To examine the psychosexual impact and disclosure experiences of women testing HPV‐positive following cervical screening.In‐depth semi‐structured interviews.Interviews were conducted with 21 women of screening age (i.e. those aged 24–65 years) in England who self‐reported testing HPV‐positive in the context of cervical screening in the last 12 months. Data were analysed using Framework Analysis.The sexually transmitted nature of HPV, and aspects relating to the transmission of HPV and where their HPV infection had come from, had an impact on women's current, past and future interpersonal and sexual relationships. Most women had disclosed their HPV infection to others, however the factors influencing their decision, and others' reactions to disclosure differed. The magnitude and extent of psychosexual impact was influenced by how women conceptualized HPV, their understanding of key aspects of the virus, concerns about transmitting HPV and having a persistent HPV infection.Increasing knowledge of key aspects of HPV, such as its high prevalence and spontaneous clearance, and the differences between HPV and other STIs, may increase women's understanding of their screening result and reduce any negative psychosexual consequences of testing HPV‐positive. Referring to HPV as an infection that is passed on by skin‐to‐skin contact during sexual activity, rather than an STI, may help to lessen any psychosexual impact triggered by the STI label. Quantitative research suggests that psychosexual distress is elevated among women testing HPV‐positive.Psychosexual concerns include where an HPV infection came from and the potential to transmit HPV to a sexual partner.Some women have concerns about disclosing HPV to a sexual partner, partly because of the stigma associated with having an STI and uncertainty about how their partner would respond.This study is the first to explore psychosexual and disclosure‐related outcomes in‐depth among women testing HPV‐positive.The sexually transmitted nature of HPV impacted women’s current, past and future interpersonal and sexual relationships.Women’s knowledge and conceptualisation of HPV influenced their response to testing positive.Infection with high‐risk human papillomavirus (HPV) is the cause of virtually all cases of cervical cancer  who self‐reported having tested HPV‐positive  in the context of cervical screening in the last 12 months. Women were eligible to take part in the study if they spoke English and were able to give informed consent.Ethical approval was obtained from University College London's Research Ethics Committee (6930/003).https://www.sarosresearch.com/), a market research recruitment agency with a database of over 300,000 participants. All women aged 24 to 65 years and living in England were invited by email to take part in the study . To assess eligibility for the study and enable recruitment of a range of women with different characteristics, women expressing an interest in the study completed a questionnaire assessing age, relationship status, ethnicity, education level, cytology result and HPV knowledge. In addition, two items from the HPV Impact Profile assessed sexual impact following their most recent cervical screening result , single (n = 10) and one woman who was dating/in a casual relationship. Women reported that they were HPV‐positive with normal cytology (n = 10) or HPV‐positive with abnormal cytology (n = 11). Participant characteristics are shown in Table Interviews were carried out with 21 women  and lasted from 21–70 minutes. Participants included women who were in a relationship …I was absolutely panic‐stricken because I thought, “Oh my God, this sounds awful. I've got a sexually transmitted disease at my age. Where have I got this from?” You know. Ohhh! I was a bit shocked cos I thought, “Well, I'm with the same person that I've been with for like a long time”. Women felt worried and concerned when they received their HPV‐positive result, because they did not know what HPV was or were concerned about developing cancer. Some felt shocked, surprised or panicked at having an STI because they had been with their current partner for several years, or had not had unprotected sex and questioned whether their result was correct because they were not ‘that sort of person’..I think it's just because you just hear so many things, don't you, of unprotected sex and you know you shouldn't do it, but I did .With future pregnancy…I think it makes you worry that everything will be alright, and then it kind of puts this weight on you if it doesn't go, you feel that it could be, you know, your fault somehow Women blamed themselves for acquiring HPV because they had had unprotected sex or had several sexual partners around the same time, or felt that potential future issues regarding fertility or pregnancy could be viewed as their fault:Women described feeling ‘dirty’, ‘horrible’, ‘grim, and infected and nasty’ when they received their result and commented on the stigma or negative connotation that is associated with ‘…anything that's sort of a bit sexually orientated’..‘I felt a bit dirty with it, like it felt like I did have some kind of infection’ .…this stereotype of someone that get, that gets HPV, people that have unprotected sex and lots of partners. And because that wasn't the case with me I think that brought up the shame. It's like oh, they probably think I'm promiscuous or… Do you know what I mean? So it was definitely uncomfortable Embarrassment and shame at having something acquired through sexual activity was mentioned, often where women conceptualized HPV as an STI or something they had got through unprotected sex. Feeling ashamed was described because they associated STIs with promiscuity, or felt that information provided about HPV implied this, and some questioned how they had got HPV when they had not had ‘loads of partners’ and ‘didn't sleep around’. The stereotype of someone who gets HPV differed from women's view of themselves which made them feel uncomfortable:.…yeah, ‘cause that's different, it takes away that kind of like dirty, shameful stigma of it I guess, if you know, knowing that it's not something that's kind of been caught or picked up. It was just a change in cells, it was, yeah. You can associate that differently I guess Receiving an HPV‐positive result felt different from receiving an abnormal cytology result due to its sexually transmitted nature and the stigma associated with this:Embarrassment and shame were mentioned by women regardless of their relationship status..…I'm now thinking like well who did I get it from, who did I, who did I pick it up from. It must have been, you know. I'm, in my head I'm thinking it must have been somebody recently because otherwise it would have been picked up on my first two smears A common response was to try to understand where an HPV infection had come from. Women in long‐term relationships were particularly confused about this. Some assumed that the infection had come from a current or recent sexual partner because previous cervical screening test results had been normal, or HPV had not been found:.I didn't know anything about it and I was quite intrigued… not intrigued by it, but I was like, oh, I wonder why I've got it, you know, or how because at that point I was like… I'd not had sex for a few years, couple of years Others felt unable to determine which sexual partner had given them HPV. Women not in a relationship questioned how they had got HPV as they had not recently been sexually active:.Um with the previous relationship um when I first had the screening and the results, it definitely did have an impact and I think that… I suppose quite young at that point and because of the lack of understanding of it, you just… you are not sure. I suppose trust plays a part in it and it makes like your partner reflect on your history and how did you get it and um did I give it or did you have it before, those sorts of questions… Women's attempts to understand the source of their HPV infection led to issues around trust of their partner, particularly among women who lacked knowledge about HPV, and this had the potential to have an impact on their relationship:…my immediate reaction to him was, “Well, what have you been up to?” [laughter] and I just thought, “Well, you've got it off somebody. It's not off me”…I was a bit like… you know, “have you been cheating on me?” [laughter]. Some women trusted their partner and did not suspect any infidelity, however others raised concerns that their partner had been unfaithful and questioned whether that was how they had acquired HPV:Questioning partners about whether they were the source of an HPV infection had an impact on relationships because partners were unhappy about being accused of infidelity, which led to tension or arguments. For one woman, perceiving her partner to be the source of her HPV infection was having an ongoing impact on her relationship..…I said to the nurse “oh am I still allowed to have sex whilst I've got this?” and she was like “yeah of course you are” and then I was like but am I gonna pass it on to someone, you know. So, erm, yeah, that, that would be my biggest concern There was uncertainty around whether it was possible to transmit HPV to a male sexual partner. Women reported feeling ‘guilty’ and worried about potentially having transmitted HPV and were concerned that there was no way of their partner knowing if they had HPV. Concern about transmitting HPV led one woman to question whether she was ‘allowed’ to have sex:.…I wouldn't like to think that I did and then if we broke up, they pass it on to someone else. I wouldn't like to think that's sort of… you know, it was a never‐ending cycle and someone else could be in the position that I'm in in the future Concerns about HPV harming or having an impact on their partners' health were predominantly mentioned by women who had only received one HPV‐positive result. A lack of understanding about the possible impact of HPV resulted in partners being hesitant to have sex. Concern about transmitting HPV to a sexual partner and them passing HPV onto someone else in the future resulting in a ‘chain of people that could be affected’ was mentioned:Whether it was possible to be reinfected with HPV, either by their current, or a future sexual partner was queried, with condoms being used to prevent reinfection. Concerns about transmitting HPV to a sexual partner were mentioned by both women in a relationship and those not in a relationship..Well, we haven't had sex since. So that's a long time…Um, I think he is… I, I would, personally, um, but my boyfriend is unsure. He doesn't wanna catch anything and then resent me for it Having HPV had an impact on some women's sexual relationships or feelings or attitudes towards sex. Reduced interest in and frequency of sex were the changes most often described. Some stopped having sex completely because of concerns about transmitting HPV:.So, for the next few weeks, I really did avoid sex, erm because I just felt grim, and infected and nasty Self‐stigma also had an impact on women's interest in sex, affecting their confidence, self‐esteem and sexual self‐image:.…I was seeing someone at the time and I just kind of assumed that they had given it to me. Erm, and it probably actually ended our relationship… I had no interest like in that, like in having sex with him again Reduced interest in having sex with someone who they perceived was the source of their HPV infection was described and this led, in one case, to a relationship ending:Concerns that a partner might end a relationship because of reduced frequency of sex less was also mentioned.Other concerns about having sex included making abnormal cells or an HPV infection worse. Experiencing pain or discomfort during or after sex was mentioned, however it was felt that the impact this had on sexual pleasure was due to physical symptoms. HPV had a positive impact on one woman's relationship, improving communication and making her and her partner appreciate each other more.The impact of HPV on sex and sexual relationships was only mentioned by women who were in a relationship.Most women had disclosed their HPV‐positive result to a partner, friends or family. Some women who were HPV‐positive with abnormal cytology chose to focus on their abnormal cytology result because of the stigma associated with having an STI..‘I kind of expected my partner to say to me, “Who do you think you caught this from?” and to want to backtrack through my sexual history’ Disclosure was described as ‘awkward’, ‘hard work’ and explaining the circumstances surrounding acquiring HPV ‘embarrassing’ because of the sexually transmitted nature of HPV. Some women gave their partner their results letter to read because they found the letter informative or reassuring. The high prevalence of HPV was mentioned during disclosure. Disclosure was a ‘nerve‐racking experience’ as women were unsure how to approach disclosure and how their partner would respond and were concerned that might want to ‘walk away’ from their relationship or question how they got HPV:.