempts are temporarily paused due to COVID-19 pandemic spread Includes two women with OncoE6 HPV 16 positive, in whom LLETZ was recommended on recall and is still incomplete Includes one case with colposcopic impression of cancer treated with hysterectomy of what eventually turned out to be <CIN2; other two cases were recalled for LLETZ due to reporting error Invasive squamous cell carcinoma with CIN3 at margins was diagnosed on on-site LLETZ leading to a recall for a repeat LLETZ with CIN3 diagnosis leading to a 2nd recall for a hysterectomy Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 9 of 13 potential under-biopsying of more subtle acetowhite le- to be explained more clearly in future self-sampling pro- sions. 16 (21.3%) of 75 histopathologic precancer cases grams. Compliance with follow-up for colposcopy was were identified, in the absence of visible acetowhitening, also very high. only on ECC (1.2% of colposcopy examinations and 0.2% The two previous phases of Project Itoju established of the screening population). This is important in light the epidemiology of HPV infection in rural Nigeria [26], of the fact that even amongst women as young as age validated a low-cost HPV test (careHPV, Qiagen) [27] 30, the SCJ was only partially visible in 8.3% and not vis- (phase 1) and attempted (ultimately unsuccessfully) to ible in 23.1% of women (31.5% total), rising to 12.5 and determine whether immunosuppression due to soil- 52.1% (64.6% total) by age 49 years (P < 0.001, chi-square borne helminth infections or other parasitoses was the for trend) (Fig. 1). Interestingly, multiple vaginal deliver- cause of high HPV prevalence among older women in ies were found to increase full SCJ visibility (Fig. 2). the region (phase 2). In the current phase of Project Among 192 women treated on-site based on colpo- Itoju, we reconfirmed the overall high prevalence of scopic impression, without awaiting histopathology re- HPV infections even after restricting the age of screen- sults, only 48 (25%) were eventually diagnosed with ing to 30–49 years of age [28]. Although the prevalence CIN2+ on histopathology (Table 3). Viewing overtreat- of HPV decreased to 16% at age 45–49 years from 19% ment from another perspective, 11% of women with his- at age 30–34 years, it was still much higher than the glo- topathologic <CIN2 were treated based on colposcopic bal average of around 5% for women age 45–54 years impression. On the other hand, 43.2% of CIN2 (16 out and consistent with what is observed for the women in of 37) and 35% of CIN3 (14 out of 40) were not treated sub-Saharan Africa [29]. Despite the high prevalence of immediately; either because of underdiagnosis [diag- HPV infection, the prevalence of precancer was less than nosed later on ECC] or a variety of programmatic issues 1% in the general population of mostly unscreened such as equipment failure. A total of 12 (1.1% of colpos- women 30–49 years old. One reason for the low preva- copy population) precancer cases needed more than one lence of precancer could be under biopsy of more subtle treatment visit in order to obtain clear margins free of acetowhite lesions [30]. It also underscores the need to high-grade findings. further study the type-specific natural history in the re- Out of 1138 (82%) patients with full clinical quality as- gion of HPV acquisition, clearance, persistence, and pro- surance review completed, 143 (12.6%) cases were rec- gression, particularly in the setting of HIV infection. In ommended for recall; mainly (113, or 9.9% of total) for this current study, we obtained samples for HPV geno- repeat ECC; commonly because the tissue was insuffi- typing from women at screening and again at colposcopy cient for diagnosis on the prior ECC (100, or 8.8% of visit. We will be testing and reporting the results of total) or because the ECC required dilatation/sedation these tests elaborating the prevalence of various HPV ge- due to stenotic os or tethered cervix (13, or 1.1% of notypes and short-term clearance of infection in subse- total). However, the overall yield of precancer from re- quent papers. We also hope to retest the women in the call for ECC was only 0.2% for the colposcopy popula- future to explore the long-term persistence of HPV tion and 0.02% for the general population of women infection. aged 30–49 years. The prevalence of HPV and precancer (particularly CIN3+) was markedly high among WLWH as also re- Discussion ported by others [28]. This was observed despite the Our results showed the feasibility and acceptability of suppressed viral load with a documented average of < 20 self-sampled HPV testing and smartphone-based cervical copies/ml. This finding was expected since HPV and image collection in Nigeria. Nevertheless, the combin- HIV can be co-transmitted and women with HIV tend ation of higher HPV prevalence (17.4%) with a much to have lower clearance of acquired HPV infections. The lower risk of precancer (0.9%) suggests the need for tri- management of HPV in WLWH is an important topic age to improve the accuracy of the screening program. beyond the scope of this article. In addition, any visual triage method would require According to the World Health Organization (WHO), restricting the upper age limit of inclusion to