HPV testing has been integrated in cervical cancer screening program. Patient-providers relationship is extremely important to improve cervical cancer screening outcomes. This qualitative study aims to understand HPV-positive women’s needs and preferences about HCPs and patient-provider communication based on their experiences of accessing primary and specialized care.We conducted 40 semi-structured interviews with HPV-positive women. Recorded interviews transcribed and analyzed using conventional content analysis approach.The analysis of the data led to the extraction of three main categories, including: provider’s communication and counseling skills, commitment to professional principles, and knowledgeable and competent provider. Women needed understandable discussion about HPV, emotional support and acceptance, receiving HPV-related guidance and advice, and some considerations during clinical appointments. Women needed HCPs to treat them respectfully, gently and with non-judgmental attitude. “Precancerous” and “high-risk” words and watching colposcopy monitor during procedure had made women anxious. Weak referral system and limited interactions among gynecologists and other HCPs highlighted by participants.The results of this study, based on the experiences and perceptions of HPV women receiving health care, contain messages and practical tips to healthcare providers at the primary and specialized levels of care to facilitate patient-provider communication around HPV. Providers need to approach the discussion of HPV with sensitivity and take individual needs and preferences into account to improve the HPV-positive women’s healthcare experience.The online version contains supplementary material available at 10.1186/s12913-021-06283-w. Human Papillomavirus is a common sexually transmitted infection, with potentially serious health consequences like cervical cancer (CC)  which isIn Iran, the national HPV vaccination program has not been started but Gardasil can be purchased around $100 in some pharmacies. The national CC screening program recommends co-testing (Pap smear and HPV testing) for women aged 30 to 59 every 5 years  was talking about."(W.16)Women made a few points that they believed would make the discussion about HPV more understandable. Women needed HCPs to use plain language, avoid underestimating and over-simplifying the disease, stick to the factual information about HPV. The terminology commonly employed by physicians, were mostly unfamiliar to women. Few women indicated that when a doctor considers HPV insignificant, they feel ignored."I was so scared. My doctor said: 'cancer patients don't mourn like you. HPV is not that important'. I think insomuch she sees cancer it’s gotten trivial for her."(W.26)To prevent misunderstanding and ensure patient awareness, participants also noted that gradually transmitting the information in multiple appointments helped them with coping and asking their questions.Several participants reported that the providers had broken the positive-results news in a way that has made them anxious and shocked."Pointing out that HPV is common and most women are unaware of their infection changes women's attitudes toward diagnosing HPV from a threat to an opportunity to prevent cancer."(Pr.8)Some patients expressed dissatisfaction with the time physicians spend to address their concerns. “Very few doctors make time for the patient and talk to them patiently. Our services are not really good compared to some developed countries.”(W.23).Few women stated that comparing the incidence of cervical cancer and HPV infections prevalence (to clarify the cancer probability in HPV-infected women) has decreased their fear of cancer. Several women were terrified after hearing the term “precancerous/high-risk”. About the initial patient’s fear of cancer, a provider stated:“No gynecologist has the patience and time to answer my questions.”(W.12).Few women reported that their primary HCPs had referred them to the specialized clinic without explaining their health problems. Women’s questions on HPV were often inadequately answered, particularly in specialized governmental crowded referral clinics. "Every patient needs a good doctor. The patient's thoughts and beliefs forms based on his/her doctor's words. My doctor treated me well and gave me hope and spirit."(W.2)According to participants’ point of view, the cold, annoying, and repulsive behaviors of the provider/doctor hinders successful follow-up and treatment. Women’s feelings and concerns needed to be accepted by their HCPs. women found the doctor’s excessive self-protection behaviors  repulsive, HCPs attributed them to the insufficient knowledge of their colleagues.Our respondents were more satisfied with compassionate providers and saw them as a source of emotional support.Women indicated that encountering this fact that “HPV has no cure”, they have felt powerless because they can’t do anything to maintain their health."I preferred my doctors to give me a tough solution/treatment rather than leaving me alone with the fact that there was no cure for HPV. Some providers have simple, useful, and inexpensive tips to strengthen immunity of patients ."(W.2) In this regard a provider stated:"When I say most HPV infections will clear up or become undetectable on their own, some women ask me: 'Why hasn't my infection cleared?' They usually ask me if there is a test for checking the immune system or what they can do to reduce the risk of cancer to the negligible levels.” (Pr.5)Although women needed advice on sexual health, the participants indicated that they feared bringing up the subject. A gynecologist-oncologist mentioned: “Due to cultural sensitivities, neither patients nor doctors are inclined to speak about sexual issues.”(Pr.1).Women with genital warts needed additional information on surgical/medical anti-wart treatments."I no longer go to screening due to the intense pain I experienced."(W.22)Before performing the colposcopy procedure, women needed verbal or written information. Few women reported that watching the colposcopy monitor during the procedure made them terrified."I can't get that frightening colposcopy picture off my mind!"(W.9)They also preferred HIV and hepatitis tests not be asked at the very first appointment.The uncomfortable or painful gynecological examination was a problem that several women described as a factor that prevented them from returning to screening.Some of the needs emerged by women with positive HPV results were related to observing professional principles. Their statements showed that some HPV-positive women had negative experiences and wanted better healthcare providers’ performances in these areas .I think OB/GYN visits require the most privacy. I went to a famous GYN's clinic which besides me, there were other patients in the room. Imagine sitting next to a complete stranger who might hear and talking about sensitive topics that are very difficult to discuss... I was so embarrassed.” (W.10)“Several women mentioned that some clinic’s secretaries have talked about their condition (HPV) in the presence of other patients.Participants noted that in addition to physicians, any provider dealing with sexually transmitted patients must be aware of and committed to privacy and secrecy.“I don’t know, maybe my doctor is doing try and error” (W.17).Women stated several doctors had recommended or prescribed unproven medications for HPV cure e.g., probiotics and herbal vaginal suppositories. Patients were not sure if it had been a research project or not. A woman stated: Participants were less satisfied with provider/physicians who highlight stigma by their judgmental behaviors and language. Women did not trust such HCPs and were reluctant to share their private information with them. Few women attributed the provider’s excessive self-protection behaviors to religious issues related to impurity.“I paid 10,000,000 IRR for a colposcopy in a private office. If my doctor had referred me here [Valiasr], I could’ve had a colposcopy for 400,000 IRR! She deliberately did not refer me here!”(W.14).Participants indicated lack of insurance coverage for diagnostic and therapeutic services. Patient pessimistic believes about financial misconducts of HCPs revealed in some interviews. "My doctor recommended a colposcopy; After she had realized I had no money for it, she stopped explaining it and did not answer my questions anymore"(W.17)Some women were upset of HCPs who treat them based on their economic status. Few women with low socio-economic status felt being rejected by their HCPs.Knowledgeable and competent HCP is among the three main needs and preferences of women infected with HPV. Participants indicated to the importance of HCPs HPV-knowledge in three main sub-categories as follows:"After my husband's genital warts, I went to a gynecologist who said: 'It doesn't matter. Since you don't have a lesion, you don't need HPV testing."(W.31)A few participants, on the other hand, reported another form of guideline discordant that some gynecologists appeared to overuse screening tests (upon the patient’s request) to provide greater reassurance. A woman stated:"My doctor has recommended me to have GYN examinations once every two months." (W.12)Our findings showed that although some women were eligible for screening, they were not invited to be screened by a midwife/gynecologist at the primary or specialized level. A 34-years-old HPV-screening eligible woman reported:"We don't know whether we should take HPV-vaccine or not." (W.6)Participants indicated that physicians’ conflicting opinions on HPV vaccination and using condoms in those already infected with HPV were among the reasons to their frequent visits. Regarding HPV transmission concerns and questions of women, a gynecologist stated:"I explain (my patients) intentionally vague about HPV transmission; I try to convince them that they could have contracted it in almost any way."(Pr.2)Several women reported their HCPs have used extra-protective equipment such as wearing three pairs of surgical gloves indicating a misconception that non-sexual transmission of the virus is serious.Women noted that uncertainties such as HPV-vaccination of HPV-infected people should be addressed in HPV-discussion to prevent them from confusing and wandering.Few women exposed misinformation conveyed by their HCPs who may lack current HPV information. A woman reported a provider has recommended her cesarean section because of her vaginal warts.“I’ve seen an ENT specialist and a dentist to get other opinions just to be on the safe side.”(W.28).More than half of the women interviewed had seen at least three doctors in the 12 months before the interview. It seems that the more women went to different clinics (offices), the more HPV-knowledge and less stress. However, this would have cost more. Women reported attending and talking to a number of different HCPs. According to participants’ point of view, there is not an optimal cross-disciplinary referral system among gynecologists and other HCPs. A woman with oral ulcers who was worried that her lesion might be an HPV-related oral cancer (or precancerous) stated: "When my partner and I realized we have genital warts, we did not know which specialist to go to. We went to an infectious disease specialist, urologist, gynecologist and dermatologist. In my opinion, the best specialist in the treatment of genital warts is a dermatologist. I wish we had gone to a dermatologist from the very beginning of my diagnosis." (W.14)Another woman expressed:In this study, experiences, needs and preferences of HPV-infected women about patient-providers communication clearly identified. Findings indicated that providers’ communication strategies are as important as content. The way that HPV-information is conveyed needs careful consideration. Similar findings showed how providers communicate women about their HPV results can have an influence on their emotional responses to HPV diagnosis . Women mhttps://CervixCa.nlm.nih.gov/RiskTables, [Our findings noted that healthcare providers should be cautious in using cancer-related terms. Similarly, ASCCP consensus (2020) is hesitant that precancer is the best definition for CIN2 or CIN3 and higher , 36. UsikTables,  could alIn response to participant’s need HCPs to spend enough time answering their questions, our findings suggested addressing current gaps and uncertainties in HPV-knowledge. Similarly, another study addressed the importance of disclosing existing limitations in HPV-knowledge with patients . AnotherIn line with our findings that women needed to be treated gently and respectfully in clinical appointments and gynecological examination, a qualitative study conducted in Tanzania (2019), indicated that the gynecological examination was an element that may hinder women from returning to screening . In the One of our findings that may be influenced by Iran’s religious context was that women viewed the protective behavior of HCPs as the rejection related to impurity (being dirty) issues. Another study indicated that HPV patients may perceive certain physician behavior as negative even though the doctor does not plan to . In a quIn a study conducted in Kenya, financial support was key facilitators of HPV-positive women’s treatment access . Due to Our study showed that knowledgeable and competent HCP is among the main needs of women infected with HPV. Provision of communication skills training for sample takers should help minimize adverse psychological responses . CounselService coordination among different levels of care was addressed in another study . In our We acknowledge the study’s limitations. Our study environment was one busy specialized center for HPV-patients so it is possible that emerged themes would be different from other clinics nationally. Therefore, caution is required when interpreting data. We should comment on the potential influence of a highly educated sample (~ 60% with university education) as another limitation. Naturally, qualitative research is not targeted at representativeness, but it seems likely that less-educated women’s viewpoints were not thoroughly explored. Some of the findings are cultural-specific and cannot be generalized to all communities.Like other qualitative studies, the generalizability and relative weight of emergent categories are not obvious. This paper follows COREQ checklist for reporting qualitative studies . ParticiThere is a need for further development of research on patient-provider communication, in particular with respect to a more solid theoretical basis, integration of methods including qualitative and quantitative methods, self-evaluations, and interaction analyses, and also concerning conducting more longitudinal studies.Women need advice on boosting immunity, vaccinations, follow-up, and sexual health.HPV-positive women’s main expectations of their healthcare providers include communication and counseling skills, commitment to professional principles, and HPV-knowledge.Exposing current HPV-knowledge limitations, gaps and uncertainties prevent women from excessive clinical referrals.Our findings, based on the experiences and perceptions of HPV women, contain messages that may provide deeper insight to HCPs working with HPV-infected people to establish evidence-based strategies to support and facilitate patient-provider communication in the primary and specialized levels of care. These practical tips will improve the quality of care for HPV-positive women, which is an integral component of cervical cancer prevention programs.Additional file 1.