…I certainly didn't want to cause erm anxiety in the relationship…So, I thought maybe on balance, I decided not to tell him. And he still doesn't know now… As it's something that your body can clear itself of quite quickly, or over a period of, you know, and it doesn't cause any symptoms, I didn't want him to think maybe I'd been having an affair [laughs] with someone or anything, which I wasn't, erm, but I just didn't want to create that atmosphere Several factors appeared to influence women's decision about whether to disclose their HPV infection to a sexual partner. Sharing health‐related information was considered normal practice for some. In contrast, others felt cervical screening was infrequently discussed and having HPV had not ‘come up’. Some women did not want others to worry about their HPV‐positive result or were concerned about the impact of disclosure on their relationship:One woman felt she would ‘probably’ tell future sexual partners but had not told her current partner.Some believed that HPV would not adversely affect their partner so there ‘isn't really any need to discuss it’. Uncertainty about whether it was possible to transmit HPV and the potential impact for men led to questions about whether disclosure was necessary. Conflicting information about disclosure online resulted in women feeling ‘quite confused and concerned about that area of my life’. Being told that HPV was sexually transmitted but receiving guidance with their results informing them that they did not need to tell their partner was ‘confusing and upsetting’..‘Erm, the letter where it said “You don't have to tell your partner,” when I read that line, I immediately thought “I would never do that, I would never not tell someone, they have a right to know.” So, instantly I thought I should tell him. I never thought, I never considered not telling him’ Although concerns about disclosure were expressed, worry about transmission or feeling that it was the right and thing to do, regardless of the guidance they had received with their results, sometimes resulted in disclosure:.‘I was like, “You haven't had any symptoms, you probably won't, but obviously keep an eye on it.” Erm and that was that really. He wasn't too bothered either because I think he was just the same as me; the people, the right people know about it, and if there's an issue, they'll find it at the next screening…’ There was little or no reaction from some women's partners when they disclosed their HPV infection, with women describing partners as being ‘pretty laid back about it all’:One woman was upset about her result and concerned about her partner's reaction, however he ‘…wasn't interested at all’ and was dismissive of her feelings.Partners ‘didn't have a clue what it [HPV] was’ and did not understand what the result meant. Knowledge of the HPV vaccine did not necessarily help them understand an HPV‐positive result.Partners were supportive and understanding following disclosure and concerned about the impact that HPV might have. Others were ‘taken aback’ and ‘overwhelmed’ and felt it was something they needed to get their ‘head ‘round’. Some partners responded defensively, or expressed concerns about transmission of HPV and the impact it might have.‘To be honest, it's not a joke, but we almost took it like that [laughter]. It didn't bother us, [laughs] because it was almost a bit like, “Oh, you've got an STI, ha ha,” it was kind of told like that. We did not, we did not see it as anything serious, erm, because I had not had any symptoms and yeah, it's quite a common thing to get, so it, we almost saw it as a bit of a joke’ .Some partners reacted with humour about the sexually transmitted nature of HPV. One woman who did not view HPV as ‘anything serious’described how she and her partner ‘almost saw it as a bit of a joke’:.‘And then later, he made this joke, erm ringing a bell and saying “unclean,” and I said to him, “I don't find that funny, I'm really not happy with this. ” And he was like, “Oh okay, sorry,” [laughs] and he actually made it, I told him about this research, and he went, “Oh, the unclean phone‐call,” and rang the little bell again. And I was like, “You're not funny, you're really not funny.”’ In contrast, another woman was upset by stigmatizing remarks her partner made about her having HPV:front .I'm just more concerned about just making sure that we use adequate protection, just for the benefit of both people, you know, myself and the guy I'm with, just to make sure that you know, you don't get anything. Not just HPV, but anything else as well, just you know, just be cautious on that Women, often those not in a relationship, questioned what they could do to prevent HPV transmission and avoid being re‐infected in the future, perhaps by using condoms:.…I would probably wait until September because, erm, my next smear is in September… And if I get a… like you know, like a negative result then, then I'd be happy to, but until then I don't, I don't feel like I want to, I don't feel like I'd want to at all… Erm, just in case I have still got it and then I pass it on to somebody else Others felt there was ‘no point’ as their partner had probably already been infected. Receiving an HPV‐positive result was described as ‘a wake‐up call’ and some women felt they would be more likely to use condoms in the future. To avoid transmitting HPV, waiting until their next screening test before having sex to see if their HPV infection had cleared was described:Asking a partner to have a sexual health check before a new sexual relationship was mentioned. For some women this was something they had done prior to having HPV but now felt it was even more important. Questions were raised about HPV testing for men and whether HPV testing was part of a sexual health screening. Women felt that if there was an HPV test for men, they might be able to prevent passing on the infection to women..Initially I probably thought oh my gosh, I never wanna have sex again, um, thinking that, you know, if you can catch it when you've used protection and you've been careful about the choice of partners, there was definitely that bit of is it worth it. Is it worth having sex if you can catch these viruses… Concerns about acquiring another STI led women to feel ‘wary’ or ‘cautious’ about future sexual relationships, particularly when they had acquired HPV despite having had a sexual health screening before starting a new relationship and using protection:I think it is something that I can put to the back of my head. Um, and, you know, I'm not gonna let it stop me but, um, I've just gotta look out for any symptoms, you know. I'm not gonna put my life on hold because of that .For others, having HPV had not affected their feelings about future relationships or sexual relationships:Several factors appeared to influence women's emotional and psychosocial responses and minimize the negative psychosexual impact of testing HPV‐positive.HPV was described as ‘…like an STD’, but also as something that was transmitted ‘…through sexual contact…but not an STI’. There was uncertainty around how HPV should be labelled (i.e. as a virus or STI). The term ‘HPV‐positive’ contributed to women's conceptualization of HPV as an STI because of the similarity to ‘HIV positive’..And obviously they're saying it's sexually transmitted but…my understanding was that also it's a bit like a herpes virus in that everybody has it in them but in some people it's activated and in other it lies dormant. Um, so I, the only thing I'm not clear on is if it's something we all have in us that is just activated now and then, by stress or hormones or whatever, or if it is actually only got from sexual transmission Women's certainty about HPV being sexually transmitted varied, with information from healthcare professionals appearing to influence beliefs. The influence other factors might have in causing HPV, such as whether all women have HPV and it is ‘activated’ by stress or hormones or having a ‘low immune system’ was described:.I think that the taboo of having an STI can be really embarrassing, err so yeah, I think probably people could be embarrassed by it. Not, not me so much, because I know, I'm in, you know, a long‐term relationship and it's just something I could have picked up… Erm I think a lot of people know what Chlamydia is, well, I didn't know what HPV was, but if someone said Chlamydia, you know what it is, and you associate it with certain… It's wrong, but you stereotype it a little bit. Whereas because I hadn't heard of HPV, to me it wasn't kind of in the same area as Chlamydia Understanding the high prevalence and asymptomatic nature of HPV and that it was something that could clear by itself without treatment appeared to minimize its impact. In contrast, a lack of knowledge also appeared to act as a buffer to negative emotional and psychosocial responses. If HPV was not part of sexual health screening then it could not be a ‘serious sexual thing’. Women who did not consider HPV an STI did not feel embarrassed or ashamed disclosing as it was just a ‘biological medical thing’ similar to an abnormal cytology result. A lack of concern about transmitting HPV because it was not ‘…like gonorrhoea or syphilis or HIV or something where you could endanger somebody else’ was mentioned..…I think I know that you can carry this virus around yourself for years and years and years and it might not come to anything and then all of a sudden… so it could have been something from years previously, as it most probably was with me Women understood that HPV could stay in the body for years or lie dormant which provided reassurance that it had not come from their current partner, reducing concerns about infidelity. However, this also caused confusion. Women questioned how long it could lie dormant for, why it had not ‘shown up’ on previous screening tests and how it could be present when they had not recently been sexually active. Beliefs about dormancy influenced women's beliefs about the source of their HPV infection:.I think what got me over it was if I was going to infect him with it, I already have. Erm and it's not something that could hurt him long‐term While some women were concerned about transmitting their HPV infection to their partner, others felt that HPV was something that was ‘more harmful to a female than it was to a male’. This belief allowed women to justify why they had not disclosed having HPV to a male sexual partner. Women felt that they would be more concerned about transmitting HPV if they were single, however believing that they had probably already transmitted HPV to their long‐term partner provided reassurance:.I think originally I'd been worried you know, like, who did I get it from or how did I get it? And now that I think like it's – it's quite normal, I don't worry about it as much Some women who had tested HPV‐positive more than once mentioned that HPV did have an impact on their sexual relationship when they received their first result. However, at the time of the interview it was no longer having an impact, partly because their knowledge about HPV had increased.We explored the psychosexual impact and disclosure experiences of women who had tested HPV‐positive in the context of HPV‐based cervical screening. The sexually transmitted nature of HPV, and concerns about transmission of HPV and where their HPV infection had come from, impacted women's current, past and future interpersonal and sexual relationships. However, psychosexual impact varied. Women's psychosexual response was influenced by how they conceptualized HPV, their understanding of key aspects of HPV such as its high prevalence and dormancy, concerns about transmitting HPV and having a persistent HPV infection.Responses to testing HPV‐positive such as questioning the source of their HPV infection, concerns about transmitting HPV to a sexual partner, reduced interest in sex and concerns about disclosure because of the stigma that is attached to having an STI are consistent with previous research  (McSherry et al., How women conceptualized HPV influenced psychosexual response. Referring to HPV as an infection that is passed on by skin‐to‐skin contact during sexual activity, rather than an STI, may help to reduce any psychosexual impact triggered by the STI label. This is an approach already being advocated by a UK‐based cervical cancer charity in an attempt to reduce stigma, fear and confusion about HPV (Jo's Cervical Cancer Trust, Testing HPV‐positive can result in adverse emotional and psychosocial responses impacting interpersonal and sexual relationships. However, some women appeared to be relatively unaffected by their HPV‐positive result. Several factors appeared to influence this. The findings suggest that increasing knowledge of key aspects of HPV, such as its high prevalence and spontaneous clearance, and the differences between HPV and other STIs, may increase women's understanding of their screening result and reduce any potential negative psychosexual consequences of testing HPV‐positive.Kirsty F Bennett: Conceptualization; formal analysis; investigation; methodology; project administration; writing – original draft. Jo Waller: Conceptualization; formal analysis; methodology; writing – review and editing. Julia V Bailey: Conceptualization; formal analysis; methodology; writing – review and editing. Laura AV Marlow: Conceptualization; formal analysis; methodology; writing – review and editing.All authors declare no conflict of interest. Click here for additional data file.


