Cervical cancer (CC) is nearly always caused by persistent human papillomavirus (HPV) infection. It is the most common cancer among women living with HIV (WLWH) and is the leading cause of cancer-related death in women in East Africa, with 10,241 new cases reported in Tanzania in 2020. In 2019, the World Health Organization (WHO) presented a global strategy for the elimination of CC as a public health problem, proposing targets to meet by 2030 for HPV vaccine coverage , CC screening  and treatment delivery, to be scaled at national and subnational levels with a context-sensitive approach. This study aims to evaluate the upscaling of screening and treatment services at a rural referral hospital in Tanzania in order to address the second and third WHO targets.This is an implementation study with a before-and-after design performed at St. Francis Referral Hospital (SFRH) in Ifakara . CC screening and treatment services are integrated within the local HIV Care and Treatment Center (CTC). The standard of care, consisting of visualization of the cervix with acetic acid (VIA) and cryotherapy has been up-scaled with self-sampled HPV testing and also involved the introduction of mobile colposcopy, thermal ablation and loop electrosurgical excision procedure (LEEP). Participants are WLWH aged 18 to 65 years. Outcome measures included the percentage of women screened, HPV prevalence and genotype, and adherence to screening, treatment and follow-up plan. Additionally, we will explore the performance of novel diagnostic tests , which share the features of being manageable and inexpensive, and thus a potential tool for effective triage in HPV high-prevalence cohorts.The study will provide relevant information about HPV prevalence and persistence, as well as reproductive and lifestyle indicators in a CC high-risk cohort of WLWH and about upscaling screening and treatment services at the level of a rural referral hospital in Tanzania. Furthermore, it will provide exploratory data on novel assays.ClinicalTrials.gov Identifier: NCT05256862, date of registration 25/02/2022. Retrospectively registered.The online version contains supplementary material available at 10.1186/s12913-023-09113-3. Cervical cancer (CC) most frequently arises from persistent infection with oncogenic high risk (HR-) human papillomavirus (HPV) types . Due to The World Health Organization (WHO) is leading a global effort to eliminate CC as a public health problem, proposing the “90–70-90” targets to meet by 2030: 90% of girls fully vaccinated against HPV by 15 years of age; 70% of women screened using a high-performance test by 35 years of age and again by 45 years of age; 90% of women identified with CC treated and given care .The United Republic of Tanzania, where this study is planned, has the highest incidence of CC in East Africa, with 10,241 new cases diagnosed in 2020, a cumulative risk of 25.3% of new cases and 24.2% of cancer-related deaths per year according to the IARC/WHO Report . Among WCC is preventable with HPV vaccination. Following a successful pilot project in 2014 in the Kilimanjaro region among girls aged 9 – 14 years, in 2018 the Ministry of Health (MoH) of Tanzania introduced nationwide vaccination as a school-based program for 14-year-old girls , reachinVIA is used to identify acetowhite lesions, which if eligible according to the WHO guidelines, are treated with cryotherapy or thermal ablation.While VIA, as well as cryotherapy for VIA positive (VIA +) minor changes can be provided effectively at a district level, many factors, including disease perception, stigma, equipment, and personnel availability can affect the uptake , 17. MorIn order to meet the WHO targets, higher coverage of screening services and scaling-up of existing screening points with more sensitive and specific methods such as HPV-testing, and therapeutic options for severe dysplasia/ICC such as LEEP within a same-day-screen & treat approach, is urgently needed –20. As oThis is a before-and after study to evaluate the impact and feasibility of upscaling CC screening and treatment services for WLWH attending a rural referral CTC in Tanzania. The main objective is to evaluate the uptake by WLWH attending screening after implementation of HPV testing on a self-sampled cervico-vaginal smear, compared to a retrospective cohort screened by VIA. HPV testing has been implemented in a bundle with: a) a smartphone integrating a mobile colposcope  for digital enhancement of VIA examination with cervix magnification and second look/quality control; b) thermal ablation in place of cryotherapy (thus avoiding the need for replenishing nitrogen gas cartridges); and c) LEEP.We adopted an uncontrolled before-and-after design to compare proportion of WLWH attending screening before and after implementing mentioned interventions. A sub-study with cross-sectional design aims to explore diagnostic performance of two novel tests: the first, QuantiGene-Molecular-Profiling-Histology (QG-MPH), is based on transcriptomic biomarker analysis, while the second is a serological assay to detect antibodies against HPV16-L1 , either The study is conducted in rural south-central Tanzania, at the St. Francis Referral Hospital (SFRH) in Ifakara, a 371-bed designated district referral hospital for the districts of Kilombero and Ulanga (Morogoro Region), serving over 800,000 inhabitants of the valley of Kilombero. Since its accreditation in 2005, a hospital-based HIV Care CTC was built up as a center for integrated care and as a research platform, the "Chronic Disease Clinic of Ifakara” (CDCI), through an international partnership between the hospital, the Ifakara Health Institute (IHI), the Swiss Tropical and Public Health Institute (Swiss TPH) and the University Hospital of Basel, Switzerland (USB) . In addiThe study recruits consenting WLWH, aged 18 to 65 years, enrolled in KIULARCO, who have been on antiretroviral therapy (ART) for at least three months. Exclusion criteria consist of reported pregnancy, known CC and/or any other condition interfering with the visualization of the cervix, and previous hysterectomy. Participation in the study will be offered as per Standard Operating Procedure (SOP) during routine visits at the CDCI, where WLWH are referred either by the triage nurse or by the attending clinician, or directly at the CC screening service in case of self-referral. Specifically trained nurses will provide, in the native language of Kiswahili, detailed explanations and a copy of the informed consent form (ICF) to eligible WLWH, which will be read aloud to illiterate patients. Any questions or concerns will be answered accordingly. If the patient is willing to participate, the ICF will be signed or marked with their fingerprint and kept in the patient’s data file. Participants will undergo screening and treatment as specified in paragraph “A rapid lateral flow assay (rLFA)  to detect anti-HPV16-L1 antibodies will be performed first, at the point-of-care (POC), on a single drop of finger capillary blood  a medical device designed for this purpose and already validated in large scale screening programs , 32.The first participants will undergo physical examination and VIA at baseline visit, in accordance with the national guidelines, as well as HPV-testing as detailed further, until 150 HR-HPV negatives have been examined as a control population for the diagnostic sub-study. Thereafter, only participants that report vaginal symptoms  and/or test positive at rLFA will undergo physical examination at baseline. Otherwise, physical examination is reserved for participants with detected HR-HPV infection, and this will take place at a second visit, due to laboratory turnaround time. Physical examination is performed as per local routine, i.e., with speculum and VIA, in accordance to WHO/IARC standard recommendations . Images The samples will be stored at room temperature and brought to the IHI laboratory, located next to the SFRH, at the end of the day. Serum will be obtained from the whole blood sample for quantification of antibodies to the HPV16-L1 DRH1 epitope . After pre-incubation of 25 µL serum with an HPV16-L1-specific reagent for 5 min, the mixture is transferred onto a lateral flow test cassette. The test result is measured 15 min later with a table-top reader .Self-collected cervico-vaginal smear samples are used for HPV testing and QG-MPH, an innovative patented assay by Charité-Universitätsmedizin Berlin (Charité), Berlin, Germany  based on QuantiGene 2.0  platform technology. QG-MPH has promising results for detecting CIN3 + with 90% accuracy , and could become a quick onsite-triage method and a pathology-replacing, molecular diagnostic assay . FurtherThe cervico-vaginal swab is fixed in a methanol-based transport medium . HPV testing takes place in two steps: DNA extraction will be performed at IHI Laboratory in Ifakara, where samples undergo temporary storage at -20 °C. Every 1–2 weeks, a batch of 200–400 samples will be transported to the IHI Laboratory in Bagamoyo for PCR-based DNA amplification with the Seegene Anyplex™ II 28 HPV Detection Test , an RT-qPCR assay designed to detect and individually genotype 19 different HR-HPV types  and 9 low risk (LR)-HPV genotypes  in 2 parallel wells. Staff training and laboratory setup for transcriptome analysis in Tanzania was recently hindered by the SARS-CoV-2 pandemic. CFX96™ Dx Real-time PCR System was not lent to the IHI branch of Ifakara because of shortages of the instrument in East Africa. A cervico-vaginal smear sample aliquot will also be shipped for further transcriptome analysis at the HPV laboratory of Charité according to the MTA .Schistosoma egg detection for GS will be performed at the IHI laboratory in Ifakara.Tissue of cervical biopsies and/or LEEP excisions will undergo histopathological studies at ORCI. Urine sediment microscopy for , using a C3 Thermo-coagulator , an easy to handle, reusable, thermo-probe device, which can run on the standard power grid or be connected to an external powerpack, and thus practical in settings where the electricity supply might be interrupted. Similar devices are available at other facilities in the region, the nearest being Morogoro Regional Hospital (ca. 231 km or five hours drive from the study site).VIA + women with minor changes are treated at the same session by thermal ablationVIA + women with major changes and/or suspicion of ICC and/or transformation zone type 3 are treated at the same visit in a separate session in the gynecology surgical theater by LEEP. LEEP is a 10-min procedure to remove a cone-shaped area of tissue from the cervix for diagnostic and/or therapeutic purposes with a thin wire loop heated by electricity. It has the potential of being curative, usually without significant adverse events . ExcisedThe follow-up plan is determined by the analysis results Fig. . If antiThe primary endpoint is screening coverage, defined as the proportion of WLWH enrolled in KIULARCO screened for CC in the 12 months period after implementation of HPV testing. Participants that undergo screening and transfer out to other CTC within the study period are also included. Secondary endpoints are listed in Table Attendance to screening and its results are routinely recorded on a card given to the patient, and on paper registries. For the purpose of this study, data are additionally recorded in an online, password-protected electronic data capture software (REDCap™). The questionnaires administered to the participants will be filled out in real time by study nurses or physicians on a tablet device, while laboratory data will be entered by the laboratory technicians as soon as they become available. Data relevant for patient care will be recorded separately in the electronic database of KIULARCO (OpenMRS v2.0.6). Participant data will be managed confidentially: pseudonymized (study ID as well as NACP ID for linkage with NACP and KIULARCO dataset) for the purpose of patient care and anonymized for scientific evaluation and further dissemination