To evaluate partial HPV16/18 genotyping as a possible biomarker to select women attending HPV‐based cervical cancer screening at higher risk to be referred to colposcopy.Population‐based cohort study.Organised cervical cancer screening programmes .Women with high‐risk HPV infection (period: 2015–2019).+) lesions.We analysed the association between partial HPV16/18 genotyping, cytology triage and histologically confirmed diagnosis of high‐grade cervical intraepithelial neoplasia (CIN3+ (any episode in the 2 years after baseline); sensitivity for CIN3+ and number of colposcopies needed for lesion detection.Detection rate (DR) and positive predictive value (PPV) for histologically confirmed CIN3+ at baseline; HPV16 and HPV18 were present in 1865 and 594 samples, respectively. The cumulative (baseline plus 1‐year repeat) cytology positivity was 42.8% and high‐grade cytology was significantly higher (P < 0.0001) among women with HPV16 infection at baseline (15.2%). The cumulative CIN3+ DR for women with HPV16, HPV18 and other HPV‐type infections was 9.8%, 3.4% and 1.8%, respectively.The study included 145 437 women screened with HPV testing by the clinically validated COBAS 4800 HPV assay (Roche). Overall, 9601 (6.6%) women were HPV+ detection while minimising colposcopy at baseline or 1‐year repeat.Partial HPV16 genotyping may play a role in triage, whereas HPV18 seems to behave much more similarly to the other HPV types and does not provide additional stratification. HPV16 genotyping combined with high‐grade cytology can be envisaged as a triage biomarker in cervical screening to maximise CIN3+ in HPV‐positive women.HPV16 genotyping combined with high‐grade cytology can be used as triage biomarker for CIN3 + in HPV‐positive women.HPV16 genotyping combined with high‐grade cytology can be used as a triage biomarker for CIN3 Cervical cancer is caused by persistent infection with high‐risk human papillomaviruses (HPV),Screening tests are used to identify women at risk of developing cancer and aim to reduce the risk of disease and associated mortality by detecting and treating precursor lesions before they progress to cervical cancer.European guidelines recommend primary HPV testing for organised, population‐based screening.Essential Health Interventions’ since 2001.In Italy, the implementation of organised cervical screening programmes has been recommended since 1996,In 2013, following the results of pilot projects including 25‐ to 64‐year‐old or 35‐ to 64‐year‐old women, the Italian Ministry of Health included HPV test every 5 years as an option for screening programmes for women aged ≥30 years.HPV infections have a transient nature, with only a small proportion of HPV‐positive women developing high‐grade cervical intraepithelial neoplasia (CIN).+). Cytology is the most frequently used triage strategy, but the management of HPV‐positive women with negative cytology is particularly challenging, as the risk of disease is too high to return these women to regular screening but too low for colposcopy referral,Therefore, all HPV‐positive results need additional triage in order to identify women with a high risk of cervical cancer and precancer  and Central (Umbria) Italy were included in the study.Veneto and Emilia‐Romagna participated with one of the three regional centralised HPV laboratories, whereas Umbria with the only one regional laboratory. Organised cervical cancer screening has been in place in all three regions for more than 20 years; HPV testing has replaced cytology as the primary test since 2015.+) and positive cytology  or worse – cyto+) underwent immediate colposcopy; women with HPV+ and negative cytology (cyto−) were referred to HPV re‐testing at 1 year. In this paper, we refer to HPV testing plus cytology triage and eventual colposcopy as ‘baseline’. After 1 year, women with persistent infection underwent colposcopy (irrespective of cytology result), whereas those with a negative HPV returned to regular screening  women were followed by repeat testing, according to the local protocols, with timing between visits being defined according to cytology grade.+ was recorded.A 2‐year period of post‐colposcopy follow up (FUP) was available and was considered for this study. Despite the availability of national indications for the FUP protocol after an abnormal Pap test and a negative colposcopy, there is no uniformity in their application,Data about HPV test, cytology, colposcopy and histological diagnosis at baseline and 1‐year repeat were extracted by the screening programmes databases. As regards post‐colposcopy FUP, only data for HPV positivity and histological diagnosis were available.HPV testing was performed by the clinically validated COBAS 4800 HPV assay , based on real‐time polymerase chain reaction (PCR) technology.The method detects 14 HPV types  and provides specific results for HPV16 and HPV18, and altogether for the other 12 types (hereafter referred to as non16/18HPV), and includes an internal quality control (beta‐globin) for each sample. In the case of multiple infections, women with at least HPV16 (including HPV18) were classified as HPV16, and women with HPV18 or co‐infected by HPV 18 and other non‐16 types were classified as HPV18. Typing was not used for triage.Cytology triage was classified according to the 2014 Bethesda reporting system: negative for intraepithelial lesion or malignancy (NILM) and epithelial cell abnormalities categorised in low grade (LG) and high grade (HG). LG includes ASC‐US and low‐grade squamous intraepithelial lesion (LSIL). HG includes atypical squamous cells, not excluding high‐grade SIL (ASC‐H), high‐grade squamous intraepithelial lesion (H‐SIL), atypical glandular cells favor neoplastic (AGC), adenocarcinoma in situ (AIS), squamous cell carcinoma and adenocarcinoma.Colposcopies were performed according to the 2011 colposcopic terminology of the International Federation for Cervical Pathology and Colposcopy (IFCPC).+ (any episode in the 2 years after baseline) of HPV16, HPV18 and non16/18HPV, and (iii) the sensitivity for CIN3+ and number of colposcopies needed for lesion detection. Histologically confirmed CIN2+ was a secondary outcome of the study.The primary outcomes of the study were (i) the prevalence of HPV types at baseline, (ii) the detection rate (DR) and positive predictive value (PPV) for histologically confirmed CIN3The parameters listed below were calculated for HPV16, HPV18 and non16/18HPV groups.+ tests/HPV tests).HPV test positivity (HPV+ (women with cyto+ at baseline/HPV+ women), overall and by cytology grade .Proportion of positive cytology among HPV+ and CIN2+ among HPV+  at baseline/HPV+ women).Detection of CIN3+ and CIN2+ of biopsy  at baseline/biopsies performed).Positive predictive value (PPV) for CIN3+ tests/HPV tests at repetition).Persistence of HPV positivity at 1‐year (HPV+ at 1‐year/ HPV tests at repetition), overall and by cytology grade .Proportion of positive cytology at 1‐year repeat  at 1‐year repeat/HPV tests at repetition).Detection of CIN3+ and CIN2+ of biopsy  at 1‐year repeat/biopsies performed at repetition).Positive predictive value (PPV) for CIN3+ and CIN2+  at baseline + 1‐year repeat/HPV tests at baseline).Detection of CIN3+ and CIN2+ of biopsy  at baseline + 1‐year repeat/biopsies performed at baseline + 1‐year repeat).Positive predictive value (PPV) for CIN3For women with a persistent HPV infection at 1‐year, we compared the HPV type detected at baseline with the HPV type at repetition.Results for HPV testing.+ detection rate.CIN3All analyses were performed for all screened women and (limited to Umbria and Veneto regions) stratifying the indicators in four age classes .+) excluding all women who had multiple HPV types detected.A sensitivity analysis was performed estimating the main outcomes , compared with a reference strategy, and the number of colposcopies needed to detect one lesion (CIN3+) were computed. The reference strategy at baseline (Strategy 1) refers to colposcopy for all HPV+ women with a positive triage cytology  or with HPV16. The reference strategy at the 1‐year repeat (Strategy 5) refers to colposcopy for all women testing HPV+.Finally, according to the study results, we identified different triage strategies of HPV positives at the baseline and for women with a persistent HPV at the 1‐year repeat. For each strategy, the relative sensitivity for CIN3χ2) and the Fisher’s Exact test.Differences in outcomes distribution among HPV types were tested using the Chi‐square test  was used for all analyses.A Core outcome sets and patient involvement are not relevant to this study.This study included 145 437 women screened with an HPV test between January 2015 and December 2017 Table . OverallThe results are presented divided by the specific time‐points of the screening episodes: baseline; 1‐year repeat and cumulative; follow up.