==== Front BMJ OpenBMJ OpenbmjopenbmjopenBMJ Open2044-6055BMJ Publishing Group BMA House, Tavistock Square, London, WC1H 9JR bmjopen-2017-02043210.1136/bmjopen-2017-020432Global HealthProtocol15061699Protocol for the study of cervical cancer screening technologies in HIV-infected women living in Rwanda Murenzi Gad 1Dusingize Jean-Claude 1Rurangwa Theogene 1Sinayobye Jean d’Amour 1Munyaneza Athanase 1Murangwa Anthere 1Zawadi Thierry 1Hebert Tiffany 2Mugenzi Pacifique 1Adedimeji Adebola 2Mutesa Leon 13Anastos Kathryn 2Castle Philip E 2 1 Rwanda Military Hospital, Kigali, Rwanda 2 Albert Einstein College of Medicine, Bronx, New York, USA 3 University of Rwanda, Kigali, Rwanda Correspondence to Dr Gad Murenzi; gadcollins@gmail.com2018 5 8 2018 8 8 e02043204 11 2017 19 6 2018 22 6 2018 © Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.2018This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.Introduction The optimal method(s) for screening HIV-infected women, especially for those living in sub-Saharan Africa, for cervical precancer and early cancer has yet to be established. Methods and analysis A convenience sample of >5000 Rwandan women, ages 30–54 years and living with HIV infection, is being consented and enroled into a cross-sectional study of cervical cancer screening strategies. Participants are completing an administered short risk factor questionnaire and being screened for high-risk human papillomavirus (hrHPV) using the Xpert HPV assay (Cepheid, Sunnyvale, California, USA), unaided visual inspection after acetic acid (VIA) and aided VIA using the Enhanced Visual Assessment (EVA) system (Mobile ODT, Tel Aviv, Israel). Women positive for hrHPV and/or by unaided VIA undergo colposcopy, which includes the collection of two cervical specimens prior to undergoing a four-quadrant microbiopsy protocol. The colposcopy-collected specimens are being tested by dual immunocytochemical staining for p16INK4a and Ki-67 (CINtec PLUS Cytology, Ventana, Tucson, Arizona, USA) and for E6 or E7 oncoprotein for 8 hrHPV genotypes (HPV16, 18, 31, 33, 35, 45, 52 and 58) using the next-generation AV Avantage hrHPV E6/E7 test (Arbor Vita Corporation, Freemont, California, USA). Women with a local pathology diagnosis of cervical intraepithelial neoplasia grade 2 (CIN2) or more severe (CIN2+) or pathology review diagnosis of CIN grade three or more severe (CIN3+) will receive treatment. Clinical performance and cost-effectiveness (eg, sensitivity, specificity and predictive values) of different screening strategies and algorithms will be evaluated. Ethics and dissemination The protocol was approved by local and institutional review boards for human subjects research. At the completion of the study, results will be disseminated to the scientific community through peer-reviewed publication and to the Rwandan stakeholders through an external advisory panel. human papillomavirus (hpv)cervical cancergynaecologycervical intraepithelial neoplasiahttp://dx.doi.org/10.13039/100000054National Cancer Institutehttp://dx.doi.org/10.13039/100001299Prevent Cancer Foundationspecial-featureunlocked ==== Body Strengths and limitations of this study We are enroling a very large sample size of HIV-infected women living in Rwanda who otherwise would probably not get cervical cancer screening. We are employing rigorous disease ascertainment protocols to minimise misclassification. Some tests, such as the Xpert HPV and the AV Avantage HPV E6/E7 assays, are being done on site in Rwanda using technologies that could feasibly be deployed there. A weakness of the study is that cervical cytology is not being included as a comparator due to financial and logistical constraints. Introduction Invasive cervical cancer (ICC) remains a significant cause of morbidity and mortality globally. Approximately 530 000 cases of and 270 000 deaths due to ICC occur annually, making it the fourth most common malignancy and cause of cancer-related death in women globally.1 In many high-income countries, ICC rates have declined by 50% or more2 due to the introduction of effective, high-coverage Pap testing (cervical cytology)-based screening programmes that include timely follow-up of screen positives, treatment of women with precursor lesions and management of cancers. Now, almost 90% of ICC and ICC-related deaths occur in low/middle-income countries (LMICs) due to a lack of resources and healthcare infrastructure needed to provide preventive services. ICC and ICC-related mortality rates are particularly high in sub-Saharan Africa, which also has the highest rates of HIV infection in the world. Now, over 12 million HIV-infected (HIV-positive) women in sub-Saharan Africa are living longer because of antiretroviral therapy, thus increasing their likelihood of dying from ICC.3 However, many of these women are already exposed to human papillomavirus (HPV), the viral cause of ICC, and will not benefit from or be targeted for prophylactic HPV vaccination. Thus, cervical cancer screening will be needed for the foreseeable future. However, setting up effective cytology for cervical cancer