increase Nigeria has only four physicians per 10,000 population program effectiveness. [31]. At present, only < 10% of women > 15 years have There was a very positive response and clear accept- ever been screened for cervical cancer in Nigeria [32]. In a ability for vaginal self-sampling for HPV testing. The dif- resource-constrained setting with a shortage of expert gy- ficult aspects of self-sampling reported by some necologic providers and infrastructure, avoidable referral participants included identifying the vaginal opening, de- to colposcopy and substantial overtreatment are not sus- termining depth of the insertion of the brush into the tainable. On the contrary, in settings with once in a life- vagina, rotating the brush inside the vagina, and putting time screening opportunity for a majority of women, the brush back into the tube after collection; these need substantial missed diagnosis and undertreatment is not Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 10 of 13 Fig. 3 Effect of image capture method on cervical appearance and limitation of visual triage method. a. Cervical images of the same cervix showing histopathologic <CIN2 (left trio) and CIN2+ (right trio), captured with (clockwise starting from upper left) a Samsung S8 smartphone camera and its flashlight, a MobileODT EVA device (Samsung J5 phone with an extra light source and a zoom lens), and a Zeiss FC150 colposcope with a beam splitter and DSLR camera (see supplement). b. Cervical images of histopathologic CIN2+ cases with squamocolumnar junction (clockwise from upper left) fully visible, partially visible, and not visible (diagnosed on ECC); captured with a Zeiss FC150 colposcope with a beam splitter and DSLR camera acceptable either. It is therefore essential that simple yet images from three different devices (Fig. 3a) has raised an accurate triage tests, separately or in combination, are important research question regarding the device portabil- available to stratify risk of precancer/cancer among HPV- ity of deep-learning based AVE algorithms due to vari- positive women such that treatment intensity can be tai- ation in color, brightness, reflection, glare imparted by lored to risk of cancer and sustained with local resources. each device camera, light source and image processing ap- In the future, we will be assessing the machine-learning plication. Training or automation to capture an in-focus, based AVE of cervical images using three different image non-blurry good quality cervical image, capturing entire collection methods. We will be reporting the results of the SCJ, for evaluation by AVE, is yet another challenge. assessment of the combination of AVE with HPV geno- It is important to note that AVE, like other visual as- typing for triage of HPV-positive women in subsequent sessment methods, requires that the cervical SCJ be vis- papers. However, preliminary analysis of the cervical ible. In the current study population, we found that the Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 11 of 13 SCJ was not fully visible for almost 64.6% of women by high risk of progression to invasion. Even though in the age 49 years. We also corroborated an earlier poorly study we managed to achieve a relatively higher rate of understood observation that women with multiple vagi- compliance with follow-up, the compliance in the real- nal deliveries were more likely to have fully visible SCJs world setting needs to be monitored and factored in the [33]. No visual screening methods or ablative treatment effectiveness of the screening programs. methods work when the transformation zone, where cer- We had to halt the recall activities under the screening vical cancer typically arises, is not visible (Fig. 3b) [12, program as the global spread of the COVID-19 pan- 34]. This could partially explain the decrease in the demic led to pausing of field efforts in Nigeria, including prevalence of precancer with age despite the high HPV a very few treatment visits still pending, and changed the prevalence, found in this study. It is, therefore, particu- risk-benefit ratio for cervical cancer screening. When we larly crucial to emphasize restricting visual approaches reopen, the first step will be to complete these treatment for screening or triage of older women to avoid giving visits. Then, moving forward in the COVID-19 era, we false reassurance to women in these age groups and to are considering the importance of self-sampled HPV avoid identifying high-risk HPV-positive women, par- testing with a sterile kit at a household level, avoiding ticularly with HPV 16, 18/45, with no available means of any mass gathering and minimizing the need for further triage. It is worth noting however that even at speculum examination. A small percentage of HPV- age 30, the SCJ was not fully visible in up to 30% of positive women with high-risk types could be triaged women, thus age-restriction to women <=49 years does and treated, spacing community clinic appointments in a not eliminate inadequacies in visual triage. The current COVID conscious manner [36]. standard practice is to collect an ECC sample whenever The limitations of this study should be noted. The the SCJ is not fully visible, to rule out cancer within the population selected for the