+: 1.28% for HPV16, 0.41% for HPV18, and 4.91% for non‐16/18HPV types (Table At baseline 9601 (6.6%) women were HPV+ women at baseline were positive at cytology triage: 44.1% for HPV16, 34.3% for HPV18 and 32.8% for non‐16/18HPV (P < 0.0001). These results were consistent among the three study regions.Overall, 35.1% of HPVP < 0.0001).An HG cytology was diagnosed in 15.2% (283/1865) of HPV16+ women versus 7.4% (44/594) of women with HPV18 and 4.8% (346/7142) of women with non‐16/18HPV . The detection rate of CIN3+ was 2.7% (256/9601): 7.9% among women with HPV16 infection (148/1865), 2.0% among women with HPV18 infection (12/594) and 1.3% among women with non‐16/18HPV infection   versus 1.5% with LG cytology (P < 0.0001).The detection of CIN3P < 0.0001) (Table Attendance with the repetition of HPV testing at 1 year was 79.3% (4943/6232). HPV positivity was recorded for 59.1% (2922/4943), with statistically significant differences according to HPV type at baseline: 67.0% for HPV16, 62.7% for HPV18 and 57.2% for non‐16/18HPV .The result of cytology at the 1‐year repeat is available for 2843/2922 (97%) women; 25.9% (737/2843) tested positive, without any statistically significant difference by HPV type at baseline. However, women with HPV16 at baseline and persistent HPV infection were twice as likely to have HG cytology compared with women with HPV18 or other HPV types , higher with positivity at baseline for HPV16 than other HPV types, i.e. 4.2% (34/811) versus 2.6% (8/311) for HPV18 infection and 0.8% (31/3821) for other HPV infection (P < 0.0001).Women with a positive HPV test at 12 months were referred to colposcopy; the attendance rate was 95.8% (2799/2922). The CIN3+ was 4.6% in women with persistent HPV16 infection at 1 year, whereas it was 1.4% in women with persistence of infection by HPV18 or other HPV types (P < 0.001).The detection of CIN3P < 0.0001).The cumulative (baseline plus 1‐year repeat) cytology positivity was 42.8% (4106/9601) and HG cytology was significantly higher with HPV16 infection at baseline  than with HPV18 or other HPV types   and it was two or three times greater for women with HPV16 infection than for women with HPV18 or non16/18HPV  .Follow‐up data were available for 2452/5148 (47.6%) women; most missing data refer to women whose follow up was not completed, mainly women in follow up after 1‐year HPV persistence. The HPV persistence was 57.5%; higher for HPV16 than for HPV18 or non16/18HPV  than with HPV18 or with non16/18HPV types .The detection of CIN3P < 0.0001).HPV positivity was 18.9% in the 25–29 age group and decreased at increasing age to 3.7% in the 55–64 group (Table + was higher in the 25–29 (3.5 per 1000 screened) and in the 30–44 age (4.0) groups and decreased to 1.6 in the 45–54 and 0.7 in the 55–64 age groups (P < 0.0001) (Table The cumulative detection of CIN3+ cases were associated with HPV16 (67% in the 2‐ to 29‐year‐olds and 60% in the 35‐ to 44‐year‐olds), whereas about half of the cases were associated with HPV16 (48% in both age groups) in older women, and non16/18 HPV types accounted for 42% and 44% of cases, respectively, in women 45–54 and 55–64 years old.In the two younger age groups (i.e. 25–44 years), about two‐thirds of CIN3+ (panel A), and sensitivity and number of colposcopies needed (NNC) to detect one lesion (panel B) for various strategies at the baseline and at 1‐year repeat. Compared with the reference strategy (highest sensitivity) for the baseline , the strategy based on HPV16 alone (Strategy 3) would detect about two‐thirds of CIN3+ . On average, 9.4 colposcopies would be needed to detect one CIN3+, compared with 12.1 under the current strategy . Referring to colposcopy, both HPV16 and non‐16HPV with high‐grade triage cytology (Strategy 4) would yield a sensitivity of 0.93 (95% CI 0.90–0.96), with the lowest NNC to detect one CIN3+ (7.7).Figure + , compared with the (current) referral of all women with persistence of HPV (Strategy 5). However, the NNC to detect one CIN3+ would be 18.9 versus 38.3.At the 1‐year repeat, referring to colposcopy women with a persistent HPV16 or a persistent non‐16HPV with positive cytology (Strategy 6) would miss 18% CIN3In this study we evaluated partial HPV16/18 genotyping as a triage biomarker for high‐risk HPV‐positive women identified within organised HPV cervical screening, in order to devise new strategies to improve its efficacy. This is especially important for the management of HPV‐positive women with negative cytology at baseline, which is particularly challenging.+ development than any other HPV type (including HPV18) at all time‐points considered . With all analyses, it emerged that HPV18 infection behaves more similarly to other high‐risk HPV types than to HPV16, in accordance with previously published results.Infection by HPV16 was associated with greater detection of high‐grade cytology, viral persistence and CIN3In particular, triage cytology at baseline was positive in almost half of women with HPV16, but in only one‐third of women with HPV18 or other HPV types. Further, about one in three of positive cytologies in HPV16 cases were HG, as compared with one in five with HPV18 and one in seven with non16/18HPV.According to the Italian screening protocol, women who test HPV‐positive/cytology‐negative are referred to repeat HPV testing after 1 year, followed by colposcopy in the case of positivity (irrespective of cytology result). Overall, nearly 60% of these women are still HPV‐positive, and viral persistence was greater in women with HPV16 than with other HPV type infection at baseline (up to 10 percentage points than for HPV18), without significant differences by age. Longer intervals between baseline and HPV testing repetition, as well as risk stratification by molecular biomarkers, have been proposed.+ was 2.7%, with a PPV for HPV+/cyto+ at colposcopy of 8.2%. The detection of CIN3+ among women infected by HPV16 was 7.9%, with a PPV of 19.6%, whereas HPV18 infection had a lower association with CIN3+ , closer to non16/18HPV types .The role of HPV16 also emerged when considering histologically confirmed high‐grade lesions. At baseline, the overall detection of CIN3+ decreased, as expected, but the association with HPV16 was confirmed (PPV of 6.4% versus 4.2% for HPV18 and 1.5% for other HPV types).At the 1‐year recall, the detection of CIN3+ further decreased at follow up, but also in this case, the relative weight of HPV16 compared with the other HPV types was further strengthened.As expected, the detection of CIN3These findings highlight that partial HPV16 genotyping may play a role in improving immediate and short‐term triage, whereas HPV18 seems to behave much more similarly to the other HPV types and does not provide additional stratification. Nonetheless, as several studies indicate that HPV18 persistence is associated with an increased long‐term risk,Our findings derive from routine clinical practice within quality‐assured population‐based organised cervical cancer programmes. Attendance was 92.3% at immediate colposcopy and 95.8% at colposcopy after 1‐year HPV persistence, and compliance with HPV repetition at 1 year was 79%. Moreover, the main results showed high levels of consistency among the three study regions, supporting generalisability to Italy as a whole, and possibly to other European countries.A limitation of our study is that not all the analyses performed for the baseline and 1‐year recall could be performed for the follow‐up outcomes  due to partial availability of the data. Moreover, as not all the women underwent colposcopy, we cannot exclude some risk of verification bias.+ at all three time‐points could be exploited within triage protocols based on partial HPV16 genotyping, to provide a more efficient risk stratification and to reduce the number of colposcopies needed to detect a lesion, as suggested by the recent literature.The close association between HPV16 infection at baseline and CIN3+ at baseline and the 1‐year repeat.Based on these findings, Figure At baseline, the most sensitive strategy  does not seem to offer the best balance between lesion detection and the need for colposcopies. On the other hand, Strategy 4 (HPV16 or non16‐HPV with HG cytology), which combines reasonable sensitivity with a considerable decrease in the required number of colposcopies, appears to be the most attractive.At 1‐year recall, Strategy 6 (HPV16 persistence or non16‐HPV with HG cytology), compared with the reference, which is the current standard in Italy, shows a saving of about 50% in the number of colposcopies needed and a reasonable loss of sensitivity.+ development, in particular HPV33, HPV 35, HPV31; additional typing might therefore be useful, especially in vaccinated cohorts.Previous studies have shown other strategies to be effective. In particular, data from the ATHENA study suggested referring to colposcopy women who were ASC‐US/HPV‐positive and LSIL or more severe cytology, irrespective of HPV result, as well as all women who were cytology‐negative/HPV16/HPV18‐positive.+ lesions, as highlighted in our study, it appears that a strategy in which HPV16 positivity is utilised as a stratification tool may answer a currently posed question as to whether it is necessary to detect all CIN3+ or just those that are most likely to progress.Given the important role of HPV16 in detecting CIN3The results of this study demonstrate that within organised cervical screening the combination of partial genotyping and HG cytology has a potential for better stratification after HPV positivity.The authors wish to thank Dr. Luca Weis for assistance in the analysis of data and helpful discussion.The authors declare no potential conflicts of interest. Completed disclosure of interests forms are available to view online as supporting information.SG conceived the study, collected and analysed the data and drafted the manuscript. MZ, CC, RF, BP locally coordinated the screening activities and retrieved the data. JB and MZ performed the statistical analyses and reviewed the manuscript. SG, DG, HF, GS, SB, CF, EC performed HPV and/or cytological analyses. ADM supervised the planning design and the data analyses and reviewed the manuscript. All authors read and approved the final version of the manuscript.The study was performed in the context of a clinical service review and therefore ethical approval was not required.None.Table S1. Regions involved in the study, with study periods, number of screened women overall and by age, and proportion of positive HPV tests.Click here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.Supplementary materialClick here for additional data file.