of results. At baseline, sociodemographic data, medical history, laboratory analysis results as well as knowledge, attitude and practice as regards CC and HPV vaccine will be captured. A data safety and management board is not planned. Data monitoring will be performed on a regular basis by co-investigators not involved in data collection. At the end of the project, the entire database, property of IHI and USB, will be archived for minimum period of 10 years. The study represents implementation research: HPV testing and established therapeutic interventions  are already available for routine care elsewhere in the country. The experimental diagnostic tests have a minimal impact (only capillary POC test) on clinical decisions and have been granted approval by TMDA. Adverse events will not be recorded. A Data Transfer Agreement (DTA) has been signed between IHI and respectively Charité and USB.For the primary endpoint, the hypothesis is that implementing a bundle of interventions comprising HPV testing on self-collected samples will improve rates of CC screening among WLWH enrolled in KIULARCO from 8 to 30%. Due to the uncertainty about the WLWH population size, this hypothesis is considered as exploratory, therefore only the precision of the proportion is estimated (screening rate), with a 95% confidence interval. The proportion of WLWH attending CC screening in the post-period will be estimated with 95% confidence interval.p-value < 0.05 (two-sided) is considered as statistically significant.For the secondary and other endpoints no hypothesis will be formulated and results will be compared in an exploratory manner. Diagnostic test accuracies  will be calculated for comparison. Accuracies will be calculated with a 95% confidence interval. Prevalence of secondary endpoints will also be estimated with 95% CI's. Diagnostic tests will be pairwise compared using McNemar test. The assessment of acceptability and feasibility will be evaluated with exploratory statistics. The agreement of raters between diagnostic interpretations will be described with kappa coefficients and polychoric correlations. All secondary parameters will be reported with descriptive statistics including minimum and maximum values, interquartile range, median, mean, SD, count, and proportion as appropriate. Comparisons between study groups will be done using Kruskall-Wallis test for metric or ordinal data and Fisher's exact test for categorical data. A All evaluations will be done with the latest version of the statistical software R.No sample size calculation was done due to the exploratory nature. For the primary outcome, we performed an estimate of the precision. Given a precision of ± 3% and assumed proportion of 30% self-sampling (enrolled subjects), a sample size of 1200 WLWH estimates a 95% confidence interval completely within the required precision. In the pessimistic case with a given a precision of ± 2% and assumed proportion of only 10% self-sampling (enrolled subjects), a sample size of 1200 WLWH estimates a 95% confidence interval completely within the required precision.Serum and cervico-vaginal smear samples will be labelled, processed, analyzed, stored at the IHI laboratory in accordance with SOPs. For the QG-MPH test and quality control of HPV testing, one cervico-vaginal smear sample per participant will be transported from Ifakara to the HPV Laboratory, Charité (DE) as per the MTA. Once all analyses have been performed, the materials will be stored for a period of 10 years and thereafter destroyed by standard internal procedures of the IHI and Charité, respectively.No patient or patient advisor was involved with study design, recruitment, or conduct.Training in colposcopy and the routine use of a mobile colposcope started in February 2020, and HPV testing on self-cervico-vaginal collected samples was implemented starting from 30.07.2021. LEEP was introduced in September 2020 and thermal ablation in October 2021. As per 31.12.2022, 1500 participants have been enrolled.This implementation project represents a micro-initiative, at rural referral hospital level, to contribute to the efforts of improving CC prevention and the care cascade in Tanzania, complying with the WHO and national recommendations regarding secondary and, in part, tertiary prevention. The choice of implementing treatment procedures along with HPV testing has an ethical rationale. The decision to substitute the already in-use cryotherapy with a thermal ablation device was made after frequent reports of cryotherapy gas supply challenges, with consequently missed treatment opportunities. The introduction of a mobile digital colposcope had the purpose of allowing second-look evaluations by collaborating clinicians, for continuous education and for quality improvement. The choice of a laboratory-based HPV testing platform over the on-site available Xpert® platform was firstly due to concurrent testing needs (esp. for tuberculosis diagnostics) which would have hampered an HPV-based single-visit screen-and-treat approach only, and secondarily because the chosen platform allows for identifying more individual genotypes. The choice of a before-and-after study design has certain limitations, in that the intervention effect may be overestimated by unrecorded, concurrent factors , which in this case are relevant to some extent, since the ideal comparison is the WHO’s second target (70% screened). Further, the low estimates of pre-intervention screening rates (8%) may reflect incomplete reporting or heterogeneous data sources. For the purpose of this study, a digital case report form (e-CRF) was developed which could be accessed via a tablet device or computer  and filled out in real-time. This should minimize bias in data collection. Considering that CC screening was already routinely offered to the study population and that reporting may be of suboptimal quality, recall bias cannot be ruled out for the patients who had previous diagnosis of precancerous lesions, although this should not affect the primary outcome. Cost of service is a major concern regarding the sustainability of the implementation project, which we plan to address in a future publication. Provided free of charge or after a symbolic financial contribution, CC screening and treatment with VIA / cryotherapy was financially supported by non-governmental implementing partners, including financial incentives to the screening team and an additional outreach program. Currently, diagnostic and therapeutic options are limited in peripheral health-care centers of Tanzania and different factors hinder access to CC screening and treatment programs. Implementing “near-home” options may substantially increase the participation rate as well as improve the adherence to the appropriate care plan, with rationalization of health-care center resources and improvement of quality of services.In addition, the study has the potential to understand the specific needs of a semi-rural patient population , of outcome of screening and treatment, and reasons for non-adherence, which are important factors for future scaling-up procedures. The results of this study will provide additional information to the Ministry of Health of Tanzania on the epidemiology of HPV infection, CC and comorbidities in the high-risk population of WLWH accessing a rural referral hospital. It might help to provide elements useful for the development of strategies in similar settings for comprehensive reproductive and sexual health care. Since the study team was already involved in CC prevention and treatment, and the provided instruments will be available for routine clinical care, patients not included in this study are expected to benefit as well. Finally, the exploratory analysis on novel tests will hopefully contribute to potential innovations in clinical practice, which, if proven effective, may contribute to a reduction in CC screening time and costs worldwide.Additional file 1: Supplementary Materials - Annex A. Annex A. Details and evidence on novel diagnostics. Annex A1. HPV16-L1 immunoassay (PrevoCheck® and PT Monitor®). Annex A2. QuantiGene Molecular Profiling Histology (QG-MPH®).Additional file 2: Supplementary Materials - Annex B. Annex B. Definitions.


This study aimed to determine the knowledge of human papillomavirus vaccination (PHV) and associated factors among primary school girls in Arba Minch town, South Ethiopia, in 2020. Hence, the levels of knowledge towards the HPV vaccination of girls were assessed and recommended to the concerned bodies. Clinically, this study could increase the acceptance of HPV vaccination after the intervention of the concerned bodies to improve adolescents’ knowledge levels. As a result, it could decrease the incidence of cervical cancer. Socially, this study may increase the research involvement of adolescents, communities, and stakeholder groups.A school-based cross-sectional study involving 537 female students was conducted on January 24, 2020. The study participants were selected by a simple random sampling technique. A pre-tested questionnaire was used to collect the data. The logistic regression model was used to identify the statistically significant variables for knowledge of the human papillomavirus vaccination.The overall knowledge level of the participants in this study was 71.7%. Their main source of information is social media (41.74%), followed by medical staff (29.69%). Age ≥15 years old, AOR =3.74, 95% CI (2.20_6.37), P-value <0.001, grade level of 7 and 8, AOR =3.98, 95% CI (2.40_6.58), P-value <0.001, mother’s educational status of secondary and more than secondary, and father’s educational status of more than secondary, AOR=13.60, 95% CI (5.69_32.53) P-value <0.001, 22.27, 95% CI (8.23_60.30), P-value<0.001, 2.18, 95% CI (1.09_4.35), P-value 0.03, respectively, and access to HPV vaccination information , P-value 0.001) were associated with overall knowledge.Nearly three-fourths of the study participants were knowledgeable about human papillomavirus vaccination. Knowledge about the human papillomavirus vaccination shows a positive association with age, education level, parents’ educational status, and access to information sources. The HPV vaccine protects against infection with the human papillomavirus (HPV). The human papillomavirus (HPV) is a virus that causes over 200 diseases, 40 of which are spread through sexual contact. More than a dozen HPV strains can cause cancer in the anal, cervical, vulvar, oropharyngeal, penile, vulvar, and vaginal locations, whereas two HPV variants can cause genital warts.6The majority of cancers (over 80%) in sub-Saharan Africa (SSA) are detected at a late stage, predominantly due to a lack of information about cervical cancer and a scarcity of prevention services, which in turn are associated with low survival rates after surgery or radiotherapy.However, in many countries, knowledge and acceptance of HPV vaccination are low. For example, knowledge in China was 15.7%,20Factors that are associated with the knowledge of HPV vaccination include information source,7Even if knowledge of HPV vaccination affects HPV vaccination uptake among adolescent girls, knowledge of HPV vaccination was limited in Ethiopia. Therefore, this study aimed to fill this information gap by assessing knowledge and associated factors of HPV vaccination among primary school girls in Arba Minch town, south Ethiopia. The primary beneficiaries of this study were adolescent girls because their levels of knowledge regarding the HPV vaccination were assessed and recommended to the concerned bodies. Clinically, this study could increase the acceptance of HPV vaccination after the intervention of the concerned bodies, such as developing age-and gender-based health education to improve adolescents’ knowledge levels based on the recommendations. As a result, it could decrease the incidence of cervical cancer, which is the second most common cancer in Ethiopia. Socially, this study may increase the research involvement of adolescents and communities