Infection by human papillomavirus (HPV) induces various cancers, mainly cervical cancer but also anal and pharyngeal cancers. Reunion Island is a French department in the Indian Ocean. Due to the history of its settlement, the island’s population has a wide variety of origins, and the population’s sociodemographic characteristics differ from those of the French mainland. These characteristics make the island’s population an original French population in its own right, particularly in terms of health. Cervical cancer incidence and mortality in Reunion Island are 10.4 per 100,000 and 4.4 per 100,000, respectively, both of which are much higher than those in mainland France. There is also a disparity in the prevalence of different HPV types, with types 33 and 52 being overrepresented and type 18 being underrepresented. However, vaccination and screening coverage in Reunion Island is low. It is important to understand the burden of infection and its risk factors in members of the young Reunionese population at risk of HPV infection to promote and evaluate the implementation of future vaccination and screening programs on a larger scale.The RUN-SurV-HPV study will have the following four objectives: (1) to describe the prevalence of HPV genotypes in a population at risk of sexually transmitted infections in Reunion Island; (2) to describe the prevalence of HPV genotypes by anatomical site, gender, and sexuality; (3) to describe the correlates and risk factors for HPV detection; and (4) to examine HPV genotypes between different anatomical sites.Chlamydia trachomatis testing at a sexually transmitted infection (STI) testing center at Reunion Island will be performed. The population included 333 women who all had vaginal and pharyngeal swabs, with 80 of them also having had an anal swab. There are 167 men who have sex with men who have had anal and pharyngeal swabs, and 120 men who have sex with women who have had a urine swab only. Results will be correlated with sociodemographic and clinical data collected routinely during the consultations. HPV detection and genotyping will be performed using the Anyplex II HPV28 detection assay (Seegene).Cross-sectional analyses of 1200 routine vaginal, anal, pharyngeal, and urinary samples collected between October 2020 to December 2022 from female and male patients aged 16 to 30 years undergoing The first polymerase chain reactions should begin in November 2023. The first results should be submitted for publication in 2024.The study will determine HPV prevalence by age, sex, male sexual preference, human immunodeficiency virus status, and STI co-infection. Collecting data from young patients not usually routinely screened for HPV infection will be a simple and reproducible way of better understanding local specificities, encouraging vaccination campaigns in the short-term, and evaluating their effectiveness in the future.DERR1-10.2196/47379 Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) in the world. There are more than 100 types of HPV, with 13 classified as oncogenic. Of these, HPV types 16 and 18 are the cause of 70%-80% of invasive cervical cancers . HPV infReunion Island is a French overseas department located in the Indian Ocean that is populated by more than 850,000 inhabitants. Due to the history of its settlement, the population of the island has very varied origins  that differ from mainland France. The geographical distance and its sociodemographic characteristics make it an original French population in its own right. In Reunion Island, cervical cancer is the fourth most common type of cancer in women. From 2013 to 2015, cervical cancer incidence and mortality were 10.4 per 100,000 and 4.4 per 100,000, respectively, making it the eighth deadliest cancer, 2 times higher than in mainland France  comparedThe first HPV vaccine was marketed worldwide in 2006. Initially, the first 2 vaccines protected against 2 genotypes (16 and 18) or 4 genotypes . Currently, the latest vaccine protects against 9 genotypes . In 2007, the French High Authority for Health (HAS: Haute Autorite de la Sante) first implemented a community-based vaccination program that recommended HPV vaccination for girls aged 11 to 14 years with the possibility of catching up until 19 years of age. In 2019, the program was extended to include boys in the same age groups . This ofThe peculiarity of this population regarding its low vaccination coverage, high cancer rate compared to the national average, diverse ethnic origins, and risk factors encourages local exploration and monitoring of HPV genotype specificity. Furthermore, a previous study in women with atypical squamous cells of undetermined significance (ASCUS) Papanicolaou test results  and cervical biopsies of high-grade histological lesions or cancer  identified that the distributions of different types of HPV in Reunion Island were not similar to those observed in mainland France  . MoreoveChlamydia trachomatis (CT). The inclusion of young patients at risk of STIs, and therefore at risk of HPV infection, including both women and men, will enable us to study a population in which the incidence of HPV infection will likely be high. The inclusion of men, whether MSM or MSW, is in line with the recent extension of vaccination to French men and women, regardless of their sexuality [In the RUN-SurV-HPV study, we want to analyze the distribution of different oncogenic HPV types  in a population of women and men at risk of STIs attending an STI testing center for exuality . Indeed,The main objective is to describe the prevalence of infection by at least 1 of the 7 high-risk oncogenic HPV genotypes contained in the GARDASIL 9 HPV vaccine  in women younger than 30 years who have had a vaginal swab and MSM who have had an anal swab for CT infection in the Reunion Island population.The secondary objectives are as follows:To describe the prevalence of infections by different high-risk  and low-risk  HPV genotypes in samples from routine vaginal, anal, or pharyngeal CT testing from women younger than 30 years; urine samples from MSW younger than 30 years; and anal samples associated with pharyngeal CT testing from MSM.To investigate HPV distribution in the same patient (women and MSM) according to the swabbed location.To describe the correlates and risk factors for HPV infection, including age, CT status, status of other STI infections, socioeconomic status, sexuality, reasons for consultation, HPV vaccination status, and human immunodeficiency virus (HIV) status, by patient and type of specimen.This protocol is for a single-center, descriptive, cross-sectional study of all samples collected from male and female patients undergoing routine CT testing at the STI testing center of University Hospital (UH) in south Reunion Island between October 2020 and December 2022.The national recommendation for CT screening in France suggests screening all woman from 15 to 25 years of age (pregnant women included) as well as those older than 25 years with risk factors and all men . In this study, samples were obtained from patients who went to an STI testing center mainly because of a recent sexual risk or symptomatic genital affection or to be able to remove the condom in a stable relationship.The following patients were included in the study:Those who consulted the STI testing center of the UH site in south Reunion Island between October 1, 2020, and December 31, 2022;Female patients younger than 30 years because the national recommendation for HPV screening is from 30 years of age and following a younger population would provide insight about the first impact of the national vaccination program;MSM  because they have a higher risk of anal cancer;MSW younger than 30 years because they are also affected by HPV-induced cancers, are included in the new French vaccination recommendations, and comprise a specific population that is able to infect women;Those who were tested for CT infection on at least 2 different anatomical sites  for women, first pass urine for MSW, and at least 2 different sites  for MSM;Those whose residual samples were available at the microbiology laboratory of the UH of Reunion Island.The following patients were excluded from the study:Those who were outside the study age range,Those who did not consent to the use of their data for research,MSW with no sites collected and women and MSM with less than 2 sites collected,Those for whom there was insufficient biological material.Social and demographic data were extracted from routine clinical records. These data were collected for each person presenting for screening via a self-administered questionnaire that was routinely administered and completed during the consultation . This quSample collection at the laboratory, the supply of sampling equipment, and the transport of the samples was carried out in compliance with the NF EN ISO 15189 standard, thus guaranteeing the integrity of the samples and their traceability.Samples were transported to the laboratory at 4 °C in eSwab transport media (COPAN Diagnostics) for swabs  and in sterile preservative-free flasks for urine. After the CT polymerase chain reactions (PCRs) were performed, residual samples were frozen at –80 °C to perform this study.Enclosures equipped with range temperature sensors (SpyRF) are metrologically monitored using a monitoring web platform software (JRI-MySirius), allowing the exploitation of alarms and data. An Ethernet modem is installed in each lab, thus ensuring radio frequency communication with the SpyRF.Samples will be tested with the Anyplex TM II HP28 Detection kit (Seegene), a semi-qualitative test for the detection of DNA and genotyping of HPV types. DNA extraction will be performed using the STARMag Universal extraction kit (Eurobio Scientific) on the STARlet (Seegene) automated system. After extraction, genotyping will be done by amplification on a CFX96 TM thermocycler (BioRad). This kit uses the human housekeeping gene, beta globin, as an endogenous internal control to verify the DNA