screening is expensive and requires a complex clinical and laboratory infrastructure that generally does not exist in LMICs.4 5 Moreover, it is now well understood that cytology has only a low to moderate one-time sensitivity for precursor lesions and therefore must be done repeatedly over many years to reduce cancer risk. Alternative strategies to address the ICC burden in LMICs, especially in SSA, must be developed and validated. Persistent cervical infections by high-risk HPV (hrHPV) types cause virtually all ICC and its immediate precursor lesions, for example, cervical intraepithelial neoplasia grade 3 (CIN3) and adenocarcinoma in situ (AIS) everywhere in the world.6 7 hrHPV causes most anal and vaginal cancers and a significant proportion of vulvar, penile and oropharyngeal cancers.8 HPV16 is the most important causal type, responsible for ~60% of ICC.9 HPV18 is the next most important, responsible for 10%–15% of ICC, including 30%–40% of adenocarcinoma of the cervix,9 which is on the rise in Western countries.10 11 Together, HPV16 and HPV18 account for ~70% of ICC, and the same 15 hrHPV types account for ~99% of ICC everywhere in the world.9 There is now overwhelming evidence to suggest that testing for hrHPV is more sensitive, although less specific, than high-quality cytology for identifying women with cervical precancer.12–16 One-time hrHPV testing can reduce the risk of ICC incidence by approximately 40% in 6.5 years compared with cytology screening,16 and ICC mortality by approximately 40% (approximately 50% overall) in 8 years compared with cytology.17 Importantly, a negative hrHPV test provides superior reassurance against CIN3+18 and against ICC,16 17 permitting safe extension of screening intervals. The WHO released cervical cancer screening and treatment guidelines in 2013, recommending two evidence-based approaches to cervical cancer screening19: (1) Use either hrHPV testing or visual inspection after acetic acid (VIA), which involves the inspection of the cervix with a speculum in place and following the application of dilute acetic acid to help identify potential CIN by its characteristic white colouring in the presence of acetic acid (acetowhite), as alternative initial screening tests instead of Pap, and (2) immediately treat those who screen positive using the screening test, rather than require diagnostic verification through colposcopy and biopsy. This approach is commonly called screen-and-treat (S&T), and is increasingly thought to be more amenable to LMIC settings. However, hrHPV testing is also a much more effective screen than VIA,17 which on a large scale appears to only downstage cancer rather than prevent it.20 Thus, the recent American Society for Clinical Oncology resource-stratified guidelines for secondary cervical cancer prevention21 22 emphasise that hrHPV testing is the preferred choice for screening, with VIA only being used until hrHPV testing becomes available, and that HIV-infected women, because of their higher risk, should be screened twice as frequently as the general (HIV-uninfected) population. Recent data in HIV-positive women living in the USA suggest that hrHPV testing may have clinical utility similar to that in HIV-negative women. Several observational studies have shown that an extended screening interval is safe in HIV-positive women who test hrHPV and Pap negative as it is for HIV-negative women.23 24 In a study of women enroled in Women’s Interagency Health Study in 2002, HIV-positive and HIV-negative women who tested hrHPV and Pap negative were at a similarly low risk of CIN2 or more severe (CIN2+) histology over a 5-year follow-up.23 In addition, no cases of histologically confirmed CIN2+ were diagnosed in the follow-up of hrHPV-negative and Pap-negative HIV-positive women aged 30–64 years who underwent routine 3-year hrHPV and cytology cotesting at Kaiser Permanente Northern California.24 Thus, both studies found very high negative predictive values (NPV) >99% in HIV-positive women who test hrHPV negative. However, how hrHPV testing can best be used to screen HIV-positive women living in sub-Saharan Africa to prevent ICC remains to be determined. Recent studies25–28 in HIV-positive women living in sub-Saharan Africa have compared hrHPV, VIA and/or Pap for the detection of cervical precancer/cancer. The results can be summarised as follows: (1) hrHPV detection was more sensitive but less specific than VIA; (2) cytology was equally or more sensitive but less specific than VIA; and (3) cytology was equally or more sensitive but less specific (vs the converse) than hrHPV testing. Results and conclusions are varied, leaving unanswered the question of what screening strategy in HIV-positive women living in sub-Saharan Africa has the greatest effectiveness and cost-effectiveness. Regardless of the screening method, most screen-positive women who go to colposcopy or are treated immediately without diagnostic verification do not have cervical precancer and cancer (positive predictive value (PPV) for screening tests are typically 10%–20%). In places like sub-Saharan Africa