as well as invite stakeholder groups such as educational institutions, health institutions, health policymakers, and nongovernmental organizations to participate in relevant interventions. The results of this study could also be a crucial source of information for health program planners and further investigators. This can benefit already existing literatures by bringing updated information.Arba Minch town, the capital city of Gamo zone, is 505 km from Addis Ababa  and about 280 km from Hawasa . There are two hospitals, two health centers, and 11 health posts providing health services. Arba Minch town has four sub-cities and eleven kebeles (the lowest administrative unit in Ethiopia). Arba Minch town has 55 kindergarten (KG) schools, where schoolchildren attend from 4 to 6 years of age before starting grade one, of which 6 are public. There are also 18 full primary schools (grade one up to eight), of which eight are public, and nine secondary and preparatory schools (grade nine up to twelve), of which five are public. The total number of students, without adding kindergarten students, registered in 2020 in Arba Minch town was 27,512, of which 14,431 were female students. For our study purposes, we used primary school students who were in grades five through eight, with a total number of 8867, of which 4758 were female students. The age distribution of female students in this grade level during our study period was 12 to 17 . Zonal and city health offices work jointly with schools to deliver the vaccination in a school-based way after creating awareness through health extension workers and other concerned bodies . Students were classified as rural or urban based on their parents’ habitations. For those students who are classified as rural, their parents live in a rural area very close to Arba-Minch town, and the students come to city only for educational purposes; they return to their parents for habitation. Some others dwell in the city with their guardians for working days only.A school-based cross-sectional studyAll girl students in primary schools of Arba Minch town, South EthiopiaAll girl students in 6 selected primary schools of Arba Minch town during the data collection period.Knowledge of HPV vaccination.Age, Grade level, School type, Educational status of family, Source of information about HPV vaccination, School based education about HPV vaccination,Good knowledge: those who answered 50% or more or 4 and above from 7 knowledge assessing questions.16Poor knowledge: those who answered below (50%) from knowledge assessing questions.16The human papillomavirus: is a group of viruses that is extremely common worldwide.23The human papillomavirus vaccine: is a vaccine used to protects against infection with the human papillomavirus (HPV).1Those girls who were attending a primary school and in grade 5 up to 8.Those girls who were ill at the time of data collection.The sample size was determined by using a single population proportion formula with the assumption of a 95% confidence level, 5% margin of error, by taking “p” (0.696) from similar studies conducted among adolescent girls,From a total of 18 primary schools found in the city (both private and public), 6 primary schools were selected by simple random sampling without replacement through the lottery method. Then the calculated sample size was allocated to each school proportionally. After getting eligible adolescent girls for each school from their homeroom teachers, we relisted to prepare a sampling frame for that school. While preparing the sampling frame, a section of given students was listed in front of their names to finally get them, and then study units of a given school were obtained by a simple random sampling technique. Finally, selected study units for given schools were requested to be in one place (to one of the sections at break time) and fill in the questionnaire through the guidance of data collection facilitators and supervisors.Annex: 3). The questionnaire was developed after an extensive review of published articles. Data collectors were selected from among the teachers of their respective schools. The questionnaire consists of socio-demographic information and knowledge of the HPV vaccination among adolescent girls of Arba Minch town. The tool was prepared with the English version and translated to Amharic to avoid missing important information; then it was translated back to English for analysis.A pre-tested, semi-structured, translated into Amharic language, and self-administered questionnaire was used  were labeled as having poor knowledge and those who answer 4 or above from 7 questions or score 50% and above were labeled as having good knowledge.Logistic regression was applied to see the association between dependent and independent variables. Independent variables, which had an association in bi-variable analysis with a p-value <0.25 were entered into the multivariable logistic regression model. Independent variables with a p-value<0.05 in the multivariable logistic regression model were considered as statistically significant factors for knowledge. The results were presented as odds ratios (OR) with 95% confidence intervals. An odds ratio with a corresponding 95% CI was used to quantify the association between a dependent variable and independent variables. The model fitness was checked by using the software application of the Hossmare and Lame show test (>0.05). The results were presented using text, and tables.Out of the 537 samples that were distributed, 516 study participants were interviewed in this study, with a response rate of 96.1%. The majority of study participants (71.3%) were Gamo in ethnicity and protestant Christians in religion (60.5%)  Gamo is Three hundred seventy-one, 71.7% CI, 95% (67.6_75.4) of the study participants were found to have good knowledge about the HPV vaccination. 69.8% of the study participants knew the targeted age group and 78.3% knew the number of HPV vaccinations to be given for the prevention of cervical cancer. The main source of information was social media (41.74%), followed by health care providers while giving health information at school (29.69%) Table 2KnLogistic regression analysis was done to assess an association between the socio-demographic characteristics of the study participants with their knowledge level towards HPV vaccination. It showed that; age, grade level, familial educational status, and information source had a positive association. That is, those who are ≥15 years old have a close to fourfold greater likelihood of having good knowledge about HPV vaccination as compared to those who are 12–14 years old. AOR =3.74, 95% CI (2.20_6.37), P-value (<0.001),Those who are in Grade 7 and 8 are close to four-fold more likely to have good knowledge about HPV vaccination as compared to those who are in Grade 5 and 6. AOR = 3.98, 95% CI (2.40_6.58), P-value (<0.001).Familial educational status: those whose mothers’ educational status is secondary close to fourteen fold and whose fathers’ educational status is more than secondary close to threefold have better knowledge about HPV vaccination than their counterparts. AOR=13.60, 95% CI (5.69_32.53) P-value <0.001), 22.27, 95% CI (8.23_60.30), P-value<0.001), 2.18, 95% CI (1.09_4.35), P-value 0.03) respectively,Those who obtained HPV vaccination information have a close to nine-fold higher likelihood of having good knowledge about HPV vaccination than those who do not obtain HPV vaccination information. AOR=8.65, 95% CI (3.92_19.07), P-value (<0.001) . HossmerThis study’s overall knowledge score was 71.7% CI 95% (67.6_75.4), which is lower than studies conducted in Australia (97.4%),The age of respondents had a positive association with knowledge. Those who are >15 years old are close to fourfold more likely to have good knowledge about HPV vaccination as compared to those who are 12_14 years old. AOR =3.74, 95% CI 2.20_6.37), P-value (<0.001), which is similar to a study conducted in Greek_6.37, P-As the education level of girls’ increases, so does knowledge of the HPV vaccination. Those who are in Grade 7 and 8 are close to four-fold more likely having good knowledge about HPV vaccination as compared to those who are in Grade 5 and 6. AOR=3.98, 95% CI (2.40_6.58), P-value <0.001). A similar finding was found in a study conducted in Jimma, southwest Ethiopia,Familial educational status . AOR=13.60, 95% CI (5.69_32.53) P-value (<0.001), 22.27, 95% CI 8.23_60.30), P-value (<0.001) 2.18, 95% CI (1.09_4.35) P-value (0.03) respectively. This is similar to a study conducted in Senegal._60.30, PInformation sources about HPV vaccination have a statistically significant association with knowledge of HPV vaccination. That is, those who obtain HPV vaccination information have a close to nine-fold higher likelihood of having good knowledge about HPV vaccination than those who do not obtain HPV vaccination information. AOR = 8.65, 95% CI (3.92–19.07), P-value <0.001, which is similar to a study conducted in the USA.This is significant both clinically and socially. Clinically, after the involvement of the relevant bodies, such as providing age-and gender-based health education to raise adolescents’ knowledge levels based on the guidelines, this study may increase the acceptance of the HPV vaccine. As a result, cervical cancer, Ethiopia’s second-most frequent cancer, could become less common. In terms of social implications, this study may boost adolescent and community participation in research as well as invite stakeholder groups like educational institutions, health institutions, health officials, and nongovernmental organizations to participate in relevant interventions. The findings of this study could be useful to health-care planners and researchers in the future. This can help to update existing publications.The quality of the study was unquestionable as it was conducted in a multidisciplinary way. In addition to this, a pretest was conducted and the relevance, consistency, and validity of the tool were checked. It was able to survey a random sample of female students, thereby increasing the generalizability of the findings to other eligible students. This study utilized a self-administered questionnaire, which increases the likelihood of respondents answering openly, hence increasing internal validity. This study provides new and updated information about adolescents’ knowledge of HPV vaccination, which can serve as a baseline for future researchers as well as health policymakers and implementers.The limitation of this study was that it did not include the girls’ parents in the study as they were decision-makers. Parents’ knowledge and attitude can influence their acceptance of HPV vaccination for their daughters, thereby affecting their actual utilization of HPV vaccination. In our country, adolescents less than 18 years old cannot decide by themselves but through their parents or guardians. Therefore, the researcher/s should consider the parents of the adolescents to cover their attitudes, acceptance, and knowledge of HPV vaccination. This study also did not cover the attitudes of the adolescents as well as the communities, which are the important factors to consider. Our study included only a few rural students due to different resource and technical problems. The rural areas are the places where there is limited access to information, so the next researcher should consider it.The overall knowledge level of study participants was 71.7%, which is still less than three-quarters. Factors positively associated with knowledge of HPV vaccination were age, grade level, and familial educational status, as well as information source. We recommend that health institutions prepare age-and gender-based health education to further improve adolescent knowledge and those educational institutions encourage female education as well as participation in health and health-related clubs. Researcher who delighted in investigating the same thematic area better to incorporate parents or guardians.