extraction, PCR reaction, and presence of cells in each sample. This real-time multiplex assay allows simultaneous amplification, detection, and differentiation of target nucleic acids of 28 HPV types .Using standard software extraction tools, throughout the study, researchers will generate a list of all the information included in patients’ records and self-administered questionnaires issued and completed on the day of collection. This anonymous information will then be extracted into a secure Excel spreadsheet (Microsoft Corp). HPV genotyping results will also be extracted into a secure Excel spreadsheet.Sample collection started in October 2020 and ended in December 2022. For women, 1436 swabs were collected from 665 women. A vaginal and pharyngeal swab was collected on the same day from 333 women younger than 30 years, 80 of whom also had an anal swab. For MSM, 738 samples were collected from 258 MSM. An anal and pharyngeal swab was collected on the same day from 167 MSM. Finally, 661 MSW provided a urine sample and 324 were younger than 30 years. Between 120-180 urine samples from MSW will be randomly selected for inclusion in the study, depending on the availability of stored samples from women and MSM. A total of 1200 samples will be tested . Due to t test or Wilcoxon test depending on the conditions of validity. To account for confounding effects and to identify factors independently associated with HPV infections, a multivariate analysis by logistic regression will be performed. The dependent variable will be HPV infection (yes or no). The independent variables will come from the self-administered questionnaire and laboratory results and will include sex, age, CT status, status of other STIs, socioeconomic status, sexuality, reasons for consultation, HPV vaccination status, and HIV status. The independent variables entered in the model will be variables for which the significance level will be P<.2 in the bivariate analysis; variables will be selected using backward stepwise regression with a significance level for removal from the model of P≥.1. To investigate whether factors associated with HPV infection differ according to sex and type of specimen, subgroup analyses will be performed by swab site and sex. McNemar tests will be used to assess differences between matched samples. Cohen κ will be calculated to check the concordance of HPV infections between matched samples from the same patient. To assess this concordance for women and MSM, individuals with samples from 3 sites will be preferred over those with samples from only 2 sites. A random selection will be made to select individuals with only 2 samples in order to meet the allowable sample size.Patient characteristics will be described in terms of numbers and percentages for qualitative variables and in terms of mean and SD, median and IQR, and range for quantitative variables. The prevalence of HPV infections will be expressed as percentages. The exact binomial method will be used to calculate 95% CIs. Analysis of primary assessment criteria will be performed per patient considering only vaginal samples for women and anal samples for MSM. For analysis of factors associated with HPV infections, associations with factors of interest will be carried out for qualitative variables by chi-square test or Fisher exact test ; comparisons concerning continuous variables will be carried out by Student (P<.05). CIs will be calculated at 95%. The statistics will be calculated with the STATA 17.0 software.The significance threshold for the tests will be 5% The expected benefits outweigh the potential risks to patients.National cervical cancer screening with HPV PCR is recommended by the HAS for individuals 30 years and older. In our study population, and in accordance with national recommendations, there is no obligation to follow up or to carry out additional examinations in the event of a positive result for HPV at the cervix-vaginal level, particularly if it is one of the oncogenic or probably oncogenic HPV types. The natural history of HPV infections is specific to the population younger than 30 years. The incidence and prevalence of HPV infections are at their highest levels due to the presence of major risk factors, such as number and frequency of new sexual partners. These infections are responsible for minor cytological lesions that evolve spontaneously and favorably in the majority of cases. Indeed, in this population, viral clearance is earlier and more marked than in older women, with 50% clearance of the infection within the first 6 months and 90% clearance within 2 years. In France, to date, there is no recommendation for HPV screening, nor is there a recommendation for treatment in the event of the discovery of an oncogenic HPV infection, whether it be anal, pharyngeal, or urinary, in women, MSM, or MSW.An information note will be given at the time of the consultation setting out the objectives of the research organized based on data collected during care.In compliance with national legislation, ethics approval has been obtained from the ethics evaluation committee of INSERM  and all procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975.Patient recruitment began in October 2022 and ended in December 2022. Approximately 1200 samples were collected from 333 women, 167 MSM, and 120 MSW. Funding was obtained thanks to the support of the University Hospital of La Reunion. The first PCRs are scheduled for November 2023. We expect to submit the first results in 2024.The results of this study, carried out in a largely unvaccinated population, will enable us to give more details on the distribution of HPV infections in the Reunion Island population before vaccination. Currently, in France, but also locally in Reunion Island, there is a desire to promote a community-based vaccination program with GARDASIL 9 to achieve a national coverage target of 80% among those aged 11 to 19 by 2030 ,18. The Vaccination has already been shown in several studies to be effective in reducing HPV infections  and precThe Swedish team of Söderlund-Strand et al  has alreThese different studies, each with their own specific design, demonstrate the value of carrying out this type of cross-sectional study repeatedly over time. With regard to Reunion Island and the prospect of repeating the RUN-SurV-HPV study in several years’ time, it will be important to monitor changes in vaccination coverage locally. The next study should be carried out sufficiently far in advance of the current one so that vaccination has had time to become more widespread in Reunion’s population. We can then hope to demonstrate the effect of vaccination with GARDASIL 9 by identifying a significant reduction in HPV infections and monitor the appearance of other genotypes in our population of women and MSM younger than 30 years.In this study, we expect to have different results from the studies already conducted in Reunion Island. The last genotyping study of HPV infections in Reunion Island was conducted from 401 ASCUS Papanicolaou tests and 94 cervical biopsies with histopathological diagnosis of high-grade lesions or cancer performed between 2008 and 2012 . After aIdentifying the prevalence of HPV genotypes by analyzing samples collected for CT testing has been done in several countries to map HPV infections and assess the impact of their vaccination programs ,24. In aThe concordance of HPV genotypes in the same individual at different anatomical sites remains controversial in the literature. Müller et al  identifiThe strengths of this study lie in the originality of its design, with a data collection method  that is not restrictive for the participants and investigators. This method makes it possible to include a large number of participants for more representative results. It is also easily reproducible over time, which will make it possible to repeat this type of study to monitor the evolution of HPV infections in the Reunion Island population.The characteristics of the study population are also interesting. First, it is composed of both women and men, which coincides with the recent extension of French vaccination recommendations against HPV to adolescents of both sexes . To our This study has several limitations. The main limitation is the selection bias of our population. The population consulting STI testing centers is a specific population that cannot be extrapolated to the general population of Reunion Island. This population has an increased risk of HPV infection, and our results may be affected by this. The fact that the patients were recruited via a free screening center where they could consult spontaneously and anonymously reduced the risk of selection bias in our population. In our study, the proportion of under-age patients is likely to be low, even though this population is the main target for vaccination. Unfortunately, despite our efforts, it is difficult to have them consulted spontaneously and to include them while respecting both the anonymity of the consultation and the legislative framework for protecting the data of minors. Finally, this study was only carried out in one screening center, which could affect the results. The results may also be affected by the way the test is performed and its performance, even though it has already been validated in several studies ,31. The This original epidemiological study, which has already proved its worth in other countries, will have rapid implications by promoting vaccination campaigns and identifying the populations most at risk of being infected by an oncogenic HPV genotype. By comparing the prevalence of HPV infection in the same person and at different anatomical sites, this study will also provide a better understanding of its pathogenicity and mode of transmission and better delineate the complementarity of certain screenings. In the long term, these studies will allow for monitoring of the evolution of HPV infections and the impact of HPV vaccination campaigns in the Reunion Island population.