Moreover, our study reveals a positive correlation between HPV presence and glioma malignancy, and a negative correlation with meningioma grading. Our results suggest that the presence of HPV seems to be significantly associated with primary tumours of the CNS and its meninges.Central nervous system (CNS) tumours have devastating effects and are recurrent, with dismal prognosis (gliomas) or life‐threatening by the compression effect (meningiomas). This disease's aetiology remains debatable. Over the last decade, the hypothesis that human viruses may be implicated in these tumours has been proposed. In this study, our aim is to examine the presence of 11 viruses in the most frequent CNS primary tumours. Using polymerase chain reaction (PCR), we assessed the viral presence in archived, paraffin‐embedded tumour tissues from 114 patients with glioma and meningioma and in the brain tissue from 40 controls lacking tumour pathology. We focused on candidate neuro‐oncogenic types (herpesviridae and polyomaviruses) and on human papillomavirus (HPV). HPV presence, for which involvement in these tumours was hardly investigated, was found to be associated with both tumour categories compared with controls (glioma,  However, since they cause morbidity and mortality that are disproportionate to their incidence, the mechanisms underpinning their aetiopathogenesis are an important topic of research. There are over 100 different histological types of primary CNS tumours, with an annual age‐adjusted incidence of 28.57 cases per 100,000 inhabitants.The most common tumours affecting the brain are brain metastases. Among primary tumours, gliomas  are the most prevalent malignant types, while meningiomas are the most common and histologically benign tumours affecting the CNS. Glioblastomas constitute 45% of malignant primary brain tumours and, according to the Central Brain Tumor Registry of the United States data (2008–2012), meningiomas account for 35% of all primary brain tumours.The aggressiveness of glioblastomas is both histological and clinical, determined by their localization within a rigid space and adjacent to vital areas; for example, patients with glioblastoma, the most aggressive glial primary tumour, have a 5‐year survival rate of 5.1% and a median survival of around 10 months.For any other tumour type, knowing the precise aetiology and pathogenesis would be an essential step to treat, cure or even prevent their onset. Little is known about the aetiology of this brain proliferative condition, and currently, the only established risk factors are ionizing radiation and some rare genetic‐related syndromes.Overall, approximately 12% of human cancers are supposed to have a viral aetiology.Regarding cancer, although EBV and KSHV are officially recognized as carcinogens, other viruses from this family have also been associated with different types of cancer, but their role in carcinogenesis remains unclear.Emerging evidence has demonstrated the presence of human CMV proteins and nucleic acids in brain tumours in both adults and children,Also, the results regarding the role of the Epstein‐Barr virus (HHV‐4) in glioma genesis are controversial.Also, HHV‐6 is potentially involved in glial oncogenesis. The ability of HHV‐6 to integrate into a chromosomal region that is highly relevant to carcinogenesis, the ability of the HHV‐6 ORF‐1 protein to bind to p53, and its detection of early and late antigens in adult primary and recurrent CNS tumours HHV‐8 or Kaposi's sarcoma‐associated herpesvirus is well known for its oncogenic role in a wide category of haematological malignancies. However, since it also has a strong neurotropism,Moreover, HHV‐2, EBV, and HHV‐6 have been detected in high‐grade glioma tissues, and HHV‐1 and HHV‐2 immunoreactivity have been found in some cases, although these were not detected in their tumour tissue.Human polyomaviruses represent a diverse group of human pathogens comprising John Cunningham virus (JCV), BK virus and simian virus 40 (SV40), which generally cause asymptomatic infection in healthy individuals. JC and BK viruses are mostly associated with cutaneous, mucosal tumours and bladder carcinoma.Regarding SV40, most studies on its presence have been performed in the sera of glioma patients without assessing its presence within the brain tissue.2In this retrospective study, we aimed to detect the presence of various potential oncogenic viruses in the most frequent tumours of the brain and their meninges (glioblastoma and meningioma) compared to patients lacking tumour pathology.2.1In this study, we enrolled 154 individuals. Fifty‐six patients were diagnosed with meningioma and 58 with glioma . Forty patients represented the control group, deceased by other causes and lacking any brain tumour. All study specimens were collected from the Neuropathology Department of the National Institute of Neurology and Neurovascular Diseases. This study was conducted following the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the National Institute of Pathology ‘Victor Babes’ (Registration number 53 of 6 December 2017). All the patients or their relatives signed the written informed consent. Only patients with primary tumours of the brain and meninges were enrolled. We excluded metastases (with biology specific to the tissue of origin), glandular  or accessory tissue (eye) tumours.The first section was stained with haematoxylin‐eosin, and a second histopathological diagnosis was established by an expert pathologist to confirm the initial classification of the cases. All tumours were classified and graded according to the WHO classification of CNS tumours.The initial series comprised all primary tumour categories of the brain, including oligodendroglioma, ependymoma and choroid plexus tumours, with a total of 200 cases. However, since most of these other entities were represented by a limited number of cases, we restricted our research to the most common and aggressive brain tumour, grade IV glioblastoma.8963.The sociodemographic and clinical data of the selected cohorts are reported in Table 2.2A number of 5–10 slides (50 μm) were cut from each sample according to the volume. Before DNA isolation, to remove the paraffin, the sections were placed at room temperature in two baths of xylene and absolute ethanol. Genomic DNA from FFPE tissue sections was isolated using a QIAamp DNA FFPE Tissue Kit , following the manufacturer's protocols. The quantity and purity of the DNA were determined using NanoDrop 2000 .The presence of HHV1 and HHV2, HHV4–5 and BK, HHV6, HHV7, HHV8, JCV and SV40 viruses was detected using dedicated Genesig Advanced kits  by qPCR, following the instructions of the manufacturer on the ABI7500 fast . Each kit comprises a positive control that contains templates for the targets in the test and an endogenous control to confirm the extraction of a valid biological template.The presence of HPV has been detected by PCR using consensus primers targeting the L1 region, GP5+/GP6+ primer set,For samples found HPV positive by auto‐nested PCR, genotyping was performed using the AMPLIQUALITY HPV‐TYPE EXPRESS v3.0  that allows the identification of the presence of HPV and the detection of 40 HPV genotypes: HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68 (68a e 68b), 69, 70, 71, 72, 73, 81, 82, 83, 84, 87, 89, 90. This kit is IVD (In Vitro Diagnostic) marked for detecting HPV in histological FFPE samples. With this method, we did not obtain PCR products after the first amplification. Thus, PCR reactions were subjected to a further 30 rounds of PCR amplification (auto‐nested PCR). In order to validate the specificity of the in‐house PCR, each experiment was performed using positive and negative controls for H39pi9p9piuprupii9PV. The positive control was represented by a DNA isolated from cervix carcinoma and the negative control by NTC (not template control) in order to verify the absence of cross contamination among the analysed samples.2.3Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS version 17.0). Continuous variable (Age) was tested with the t test. For categorical variables, the frequency, with percentages, was reported and assessed for the two groups by chi‐square test if the number of patients in a subgroup was ≥5, and when the number was less than 5 in any cell, the Fisher exact test was applied.3n = 56), glioma  and control (n = 40). The groups were homogenous for sex but not for age since the control group was older than the experimental group (p < 0.05). Regarding grading, in the meningioma group, most of the tumours were of grade I (67.86%), followed by grade II (26.78%), and only 5.36% were of grade III. The glioma group comprised 75.86% glioblastoma, grade IV, and 24.14% of astrocytoma. Among these, 64.28% were diffuse astrocytomas (grade II), and 35.72% were anaplastic astrocytomas (grade III).A total of 154 individuals were involved in the study, including 79 females (51.3%) and 75 males (48.7%). The mean age of the study population was 58.89 years (SD ±14.01). The participants were grouped into three groups according to the diagnosis of meningioma .p = 0.032    Figure . No stat) Figure . In the ) Figure .Regarding localization, no statistical difference was observed between convexity meningioma (9 HPV‐positive cases out of 35) and basal meningioma (5 HPV‐positive cases out of 21) , demonstrating the presence of HPV. However, none of the 40 genotypes tested were found in our groups.An analysis examining the HPV frequency in age categories (age ≤60 and age >60) was conducted in the entire selected cohort and in the three groups. No statistical difference was found between the two age categories in any analysed group Table .The distribution of HPV‐positive and HPV‐negative cases in more stringent age categories is reported in , the number of HPV‐positive cases was higher in the female group (26.6%) than in the male group (14.7%) but without statistical significance . This trend of difference between males and females was also observed in the control, glioma and meningioma groups, again without statistical significance. . No statistical difference was found regarding the presence of this virus among the groups. One of the positive patients with glioblastoma had a co‐infection with HPV and another with HHV6.HHV6 was detected in the same percentage as HHV4, but with a different distribution, with four positive cases in the control group, 1 in the meningioma group, and 1 in each glioblastoma and astrocytoma subgroups. Again, no statistical differences among the groups were shown. Of note, the patient with glioblastoma HHV6 positive presented with co‐infection with HPV.HHV7 was detected only in one patient with meningioma with HPV co‐infection, whereas HHV1–2, HHV5‐CMV, HHV8, JCV, BKV and SV40 were not detected in the control group or in the patient group. All the results are shown in Table 4p = 0.032 and p = 0.032, respectively). Of note, HPV positivity in the glioma group was more present in the