The relationship between human papillomavirus (HPV) and cervical lesions has been extensively elucidated, but infection with multiple genotypes is less investigated due to methodology limitations. In the current study, with a method of genotyping 21 HPVs in a routine cervical screening population, we aimed to investigate the prevalence and diversity of HPV infections in Chinese women and further evaluate the impact of multiple infections of HPV on cervical lesion progression.  Totally, 73,596 patients who underwent 21-genotype HPV testing from January 2018 to April 2019 were retrieved from the database of the Department of Pathology, Obstetrics and Gynecology Hospital of Fudan University. HPV testing was performed by real-time PCR assay, including 13 high-risk HPVs (hrHPV), 5 potential hrHPVs, and 3 low-risk HPVs. P < 0.001), indicating that mixing with other genotypes alleviated pathogenicity. The infection of HPV52, 53, 56, 51, 39, 66, 59, 68, and 35 showed an opposite pattern, indicating that they were less likely to be pathogens individually. All other types showed no significant differences, indicating the capability of pathogenesis independently. HPV26 showed a higher OR for CIN2+ than most traditional hrHPV genotypes. The vial load and the percentage of HPV16 showed positive correlation with the severity of cervical lesions.  Of the 17,079  hrHPV- or potential hrHPV- (hr/phrHPV-) positive cases, 26.3% had multiple infections. Women younger than 25 and older than 65 were more prone to multiple infections. Of the hr/phrHPV-positive cases involving cervical intraepithelial neoplasia (CIN) 2 or worse (CIN2+), HPV73, 53, and 66 (=59) were the top three genotypes most likely to be included in multiple infections, while HPV16, 18, and 58 were the 3 least. Patients with single infection of HPV16 had higher incidences of CIN2+ than those with multiple-infection pattern ( Extensive genotyping identified 3 most frequent genotypes, HPV16, 52, and 58, in CIN2+ of Chinese population. HPV16 mixing with other genotypes alleviated its pathogenicity. The vial load and the percentage of HPV16 were positively correlated with the severity of cervical lesions. HPV26 may be considered as a hrHPV, which needs to be evaluated and confirmed by more cases. Cervical cancer has the highest incidence of the three major malignant tumors in female reproductive system worldwide. There are 604,127 new cases of cervical cancer worldwide in 2020, of which 109,741 are from China . HPV infThe relations between the virus and cervical lesions have been extensively elucidated, but multiple-genotype infections are less investigated due to methodology limitations. The results from various population studies have shown that multiple-HPV-genotype infections are frequently encountered, and cervical lesions are not always caused by a single HPV genotype –10. DiffIn this study, with a method of genotyping 21 HPVs in a large quantity of cases, we investigated the prevalence and diversity of HPV infections in a routine cervical screening population in Shanghai, eastern China. We analyzed the relationship between multiple infections of various HPV genotypes and cervical lesions and aimed to further evaluate the impact of multiple-HPV infections on cervical lesion progression.With Institutional Research Review Board approval, the data for 73,596 outpatients who underwent 21-genotype HPV testing from January 2018 to April 2019 were retrieved from the database of the Department of Pathology, Obstetrics and Gynecology Hospital of Fudan University (OGHFU), including 64,534 with Pap cotest results, and 17,394 had a histological diagnosis within 6 months after HPV testing.TOP3A DNA. The BMRT real-time PCR assay utilizes eight tubes for one sample with three different genotypes in each tube including a reference control. In this study, only 13 types of hrHPVs and 5 types of potential hrHPVs were included in the analysis of multiple infections. Some studies have reported that the BMRT assay seemed to be a good alternative approach for HR-HPV testing, due to its high level of automation and ability to quantify HPV-16, HPV-18, and other HR-HPVs [All HPV testing was performed in the Department of Pathology at OGHFU, according to the manufacturer's instructions. HPV testing was performed by BMRT real-time PCR assay , which genotypes 13 high-risk HPVs (hrHPVs) , 5 potential hrHPVs , and 3 low-risk HPVs  separately, and the titers were estimated by comparing to the amount of human  HR-HPVs . AnotherSpecimens for LBC (liquid based cytology) were obtained by a plastic spatula with an endocervical brush and were placed in transport medium. LBC was prepared via the SurePath method (BD Diagnostics) and was interpreted using the 2001 Bethesda classification system: no intraepithelial lesion or malignancy (NILM), atypical squamous cells-undetermined significance (ASC-US), atypical glandular cells (AGC), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), atypical squamous cells-cannot exclude HSIL (ASC-H), adenocarcinoma in situ (AIS), squamous cell carcinoma (SCC), and adenocarcinoma (ADC). Laboratory workload standards, quality control practices, and cytology-histology correlation reviews were all performed in accordance with current CAP Laboratory Accreditation Program checklists. All Pap test screenings and final interpretations were performed by pathologists in the Department of Pathology at OGHFU.χ2 test was used to compare the ratios among the groups. The Kruskal–Wallis test and post hoc pairwise test were used to compare vial loads among groups. Logistic regressions were performed to calculate the odds ratio (OR) and 95% confident interval (CI) to assess the association of HPV subtypes and age categories with CIN2+ histopathological results . The results were visualized by the R program using the ggplot2 package (R Core Team). Two-tailed P < 0.05 was considered statistically significant.Statistical analyses were performed by using IBM SPSS Statistics 20 software . The In total, 73,596 outpatient cases were included. The age of these women ranged from 13 to 94 (mean age: 39.0 ± 11.6) years old (yo). A total of 64,534 women had Pap cotest results, of which 9.9% had low-grade cytological abnormalities (LSIL or ASC-US), and 1.3% had high-grade cytology . A total of 17,394 women had a histological diagnosis within 6 months after HPV testing, of whom 7.7%  had CIN2 or worse (CIN2+) lesions, and 92.3%  had less than CIN2 lesions (not CIN2+), including 17.9%  with CIN1.χ2 = 449.390, P < 0.001), and the multiple-infection rate of hr/phrHPV was 36.6%, 27.0%, 20.7%, 23.5%, 32.1%, and 37.0%, respectively  . When potential hrHPVs were added, the overall infection rate was 23.2% , and 26.3%  of which had multiple infections with as many as 9 types of hr/phrHPV. The prevalence of each genotype in descending order was HPV52>16>58>53>56>39>51>66>68>59>33>18>31>35>45>82>73>26 . In hr/p< 0.001) . Women aIn patients with CIN2+, the prevalence of hrHPV and hr/phrHPV was 87.3% (1142/1308) and 88.9% (1163/1308), respectively. The prevalence of each genotype in CIN2+ patients in descending order was HPV16>52>58>33>31>18=53>56>51>39>59=66>68>35>26=45>82>73, and among which, the single-infection rate in descending order was HPV16>58>52>33>18>31>51>56>39=26=45>53=68=82>35>59=66>73 . HPV16, In hr/phrHPV-positive cases with CIN2+, the descending order of the proportion of multiple-infection was HPV73>53>66=59>68>39>56>35>51>82>26=45>52>33>31>58>18>16. Furthermore, only HPV16, 18, and 58 were more likely to be included in single-infection than multiple-infection in patients with CIN2+, while all other types were the contrary. And HPV73, 53, and 66(=59) were the top three genotypes most likely to be included in multiple infections .P < 0.001), indicating that mixing with other genotypes alleviated its pathogenicity. HPV52, 53, 56, 51, 39, 66, 59, 68, and 35 showed the opposite pattern (P < 0.001), indicating that they were less likely to be pathogens individually. All other types showed no significant differences, indicating the capability of causing diseases independently  for CIN2+, followed by 18, 35, and 58 . In the To date, 20–50% of HPV-positive women have been reported to be infected with multiple HPV types , 30–32. A study showed that clade A9  was significantly less likely to be involved in multiple infections than all other clades [In agreement with the previous research, we found that patients with single infection of HPV16 had higher incidences of CIN2+ than those with multiple-infection pattern , indicatStudies have shown that HPV16 is the most frequently detected type and often presents with a higher viral load in cervical squamous cell carcinoma, suggesting that it could be responsible for the pathogenesis of the lesions in the majority of cases . In the In conclusion, extensive genotyping identified the 3 most frequent genotypes, HPV16, 58, and 52, in CIN2+ of Chinese population. Women aged younger than 25 years and older than 65 years were most prone to multiple infections. Among the hr/phrHPV-positive CIN2+ cases, HPV73, 53, and 66 (=59) were the top three genotypes most likely to be included in multiple infections, and HPV16, 18, and 58 were the 3 least. HPV26, a potential hrHPV genotype, showed a higher OR for CIN2+ lesions than most traditional hrHPV genotypes. When HPV16 mixed with other genotypes, its pathogenicity was alleviated. The vial load and the percentage of HPV16 showed a positive correlation with the severity of squamous lesions, supporting the value of extensive genotyping and viral titer quantitation. However, there are still some limitations in this study. Most of the patients in our study were from East China, and a few were from Northeast, Central, and South China. So, it cannot represent the overall prevalence of China. In addition, the number of cases of some genotype infection in CIN2+ is small, resulting in certain limitations of some results. More cases need to be accumulated for research in the future.