Despite the known benefits of preventing human papillomavirus (HPV)–related cancers, HPV vaccine coverage is low in the United States. Grounded in Social Ecological theory, we assessed the macro-level (state) and meso-level (organization) factors associated with HPV vaccine initiation and up-to-date.Data from 2020 National Immunization Survey-Teen were used to study a sample of 20 163 US adolescents (aged 13-17 years). The data were collected from each teen’s parents or guardians and health-care professionals. Weighted prevalence estimates were calculated, and multivariable regression analyses were conducted.The prevalence of HPV vaccine initiation was 75.1% and of remaining up-to-date was 58.6%. At the macro level, teens living in states with high and moderate religiosity had lower odds of HPV vaccine initiation  and up-to-date  than states with low religiosity. At the meso level, when none of their healthcare professionals ordered vaccine from the state, teens had lower odds of initiation  and up-to-date  than teens whose healthcare professionals ordered vaccine from the state. In addition, race and ethnicity, age, mother’s education level, household income, well-child examination status, and doctor’s recommendation were significantly associated with HPV vaccine uptake.A multiprong approach is needed to address religious and systemic barriers to HPV vaccination and expand healthcare professionals’ access and enrollment in state vaccine initiatives, such as the Vaccine for Children program. Human papillomavirus (HPV) is responsible for common infections such as genital warts; papillomatosis; and multiple cancers, including oropharyngeal and anogenital cancers . Yearly,Despite the benefits, HPV vaccination coverage is suboptimal in the United States . AlthougVaccination initiation and being up-to-date do not depend on individual factors alone. The Social Ecological theory posits that individuals and the social environment are interconnected, and health behavior results from an interplay of factors at the individual level and the social environment  . HPV vacIn addition to the macro-level factors, health behavior is affected by meso-level factors, such as the vaccine procurement process in the practicing organizations and facility type. To address the cost-related barriers to vaccination coverage, the Vaccines for Children (VFC) program was created . StudiesBecause HPV vaccination and initiation studies have focused primarily on individual factors, there is a paucity of information about the role of macrostructural (state religiosity) and mesostructural (VFC enrollment/facility type) factors on vaccination coverage. Therefore, this study was grounded in Social Ecological theory to comprehensively understand the interplay of barriers and facilitators with respect to HPV vaccination, focusing on religiosity at the state level and source of vaccine procurement and facility type at the organizational level. We hypothesized that after adjusting for other individual-level factors, 1) highly religious states have low odds of HPV vaccination initiation and being up-to-date, 2) patients visiting all nonpublic facilities have higher odds of initiation of and being up-to-date with HPV vaccination, and 3) teens whose healthcare professionals procure vaccine from the state  have higher odds of initiation of and being up-to-date with HPV vaccination.The current study is based on a comprehensive dataset that included individual and state-level data. The individual-level data for the study were obtained from the 2020 National Immunization Survey-Teen (NIS-Teen 2020), a population-based survey that collected data on teenage vaccination from parents and guardians as well as healthcare professionals in all 50 states, the District of Columbia, and some US territories. The parents were interviewed regarding their teen's vaccination history and families’ sociodemographic factors by telephone. Then, each teen’s healthcare professional was contacted  to collect data on the immunization history using a mailed questionnaire. The details of the NIS-Teen 2020 study methodology were reported in previous publications . The NatThe current study had 2 primary outcomes—HPV vaccination initiation and being up-to-date—that were obtained from clinician-verified vaccination history. Initiation of HPV vaccination was defined as receiving at least 1 dose of the HPV vaccine. The second outcome variable, being up-to-date, was defined according to the 2016 guidelines of the Advisory Committee on Immunization Practices based on 2 criteria: 1) receipt of 3 or more doses or 2) receipt of 2 doses of the HPV vaccine when the first shot was administered before the patient was 15 years of age .Information about the macro-level predictor, state religiosity, was obtained from the Religious Landscape Study by the PEW Research Center , developMeso-level predictors included 1) the type of facility in which the health-care professional practiced, which was assessed using the question “Which of the following describes this facility”  and 2) physicians ordering vaccine from the state. The second variable was used as a proxy to measure participation of healthcare professionals in the VFC program, assessed based on responses to the question “Do the teen’s providers order vaccines from state/local health department?” Given that some teens have multiple health-care professionals, this variable was categorized as “All providers,” “Some but possibly or definitely not all providers,” or “No providers” order vaccine from the state or local health department.The following individual characteristics of teens were also assessed as predictor variables:Receipt of a well-child exam. This characteristic was measured using the question “Did teen have an 11-12-year-old well child exam or check-up?” Responses included in the study were “yes” or “no.”Recommendation of HPV vaccine by a health-care professional. This characteristic was measured using the question “Has a doctor or other health care professional ever recommended that teen receive HPV shots?” Responses included “yes,” “no,” and “don’t know.”Respondents’ relationship to teen. This characteristic was measured using the question, “What is your relationship to the teen?” Responses included “Mother or female guardian,” “Father or male guardian,” “Grandparent,” and “Other family members/friend.”Additional individual characteristics included in the analysis were the teen’s age (continuous variable), sex (binary variable), race and ethnicity , family’s poverty status , and mother’s education level . Race and ethnicity were combined as a single variable based on the current literature ,13,33,34P < .05 for all tests included in this study. All statistical analyses were performed using R, version 4.2.3 .All analyses were survey weighted to account for the complex survey design in the NIS-Teen 2020 and allow for the representativeness of the demographics of the US population. Survey weights accounted for nonresponse, nonresolution of telephone numbers, subsampling of 1 age-eligible child per household, and multiple telephone lines in the home. Moreover, adjustments were made after stratification on the basis of the respondents’ sociodemographic characteristics and missing clinician data. The weighted prevalence of initiation of and being up-to-date with HPV vaccination were estimated. A survey-weighted multivariable logistic regression was conducted to determine the key factors associated with the initiation of and being up-to-date with HPV vaccination. The variables included in our model were selected a priori on the basis of previous literature and the study objective. We did not conduct any model selection. Because missingness for the variables included in our study was less than 2%, we conducted a complete case analysis. We also mapped the relationship between state religiosity and HPV vaccine initiation and up-to-date status across the US states. We set statistical significance as a 2-sided The overall initiation rate for the HPV vaccination series among adolescents was 75.1% . The iniP < .001) and 32% lower odds  of HPV vaccine initiation, respectively.In the multivariable regression analyses , at the P = .002) than when all the teen’s health-care professionals ordered vaccine from the state.At the meso level, when none of the teen’s health-care professionals ordered vaccine from the state, teens had 32% lower odds of initiation  and moderate religiosity had 26% lower odds of being up-to-date  than teens living in states will low religiosity. At the meso level, teens had 24% lower odds of being up-to-date  when none of the teen’s health-care professionals ordered vaccine from the state or local health department than when all health-care professionals of the teen ordered vaccine from the state or local health department. At the individual level, teens who were non-Hispanic Black or Hispanic, lived in households below the federal poverty level, had received a well-child examination at 11-12 years of age, and had received a doctor’s recommendation for the HPV vaccine had higher odds of being up-to-date. The association between age and being up-to-date with HPV vaccination was also found statistically significant. Finally, sex, mother’s education level, and facility type were not statistically significant variables for being up-to-date with HPV vaccination . This finding corroborates studies indicating that as more physicians enrolled in the VFC program, their cost-related barriers were reduced significantly, encouraging them to recommend the HPV vaccine . We alsoAfter controlling for the macro-level factor (state religiosity) and meso-level factors (VFC program enrollment and facility types), we found that at the individual level, the healthcare professional’s recommendation had the greatest impact on increasing the odds of initiation as well as being up-to-date compared with all the factors studied in the model. This finding resonates with the existing literature, which found that healthcare professional recommendation was statisticaly significant and positively associated with HPV vaccine initiation and being up-to-date ,25,34,51This study had some limitations. Because it was a cross-sectional study, causality cannot be determined. Moreover, each teen’s household data were based on self-report, which increased the risk of social desirability and recall bias. Furthermore, religiosity was at the state level because NIS-Teen 2020 did not collect data on religiosity. The significant inverse association between state-level religiosity and individual-level HPV vaccine uptake found in our study, however, reaffirmed that religiosity shapes the collective consciousness of society at large, which in turn affects individual vaccine decisions. The response rate of NIS-Teens 2020 national data was low, but the NIS-Teen 2020 study incorporated weights adjusting for nonresponse for household interviews and insufficient clinician data . ImportaHPV vaccination can reduce the incidence of HPV-associated cancers if the population receives the recommended dosage. Rising oropharyngeal cancer rates and existing challenges with high mortality from cervical cancer call for more consolidated efforts to increase HPV vaccination initiation and being up-to-date. Based on our findings, we recommend a multiprong approach that includes a faith-based HPV vaccine intervention involving religious leaders, reduces the administrative barriers in vaccine cost reimbursement, and provides training to the healthcare community to increase vaccine recommendation.pkad068_Supplementary_DataClick here for additional data file.