glioblastoma subgroup (29.5%) compared to the controls (p = 0.012).In this study, we explored the presence of infectious agents belonging to the Herpesviridae family , polyomaviruses JCV, BKV, SV40 and HPV in patients with the most common brain tumours (glioma and meningioma) compared to the control group. We found that the most frequent virus in our analysed cohort  was represented by HPV, with 20.78% of positive cases. Specifically, HPV was detected in 25% of meningioma cases, 25.86% of glioma cases and 7.5% of control samples. Hence, HPV DNA presence was statistically more frequent in patients with glioma and meningioma compared to the controls , followed by grade II (13.3%), and no cases in grade III  tumours. This could be due to genetic abnormalities specific to each meningioma group , which could interfere with the viral infection. Since the spectrum of histological types of meningeal tumours is remarkably wide, ranging from epithelioid cells to mesenchymal ones (as is the fibrous variant), such particular interactions between the cell of origin (the arachnoid cap cell) and HPV are supposed to generate mostly benign categories of meningothelial proliferation.No statistical association was observed between the other selected viruses and the brain tumours analysed in our study.Our results on HPV in glioma and glioblastoma parallel those reported by Vidone and collaboratorsAgain, in line with our findings, Hashida's study did not show a correlation between CMV and HHV‐8 or EBV presence with glioblastoma.Regarding viruses belonging to the herpesviridae family, our results showing a lack of association between CMV and brain tumours parallel what was found by Holdhoff and collaborators,Herpes viruses, mainly HHV‐6, followed by HHV‐1, HHV‐3, EBV, HHV‐2 and CMV, are present in relatively high percentages in the facial or trigeminal ganglia in the general population,Finally, in our cohort, the presence of polyomaviruses  was not detected. Data from the literature affirm that, in adulthood, a large percentage of the population has been infected with JC and BK viruses. Indeed, a seroprevalence of 39% for JC and 82% for BK was detected in other studies, with the profile of young subjects being similar to adults, demonstrating that primary exposure occurs precociously in life.5Overall, our findings, although based on a small number of cases, show a significantly increased frequency of HPV in glioblastoma and meningioma compared to normal brain tissue and suggest the non‐involvement of herpes and polyomaviruses in the investigated brain tumours. However, the detection of a virus within the tumour tissue is not sufficient to affirm its role as a causative agent. Further studies on larger cohorts are needed to clarify the role of these infectious agents in brain tumours and to develop new preventive or therapeutic strategies. Since HPV has been found to have prognostic implications in glioblastomas, and the HPV vaccine in order to prevent cervical cancer cases has established success, a brain tumours prevention through vaccine may be considered.The authors declare that they have no competing interests.Dorel Eugen Arsene: Conceptualization (lead); Resources (lead); Supervision (lead); Writing‐original draft (lead). Elena Milanesi: Data curation (lead); Investigation (lead); Methodology ; Writing‐review & editing . Maria Dobre: Formal analysis (lead); Investigation (lead); Methodology ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


A further blinded independent test set (n = 14) was used to validate the model, and sensitivity of 90% and specificity of 100% were achieved. Improved triage would allow women with a high-risk persistent HPV infection to be referred for immediate treatment, while women with a low-risk transient infection could avoid overtreatment.Persistent high-risk human papillomavirus (HPV) infection can lead to cervical precancer and cancer. Recently, HPV testing has been introduced for primary cervical screening, but the HPV DNA test cannot distinguish between transient and persistent HPV infection. Thus, there is an unmet clinical need to develop a new test to identify women with a high-risk persistent HPV infection. Raman spectra were recorded from cervical smear samples  using the Cobas 4800 and APTIMA HPV test, respectively. Raman spectra were recorded from single-cell nuclei and subjected to partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 14). Sensitivity of 85% and specificity of 92% were achieved for the classification of transient and persistent HPV infection, and this increased to 90% sensitivity and 100% specificity when mean sample spectra were used instead of individual cellular spectra. This study showed that Raman spectroscopy has potential as a triage test for HPV-positive women to identify persistent HPV infection.The role of persistent high-risk human papillomavirus (HPV) infection in the development of cervical precancer and cancer is now well accepted, and HPV testing has recently been introduced for primary cervical screening. However, the low specificity of HPV DNA testing can result in large numbers of women with an HPV-positive result, and additional triage approaches are needed to avoid over-referral to colposcopy and overtreatment. The aim of this study was to assess Raman spectroscopy as a potential triage test to discriminate between transient and persistent HPV infection. HPV DNA status and mRNA status were confirmed in ThinPrep Cervical cancer ranks fourth for both incidence and mortality with an estimated 570,000 cases and 311,000 deaths worldwide in 2018 [Persistent infection with high-risk human papillomavirus (HPV) is accepted as the major cause of the development of cervical precancer and cancer . Over 10HPV testing has recently replaced cytology as the primary cervical screening method in many countries, including Ireland . HPV tesCytology-based triage, with and without HPV16/18 genotyping, is generally used to identify women with low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). However, cytology alone has a sensitivity of only 59–70% for detection of HSIL due to sampling, technical, and/or inter-observer errors mainly associated with the subjectivity of the morphological assessment .Thus, there is an unmet need for additional triage tests that can identify disease-specific biomarkers. A number of molecular biomarkers have recently been investigated to detect transforming HPV infections including E6/E7 mRNA, methylation markers, miRNAs, and protein biomarkers, such as p16INK4a, Ki67, minichromosome maintenance protein 2 (MCM), and topoisomerases type IIa (TOP2A) .As an alternative approach to single molecular biomarkers, Raman spectroscopy can provide a biochemical fingerprint of a cell or tissue ,13 and, Jess et al.  showed tn = 60); then, on the basis of HPV DNA and HPV mRNA test results, a classification model was developed to identify transcriptionally active HPV infections. A further blinded independent test set (n = 14), which was completely separate from the training set, was utilized to validate the model, and the results were compared to cytology, histopathology, and HPV DNA and mRNA test results.To our knowledge, no studies to date have investigated the potential of Raman spectroscopy for detection of transcriptionally active HPV infection. Thus, the main aim of this study was to assess Raman spectroscopy as a potential triage test for HPV-positive cervical samples. Raman spectra were recorded from liquid-based cytology (LBC) samples prepared as ThinPrep slides (n = 30) and HPV DNA-positive mRNA-negative samples represented the non-transcriptionally active form (n = 30).Residual material from cervical smear samples collected in PreservCyt solution was obtained from women attending the colposcopy clinic at the Coombe Women and Infants University Hospital (CWIUH), Dublin, Ireland, for a routine colposcopy visit. Women gave written informed consent for their sample to be used for this research study. Ethical approval was obtained from the CWIUH Research Ethics Committee (Study No. 28-2014). A subset of samples was obtained from the residual cervical smear samples from women enrolled in the CERVIVA HPV Primary Screening Trial. Ethical approval for this was obtained from the ICGP Research Ethics Committee. This study is part of a larger study underway within CERVIVA and, thus, samples were specifically selected that were HPV DNA/mRNA-positive or HPV DNA-positive only. A total of 60 cervical LBC samples were used for this study to develop the classification model . HPV mRNHPV DNA status was confirmed using the HPV Cobas 4800 test which detects the presence of hrHPV DNA. This test detects HPV16 and HPV18 separately, as well as a pool of 12 other high-risk types .HPV mRNA status was confirmed using the Aptima HPV Assay. The Aptima HPV assay is a target amplification nucleic acid probe test for the in vitro qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 high-risk types of HPV (16/18/31/33/35/39/45/51/52/56/58/59/66/68). The Aptima HPV assay does not discriminate between the 14 high-risk types.n = 14), selected as above but completely separate from the training set, was used as a test set to validate the classification model and consisted of a mixture of non-transcriptionally active and transcriptionally active HPV infected samples.A further set of blinded samples . The ThinPrep processor homogenizes the sample by spinning the filter, creating shear forces that break up any clumped material . The cells are then transferred onto the TransCyt filter and transferred onto a glass slide to produce a circular monolayer of cells approximately 20 mm in diameter. The slide is then ejected into a fixative bath of 95% ethanol.2O2 at room temperature for 3 min, followed by a 70% solution of industrial methylated spirits (IMS) for 3 min, followed by multiple dips into 100% IMS to remove any remaining cellular debris and H2O2, before being air-dried.The slides then underwent a pretreatment step to remove any molecular contamination by hemoglobin, which obscures several features of the cellular spectrum as described previously . Briefly−1 line of silicon before sample acquisition. Calibration spectra of 1,4-bis(2-methylstyryl) benzene  were also recorded at the time of each sample acquisition. All spectra were subsequently wavenumber calibrated using in-house developed procedures in Matlab v.9.3 . For sample acquisition, a Raman spectrum was acquired for each cell from the nucleus in the fingerprint region, 400 to 1800 cm−1, with an integration time of 30 s averaged over two accumulations. As spectra from the cell nucleus have been found to be more reproducible and consistent than spectra from the cell cytoplasm [A HORIBA Jobin Yvon XploRA system , with a 532 nm diode laser source, which incorporates an Olympus microscope BX41 equipped with a X100 objective  was utilized to record Raman spectra. Laser power was set to 100% resulting in 16 mW at the objective, and the confocal