HPV16 causes approximately 60% of uterine cervical cancer and 95% of HPV-driven oropharynx cancers. Despite both being HPV-associated, these tumors are very different. We directly compare integration of the HPV16 genome into the human genome in these two diseases, finding that the viral gene E2 is frequently lost in cervical cancer, but usually maintained in oropharyngeal cancer. We also found that oropharyngeal cancers with integration have many more integration sites per tumor and these more frequently occur in genomic regions with a high density of genes.Squamous cell carcinoma of the oropharynx caused by HPV type 16 (HPV16+ OPSCC) is the most common HPV-associated malignancy in the USA and has many molecular differences from uterine cervical squamous cell carcinoma (UCSCC). Our understanding of HPV oncogenesis relied on studies of UCSCC revealing a consensus model reliant on HPV integration with a loss of E2. Here, we compare patterns of HPV integration in UCSCC and OPSCC by analysis of affinity capture sequencing of the HPV16 genome in 104 OPSCC and 44 UCSCC tumors. These cohorts were contemporaneously sequenced using an identical strategy. Integration was identified using discordant read pair clustering and assembly-based approaches. Viral integration sites, structural variants, and copy losses were examined. While large-scale deep losses of HPV16 genes were common in UCSCC and were associated with E2 loss, deep copy losses of the HPV16 genome were infrequent in HPV16+ OPSCC. Similarly, structural variants within HPV16 favored E2 loss in UCSCC but not OPSCC. HPV16 integration sites were non-random, with recurrent integration hot-spots identified. OPSCC tumors had many more integration sites per tumor when compared to UCSCC and had more integration sites in genomic regions with high gene density. These data show that viral integration and E2 disruption are distinct in UCSCC and OPSCC. Our findings also add to growing literature suggesting that HPV tumorigenesis in OPSCC does not follow the model developed based on UCSCC. Papillomaviruses are species-specific, non-enveloped, double-stranded DNA viruses with an about 8kb circular genome protected by a 55 nm icosahedral capsid ,2,3,4. HCervical cancer, the second most common cancer in woman and with a gradually decreasing incident rate in the United States, is usually associated with HPV. Development of cervical cancer is a multistep process that includes initial HPV infection followed by persistent infection resulting in a relatively well-established progression from pre-cancerous cervical intraepithelial neoplasia (CIN) to malignant lesions . In earlAs the opposite of cervical cancer because it is constantly increasing in the USA, oropharyngeal cancer is detected mostly in men, with male-to-female ratio of about 4:1, and roughly 80% of this disease is driven by HPV. HPV tumorigenesis in the oropharynx has not been as intensively investigated, and it most likely differs from cervical lesions due to unique features of tonsillar epithelia. The epithelia of the lingual and palatine tonsils, the regions where HPV-associated cancer arises , are lymTRAF3 and CYLD genes that are rare in uterine cervical cancer [In addition to epidemiological differences, although initiated by the infection of HPV, cervical and oropharyngeal malignancies are characterized by several biological and clinical dissimilarities that highlight distinct HPV-mediated carcinogenesis . First, l cancer . The NF-l cancer ,28,29,30l cancer .Both integrated and episomal HPV DNA occur in OPSCC and cervical malignancies, with some tumors containing only integrated HPV DNA, others containing only episomal DNA, and a third group containing both ,33,34,35Important differences between HPV driven oropharyngeal and uterine cervical carcinomas continue to emerge; however, no comprehensive study has compared HPV integration sites and HPV structural genomic aberrations present in these two cancers. To define similarities and differences, we used identical ultra-deep targeted sequencing of HPV16 from 104 oropharyngeal and 44 uterine cervical tumors. Here, we present an in-depth investigation that reveals drastic dissimilarities in HPV integrations and structural genomic aberrations in OPSCC and UCSCC. These findings emphasize their individual biology and suggest that HPV-driven carcinogenesis is different between these tumor types.Human Subjects—DNA sequencing of data was performed as a part of the UNCseq tumor sequencing program. Tumor sample identifiers and genomic data were derived from the clinical trial LCCC1108: Development of a Tumor Molecular Analyses Program and Its Use to Support Treatment Decisions. This IRB-approved trial opened in 2011. All studies were done with the approval of our Institutional Review Board, patient participation required written informed consent, and all studies were conducted in accordance with recognized ethical guidelines as described in U.S Common Rule. By means of a chart review of electronic medical records, demographic information was obtained for each study subject, including age, gender, race, and smoking history. The clinical stage at presentation according to the AJCC staging system (AJCC 8th edition) was recorded considering that many patients did not receive pathological staging.DNA Isolation, Library Preparation, and Sequencing—A pathologist examined H&E-stained slides from each case to confirm the diagnosis of squamous cell carcinoma. Automated DNA extraction was from FFPE tissue sections using the Promega Maxwell MDx16TM instruments (Promega) and then fragmented by sonication. Subsequent quality assessments were performed by ultraviolet absorbance and quantity assessments. During DNA isolation and library preparation, DNA concentration was measured by fluorometry and DNA quality was evaluated using the Agilent 2100 Bioanalyzer high sensitivity assay. DNA libraries were pooled for deep sequencing using an Illumina HiSeq2500TM sequencer. The UNCseq targeted sequencing platform involves sequencing exons of a custom list of 650 human genes (covering 3.4 M bases) and 10 pathogen genome segments in fixed or frozen cancer tissue and matched germline DNA from consenting local patients. This custom sequencing platform provided deep coverage of all HPV16 open reading frames. For general metrics on human gene sequencing quality, read depth, and coverage statistics, please see our recent report which reviewed these features of the data set [data set . ExcludiInclusion Criteria—The UNCseq database was queried for p16+ tumors originating from the anatomic oropharynx (tonsil or tongue base) with available tumor sequencing data as well as data on stage, treatment strategy, clinical outcome, and histopathology available. HPV16 positivity was confirmed by DNA sequencing reads which mapped to the HPV16 genome (see above comments). Patients were excluded from clinical analyses if tumors were not p16+. The UNCseq database was also queried for squamous cell carcinomas of the uterine cervix; HPV16 positivity was confirmed by DNA sequencing reads which mapped to the HPV16 genome . For both tumors from the oropharynx and uterine cervix, tumors with non-HPV16 genotypes were excluded from the study.Assigning HPV16 Positivity and Viral Copy Number Analysis—Raw reads were aligned to the human genome plus a comprehensive library of HPV virus sequences, using compiled reference sequences from the ViFi analysis pipeline [10(ReadsHPV16/ReadsHG19) > −4 was used as an empiric cut off for HPV positivity. This threshold was selected based on an obvious bimodal distribution in the data; see our prior publication justifying this criterion [10(ReadsHPV16/ReadsHG19) calculated as above was considered as a relative metric of the HPV16 copy number.pipeline . An HPV1riterion . The Samriterion . For HPVIntegration Site Calling—Raw reads were aligned to the human genome plus a comprehensive library of HPV virus sequences, using compiled reference sequences from the ViFi analysis pipeline [pipeline . The ViFpipeline . Breakpopipeline . AlignmeAssigning Gene Effects—The R SMITE [ R SMITE  and Geno R SMITE  packages R SMITE , we usedStructural Variant Calling and Analysis—We also considered structural variants which joined disparate aspects of the HPV16 genome. To distinguish these form HPV16–human structural variants , we will refer to these events as HPV–HPV structural variants. HPV–HPV structural variants were identified from the svABA analysis as described above. HPV–HPV structural variants were only considered if they passed svABA quality filters. Additional filtering to assess for biologically relevant structural variants was performed based on variant allele frequencies (VAFs) estimated by svABA. The thresholds for VAF filters are described in the results section.Viral Deep Copy Loss Analysis—Considering that HPV+ cancer cells often contain multiple copies of the viral genome, the loss of genomic material in some copies of the virus might result in a decreased copy number  of some viral genes. However, loss of genomic material in all or the vast majority of copies, or deep copy loss, would be expected to result in the most prominent functional differences. In this manuscript, we focused on copy number analysis on regions of deep viral copy losses, which represent the complete or nearly complete loss of the genomic regions in question. To identify these events, nucleotide-specific estimates of read depth were acquired using the Samtools depth function [function . Relativn = 104) and uterine cervical  cancers (see p < 5 × 10−8). Amongst tumors with HPV16 integration, OPSCC tumors had significantly more integration sites per tumor (see p < 0.001). Integrations in areas of the genome with the highest gene density (top 5%) were also enhanced in OPSCC as compared to UCSCC . The regions of deep loss identified are displayed in We also investigated structural variants (SVs) within HPV16 itself (HPV–HPV SVs). These variants were identified by the svABA pipeline as passing the default quality filter, and were then further filtered by variant allele frequency to look for potentially biologically relevant variants A. There Taken together, these results indicate that both integration and HPV–HPV SVs are selected for E2 loss in UCSCC, but not OPSCC. Conversely, the higher frequency of integration sites per tumor and preferential integration in a gene-dense area of the genome, suggests that integration sites in OPSCC are selected for the oncogenic properties resulting from effects on the host genome.Our direct comparison of HPV integration in oropharyngeal and cervical tumors revealed several important dissimilarities supporting the idea that HPV-driven carcinogenesis is different between these cancers. Below, we summarize major points highlighting their discrete biology.The Different Role of E2 in HPV Carcinogenesis—Integration of the HPV genome into the human genome is an established hallmark of HPV-mediated carcinogenesis. Given that the HPV E2 protein negatively regulates expression of viral oncoproteins E6 and E7, another hallmark of this model is E2 loss. This HPV carcinogenesis model was developed from analysis of UCSCC, and our analyses of UCSCC are supportive since integration was detected in the vast majority and E2 was the HPV gene most commonly altered by deletion (30%) and SVs (40%). On the other hand, integration was less frequent in OPSCC, and even in integrated tumors, E2 loss by deep deletion or HPV–HPV SVs was significantly less frequent in OPSCC, where L2 was found to be the HPV gene most frequently altered by deletion and SVs  has been demonstrated in both OPSCC and UCSCC ,12 with MYC ,50, MYB 0 Kb has MYC [MYB , PVT1 [5YB [PVT1 ,52,53, R [RAD51B , EMBP1 [B [EMBP1 , CD274/P74/PD-L1 , and SOX2, FGFR2 , PD-L2 [2 [PD-L2 , TRAF3 [2 [TRAF3 , BRCA1, A1, SPOP , and BCLP [BCL-2  Our resu4A2, MYC , LRP1B [C [LRP1B ,62,63, a[MACROD2 . Unfortu[MACROD2  and UCSC[MACROD2 ,65. Our NLGN4Y , insertions of tandem viral repeats (Type II), and tandem repeats of hybrid viral–human DNA (Type III) . UnfortuOur study was limited in the ability to resolve some complexities of integration events, primarily due to the targeted affinity capture technique, which does not allow unbiased analysis of the copy number of small regions of non-target human genomic DNA, as is possible with whole genome sequencing. More specifically, because of this, we were not able to determine if the integration sites detected were related to chromosomal or extrachromosomal DNA, although other groups using whole genome sequencing have reported detecting human–HPV hybrid extrachromosomal DNA in OPSCC tumors .Clinical Implications of HPV16 Integration in OPSCC—We did not find any prognostic relationship to HPV16 viral integration in HPV+ OPSCC in this study. However, this is a frequently discussed association in the literature, with most studies suggesting that integration is a poor prognostic marker [c marker ,86, but c marker ,87. We nc marker . Interesc marker ,86, whilc marker ,36,87; tWe directly compared 44 cases of HPV16+ UCSCC and 104 cases of HPV16+ OPSCC. E2 loss was common in HPV+ UCSCC, mediated by frequent copy number losses or structural variant breakpoints. Conversely, E2 loss was not selected for in HPV+ OPSCC. Tumors with no evidence of integration represented ~25% of both UCSCC and OPSCC cases. Amongst tumors with genomic integration, OPSCC tumors had many more integration sites per tumor, and were more likely to have integration sites in high gene density areas of the genome. These data highlight clear differences between HPV16-associated UCSCC and OPSCC related to the physical state of the HPV16 genome, and there may be implications for distinct HPV-associated carcinogenesis in the two diseases.