that lack necessary infrastructure and personnel such as pathologists,29 excessive referral to colposcopy is problematic. Although WHO recommendations for S&T will hopefully overcome this bottleneck and increase the number of women living in LMICs who get screened, many countries may not adopt current S&T strategies because of concerns of low specificity and overtreatment, resulting in increased costs, unnecessary patient discomfort and concern and wasting valuable healthcare resources that could otherwise be used to expand access to screening. Thus, methods to increase the accuracy of screening by reducing the numbers of women having colposcopy and biopsy or getting treated immediately in this context are highly desirable as they will likely increase the uptake of cervical cancer screening. In order to improve the specificity of screening tests, secondary tests (biomarkers) are used following a screen-positive result with women who test positive for the triage undergoing further management (eg, colposcopy or immediate treatment) and those who test negative typically being deferred to further evaluation in 6–18 months to allow hrHPV infections to clear. There are several very promising biomarkers that might be used to improve the specificity and PPV of the screening tests.24 Given that HIV-infected women are more likely to test hrHPV positive,30–34 it is important to validate a triage strategy of using a secondary biomarker that sensitively and specifically rules in women with cervical precancer and cancer among the HIV-positive, hrHPV-positive women. We are therefore conducting a cervical cancer screening study of >5000 Rwandan women, ages 30–54 years, living with HIV infection. We are evaluating different screening tests (hrHPV DNA and VIA), those recommended by the WHO for cervical cancer screening,19 and different triage tests and biomarkers for the management of screen-positive women (E6/E7 oncoprotein detection, p16INK4a immunocytochemistry and hrHPV viral methylation). Screen-positive women undergo a rigorous colposcopic evaluation with multiple biopsies taken and the biopsies will undergo pathology review, to minimise the misclassification of endpoints. The primary objective of the study is to determine and compare clinical performance (Sensitivity (SE), Specificity (Sp), PPV and NPV) and cost-effectiveness for identifying HIV-positive women with CIN3+ and CIN2+ of different cervical cancer screening and management algorithms. Methods and analysis Study design and population We are recruiting those women receiving care for confirmed HIV infection at health centres and various hospitals operated by the Ministry of Health or Rwanda Military Hospital (RMH) during 2016–2018 (table 1). Sites were selected in collaboration with Rwanda Biomedical Centre, which calculated estimated numbers of potentially eligible women using data from their HIV database (OpenMRS-Open Medical Records System; http://openmrs.org/). The total of 7614 was the estimated number of women eligible for the study according to the inclusion/exclusion criteria (see below) at the beginning of the study in 2016. From each site, a convenience sample of women are being recruited to participate. Table 1 Recruitment sites and estimated eligible population Province Site Type of site Potential participants per site (approximate) Kigali Cor-unum HC Public Health Centre 1405 Kigali Kimironko HC Public Health Centre 1227 Kigali Rwampara HC Public Referral Hospital 1098 Kigali Kicukiro HC Public Health Centre 1054 Kigali Kacyiru HC Public Health Centre 905 Kigali Gikondo HC Public Health Centre 925 Kigali Rwanda Military Hospital Public Referral Hospital 300 Kigali WEACTx for Hope Private HIV Clinic 500 Kigali Busanza HC Public Health Centre 100 Kigali Nyarugunga HC Public Health Centre 100 Total 7614 Inclusion criteria Inclusion criteria include (1) living and receiving HIV care in Rwanda, (2) ages 30–54 years, (3) confirmed HIV positive based on medical records, (4) no prior cervical cancer screening, (5) no history of ICC and (6) willing, able and competent to provide written, informed consent. We are extending age range beyond that of the age range (30–49 years) recommended by the WHO for cervical cancer screening19 because there is limited evidence for the optimal upper age for cervical cancer screening of HIV-infected women. Exclusion criteria Exclusion criteria, in addition to not meeting the inclusion criteria, include (1) pregnant, (2) signs of abnormal, non-menstrual bleeding suggestive of ICC, (3) without a cervix due to hysterectomy and (4) not sufficiently healthy to participate in a research study based on the judgement of the clinicians. Excluded women are being advised to seek routine cervical cancer screening through government programmes. Prior to initiation of enrolment at a specific clinic, the local HIV care provider team identifies potentially eligible women at their routine clinic visits and offers them enrolment. Women indicating interest in the study are then registered by our research nurses using the eligibility criteria checklist. All