study was a volunteer popu- endocervical canal. But an ECC needs an interpretation lation of women residing in the university town of Ile- by an expert histopathologist, which is challenging in Ife, which may have a lower HPV prevalence than the low resource settings. Difficulty in collecting ECC in general population in Nigeria. Also, HC2 is not generally women with a stenotic cervical os and insufficient tissue used for self-sampling, as it is slightly less sensitive for in the ECC sample to rule out carcinoma are other chal- detection of precancer than PCR-based HPV test lenges with ECC. Thus, the development of low-cost methods [37]. A moderate amount of cross-reactivity simple triage alternatives for ECC remains an unsolved against other genetically related but less oncogenic HPV challenge for improving cervical cancer screening pro- types with HC2 is well-documented. This may partly ex- grams. Research is also needed on simpler alternatives plain the low precancer to HPV ratio found in this study. for LLETZ, that could be performed easily by a general Despite the known limitations of HC2, its operational physician or a nurse without the need of an expert simplicity, and easy trainability, as supported by virtually gynecologist, particularly in low-resource settings where trouble-free operation of HC2 throughout the study sup- the needed resources are in short supply especially in ported its use. rural underserved areas. In future work we will assess misclassification by It is worth noting that even though the yield of pre- retesting residual screening samples with a more sensi- cancer from ECC at colposcopy visit was 3.2% (21% of tive whole-genome sequencing method [20]; review and total precancers diagnosed on ECC, possibly due to not recall women with highest risk HPV types and potential taking enough biopsies of subtle acetowhite lesions), the missed biopsies of subtle acetowhite lesions; and additional yield of precancer from the women recalled digitalize the histopathology slides for a second review, for insufficient ECC or difficult ECC collection was only particularly for borderline cases. 0.02% for the overall screened population. Another study has also noted overall low yield of ECC with an increase Conclusion in proportionate additional yield of ECC when fewer bi- A cervical cancer screening program using self-sampled opsies are taken [35]. It is worth exploring the cost- HPV testing, with colposcopic immediate management effectiveness of recalls for ECC to avert a very low risk of women positive for HPV, is feasible in Nigeria but re- of adenocarcinoma in low-resource settings where no sults in both over-and under-treatment. There are newer organized follow-up is available. Any recommendation HPV tests entering the marketplace that cost less than for follow-up outside the study in these settings is likely ten dollars per test, take less than an hour to perform, to remain a theoretical reassurance. In this regard, there and provide genotyping, suggesting that a self-sampled were a total of 12 women (1.1% of colposcopy popula- HPV based screening program would be feasible in this tion) who needed multiple rounds of treatment in order population in the future [38]. Having proven feasibility, to obtain clear margins on excision. These are women we are now evaluating the accuracy and efficacy in diag- with confirmed high-grade lesions and hence also at nosing CIN2+ of smartphone-based automated visual Desai et al. Infectious Agents and Cancer (2020) 15:60 Page 12 of 13 evaluation of cervical images combined with HPV geno- Author details Division of Cancer Epidemiology and Genetics, National Cancer Institute, typing as an assistive strategy to improve visual triage. NIH, Rockville, USA. Oak Ridge Institute of Science and Education, Oak Ridge, USA. Department of Obstetrics and Gynecology, Obafemi Awolowo University Teaching Hospital, Ile Ife, Nigeria. Lagos University Teaching Supplementary information Hospital, Lagos, Nigeria. Rutgers New Jersey Medical School and Cancer Supplementary information accompanies this paper at https://doi.org/10. Institute of New Jersey (CINJ), Newark, USA. Hela Health, Tel Aviv, Israel. 1186/s13027-020-00324-5. 7 8 Global Health Labs, Bellevue, USA. Albert Einstein College of Medicine, New York City, USA. National Library of Medicine, NIH, Bethesda, USA. Additional file 1. Received: 28 July 2020 Accepted: 22 September 2020 Abbreviations AUC: Area under the curve; AVE: Automated visual evaluation; CD4: Cluster of differentiation 4; CI: Confidence interval; CIN: Cervical intraepithelial References neoplasia; COVID-19: Coronavirus disease of 2019; DNA: Deoxyribose nucleic 1. Arbyn M, Weiderpass E, Bruni L, de Sanjosé S, Saraiya M, Ferlay J, et al. acid; DSLR: Digital single lens reflex; ECC: Endocervical curettage; Estimates of incidence and mortality of cervical cancer in 2018: a worldwide EVA: Enhanced visual assessment; HC2: Hybrid capture-2; HIPAA: Health analysis. Lancet Glob Heal. 2020;8(2):e191–203. Insurance portability & accountability act; HPV: Human papillomavirus; 2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. 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Published: Oct 14, 2020

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