We systematically reviewed the diagnostic accuracy of cervical cancer screening and triage strategies in women living with HIV (WLHIV).Cochrane Library, Embase, Global Health and Medline were searched for randomised controlled trials, prospective or cross-sectional studies published from database inception to 15 July 2022 reporting diagnostic accuracy of tests in cervical cancer screening and triage of screen-positive WLHIV. Studies were included if they reported the diagnostic accuracy of any cervical cancer screening or triage strategies for the detection of histologically-confirmed high-grade cervical intraepithelial neoplasia (CIN2+/CIN3+) among WLHIV. Summary data were extracted from published reports. Authors were contacted for missing data where applicable. Sensitivity and specificity estimates for CIN2/3+ were pooled using models for meta-analysis of diagnostic accuracy data. Study quality was assessed using the QUADAS-2 tool for the quality assessment of diagnostic accuracy studies. PROSPERO registration:CRD42020189031.2=65%I) for CIN2+ and 65.0%  for CIN3+; specificity for <CIN2 was 73.8% . Cytology was similarly variable . In 28 studies, sensitivity of tests targeting 14-HR-HPV types was high  for CIN3+); but specificity for <CIN2 was low . Restriction to 8-HR-HPV increased specificity  with no significant change in sensitivity . VIA triage of 14-HR-HPV positive women decreased sensitivity for CIN2+ compared to HPV-DNA test alone .In 38 studies among 18,737 WLHIV, the majority (n=19) were conducted in sub-Saharan Africa. The pooled prevalence was 12.0% (95%CI:9.8-14.1) for CIN2+ and 6.7% (95%CI:5.0-8.4) for CIN3+. The proportion of screen-positive ranged from 3-31% ; 2-46% ; 20-64% (high-risk[HR]-HPV DNA). In 14 studies, sensitivity and specificity of VIA were variable limiting the reliability of pooled estimates. In 5 studies where majority had histology-confirmed CIN2+, pooled sensitivity was 56.0% (95%CI:45.4-66.1; HPV-DNA based approaches consistently showed superior sensitivity for CIN2+/CIN3+ compared to VIA or cytology. The low specificity of HPV-DNA based methods targeting up to 14-HR-HPV could be improved significantly by restricting to 8-HR-HPV with only minor losses in sensitivity, limiting requirement for triage for which optimal approaches are less clear.World Health Organisation; National Cancer Institute; European Union's Horizon 2020 and Marie Skłodowska-Curie Actions programme. The diagnostic accuracy of cervical cancer screening strategies for CIN2+ detection is well established in the general population, but is less certain for women living with HIV (WLHIV). We conducted a systematic review of the evidence on the diagnostic accuracy of cervical cancer screening strategies for cervical precancer detection in WLHIV. We searched Cochrane Library, Embase, Global Health and Medline databases for studies reporting diagnostic accuracy of any cervical cancer screening strategy to detect cervical precancer  without language restriction from inception up to 15 July 2022 using search terms “cervical precancer” AND  AND “HIV”.2=32%I) compared to VIA  or cervical cytology . Despite the lower specificity of HPV-DNA based methods targeting up to 14 high-risk HPV genotypes , a restricted genotype approach targeting 8 high-risk HPV genotypes most associated with cervical cancer maintained high sensitivity for cervical precancer and increased specificity . VIA triage of 14-HR-HPV positive women decreased sensitivity compared to HPV-DNA test alone . Analysis of the sources of study heterogeneity using the QUADAS-2 tool highlighted the inferior performance of the commonly used visual inspection method compared to HPV-DNA test, linked with individual study design and variability in approaches to training and quality assurance for VIA.We found that HPV-DNA based tests had superior and more reproducible sensitivity  launched a global strategy to eliminate cervical cancer as a public health problem, which means to reduce the annual incidence below 4 cases per 100,000 women.Invasive cervical cancer is the second most common cancer and a leading cause of cancer-related death in women living in low- and middle-income countries.Cervical cancer screening strategies commonly used in resource-limited settings including visual inspection of the cervix or cervical cytology have shown variable diagnostic accuracy to detect cervical precancer due to the requirement for intensive training, equipment and quality assurance.,The diagnostic accuracy of cervical cancer screening strategies for CIN2+ detection is well established in the general population, but is less certain for women living with HIV (WLHIV). Previous reviews have summarised the diagnostic accuracy of cervical cancer screening strategies in WLHIVIn July 2021, the World Health Organization published an update to its guidelines for screening and treatment of cervical pre-cancerous lesions for cervical cancer prevention for women in the general population and women living with HIV. HPV DNA tests were suggested as the primary screening test rather than VIA or cytology for both the general population of women and women living with HIV.Our aim was to systematically review and conduct a meta-analysis of the diagnostic accuracy of cervical cancer screening tests for the detection of cervical precancer and above among WLHIV and conduct a structured assessment of study quality and sources of heterogeneity. Screening and triage tests evaluated included visual inspection using acetic acid [VIA], or Lugol's iodine [VILI], cervical cytology and HR-HPV-DNA tests [full and restricted genotyping]). Secondary objectives include evaluation of the modifying effect of HIV-related factors on the diagnostic accuracy of these strategies, and evaluation of the comparative accuracy of HPV-DNA tests compared to VIA or cervical cytology.We searched the Cochrane Library, Embase, Global Health and Medline databases for publications without language restriction . Reference lists of review articles and all articles identified in the systematic search were checked. The search was conducted up to 15 July 2022, without restriction on start date. All abstracts were screened and assessed for eligibility by one author (HK). Full-text copies of relevant publications were assessed for eligibility independently by two authors .Studies were included if they reported the diagnostic accuracy of any cervical cancer screening or triage strategies for the detection of histologically-confirmed CIN2+ or CIN3+ or invasive cervical cancer (reference or gold standard method for outcome ascertainment), irrespective of age at time of screening. Studies were included if there was histological verification of disease among any of: (i) colposcopy abnormal women only; or (ii) screen-positive women only (irrespective of screen test used); or (iii) all enrolled women . In studies that corresponded with (i) and (ii), women who did not have biopsy taken because they were either screen negative for one or more tests or colposcopy negative were considered as negative for CIN2+ (i.e. <CIN2).The index test could include any of the following: VIA (naked eye), VILI, VIA using digital cervicography (VIA-DC), combination VIA/VILI (either test positive), automated or assisted visual evaluation, cervical cytology and any HR-HPV DNA based tests targeting up to 14-HR-HPV types (HPV16/18/31/33/35/39/45/51/52/56/58/59/66/68) using either self-sample or provider-sample. A restricted genotype approach targeting 8-HR types (HPV16/18/31/33/35/45/52/58) was evaluated for tests that provide genotype level data as these types are most commonly associated with invasive cervical cancer, irrespective of HIV status.Eligible studies could include women with or without HIV, but must have provided data stratified by HIV status. For publications that reported results from the same cohort, but at different follow-up visits, the publication that gave the most complete set of results was included. There was no restriction on age at screening visit. From the consensus list, data were independently extracted by two authors  using a standardized form. In event of discordance, consensus was reached following detailed discussion with a third author (MA). Authors were contacted for missing data on diagnostic accuracy data where applicable.metadta, a procedure in Stata implementing bivariate fixed- and random-effects model for pooling of diagnostic test accuracy taking the intrinsic correlation between sensitivity and specificity into account and allowing inclusion of multiple covariates.metadta was applied to assess the impact of other covariates on diagnostic accuracy . Heterogeneity was quantified using the I2 measure.metaprop was used to perform meta-analyses of proportions  in Stata.Pooled sensitivity and specificity estimates were obtained using https://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42020189031.Study quality was assessed using the QUADAS-2 tool for the quality assessment of diagnostic accuracy studies.The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. or decision to submit the paper for publication. HK, IJ and MA had access to and verified the data, and HK was responsible for decision to submit for publication.The search yielded 8,622 publications  among wh,,The majority of studies were cross-sectional n=29) and conducted in Sub-Saharan Africa  when guidelines recommended initiation of ART at defined CD4+ T-cell count, but before the universal ART era (pre-2015) when ART is recommended at time of HIV diagnosis, irrespective of CD4+ T-cell count. The median CD4+ T-cell count ranged from 271 to 592 cells/µl. No studies reported prior HPV vaccination of enrolled WLHIV.,47,,[29,,,39,42,48,53,,36,42,48,26,30,34,,,47,,,30,,,,45,48,42,,,51,57,30,,,42,47,53,,36,,42,,,,55,49,45,Twenty studies39,47,, the majoThe range of quality judgements is summarised for 12 QUADAS items in Appendix, pages 9-14. For 14 studies evaluating VIA, a good quality score was given in 64% (108 of the 14*12 judgements). Low and unclear study quality scores  were noted in 16% and 20% of the judgements. In 23 studies evaluating cervical cytology, the corresponding proportions were 54%, 23% and 23% for high, low and unclear, respectively. In 28 studies evaluating HPV-DNA, the corresponding proportions were 64%, 18% and 18% for high, low and unclear, respectively. For studies evaluating VIA and cytology, the study quality was judged as low for the following items: quality control of index test, misclassification of disease avoided, partial verification avoided and representativeness of the included participants. For studies evaluating HPV-DNA tests, the study quality was judged as low for items related to misclassification of disease, partial verification and representativeness of the included participants.,,,39,,,,,,,2I=65%; In 14 studies,2=68%)I for CIN2+ (range: 57.5-100.0%) and 88.7% (2=52%I) for CIN3+ (range: 64.7-100.0). Specificity for <CIN2 was highly heterogeneous .Cytology ASCUS+ had pooled sensitivity of 85.1% (2I=45%) for CIN2+ and 92.5% (2I=32%) for CIN3+ (2=92%I) but increased with decreasing prevalence of HR-HPV among study participants  was similarly heterogeneous as it was for VIA alone . In all studies, VIA operators were reported to be blinded to the HPV test result. Similar findings were observed for cervical cytology following an HPV-positive test in 5 studies,In six studies,45,Compared to a scenario where women underwent screening with 14-HR-HPV-DNA test without triage in six studies,2I=6%), the proportion of women who underwent biopsy and histology verification was low (range: 8% to 23%). Cytology HSIL+ had the highest joint sensitivity and specificity in studies where the majority of women had histological verification of disease, and in settings with external quality assessment (EQA) programmes in place. In three studies with biopsy rate between 74% and 100%, two of which had established cytology programme in place,2=15.8%I) and specificity was 89.0%  (data not shownI).The main variation in study methodology for studies evaluating VIA and cytology was linked to endpoint ascertainment . Sub-group meta-analyses were conducted according to the proportion of women with cervical biopsy and histology verification of disease, as previously described for VIA. Few studies,The accuracy of HPV-DNA test was influenced by participant selection, namely factors associated with immunosuppression . In five studies that allowed comparison of the diagnostic accuracy of HPV-DNA tests by HIV status,50In age-stratified analyses, sensitivity of VIA for CIN2+ was lower in older compared to younger women in two studiesIn this review of studies evaluating the performance of cervical cancer screening approaches for cervical precancer detection in WLHIV, we found that HR-HPV-DNA tests demonstrated consistently higher sensitivity and identified more cervical precancer cases than VIA or cervical cytology. However, approximately half of all WLHIV had a positive HR-HPV-DNA result, with greatest proportion in sub-Saharan Africa settings where HR-HPV prevalence is highest,,,Of the estimated 19.3 million WLHIV in 2020,Although HR-HPV-DNA tests had the highest sensitivity of all screening approaches to detect CIN2+/CIN3+, its low specificity presents a challenge in a high HPV prevalence setting. An analysis evaluating the relationship between HR-HPV prevalence and the specificity of HR-HPV DNA testing to rule out CIN2+ reported that for a 10% increase in HR-HPV prevalence, HPV-DNA test specificity decreased by 8%.Cervical cytology had variable sensitivity and specificity as observed for VIA, possibly resulting from differences in operator experience, training and implementation of EQA. In settings with cytology-based screening programmes incorporating EQA, HSIL+ had the best combination of sensitivity and specificity and may be an option for triage of HR-HPV positive WLHIV.This is the first review to estimate the diagnostic accuracy of commonly available cervical cancer screening strategies for WLHIV and has investigated the sources of heterogeneity in performance, in particular the association of HIV related factors on HPV-DNA test specificity. This review has several limitations. Due to the very large methodological and statistical heterogeneity observed in the studies we reviewed, we cannot consider the pooled accuracy estimates of VIA or cytology as reliable. We did however attempt to adjust for differences in gold standard verification approaches in stratified analysis. Further, the relative accuracy estimates comparing screening tests are less likely to be influenced by these methodological differences as only those studies that conducted direct head-to-head comparison of tests were included. We were also unable to assess the impact of quality of training and QA on the diagnostic accuracy of VIA as this was not consistently reported across studies. Few (20%) studies reported on a priori sample size calculations to estimate precision and power to achieve study objectives, an essential item for reporting of diagnostic accuracy studies according to the 2015 Standards for Reporting Diagnostic Accuracy (STARD) statement,Of the current screening strategies available, high performance tests such as HR-HPV-DNA tests demonstrated consistently higher sensitivity than VIA or cytology among WLHIV. Despite the lower specificity of HPV-DNA based methods targeting up to 14 HR types, limited evidence suggests that a restricted genotype approach may increase specificity, resulting in fewer referrals and clinic visits thereby reducing inconvenience to women and costs to the service. With earlier ART initiation and HIV virological control in the universal ART era, WLHIV may maintain a functionally complete mucosal immune response leading to a more rapid clearance of HR-HPV. Consequently, the diagnostic accuracy of HPV-DNA based tests may have improved specificity as observed in the general population. Optimal triage options for HPV-positive WLHIV remain unclear. More evidence is needed on the use of molecular diagnostic tools alone or in combination with visual methods or artificial intelligence among WLHIV, particularly in resource-constrained settings. Greater efforts are needed to make current diagnostic technologies more accessible and affordable in settings where the risk of cervical cancer and its associated morbidity and mortality are greatest,HK, NB, SD, MA and SdS conceptualised the study, and developed the research protocol; HK and IJ identified articles for full-text review; HK and IJ and MA extracted data from studies that matched inclusion criteria; HK, IJ and MA had access to and verified the data; MC, SG, HS, XX and PM provided raw data for additional analyses; HK and MA did the statistical analyses with input from MC, NB and SD; HK, IJ, MC, PaM, SG, HS, XX, MS, NB, PM, SD, MA and SdS contributed to the writing of the manuscript; HK was responsible for decision to submit for publication.The dataset is available at Mendeley online repository at DOI: 10.17632/cn53hzsh5p.2.SdS is a member of the Data and Safety Monitoring Board of a screening trial in Zambia led by IARC evaluating cervical precancer recurrence after treatment and is on the Advisory Board of the HPV Prevention and Control Board. All other authors declare no competing interests.


Providing adequate information to parents who have children eligible for human papillomavirus (HPV) vaccination is essential to overcoming vaccine hesitancy in Japan, where the government recommendation has been suspended. However, prior trials assessing the effect of brief educational tools have shown only limited effects on increasing the willingness of parents to vaccinate their daughters.The aim of this trial is to assess the effect of a cervical cancer survivor’s story on the willingness of parents to get HPV vaccination for their daughters.In this double-blinded, randomized controlled trial (RCT) implemented online, we enrolled 2175 participants aged 30-59 years in March 2020 via a webpage and provided them with a questionnaire related to the following aspects: awareness regarding HPV infection and HPV vaccination, and willingness for HPV vaccination. Participants were randomly assigned (1:1) to see a short film on a cervical cancer survivor or nothing, stratified by sex  and willingness for HPV vaccination prior to randomization (yes vs no). The primary endpoint was the rate of parents who agreed for HPV vaccination for their daughters. The secondary endpoint was the rate of parents who agreed for HPV vaccination for their daughters and the HPV vaccination rate at 3 months. The risk ratio (RR) was used to assess the interventional effect.Of 2175 participants, 1266 (58.2%) were men and 909 (41.8%) were women. A total of 191 (8.8%) participants were willing to consider HPV vaccination prior to randomization. Only 339 (15.6%) participants were aware of the benefits of HPV vaccination. In contrast, 562 (25.8%) participants were aware of the adverse events of HPV vaccination. Although only 476 (21.9%) of the respondents displayed a willingness to vaccinate their daughters for HPV, there were 7.5% more respondents in the intervention group with this willingness immediately after watching the short film . In a subanalysis, the willingness in males to vaccinate daughters was significantly higher in the intervention group ; however, such a difference was not observed among females . In the follow-up survey at 3 months, 1807 (83.1%) participants responded. Of these, 149 (8.2%) responded that they had had their daughters receive vaccination during the 3 months, even though we could not see the effect of the intervention: 77 (7.9%) in the intervention group and 72 (8.7%) in the control group.A cervical cancer survivor’s story increases immediate willingness to consider HPV vaccination, but the effect does not last for 3 months. Furthermore, this narrative approach to parents does not increase vaccination rates in children eligible for HPV vaccination.UMIN Clinical Trials Registry UMIN000039273; https://tinyurl.com/bdzjp4yf To eliminate cervical cancer, the World Health Organization (WHO) set a future goal that 90% of girls worldwide would be vaccinated for the human papillomavirus (HPV) by the age of 15 years, by the year 2030 . As a reIn many countries, vaccine hesitancy is a crucial public health issue that governments continue to struggle with ,10. VaccProviding brief scientific information regarding HPV vaccination through websites has been shown to be an effective way of disseminating the importance of vaccination , with thThe aim of this study is to assess whether a cervical cancer survivor’s story could change parents’ minds about HPV vaccination. We plan to build up the theory of a better way to change people’s minds and behaviors to overcome vaccine hesitancy. Little is known about evidence-based interventions promoting the prevention of HPV, so this trial could provide a novel insight into digital educational methods for promoting disease prevention through vaccination to the general public.A total of 2175 participants were recruited in March 2020 via a webpage dedicated to this trial. These were registered members of the research panel owned by NTT Com Online Marketing Solutions Corporation  and used for consumer satisfaction, marketing, and academic research . More thBasic information about the benefits and adverse effects of HPV vaccination was impartially provided to all participants on a 2-slide handout prior to randomization, with the aim of sharing scientific nonnarrative information. On the first slide, the perceptions of the Japanese Ministry of Health, Labour and Welfare (MHLW) regarding adverse effects after receiving HPV vaccination was provided . The fol“Most vaccinated people experience pain and swelling on the arm where the shot is given.”“Severe adverse effects, such as unspecified body pain and sudden loss of strength, are rarely reported and are thought to be due to a functional disorder with unknown specific cause.”“Healthcare providers can access information on the website of MHLW regarding individuals who received the shot and developed continuous adverse effects.”On the second slide, 3 points regarding cervical cancer and HPV vaccination were provided:“In Japan, approximately 10,000 people a year are diagnosed with cervical cancer, while about 3,000 people die annually.”“Many developed countries recommend HPV vaccination in adolescence as a national prevention program.”“The HPV vaccine, which the World Health Organization (WHO) reports to be safe, can lead to the prevention of 70% of cervical cancers and other HPV-related cancers .”In this study, a 4-minute-long short film was used as the intervention. In this film, a cervical cancer survivor who had undergone radical hysterectomy with bilateral salpingo-oophorectomy and pelvic lymphadenectomy talked about her experience—from the beginning of the diagnosis to the sequelae of first-line therapy. She was a singer, songwriter, and actress in Japan. Seven messages were inserted as subtitles throughout her talk. The added subtitles were as follows:“She was diagnosed with cervical cancer at 31 years old.”“In Japan, approximately 10,000 people a year are diagnosed with cervical cancer and about 3,000 people die annually.”