hole, coupled to a slit aperture of 100 μm, was set at 100 μm. Raman signals were detected using a spectrograph with a 1200 g/mm grating coupled to a charge-coupled device  and the spectrometer was controlled by Labspec V6.0 software. The spectrometer was calibrated daily using the 520 cmData preprocessing was performed using Matlab software  and in-house scripts, including smoothing , baseline correction (rubberband), and vector normalization. A non-negative least-squares (NNLS) method was used for glass correction as described previously ,29. The n = 14). Firstly, all spectra were tested individually and, secondly, the mean spectrum of each patient sample was tested. The positive class comprised samples with a transcriptionally active HPV infection because the aim of screening is to identify patients most at risk who will require further treatment. The negative class comprised samples with a non-transcriptionally active HPV infection because they are deemed to be at a lower risk of developing cervical cancer in the next 5 years and can return to routine screening.For further validation of the model, a blinded independent test set was employed (−1 (nucleic acids), 1238 cm−1 (amide III), and 1670 cm−1 (amide I). A classification model based on the Raman spectral data was developed using PLS-DA with LOPOCV. The scatter plot shows that latent variable LV1 mainly contributed to the differentiation of samples with non-transcriptionally active and transcriptionally active HPV infection (−1 (amide I). Although a shift around 1002 cm−1  can indicate calibration issues, a rigorous wavenumber calibration procedure was carried out; therefore, it is likely that this is a protein-related difference between the two groups. A sensitivity of 88% and a specificity of 88% were achieved by PLSDA classification with LOPOCV . Samples were specifically selected for the training set as HPV-positive and high-grade HSIL cytology, and half of the samples (n = 30) tested positive for HPV mRNA. The mean Raman spectra and LV1 loadings showed that the discrimination was mostly based around increased nucleic acids , decreased glycogen , and changes in protein features , indicating increased proliferation and altered protein expression due to the overexpression of E6/E7 viral proteins in the samples with a transcriptionally active HPV infection. These discriminating spectral features are consistent with previous studies on exfoliated cells showing discrimination between samples with negative cytology and HSIL cytology [In this study, the aim was to investigate the use of Raman spectroscopy as a triage test for HPV-positive cases. Initially, Raman spectra were recorded from a training set consisting of cervical LBC samples . Raman spectra were recorded from this test set, and both individual cellular spectra and mean sample spectra were used to test the model. Both methods provided successful validation of the classification model with nine out of 10 samples with a transcriptionally active HPV infection and four out of four samples with a non-transcriptionally active HPV infection classified correctly. Sensitivity of 85% and specificity of 92% were achieved using individual cellular spectra, and this increased to 90% sensitivity and 100% specificity using mean sample spectra. The variability within the dataset was reduced by utilizing the mean spectra, resulting in an increase in sensitivity and specificity.This classification model was further tested using a blinded test set  affects the number of cells classified into each category. LSIL samples that are HPV DNA/mRNA-positive need to be included in future studies to see if this affects the correct classification of individual cellular spectra.Notably, very good agreement was observed among the cytology, histology, HPV tests, and the Raman classification result. The majority of samples that were classified as having a transcriptionally active HPV infection were both HPV DNA-positive and mRNA-positive and were reported as HSIL on cytology. The cytology result was confirmed as CIN2/3 by histology in all but one sample. Four out of five samples that were classified as having a non-transcriptionally active HPV infection were HPV DNA-positive but mRNA-negative and were reported as either negative or LSIL on cytology. All four samples were negative on histology. Interestingly, the sample that was classified incorrectly as having a non-transcriptionally active HPV infection was HPV DNA-positive and mRNA-positive but was reported as LSIL on cytology and confirmed as CIN1 on histology. A limitation of our study is that the training set consisted only of HSIL cases; hence, future work would need to include LSIL cases to fully train the classification model.An additional limitation of this study is the relatively low number of samples with a non-transcriptionally active HPV infection in the blinded test set. As the main source of sample recruitment was a colposcopy clinic, where most patients were referred for further assessment of a HSIL cytology result, the majority of patients had a transcriptionally active HPV infection. This proof-of-principle study provides sufficient evidence to warrant a larger study in a screening population where HPV is the primary screening test and the clinical value of Raman spectroscopy for HPV triage can be realized. This would provide a larger sample base of samples with non-transcriptionally active HPV infection for testing compared to colposcopy-based recruitment.In conclusion, this study showed that Raman spectroscopy has potential as a triage test for HPV-positive women to identify transcriptionally active HPV infections, although further work is necessary in a HPV primary screening setting. Unnecessary referral of women with a non-transcriptionally active HPV infection for cytological evaluation could be prevented by an improved triage test, while women with an increased risk of disease are referred to colposcopy. Women most at risk can be identified using a Raman spectroscopy-based test without the subjectivity of morphological assessment. The method can be performed on the same initial Pap test, and, if required, cytology and/or immunocytochemistry for molecular biomarkers can be carried out following the Raman spectral analysis.


Coronary luminal dimensions change during the cardiac cycle. However, contemporary volumetric intravascular ultrasound (IVUS) analysis is performed in non-gated images as existing methods to acquire gated or to retrospectively gate IVUS images have failed to dominate in research. We developed a novel deep learning (DL)-methodology for end-diastolic frame detection in IVUS and compared its efficacy against expert analysts and a previously established methodology using electrocardiographic (ECG)-estimations as reference standard. Near-infrared spectroscopy-IVUS (NIRS-IVUS) data were prospectively acquired from 20 coronary arteries and co-registered with the concurrent ECG-signal to identify end-diastolic frames. A DL-methodology which takes advantage of changes in intensity of corresponding pixels in consecutive NIRS-IVUS frames and consists of a network model designed in a bidirectional gated-recurrent-unit (Bi-GRU) structure was trained to detect end-diastolic frames. The efficacy of the DL-methodology in identifying end-diastolic frames was compared with two expert analysts and a conventional image-based (CIB)-methodology that relies on detecting vessel movement to estimate phases of the cardiac cycle. A window of ± 100 ms from the ECG estimations was used to define accurate end-diastolic frames detection. The ECG-signal identified 3,167 end-diastolic frames. The mean difference between DL and ECG estimations was 3 ± 112 ms while the mean differences between the 1st-analyst and ECG, 2nd-analyst and ECG and CIB-methodology and ECG were 86 ± 192 ms, 78 ± 183 ms and 59 ± 207 ms, respectively. The DL-methodology was able to accurately detect 80.4%, while the two analysts and the CIB-methodology detected 39.0%, 43.4% and 42.8% of end-diastolic frames, respectively (P < 0.05). The DL-methodology can identify NIRS-IVUS end-diastolic frames accurately and should be preferred over expert analysts and CIB-methodologies, which have limited efficacy.The online version contains supplementary material available at 10.1007/s10554-021-02162-x. Intravascular ultrasound (IVUS) is the preferred intravascular imaging modality for the evaluation of coronary plaque burden and the efficacy of emerging therapies targeting plaque evolution . GrayscaIt is hypothesised that a DL-based methodology will be able to detect end-diastolic frames in IVUS datasets and may be superior to human experts and previous automated methodologies. The aims of this study are: (a) to develop and train a novel DL-based methodology for automated detection of end-diastolic frames in IVUS sequences, (b) examine the reproducibility of expert analysts in detecting end-diastolic IVUS frames and (c) validate the performance of DL, the expert analysts and an established, conventional image-based (CIB)-methodology in detecting end-diastolic frames in patients with co-registered IVUS images and ECG-recordings.Twenty coronary arteries from six consecutive patients in sinus rhythm who were recruited to the “Evaluation of the efficacy of computed tomographic coronary angiography (CTCA) in assessing coronary artery morphology and physiology” study (NCT03556644), were included in the present analysis. The rationale, study design, and inclusion and exclusion criteria have been presented in detail elsewhere . BrieflyNIRS-IVUS imaging was performed using the 50 MHz Dualpro system  according to a standardised protocol. After administration of 400mcg of nitrates the NIRS-IVUS probe was advanced to the distal part of the vessel and an angiographic projection was obtained under contrast agent injection to identify the location of the probe. The probe was pulled back at a constant speed of 0.5 mm/s using an automated pullback device (frame-rate: 30 frames per second (fps)). A concurrent ECG tracing was recorded at the time of pullback. The NIRS-IVUS data and the ECG tracing were imported in a viewer-mixer that enabled simultaneous visualisation of the two recordings; the displays of the viewer-mixer were recorded at 120fps and digital video files were transferred to an offline workstation for further analysis.Analysis was performed by two well-trained analysts (RB and YK) who have segmented > 375,000 NIRS-IVUS frames. The two analysts reviewed the digital-video files that simultaneously portrayed the NIRS-IVUS pullback and the ECG tracing and identified each NIRS-IVUS frame that corresponded to the peak of the R-wave as an end-diastolic frame. These ECG estimations were checked by a third analyst (CVB); any conflicts were resolved by consensus.Two months after this process the two analysts reviewed the original NIRS-IVUS sequences blindly to the ECG and manually identified the end-diastolic frames; in this analysis, it was assumed