INK4a are also modulated. The combined detection of HPV oncogenes and cellular biomarkers indicative for dysplasia could be informative and convey better specificity than the current HPV tests that cannot discriminate transient infection from dysplastic changes. Methods: The QuantiGeneTM 2.0 Plex Assay platform was chosen for the effective multiplexing and quantitative detection of seven HPV-E7 mRNA targets  and the cellular mRNA of p16INK4a as a biomarker for HPV-induced transformation. Actin-beta (ACTB) and hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) were included as reference markers. Sequences for the specific capture and detector probes were customized and developed by ThermoFisher and formulated as a QuantiGene proof-of-concept (QG-POC) plex-set. The crude lysates of the HPV-positive cervical cancer cell lines CaSki (HPV16), HeLa (HPV18), MRHI-215 (HPV45), Erin59 (HPV59), ME180 (HPV68), and the HPV-negative cell line C33A, as well as liquid-based cytology smear samples (n = 441) were analyzed. The study was a proof-of-concept evaluating the feasibility of the platform. Logistic regression and receiver operating characteristic (ROC) analyses were performed to test for the sensitivity and specificity of HPV detection and dysplastic stage discrimination. Results: A QG-POC assay specifically and sensitively detects the HPV-E7 mRNA of seven different genotypes with an assay linearity between 20 and 13,000 cells. Cellular mRNA was detected from the crude lysates of cell lines and of cellular material from clinical liquid-based cytology smear samples. By combining HPV-E7 and p16INK4a expression normalized to ACTB, high-grade dysplasia (HCIN) and invasive cervical cancer (CxCa) were detectable, discriminable, and correlated to the biomarker expression strength. The ROC analysis from the multivariate logistic regression model including HPV-E7 and p16 INK4a resulted in an AUC of 0.74, at the optimal cut-off  for HCIN detection. CxCa was detected with an AUC of 0.77 . Conclusions: The QG-POC assay is sufficiently sensitive to detect and quantify HPV-E7 and cellular mRNA species. Multiplexing allows the specific detection of at least 10 analytes in a single reaction. Determining the abundance of E7 and p16INK4a transcripts when normalized to ACTB is informative about the presence of cervical dysplasia and potentially discriminates between low-grade and high-grade dysplasia and invasive cervical cancer. Further studies including more HPV genotypes and biomarkers are warranted.Background: Persistent infection with human papillomavirus (HPV) can lead to cervical cancer (CxCa). During the progression to CxCa, the expression of HPV oncogenes E6 and E7 is upregulated. In turn, cellular proteins such as p16 Cervical cancer (CxCa) is one of the most common female cancers, with an incidence of 600,000 new cases per year worldwide. The highest burden of disease is carried by low-and-middle-income countries (LMIC) where CxCa is frequently the leading cause of cancer deaths among women . CxCa isThe implementation of organized screening programs based on cytology testing has reduced the incidence and mortality of cervical cancer drastically . HoweverINK4a [INK4a overexpression correlates with hrHPV infection and with dysplasia severity which has been shown for mRNA and protein levels [INK4a expression rarely progresses [INK4a, may therefore be promising for the detection of high-grade lesions and the progressive potential of low-grade dysplasia. It was shown for example, that the detection of the p16INK4a protein with an ELISA has a high sensitivity for detecting CxCa [The progression to CxCa proceeds via various pre-malignant stages, histologically classified as cervical intraepithelial neoplasia (CIN) grades 1, 2, and 3. Cytological classification discriminates these stages as low- or high-grade squamous intraepithelial lesions (LSIL or HSIL) . During INK4a . This p1n levels ,15. Moreogresses . The quaing CxCa . The incTM 2.0 Plex Assay platform technology for the multiplexed detection of HPV-E7 and cellular mRNAs from cultured cervical carcinoma cell lines and clinical smear specimens in a proof-of-concept study. The method uses the Luminex suspension bead array platform for read-out and branched DNA (bDNA) technology for signal amplification [Here, we report the utility of the QuantiGenefication ,22. The TM 2.0 Plex Assay  for the specific detection and sensitivity of the mRNA targets HPV-E7 and p16INK4a. The cellular mRNAs actin-beta (ACTB) and hypoxanthine phosphoribosyltransferase 1 (HPTR1) were used for normalization and quality control purposes. The cell lines CaSki (HPV16), HeLa (HPV18), ME 180 (HPV68), and C33A (HPV-negative) were obtained from American Type Culture Collection Inc.  and CERV-215 (HPV45) was obtained from Cell Line Service . The cell line Erin59 (HPV59) was kindly provided by Dr. D. Brown  [2 atmosphere until 70–80% confluence. The cell numbers were determined using a Countess automatic cell counter .Established HPV-positive CxCa cell lines were used to test the QuantiGeneIN, USA) . The cel® solution  during the PIPAVIR study. Patients aged 30–60 years were recruited at an outpatient colposcopy clinic. Signed informed consent and ethical approval were obtained from the patients and the Bioethics Committee of the Medical School of Aristotle University of Thessaloniki/Greece (33–31/8/2012) and from the Ethikkommission der CHARITE-Universitätsmedizin Berlin/Germany (EA1/168/13), respectively. For each patient, an ectocervical and endocervical smear sample was collected using a CervexBrush®  and a CytoBrush® according to the manufacturer’s instructions, respectively. Cells were immediately fixed in ThinPrep containers (Hologic) containing 20 mL of PreservCyt® solution. The cytology evaluation was performed by a central study cytopathologist [n = 441) was selected from the main study, representing the different dysplasia stages, and including HPV16, 18, 31, 45, 59, 68, and HPV6 positively tested samples plus HPV-negative samples.Liquid-based cytology smear samples were collected into a PreservCythologist . The res EA1/168/, respecthologist . A subseHPV genotyping was performed by Multiplexed Genotyping Assay (MPG assay) targeting the L1 gene sequence using a broad-spectrum GP5+/GP6+-bio primer set ,27. The TM 2.0 Plex Assay , a custom plex-set was designed named QuantiGene proof-of-concept (QG-POC) targeting the following gene transcripts: HPV6-E7, HPV16-E7, HPV18-E7, HPV31-E7, HPV45-E7, HPV59-E7, HPV68-E7, and the generically upregulated cellular biomarker p16INK4a , and incubated at room temperature (RT) for 30 min. From the clinical Thinprep samples, 2 mL were centrifuged, the supernatant discarded, and the pellet lysed in 100 µL of the working lysis buffer for 30 min at RT  was applied. The effect sizes were rated according to Cohen (1992) [All statistical analyses were performed using R software . For clen (1992) . For comn (1992) . The reln (1992) . The unin (1992)  for the n (1992)  for the n (1992) . Accordin (1992) . For theINK4a in all the cell lines investigated, the genotype-specific HPV-E7 signals appeared positive in the respective cell lines , and the highest in ME 180 for HPV68-E7 with 17,774 MFI. ME 180 also had the highest E7 mRNA strength when normalized to ACTB, which was regarded a marker for cellularity. The equivalent number of cells analyzed resulted in quite different MFI values in the different cell lines, both with and without normalization to ACTB. For example, the expression strength of the HPV-E7 mRNA detected in CaSki is one order of magnitude lower than in HeLa. This suggests that different target mRNAs may have different copy numbers in the cell lines investigated, which has been described before in cervical cancer cell lines [To verify the specificity of the designed QG plex-set for the nominal mRNAs and to preclude any cross-reactions between the HPV genotypes and/or cellular RNAs, we tested the lysates generated from 4000 cells of HPV-positive cervical cancer-derived cell lines CaSki (HPV16), HeLa (HPV18), CERV-215 (HPV45), Erin (HPV59), ME 180 (HPV68), and, as a specificity control, C33A (HPV-negative). While the expression over background was detected for the cellular mRNAs of ACTB, HPRT1, and p16ll lines . Generalll lines .6 cells/mL per HPV-positive cell line) and of the HPV-negative cell line C33A. The HPV–cell-line mix and the C33A-cell lysates were serially diluted at a ratio of 1:3 starting from 40,000 cells and subjected to a QG-POC assay  the signal linearity was still given. This illustrates the wide range of cell concentrations that can be analyzed by this assay platform. For HPV45-E7 the assay linearity was given between 400 and 13,000 cells in the HPV–cell-line mix. This probe set for HPV45-E7 had shown an unspecific signal before and this again was seen in the C33A cells. As can be seen in INK4a, and all HPV-E7 mRNAs were increasing with the same slope with increasing cell numbers. This allows the normalization of the signals within the linear range of each target mRNA to the cellular reference gene, and thus to the cellularity of the sample.After having established that the target mRNA sequences were specifically detected, and sufficiently strong signals of >1 order of magnitude above the LOD were detected when 4000 cells were tested, the assay sensitivity was further explored. A limiting dilution experiment was conducted by titrating the lysates of a cell-line mix  years fixed in PerservCyt (Thinprep) were tested in this proof-of-concept study. The patients included in the diagnostic evaluation of the QG-POC assay were clinically classified using the ASSIST score as described by Agorastos et al. , which cOf the 441 patients included, 400 had tested positive by MPG for at least one of the following genotypes: HPV 6, 16, 18, 31, 45, 59, and 68. Additional HPV genotypes may have been present but were not regarded in this analysis. Importantly, we also wanted to investigate if the designed probe sets were functional and specific for the two genotypes hrHPV31 and lrHPV6, for which no cell lines were available. Therefore, clinical samples which tested positive for HPV6 or HPV31 by the MPG assay were also included. Due to the unspecific cross-reactivity of the HPV45-E7 probe set, HPV45-E7 was excluded from further analyses. A total of 41 women tested HPV-negative for any HPV infection. Out of the 400 HPV-positively tested patient smears, 223 had multiple infections, with 500 HPV infections for the included HPV genotypes in total. HPV16 was the most common type (73.0%), followed by HPV59 (12.0%) .n = 39/41). In total, the QG-POC assay found 348 HPV infections in 269 patients. A total of 57 patients tested positive for multiple HPV infections, with two samples testing positive for all six of the included HPV genotypes. The most frequently detected genotype by the QG-POC assay was HPV16 in 44.0% of the samples (n = 194), followed by HPV59 . The proportion of HPV-negative tested samples in the QG-POC assay decreased with a higher dysplasia severity for the normal clinical groups, LCIN, HCIN and CxCa, of 60.0%, 36.2%, 22.6%, and 9.1%, respectively . Convers9 12.2%; . The sam9 12.2%; .p < 0.001) which represents a fair agreement (p < 0.001). Assay concordance for HPV18-E7 had the best Cohen’s kappa with 0.70 , again a substantial agreement. Although HPV68-E7 had a high concordance between assays with 95.92%, no agreement was found with a Cohen’s kappa