“Undergoing surgery wasn’t the end of suffering.”“The burden of cervical cancer was greater than expected and is relatively seen in younger ages.”“Cervical cancer can be prevented by HPV vaccination and Pap smears.”“A lot of children have lost their chance to prevent cervical cancer in Japan.”“Why don’t we take action for our children’s future?”We did not use any psychosocial theoretical frameworks to manipulate the participants’ mindset or behavior. The short film was produced by Ideas and Effects, Ltd., and supervised by the first author (YS) and the last author (EM), for use in educational campaigns.Participants were randomly assigned (1:1) to each group using a web-based randomization procedure. The randomization with minimization was stratified by sex  and willingness toward HPV vaccination prior to randomization (yes or no). Randomization was performed using the web research system of the NTT Data Institute of Management Consulting, Inc. The participants and investigators were double-blinded to the study distribution. Once the upper limit of each stratum was reached, new participants could not be added to the web system. This ensured a uniform distribution of stratification factors. In the intervention group, we provided the narrative short film prior to answering questions related to the willingness to prevent HPV, following consent for the online study.The participant demographics included sex , age group , willingness to use HPV vaccination prior to randomization (yes or no), marital status, number of children, education, household income, and tobacco use. The individual background information already existed in the research panel database prior to our study, except for the level of willingness to use the HPV vaccine. Information regarding the HPV vaccination history of respondents and a previous Papanicolaou (Pap) test for female participants was also collected for this study. In total, data of marital status, household income, and education level were collected from 1550 participants.The participants completed a 7-item awareness questionnaire to determine HPV awareness as background information. They were instructed to answer either “Yes, I have heard of it” or “I haven’t heard of it” for each question. We defined those who answered “I haven’t heard of it” for all questions as the no-awareness group, whereas those who answered “Yes, I have heard of it” for at least 1 question were defined as the normal-awareness group.The awareness questions (AQs) were as follows:AQ 1. It is possible to detect both cancer and precancerous lesions through cervical cancer screening.AQ 2. Sexual experience is associated with HPV infection.AQ 3. Cervical cancer screening is necessary for women even after vaccination.AQ 4. I have heard of the benefits of the HPV vaccine.AQ 5. I have heard of the adverse events associated with the HPV vaccine.AQ 6. HPV can cause anal cancer and oropharyngeal cancer in males, other than cervical cancer in women.AQ 7. HPV vaccination is included in the national immunization program (routine vaccination schedule) and publicly funded for children from sixth-grade elementary school students to first-grade high school students (12-16 years of age). However, the MHLW suspended a proactive recommendation for HPV vaccination due to the suspicious relationship between the vaccine and unspecific chronic pain.The willingness questions (WQs) after the intervention were as follows:WQ 1. Would you consider getting your daughter vaccinated against HPV? (Yes/No/I’m not sure)WQ 2. Would you consider undergoing a Pap smear? If male, would you want your family member or partner to undergo a smear test? (Yes/No/I’m not sure)WQ 3. Do you think the HPV vaccination program should be actively recommended by the government? (Yes/No/I’m not sure)WQ 4. Do you plan to inform family members, friends, or others about cervical cancer prevention and screening ? (Yes/No/I’m not sure)WQ 5. Are you going to make an appointment for HPV vaccination for your daughter as soon as possible? (Yes/No)The follow-up questions (FQs) after 3 months were as follows:FQ 1. Has your daughter been vaccinated against HPV after the first-round questionnaire? (Yes/Only an appointment/No)Participants who answered “No” were subsequently tasked to answer the following items:FQ 1’. Would you consider getting your daughter vaccinated against HPV? (Yes/No/I’m not sure)FQs 2-5 were identical to WQs 2-5.Participants who answered “Yes” or “Only an appointment” in FQ 1 were tasked to answered only FQs 2-4.χ2) test, Student t test, and risk ratio (RR) were used for statistical analyses of background characteristics, baseline knowledge level, and primary/secondary outcomes. The background characteristics and baseline knowledge between both groups were not significantly different, so we did not adjust the present variables. All statistical analyses were performed using SPSS Statistics version 28 (IBM). The sample size was calculated as 80% power to detect a 10% effect in the intervention group (increased from 25% in the control group to 35% in the intervention group) with a 2-sided P value of .05. Statistical significance was set as less than .05. The hypothetical baseline willingness rate was determined based on our previous studies; 23.6% of participants who were provided with information regarding the benefit of HPV vaccination considered getting their daughter vaccinated [The chi-square . The trial registration number was UMIN000039273. We received research funding from the Japan Agency for Medical Research and Development (Grant 19ck0106369h0003). The website construction and web-based survey were outsourced to the NTT Data Institute of Management Consulting, Inc. The participants received JPY 25 (~US $0.23) and an additional JPY 5 (~US $0.045) if they joined the follow-up survey.A total of 2175 participants were recruited. Stratifying factors, such as sex and willingness to undergo HPV vaccination prior to randomization, were evenly allocated . The retFor AQ1 to AQ7 on HPV and HPV awareness, there were no significant differences in the recognition rates across all 7 questions between the intervention and control groups . Only 33Only 476 (21.9%) parents displayed a positive attitude toward HPV vaccination for their daughters (WQ 1). Compared to parents in the control group, an additional 7.5% parents responded affirmatively in the intervention group ; see The comparison of attitudes toward HPV vaccination and awareness regarding the prevention of cervical cancer according to sex are shown in P<.001).Differences between sexes were identified in all questions. Fathers were more likely to have an affirmative attitude toward HPV vaccination for their daughters in the intervention group . Additionally, fathers in the intervention group were more likely to have affirmative attitudes regarding other areas of awareness, such as undergoing a Pap smear, desiring the recommendation from the government, and disseminating HPV vaccination information to someone by social media . In an oIn addition, 650 (71.5%) of 909 mothers knew at least 1 of the items regarding HPV vaccination, while only 508 (40.1%) of 1266 fathers were aware of at least 1 item . HoweverP=0.497). We could not find any effect of the intervention on the subsequent 4 questions (FQs 2-5), although the effect was seen in the first survey.At the follow-up survey after 3 months, 368 (16.9%) parents did not answer the online survey . The remThis web-based RCT showed that showing a 4-minute-long film on the story of a patient with cervical cancer increases parents’ willingness to consider HPV vaccination for their daughters. Furthermore, the willingness to undergo cervical cancer–screening tests and to disseminate HPV vaccination–related information about what they saw and felt also increased in the intervention group. These effects were observed in the fathers’ cohort but not in the mothers’ cohort. This result was similar to our prior RCT, which showed that a brief educational tool using the importance of HPV vaccination increases the willingness to consider the vaccination for their daughters and sons , so it sSimilar to the prior RCT, a possible reason the educational intervention was more effective among men could be that women had more existing awareness about HPV and HPV vaccination and had more negative sentiments against HPV vaccination . In factIn the follow-up survey, we did not find a significant intervention effect on HPV vaccination for the participants’ daughters. There may be some reason for this. First, this awareness change happened to just 1 parent. The decision-making process with another parent or daughter would be needed to take an action for vaccination. Therefore, this type of indirect intervention could have some limitations in its impact. Surprisingly, a total of 8.2% of the participants had their daughters vaccinated after participation in this trial, which was higher than the general HPV vaccination rate in Japan at the time of the study ,6. The iAlthough face-to-face educational approaches with parents might be an effective way to improve awareness and understanding of the vaccination , we needTwitter ,31,32, IAlthough this study displayed a promising effect on overcoming vaccine hesitancy, we need to consider several limitations. First, the cohort of the specified internet survey population used in this study may have some selection bias. The cohort has a higher educational background and is wealthier than the general Japanese population; approximately JPY 5.16 million is the average household income . AdditioSecond, interventions using this kind of awareness material are usually not universal, that is, the effect may vary when other materials are used. In particular, the effect might change depending on the individuals in the film, the content of the anecdotes, and the length of the film.Third, although this trial was designed to assess the sustainability of awareness change and concrete behavior for HPV vaccination, we could not incorporate the actual vaccination records into our research. Therefore, the reported vaccination rate might be different from the actual vaccination rate.Fourth, there was some potential social desirability bias that affected the effectiveness of the intervention at the first survey; on the contrary, the effect could not be seen in the follow-up survey. We have to take a look at the effectiveness of the film, considering the existence of a bias away from null.Given that this trial implemented under the suspension of proactive recommendation by the Japanese government, we have a tentative plan to conduct the same trial after resuming the national vaccination program. In addition, there were potential negative impacts in the study period, which was carried out during the COVID-19 pandemic. A lockdown was not declared in Japan during the study period; however, a portion of participants might have refrained from going to the hospital for nonurgent vaccination.This study demonstrated a positive immediate effect on the willingness for HPV vaccination in parents who have daughters, following intervention using a short film on cervical cancer, especially among fathers. Such an approach is promising for overcoming the hesitancy toward HPV vaccination. Additionally, this RCT showed the importance of the father’s role in improving the HPV vaccination rate and overcoming vaccine hesitancy.An anecdotal cervical cancer survivor’s story increases the willingness of Japanese parents to consider HPV vaccination for their daughters. However, this type of intervention might not sustain their motivation months afterward.