that the NIRS-IVUS frame with the minimal lumen motion before the largest movement of the lumen in relation to the NIRS-IVUS catheter corresponded to the end-diastolic frame. The 1st-analyst performed the analysis twice (3 weeks apart) to report intra-observer variability while the estimations of the 2nd-analyst were used to report inter-observer variability.To explore the effect of frame rate on the estimations of the two analysts, a second analysis was performed in NIRS-IVUS sequences with a frame rate of 15fps; these data were obtained after excluding 1 of every 2 frames from each sequence. Again, intra- and inter-observer variability were reported and their estimations compared to ECG estimations, which were calculated by halving the frame number of the initial ECG estimations.CIB-gating was performed in the NIRS-IVUS data using a special version of the QCU-CMS software  by implementing an established solution, the only one that has previously been incorporated in a user-friendly software package for IVUS analysis . This apWe propose a novel DL-methodology for identifying end-diastolic frames in NIRS-IVUS sequences. The methodology consists of several steps Fig. . First, This sequence was processed by the DL-methodology which consists of a network model designed in a bidirectional gated-recurrent-unit (Bi-GRU) structure , capable of analysing data sequentially in both temporal directions, taking advantage of patterns extracted from frames before and after the end-diastolic frame . This apThe choice of loss function used for the Bi-GRU model was mean square error and learning rates in all experiments were set to Testing of the DL models was performed using the leave-one-out cross-validation approach—all pullbacks performed in the LAD, or the LCx, or the RCA were used for training apart from one vessel from each vessel type (LAD/LCx/RCA) that was used for testing. This process was repeated sequentially leaving a different vessel out from the training set until all vessels were used for testing. Similar to the training set, a 64-frame segment was generated that swept the entire NIRS-IVUS sequence. For each segment, the probability of the 32nd frame to be the end-diastolic frame was calculated and the frames with the highest probability amongst neighbouring frames were selected and constituted the network output. The DL-methodology analysed the NIRS-IVUS images twice and its intra-observer variability was reported.Numerical variables are presented as mean ± standard deviation (SD) while categorical variables are reported as absolute values and percentages. Comparison between categorical variables was performed using the Chi-square test. Bland–Altman analysis was used to assess the intra- and inter-observer variability of the analysts and compare their estimations and the estimations of the CIB- and DL-methodology against the end-diastolic frames identified by the ECG. The root mean square error (RMSE) between the estimations of the analysts, the CIB, the DL-methodology and the ECG were computed and compared. Considering that the temporal resolution of NIRS-IVUS imaging is 33.3 ms (30fps) we used a fixed cut-off range of ± 100 ms from the peak of R-wave on ECG to define correct end-diastolic frame detection Fig. . In the The baseline characteristics of the included patients are shown in Table The representative analysis time for an analyst identifying end-diastolic frames manually for a representative 5000-frame vessel was approximately 15 min. Automated methodologies were significantly quicker: the CIB-methodology required 135 s while the DL-methodology required 15 s for the same number of frames to be analysed.The 1st-analyst identified 3144 end-diastolic frames; 3034 estimations corresponded with the ECG-defined cardiac cycle; 133 estimations could not be matched with the ECG estimations as there were other annotations closer to the peak of R-wave, while in 110 cases the analyst failed to identify an end-diastolic frame for a cardiac cycle. Similar results were reported when analysis was repeated by the 1st-analyst or when end-diastolic frame detection was performed by the 2nd-analyst , the 2nd-analyst 1333 (43.4%), the CIB-methodology 1306 (42.8%) and the DL-methodology 2401 (80.4%) frames  and inter-observer  variability was higher for the estimations of the analysts when compared to the CIB- and DL-methodologies that were again perfectly reproducible  was higher than the agreement between ECG and 1st-analyst (76 ± 315 ms), 2nd-analyst (46 ± 186 ms) or CIB-methodology (13 ± 223 ms). These findings were consistent in all three coronary arteries. RMSE analysis again confirmed the superiority of the DL-methodology  expert analysts have limited accuracy and reproducibility in identifying end-diastolic frames, (2) the CIB-methodology, while more reproducible, has similar accuracy to expert analysts, (3) the efficacy of the analysts and CIB-methodology varies dependent on vessel type and (4) the DL-methodology is superior to expert analysts and the CIB-methodology, and has an excellent accuracy in identifying end-diastolic frames in all three coronary arteries.The merits of analysing IVUS images obtained at the same phase of the cardiac cycle to accurately quantify volumetric changes in atheroma burden in longitudinal IVUS studies are well recognised. Previous studies have demonstrated that the lumen area can vary significantly, by as much as 10% in disease-free segments, during the cardiac cycle , 5. MoreHardware designed to selectively acquire gated IVUS frames  and autoIn the present study, we validated the efficacies of a CIB-methodology and expert analysts in 3,167 end-diastolic frames from 20 vessels assessed by NIRS-IVUS imaging against an ECG-defined reference standard. We found that the analysts had high intra- and inter-observer variability in detecting end-diastolic frames and that there were significant differences between their estimations and the ECG and that their performance varied between vessels. Both analysts performed better in the LAD and worse in the RCA where there is a higher relative motion of the lumen with regards to the catheter. In our analysis, the CIB-methodology was perfectly reproducible but its performance in detecting end-diastolic frames was limited and not different from the experts’. Significant variations were noted in the performance of the CIB-methodology in different vessels, with better performance in the LAD compared to the LCx and RCA.Conversely, the proposed DL-methodology trained on the reference standard, taking into account the different patterns of motion in different vessels, appeared superior to the analysts and CIB-methodology with excellent accuracy in detecting end-diastolic frames overall and was not affected by the type of vessel.The results were not different when we took into account the variability in the R-R interval between patients and used a cut-off of ± 10% of the R-R to define accurate end-diastolic frame detection. DL was again superior to the analysts and the CIB-methodology but it had a smaller accuracy than the one reported with the fixed cut-off of ± 100 ms. This should be attributed to the fact that the average heart rate of the studied patients was 73 ± 18 bpm but also to the limited temporal resolution of NIRS-IVUS imaging (33.3 ms). For the latter reason we believe that the fixed cut-off may allow a more accurate estimation of the performance of the DL-methodology especially in patients that had an increased heart rate during pullback.The fixed cut-off that we used in our study also allowed us to examine the performance of the DL-methodology in IVUS sequences with a frame-rate of 15fps. The analysts had poor reproducibility and, similar to the CIB-methodology, limited efficacy in detecting end-diastolic frames while the DL-methodology did not appear to be affected by the lower spatial resolution and had excellent performance. These findings highlight the potential value of DL in processing intravascular imaging data acquired at a higher pull-back speed, or a lower frame-rate such as the NIRS-IVUS images acquired by the MC-9 system which obtains images at 14fps or the upcoming IVUS-photoacoustic imaging catheters that are expected to acquire images at a lower frame-rate than conventional IVUS systems .Another advantage of the developed DL-methodology is that it is rapid, enabling processing of large datasets within a few seconds: analysis of an IVUS sequence of 5000 frames takes less than 15 s in a computer with an Intel-i5 processor and 16 GB RAM. The proposed approach has also been incorporated in the user-friendly QCU-CMS software, widely used for the processing of intravascular imaging data and this is likely to facilitate its utility in research. Moreover, the DL-methodology is expected to have applications in future computational fluid dynamic (CFD) studies assessing the implications of the local hemodynamic forces on plaque evolution. Recently, convincing evidence has emerged highlighting the role of coronary blood flow patterns on plaque progression and destabilisation , 31, whiThe present study has several limitations that should be acknowledged. Firstly, the number of vessels included was small and thus we could not examine the implications of the erroneous estimations of the analysts and of the CIB-methodology on the quantification of the lumen and vessel volumes . SecondlAll included patients were in sinus rhythm, with a heart rate between 45 and 85 beats-per-minute and had a relatively stable R-R interval; nevertheless, premature atrial or ventricular ectopics were often seen during the pull-back and were included in the analysis. Therefore, the developed DL-methodology should be used only in patients in sinus rhythm as it is uncertain how significant rhythm variability might affect its performance. Moreover, the present analysis excluded stented segments where the friction effect between the catheter and the protruded struts may affect the movement of the lumen with regards to the IVUS probe—further research is needed to examine the performance of the proposed methodology in such segments. Finally, this study did not examine the value of IVUS-gating in the accurate quantification of atheroma burden compared to non-gated IVUS; this should be tested in a large-scale appropriately sized study.In conclusion, the proposed DL-methodology is capable of detecting end-diastolic frames in 50 MHz NIRS-IVUS datasets of all three coronary arteries rapidly and reproducibly, with greater accuracy than both expert analysts and a CIB-methodology. The DL-methodology’s performance is unaffected by the rate at which IVUS images are acquired. These features render it a useful tool for the analysis of IVUS datasets in longitudinal studies and in studies that fuse IVUS and X-ray angiographic data to assess the implication of the haemodynamic forces on plaque evolution.Supplementary Information 1 (DOCX 27125 kb)Below is the link to the electronic supplementary material.