eligible and willing women at that site enrol and receive screening before moving to another site. The study team of at least two research nurses schedules 12 to 15 women 3 to 4  days a week by calling them and confirming appointments over the telephone. Two teams of nurses are in the field, meaning that two cervical cancer screening clinics can be run simultaneously. Enrolment visit The study participant flow is summarised in figure 1. Enrolment visits, including pelvic examinations with VIA and specimen collection, are being done entirely by a team of two study nurses. During their enrolment visit, women are being educated on cervical cancer risk factors, mainly HPV infection, and why they are more at risk to develop ICC than HIV-uninfected women. They also are being allowed to ask questions before they commit to participating in the study. Women are then being asked to provide informed, written consent to participate in the study using a printed consent form. Those who provide consent complete a short nurse-administered questionnaire (see online supplementary appendix I) on cervical cancer risk factors and sociodemographic characteristics using a data capture screen in Microsoft Access. The questionnaire collects information on basic sociodemographics, factors associated with acquiring HPV (eg, marital status and recent and lifetime number of sexual partners), factors associated with increased risk of progression of hrHPV infection to precancer and cancer (eg, smoking and other tobacco use, parity and oral and other contraceptive use) and other infections common in Rwanda such as malaria35 and tuberculosis36 that have been previously reported to be associated with precancer among hrHPV-infected women. The questionnaire was not pretested. 10.1136/bmjopen-2017-020432.supp1Supplementary data Figure 1 Study design. CIN2/CIN3, cervical intraepithelial neoplasia grade 2/cervical intraepithelial neoplasia grade 3; hrHPV, high-risk HPV; VIA, visual inspection after acetic acid. Enroled women then undergo a pelvic examination, with VIA and a single cervical exfoliated (‘Pap’) specimen collected and placed into 20 mL PreservCyt (Hologic, Bedford, Massachusetts, USA) which is then being sent to the laboratory at RMH for hrHPV testing. Finally, a portable colposcope (http://www.mobileodt.com/; MobleODT, Tel Aviv, Israel) is being used for digital cervicography (comparable to VIA with magnification) and the image is being captured and saved for quality control and research, and to develop a digital library. Colposcopy visit Screen-positive women (women who test hrHPV and/or VIA positive) are being called using a telephone as soon as the hrHPV result is available and being invited to return for colposcopy within 1 month. All screen-positive women receiving colposcopy will be having two additional specimens collected, one into PreservCyt for the evaluation of other molecular biomarkers (genotype-specific hrHPV viral methylation and load, and p16/Ki-67 immunocytochemistry CINtec PLUS Cytology Kit (Roche, Tucson, Arizona, USA)) and a second as a dry swab for HPV16, 18, 31, 33, 35, 45, 52 and 58 E6/E7 oncoprotein detection by the next-generation lateral flow hrHPV oncoprotein test from Arbor Vita Corporation (Fremont, California, USA) being included in this study as a triage for screen-positive women to identify those women who are at higher risk of having CIN3+. The residual PreservCyt specimens from both the screening and colposcopy visits are being stored at −20°C, creating a biobank in Rwanda for future retrospective evaluations of promising new biomarkers and tests. After specimen collection, a colposcopic evaluation of the cervix is being done with a modified version of the four-quadrant microbiopsy procedure being performed.37 Compared with the standard biopsy, the microbiopsy protocol improves disease ascertainment and reduces biases related to selecting the most visually obvious acetowhite lesions while removing less tissue (~13 mm2 for four microbiopsies vs ~28 mm2 for one standard biopsy). Modifications to the standard four-quadrant microbiopsy procedure are: (1) endocervical curettage is being taken only for those women whose squamocolumnar junction is not entirely visible and the lesion extends into the endocervical canal; and (2) standard-size biopsies of very large lesions are being taken to increase the likelihood that the most severe area is being biopsied. Pathology Biopsies are being processed in a single cassette so that a single slide has a section from all biopsies taken. Biopsies are being read by a local pathologist at RMH and Dr Hebert or another pathologist at Montefiore Medical Centre, Bronx, New York, USA. Women receiving a diagnosis of CIN2+ by the Rwandan pathologist (TZ) or, as a safety precaution, CIN3+ diagnosis by Montefiore pathologist (TH) are receiving treatment38: (1) CIN2, CIN3 or AIS are being referred to study doctors to undergo an excision procedure (eg, loop electrosurgical excision procedure (LEEP) or cold-knife cone (CKC)) and (2) ICC are being referred to RMH Hospital for care. Women with