of −0.02, meaning the same samples were not detected positive by MPG assay and QG-POC assay for this HPV genotype. This was identified as a technical problem due to the probe design for the MPG and QG-POC assays (see discussion). The lowest concordance of 65.53% was found for HPV16-E7, with, however, a fair agreement (Κ= 0.35 (CI95% 0.26–0.43), p < 0.001). Since the QG-POC assay is a probe-based assay with no target amplification and only approximately 400-fold signal amplification, it was clearly less sensitive than the MPG assay, which is a PCR-based target amplification endpoint assay. Most clinical samples included in this study were positive for HPV16 by MPG assay and were associated with different ASSIST scores. Of note, non-detected HPV16-E7 by QG-POC assay was predominantly in the clinical group classified as “normal” with an ASSIST score of 0, or LSIL with an ASSIST score of 1, at 59.3% and 43.75%, respectively, and for both groups taken together as ≤LCIN, at 54.6%. For the ASSIST scores of 2 and 3, non-detected HPV16-E7 was 25.2% and 20%, respectively, and taken together as ≥HCIN, HPV16-E7 was negative only in 24.8%. This might correlate with the differential oncogene expression strength in dysplasia of progressive severity.Despite the low sample number for some HPV genotypes, we performed a correlation calculation since this was an exploratory study to obtain an impression of the QG-POC assay’s performance. Overall, positivity for the HPV genotypes included was lower in the non-target-amplifying probe-based QG-MPH assay than in the highly sensitive PCR target-amplifying MPG assay. The agreement between the MPG assay and QG-POC assay had a concordance of 70.29% for the six included genotypes  omitting the HPV45 data due to the unspecific probe set cross-reactivity. The correlation by Cohen’s kappa was 0.27 , 58 had a low-grade cervical intraepithelial neoplasia , 195 had high-grade cervical intraepithelial neoplasia  and 11 patients were diagnosed with invasive CxCa (ASSIST score 3)  .INK4a as biomarkers for hrHPV infection and transformation were correlated with the ASSIST scoring to investigate their information content for dysplasia grading. Spearman’s rho correlation was performed and showed that the expression of the “leading HPV” and of p16INK4a correlated positively with the ASSIST scoring, with an rs of 0.47  and 0.34 , respectively, which is rated as a medium-strong effect [INK4a, there was no difference between the normal and the LCIN group (ASSIST score 0 and 1), i.e., very low to undetectable expression. In the HCIN and invasive carcinoma groups (ASSIST score 2 and 3), however, expression is markedly increased in both markers. Statistically significant differences between the normal/LCIN versus HCIN/CxCa severity grades were seen , with an OR of 5.81 , 6.034 , and 4.53 , respectively. Because of the low numbers of patients testing positive for HPV68 and the potential probe problem, HPV68 was excluded from the regression analysis , 0.68 (CI 95% 0.63–0.72), and 0.74 (CI 95% 0.70–0.79), respectively. The sensitivity with the best cut-offs for these markers was 64.0%, 47.6%, and 63.6%, and the specificity was 68.9%, 84.7%, and 75.5%, respectively, in the sample sets investigated  and 1.51 , and an AUC of 0.69 (CI 95% 0.48–0.91) and 0.79 (CI 95% 0.62–0.97), respectively  allows for the effective multiplexing of up to 80 targets with an ELISA-like workflow in a 96-well format, which reduces workload and costs. A crude lysate can be used as a sample, which obviates the isolation steps performed, for example, in PCR methods and thus makes the method less error and contamination prone [The QG-POC assay was found to be a quick and easy method for the detection and quantification of mRNAs. The QuantiGeneon prone . The robINK4a, along with the hrHPV-positive and the hrHPV-negative cell line C33A as well. For the MFI signals for the reference mRNA, ACTB and HPRT1, the biomarker p16INK4a and all HPV-E7 mRNAs increased with increasing cell numbers with the same slope. This is important because it allows for the normalization of the measured MFI values of the target mRNA to the reference markers ACTB and HPRT1, and thereby to the cellularity of the sample. This normalization results in a semi-quantitative relative MFI (rMFI) value which was used for further calculations. The probe set of HPV45-E7 showed unspecific positive signals in cell lines positive for another genotype and in high concentrations in the HPV-negative cell line C33A and was therefore excluded from further analyses. In subsequent studies the HPV45-E7 probe set needs to be redesigned and tested.In this study we demonstrated that the QG-POC assay shows, in general, functionality on cultured cell line material. It detects the target mRNA sequences specifically with a sufficiently strong signal >1 order of magnitude above the LOD. Serial dilution experiments showed a very good assay sensitivity to detect as few as 10–50 cell equivalents. Assay linearity and therefore quantifiable results were given for all markers over three to four orders of magnitude of cell concentration, which allows for the measurement of samples with a wide range of cell concentrations. All the cell lines used in this study tested positive for p16INK4a (data not shown), which potentially indicates a lower true dysplasia severity. The dysplasia severity could be overestimated by cytology and colposcopy as the histology results were missing in this study for many of the patients. Furthermore, only about 30–50% of the HSIL samples (CIN3) will eventually progress towards invasive CxCa over up to 30 years. The other half persists or regresses [INK4a expression may better illustrate the biology of the lesion than viral DNA presence or morphology-based diagnostic methods and potentially may show a prognosis for the development of the respective lesion.After establishing the assay principle with cultured cell lines, the study was extended to clinical smear samples. We found that HPV genotyping is generally possible from a crude lysate of clinical liquid-based cervical smear samples fixed in PreservCyt, and that the natural cervicovaginal matrix of these does not compromise the results. The QG-POC assay identified less HPV infections and more samples tested negative compared to the PCR-based MPG assay. This was expected since the MPG assay is a highly target-amplifying, endpoint PCR-based test that detects DNA, while the QG-POC assay is only signal-amplifying and detects mRNA. This means the method itself is less sensitive than the PCR and only detects HPV-E7 if the host cell is really infected and actively transcribes the oncogene to sufficient mRNA copy numbers. Therefore, the QG-POC assay evaluates more samples from the normal or HPV-negative LCIN sample groups than the MPG assay. However, the QG-POC assay also missed two HPV16-positive CxCa and 38 HPV16-positive HCIN samples, where the mRNA copy numbers should be high. Interestingly, the samples detected as HPV16-E7-negative by QG-POC also showed a significantly lower expression of p16egresses . HPV16-EIn total, a fair agreement between the MPG and QG-POC assays was found, with an overall concordance of 70.29% for the six included and analyzed genotypes . Potentially, further improving the sample lysis conditions could lower the background signals and increase the assay sensitivity in clinical smear samples, and this should be optimized especially for HPV16-E7 detection. In addition to the lower HPV-positivity, Cohen’s kappa showed no agreement for HPV68-E7 between the MPG and QG-POC assays. Two genetical subtypes exist, termed HPV68a and HPV68b . It has INK4a in HCIN and CxCa samples compared to samples classified as normal without lesions and LCIN. This association has been described in studies before and has led to the hypothesis that testing the amount of mRNA is indictive of the presence of dysplasia and its severity [INK4a, HPV16-E7, and the leading HPV-E7 with high-grade lesions (HCIN+) and remarkable ORs between 4.5 and 6 was found. Furthermore, the ROC analysis showed that these biomarkers have the potential to discriminate between high-grade dysplasia and low-grade dysplasia, with AUC values of 0.71 for p16INK4a, 0.68 for HPV16-E7, and 0.74 for the leading HPV-E7. Combining the biomarkers p16INK4a and the leading HPV-E7 in a multivariate logistic regression model allowed a discrimination of the clinical stages for the border ≤LCIN and HCIN with an AUC value of 0.74 . For the discrimination of ≤HCIN and CxCa, the association between the expression strength of p16INK4a and the leading HPV-E7 had an OR of 2.08 and 1.51, and resulted in an AUC of 0.69 and 0.79, respectively. The combination of these markers into a multivariate regression model increased the detection of CxCa with an AUC of 0.77, and with a sensitivity of 81.8% and a specificity of 77.4% at the point of best cut-off.The quantification of the target mRNA is a means of evaluating whether the transcription and higher expression of the viral oncogenes corresponds to transformation events. We observed a significantly increased expression of HPV-E7 and of p16severity ,15,42. UINK4a in a multivariant regression model resulted in a higher sensitivity (70.4%) compared to the sensitivity for the biomarkers alone . However, specificity and assay accuracy did not benefit from this combination. The expression of p16INK4a is directly affected by the expression of HPV-E7, so both markers are not independent parameters, and therefore the combination of these biomarkers is not necessarily advantageous [INK4a is that its expression increases in an age-dependent manner, regardless of the presence or severity of dysplasia [s = 0.15, p = 0.014) of p16INK4a expression with the age of the patients was observed for all samples. This could be problematic for a universal cut-off development, as a woman at a higher age could have a false-positive test result, which would need to be validated. This naturally increased p16INK4a expression, however, was less intense than that induced by hrHPV infection (INK4a, other cellular biomarkers such as Ki67, MCM2, Topo2α, and hTERT have been described as potential markers for cervical dysplasia detection, triage, and diagnosis [TM 2.0 Plex Assay platform enables the multiplexed evaluation of up to 80 targets simultaneously in one single well. Therefore, the inclusion of additional hrHPV genotypes and virus-independent cellular biomarkers is possible to find the best biomarker combination for dysplasia detection and severity grading.This proof-of-concept study used a restricted number of HPV genotypes in combination with only one cellular biomarker. Combining the leading HPV-E7 and p16ntageous . A furthysplasia . Furthernfection . The addiagnosis ,45. FurtBoth commercial DNA and mRNA-detecting HPV tests, have a higher sensitivity for detecting CIN2+ and CIN3+, but display lower specificity than this experimental QG-POC assay ,47. ThesIn this proof-of-concept study we demonstrated the high potential of the QG-POC assay to detect cervical dysplasia and simultaneously indicate its severity in one step from a liquid-based cytology cervical smear sample. Further studies are warranted to investigate if the integration of other biomarkers further improves the assay specificity and diagnostic classification. Additionally, a study with more and better characterized samples is needed to develop risk scores and validate the best marker combination.