In the female population, 10 years after the start of the national immunization program, we observed a reduction in the prevalence of vaccine types and the number of circulating genotypes, especially in the “vaccine-eligible” cohort. The frequency of HPV vaccine types increased with age, particularly in males in the “pre-screening” and “screening” cohorts. Our study highlights the importance of monitoring HPV infection in both genders, to validate the effect of the HPV vaccination program.Human papillomavirus infection is a cause of the development of invasive cervical cancer. Three types of vaccine are currently available to prevent precancerous/cancerous lesions due to persistent infection, which is supported mainly by 7 different high-risk genotypes. The aim of this pilot study was to acquire preliminary data on type-specific prevalence 10 years after the implementation of the HPV vaccination program in Italy, in order to subsequently plan appropriate observational studies in the Italian population. First-voided urine samples were collected from 393 consenting subjects, both females and males, aged 18–40 years, and HPV DNA was detected by PCR amplification of a 450 bp L1 fragment. All amplified products were genotyped by means of the Restriction Fragment Length Polymorphism (RFLP) method. The female population was divided into three cohorts  according to the preventive intervention scheduled by age; males were included in the same three cohorts according to their year of birth. The overall prevalence of HPV infection was 19%, being higher in females than in males (22.1% vs. 13.6%,  It is well known that human papillomavirus (HPV) infection is the most common sexually transmitted infection worldwide ,2. Soon HPVs are small, double-stranded DNA viruses that infect the mucosa and epithelium. More than 200 HPVs have been fully characterized ,5 and arThe low-risk types, HPV 6 and HPV 11, are responsible for about 90% of anogenital warts and benign/low-grade abnormalities in the genital district , and areBefore the introduction of HPV vaccination, periodic Pap testing in women aged 25–65 years was the main way to prevent cervical cancer. Today, three prophylactic HPV vaccines targeting high-risk HPV types are available in many countries: 2-, 4- and 9-valent vaccines. All these prophylactic HPV vaccines protect against HPV-16 and HPV-18. Furthermore, the 4-valent HPV (4vHPV) vaccine also protects against HPV-6 and HPV-11, and the 9-valent HPV (9vHPV) formulation also against HPV-31, HPV-33, HPV-45, HPV-52 and HPV-58 .Data from recent surveillance systems and studies demonstrate that HPV vaccines are effective in preventing infection and diseases (vaccine efficacy 93% to 100%) ,14 and tMore than 70 countries, including 33 belonging to the WHO European Region, have introduced HPV vaccination into their national immunization programs for girls, and 11 countries have done so for boys .In 2007, Italy initiated a national immunization strategy of active call and free-of-charge vaccination for girls aged 11–12 years ,17. SubsAs yet, no comparison has been made between the prevalence of HPV before the introduction of the national HPV immunization program a decade ago and the current prevalence. The overall aim of the present study was to acquire preliminary data on type-specific prevalence 10 years after the implementation of the HPV vaccination program in Italy, in order to subsequently plan appropriate observational studies in the Italian population.The study protocol, which was used by all research units, was approved by the Ethics Committee of the Liguria Region (LHU) Genoa, Italy (protocol n. 44/12). Written informed consent to participate in the study was obtained from all subjects.In accordance with the study protocol, we enrolled all consecutive eligible women and men aged 18–40 who spontaneously accessed sports medicine centers and universities located in three cities in different Italian Regions: Milan (Lombardy), Genoa (Liguria) and Camerino (Marche).Females were divided into three cohorts according to their status regarding the prevention of HPV-related diseases: a vaccine-eligible cohort, which consisted of girls born between 1997 and 2001 and belonging to the first vaccination target cohort; a pre-screening cohort, comprising females born between 1991 and 1996, who were still too young to be called for a Pap test; and a screening cohort, consisting of girls born before 1990, who were eligible for a Pap test screening call.Males were included in the different cohorts according to their years of birth. Recruitment was conducted between February 2017 and October 2019 and each participant was invited to provide an anonymous self-collected urine sample.In Italy, the median age of sexual debut is 16 years in both sexes ,20. All g for 20 min at RT to obtain the cellular component. This phase was transferred to a new 1.5 mL collection test tube and centrifuged at 16,000× g for 15 min at RT. The pellet obtained was resuspended in 1 mL of PBS and aliquots were stored at −20 °C [First-voided urine samples were collected in sterile containers, kept at room temperature (RT) and processed within 6–8 h at each participating center. Briefly, 15 mL of each sample was centrifuged at 3800× t −20 °C  until sh® easyMAG™ automated  method in accordance with the standard protocol with off-board lysis. The concentration and purity of extracted DNA were evaluated by means of a spectrophotometer . DNA quality was assessed by amplifying a 268 bp (base pair) segment of the ubiquitous β-globin gene.HPV DNA was extracted from 200 μL of concentrated urine samples by means of the NucliSENSHPV DNA was detected through PCR amplification of a 450 bp L1 fragment by means of the degenerate primer pair ELSI-f and ELSI-r . The ampThe sample size was calculated according to data published by the HPV Information Centre , to estiOverall, an estimated prevalence of 15% in males and females was considered: 18% in subjects aged <25 years and 11% in those aged 25–40 years .The sample size was calculated by means of the following formula, considering a 95% confidence interval (95% CI) with an absolute precision of ±5%:The sample size of the overall population was estimated to be 196 subjects, for the population under 25 years was 227, and for those between 25 and 40 years of age was 150 subjects enrolled.As the HPV vaccination strategy was the same across the country, with no differences among regions, a comprehensive analysis was conducted.p-value < 0.05 was considered statistically significant (2-tailed test). Statistical analysis was performed by means of OpenEpi, version 3.01 [Descriptive statistics (median and IQR) were applied to describe the entire study sample. HPV prevalence was expressed as a crude proportion with corresponding 95% confidence intervals (95% CI) and calculated by means of the Mid-p exact test, assuming a normal distribution. A ion 3.01 .A total of 393 subjects participated in this study and provided an anonymous self-collected urine sample. The study population included 253 (64.4%) females and 140 (35.6%) males and the median age was 23 years (range: 18–40). Of the 393 participants, 220 were recruited in Milan, 115 in Genoa and 58 in Camerino .p = 0.03729).Of the 393 participants, 75  tested HPV DNA-positive: 56 of the 253  females and 19 of the 140  males  were not typable (NT). Overall, 111 different infections sustained by 34 different genotypes were identified. A single genotype was detected in 71.83%  of the HPV DNA-positive samples, and 74.51%  of the genotypes identified belonged to the HR clade. The remaining 20 HPV DNA-positive urine samples  showed multiple infections, and 95%  of these were sustained by at least one genotype belonging to the HR clade. Multiple infections were detected in 16 of 56 (28.57%) infected females and in 4 of 19 (21.06%) infected males. HPV infections and HPV infecting types, broken down by the three different cohorts, are shown in The numbers of HR and LR types observed were lower in the vaccine-eligible cohort than in the pre-screening and screening cohorts, though HPV-26, HPV-30, HPV-54 and HPV-72 were only detected in this cohort and a higher prevalence of HPV-52, HPV-56 and HPV-59 was observed.p = 0.02931, The prevalence of vaccine types was lower in the vaccine-eligible cohort than in the other cohorts. Indeed, the frequency of HPV vaccine types was significantly lower in the vaccine-eligible cohort than in the screening cohort  estimate that at least half of all sexually active individuals will acquire HPV at some point in their lives and that at least 80% of women will acquire an HPV infection by the age of 50 .While most HPV infections do not cause symptoms and resolve spontaneously, persistent infection may lead to the development of a range of conditions of the reproductive tract, including precancerous lesions that may progress to cancer, both in women and men.Human papillomavirus is a necessary (but not sufficient) cause of invasive cervical cancer. Three vaccines  with prophylactic indications are currently available to prevent precancerous lesions and cancers due to persistent infection supported by up to 7 different high-risk genotypes . The 4-valent and 9-valent vaccines also offer protection against genital warts and low-risk lesions caused by HPV-6 and HPV-11 .Since 2008, HPV vaccination has been offered free of charge to 12-year-old girls in Italy, and 70.6% of eligible subjects belonging to the first vaccinated cohort  have received at least one vaccine dose . The higIn the present study, the prevalence of HPV infection was evaluated in urine samples collected in 2017–2019 from subjects of both sexes. Cervical brushing still is the gold standard for HPV detection; indeed, a partial discordance between paired urine and cervical samples was observed in PCR-based genotyping results. Nevertheless, urine sampling is a highly sensitive method and a viable alternative to cervical brushing in women and urethral swabs in men, in order to detect and characterize HPV infection. Moreover, urine sampling is easy to carry out and offers the unquestionable advantage of non-invasiveness and the possibility of reaching a high adherence rate among women, men and vulnerable populations in the youngest age groups. Therefore, several studies have suggested that urine-based HPV detection is a suitable and effective tool for both epidemiological surveillance and screening ,31.The worldwide prevalence of HPV among women with no evidence of cervical lesions has been estimated at around 12% . Our resTo date, few data are available on the epidemiological status of HPV infection in males without clinical manifestations. A review published prior to the introduction of HPV vaccination in men found an HPV prevalence among sexually active men ranging from 1% to 84% worldwide. In a study of men aged 19–82 years (median age 46 years) from Brazil, Columbia, Thailand, the Philippines and Spain, the HPV prevalence was 16% . In the Our data showed a higher prevalence of HPV infection in females in the vaccine-eligible cohort (23.1%) and in males in the screening cohort (19.7%). These findings are in agreement with those of a systematic review of the prevalence of genital HPV in men over 18 years of age, in which this prevalence was seen to peak in older men, but not in older women .Ten years after the introduction of HPV vaccination for the female population, HPV-16 is still the most frequently observed genotype . HoweverIt is noteworthy that a reduction in the number of HR-HPV and in the circulation of HPV vaccine types  was observed in the vaccine-eligible cohort in comparison with previous studies conducted in the same area in the pre-vaccine era ,39. No vIn the other cohorts, the frequency of HPV vaccine types  increased with age in both sexes, but the preventable fraction remained around 90%. These data suggest that catch-up programs should be implemented in order to vaccinate young women and young men beyond the 12-year-old cohort. The highest prevalence of vaccine types was observed in the screening cohort, especially in the male population, and this finding underlines the importance of including men in immunization programs.In this study, we observed an overall prevalence of genital HPV infection of 19%, which was higher in females than in males. In the female population, ten years after the start of the national immunization program, we observed a reduction in the prevalence of vaccine types and also in the number of circulating genotypes, especially in the vaccine-eligible cohort. The absence of vaccine types in the male vaccine-eligible cohort could be evidence of a herd effect, despite suboptimal vaccination coverage (about 70% with three doses).None of the men in our study had been vaccinated ; our data are therefore essential to understanding the future effects of vaccination in this population.The limit of this study is that the number of subjects enrolled was not large enough to be fully representative of the Italian population, and hence to precisely assess the impact of vaccination in Italy. However, this was a pilot study to evaluate the suitability of urine sampling in a monitoring context and in a larger population.Our study highlights the importance of vaccinating both women and men in order to achieve good vaccination coverage and to protect males from HPV-related diseases. It also underlines the importance of including catch-up vaccination for women up to age 26 in the national immunization program, an approach that could be implemented by taking advantage of the Pap test call.