Self‐sampling human papillomavirus (HPV) testing has been introduced for cervical cancer screening worldwide. In Japan, there are two types (brush and sponge) of HPV self‐sampling devices. However, the recommended type for cervical cancer screening remains unclear. This study aimed to evaluate the feasibility of the HPV self‐sampling device–sponge type (HSD‐ST). Additionally, we aimed to examine the positive rate (sensitivity) for cervical intraepithelial neoplasia (CIN) 2 or worse using the HSD‐ST. Finally, we aimed to perform a questionnaire survey regarding the usability of the HSD‐ST.We included 165 women who underwent HPV testing at one of three gynecologic clinics. First, the women used the HSD‐ST and completed a questionnaire regarding its usability. Subsequently, they underwent physician‐sampling HPV testing and cytology. We examined the agreement rate of HPV positivity between self‐ and physician‐sampling HPV testing.The HPV‐positive rates of self‐ and physician‐collected samples were 59.4% and 62.4%, respectively, with an overall concordance rate of 88.5% and a calculated kappa coefficient of 0.76, indicating high concordance. Moreover, the positive (sensitivity) rates for CIN2 or worse were 81.4% and 89.8% in the self‐ and physician‐collected samples, respectively.Our findings demonstrated the feasibility and usability of the HSD‐ST. High‐risk human papillomavirus (HPV) is a major cause of cervical cancer. Moreover, most cervical squamous cell carcinomas (>90%–95%) are related to HPV.In 2009, the Japanese Guideline for Cervical Cancer Screening recommended cytology alone.There are two types of self‐sampling devices (brush and sponge). However, the recommended type for cervical cancer screening remains unclear. This study aimed to evaluate the feasibility of the HPV self‐sampling device–sponge type (HSD‐ST). Moreover, we aimed to examine the sensitivity of HSD‐ST for CIN2 and perform a questionnaire survey of the usability of the HSD‐ST.We included women who visited one of the three participating gynecologic clinics where colposcopy was available; moreover, the study population was suspected of being at a high risk of HPV infection. Women aged 20–50 years were included. We excluded women during menstruation or within 1 week after menstruation, pregnant women, and those who had undergone total hysterectomy. Finally, 165 women were enrolled from May to November 2020. All participants provided written informed consent.The participants were provided with the HSD‐ST  Figure , the resCytology and histological results were received within 3 months and 1 year, respectively, before obtaining informed consent. Cytology and histological tests were performed when considered necessary by the physician, if participants were suspected of having cervical cancer on the day informed consent was provided or if participants were suspected of having cervical cancer based on the HPV status after the day of informed consent.Cytological findings were classified using the Bethesda System and histological findings were classified based on the General Rules for Clinical and Pathological Management of uterine cervical cancer fourth edition.The study conformed to the ethical guidelines for medical and biological research involving human participants. The study protocol, informed consent sheet, and other ethics‐related documents were approved by the ethics committee of Kitamachi Clinic and the head of each medical institution (Project no. CMI07279).Most participants were not accustomed to using self‐sampling devices. Since the type of device may influence the results, we only used the HSD‐ST Figure . The phyHPV testing of self‐ and physician‐collected samples was performed using the Cobas 4800 HPV system (Roche Diagnostics KK) at CMIC Pharma Science Co., Ltd.Further, HPV DNA testing of cervical cells was performed. The levels of high‐risk HPV genotypes, that is, HPV16, HPV18, and other HPV genotypes , were measured.The primary endpoint was the overall agreement rate between self‐sampled and physician‐sampled test results, which was calculated as follows: the sum of positive and negative concordant samples/total number of samples. The secondary endpoints were the positive and negative concordance rates calculated using the physician‐sampling method for HPV testing as the benchmark. The positive concordance rate was calculated as the proportion of positive concordant samples to positive physician‐collected samples while the negative concordance rate was calculated as the proportion of negative concordant samples to negative physician‐collected samples. We performed stratified analyses of the self‐ and physician‐sampling test results.F distribution). Paired nominal data were tested using McNemar's test or binomial test, as appropriate.Statistical analyses were performed using SAS Ver.9.4. The overall, positive, and negative concordance rates were evaluated using the kappa coefficient and the confidence interval based on the Clopper–Pearson exact method  and 88.7% (95% CI: 78.1%–95.3%), respectively. These results demonstrated strong agreement between the two sampling methods.The overall concordance rate between physician‐ and self‐sampling HPV testing was 88.5%  , the kappa coefficient was 0.81 (95% CI: 0.72–0.90), and the p value for the McNemar test was 0.08. Tables Results of self‐ and physician‐sampling according to HPV genotype was shown on Table 22 Table . For HPVAmong the 162 women who underwent cervical cytology, there were 71 and 91 women with normal (NILM) and abnormal cytology (other than NILM) Tables  and 4b, The HPV‐negative and ‐positive concordance rates were high in women with normal and abnormal cytology. Tables The HPV‐positive rate was calculated for 82 women with cytology tests performed within 1 year from the day of informed consent Table . For womMost participants  were using the HSD‐ST for the first time . The calculated kappa coefficient was 0.76, indicating good consistency. Previous studies reported that the calculated kappa coefficient for HSD‐ST and HSD‐BT was 0.7–0.8.In our study, the overall concordance rate between self‐ and physician‐sampling was 88.5%, with the McNemar test revealing no significant between‐method difference  for abnormal cytology. In our study, there was no difference in the positive rate (sensitivity) between HSD‐ST and physician‐sampling . The corresponding values in a previous study were 51.4% (19/37) and 56.8% (21/37), respectively,It is important to determine the positive rate for CIN2 or worse using the HSD‐ST to determine its feasibility in cervical cancer screening programs. In our study, the sensitivity rates for CIN2 or worse were 81.4% (48/59) and 89.8% in self‐ and physician‐sampling, respectively, exhibiting no between‐method difference in sensitivity. In 2018, Arbyn et al. conducted an updated meta‐analysis on 56 accuracy studies and 25 participation trials, regardless of the device.The limitation of this study is not to directly compare that the feasibility of HSD‐ST and HSD‐BT were not directly compared, but the performance of HSD‐ST was evaluated based on the results of physician‐sampling. This study serves as a first step toward the introduction of HSD‐ST into the cancer screening program in Japan. The next research step is confirming that HSD‐ST is non‐inferior to HSD‐BT, which is the main self‐sampling device, regarding the cancer detection rate among women visiting gynecologic clinics. The final step will be directly comparing the cancer detection rate between HSD‐ST and HSD‐BT in cervical cancer screening programs.In conclusion, this is the first study to show the positive rate (sensitivity) for CIN2 or worse using HSD‐ST. We observed an extremely high concordance rate and comparable positive rate (sensitivity) for CIN2 or worse between HPV self‐ and physician‐sampling testing. Our findings demonstrate the feasibility and usability of the HSD‐ST. Introducing the HSD‐ST in cervical cancer screening programs could improve the participation rate. A future study comparing HSD‐ST and HSD‐BT in the cervical cancer screening program is required.None.The authors declare no conflicts of interest other than those noted in acknowledgments or funding information.