To estimate the prevalence of anal HPV infection, genotype distribution, intraepithelial neoplasia (AIN) and correlates in a cohort of HIV-infected patients attending at Sexually Transmitted Infections (STI) clinic in Brazil.A descriptive analysis was performed which includes, demographic, behavioral and clinical data. Anal specimens from HIV-positive men and women were collected during a regular visit and they were used for cytology and histopathology tests, as well as for HPV molecular identification.3 was a protective factor for the HPV16/18 (p = 0.048) and multiple infections (p = 0.023), and the undetectable viral load and HAART treatment were both protective for any HPV (p = 0.010), HR-HPV (p = 0.091) and multiple infections (p = 0.006). Abnormal anoscopy was found in 23.7% (53/223) of the total number of patients, and this was significantly associated with all types of investigated HPV infections (p<0.0001).A total of 223 patients  were enrolled in the study and, HPV was identified in 68.6% of the sample. The frequency of HR-HPV, HPV16/18 and multiple HPV infection were similar in both groups. The upstream regulatory region (URR) sequencing was carried out in 38 samples identified as HPV16-positive, and European variants were the most frequent (69.2%), followed by Africans (25.6%) and Asiatic-Americans (5.1%). Having more than 20 sexual partners was associated with multiple HPV infection (p = 0.000) while, anal sex and the first intercourse before 15 years of age was a risk factor for any HPV infection (p = 0.001). Being MSM (men who have sex with men) was a risk factor for any HPV and multiple infections (p = 0.002). The CD4 count >500 cells/mmIn this study, anal HPV infection was common among young HIV-positive men and women, particularly in MSM. Anal cancer screening in patients at risk, such as those who are HIV-positive, and mainly those with anal HPV infection and a history of STI, will increase the likelihood of detecting anal intraepithelial neoplasia. Human papillomavirus (HPV) is a common sexually transmitted infection (STI)that can be categorized into two groups, low risk (LR-HPV) and high risk (HR-HPV) with respect to their risk of progression to malignancy . Latent Several studies identified a fluctuating HPV prevalence around the world, which could be due to some population characteristics, such as age and behaviors or to the sensitivity of the HPV DNA tests ,8,9. HR-The role of HPV in cervical lesions is well documented, but data of anal intraepithelial neoplasia is gaining importance more recently, in both men and women. In men, HPV infection has been strongly associated with anal cancer with approximately 88% of the anal squamous cell cancers occurring annually worldwide . The preThe relationship between HIV and malignancies has been described, being significantly associated with anal cancer when comparing the general population . AlthougThis is a cross-sectional study of HIV-positive men and women (18–69 years of age) attending a public STI/HIV screening and treatment center in Vitória, Brazil, from March 2013 to February 2016. The Ethics Committee of Universidade Federal do Espírito Santo (UFES), Brazil approved this study. Participants signed a written informed consent before being included in the study. They were invited to answer an interview about socio-demographic, behavioral and clinical data and performing an anal examination, including sample collection.A 20-minutes face-to-face interview was conducted with the use of a standardized questionnaire. Enrolled patients answered the interview questions, which included demographic ; behavioral  and clinical data , CD4 counts and viral load).Sample collection was undertaken using a cytobrush inserted 4.0 cm into the anal canal and performing a spiral motion to seize samples from the entire circumference of the anal canal. The samples were spread onto a microscope slide and stained using the Papanicolaou method. The classification of cytological findings was undertaken according to the Bethesda system of cervical cytology . The speAnal samples were obtained using cytobrush and placed in 2 ml tube containing TE buffer  and store at -70°C. The DNA was isolated using a QIAamp DNA Mini Kit™ kit  according to the manufacturer's instructions. The HPV DNA was detected by amplification with PGMY09/11 primers , and theSubjects with any grade of anal cytological abnormality or anal HR-HPV were referred for examination using high-resolution anoscopy (HRA). The examination was performed with a colposcope, and a gynecologist with expertise in colposcopy performed all HRA procedures. A cotton ball with 3% acetic acid was used while anal mucosa was observed and the anoscope was moving out of the anal canal. The results of HRA were considered positive or negative for aceto-white lesions, dots, mosaics, atypical vessels following the Consensus terminology of 2016 IANS International Guidelines for Practice Standards in the Detection of Anal Cancer Precursors . Punch aFrequency distribution and descriptive statistics were used to describe the study variables. Estimated prevalence and 95% confidence interval for any HPV type, HR-HPV, HPV16/18 and multiple HPV infection were calculated. Descriptive statistics were calculated in order to describe the most prevalent genotypes in the study. Chi-square analysis or Fisher's exact test were used to evaluate differences in categorical outcome measures. Socio-demographic characteristics were stratified into two groups by gender. Data were coded and stored anonymously in a database. The statistical software SPSS v. 20.0  was used for the analyses.Condyloma acuminatum (22.9%), followed by syphilis (16.6%), and 27.7% had more than one STI. The HPV test was positive in 68.6% (153/223) of patients, 71.3% (57/80) among men, and 67.1% (96/143) among women.Anal samples were collected from 223 HIV-seropositive patients, being 80 men and 143 women, and the median age was 40.0 years (SD = 11.20). Women and men presented the same average with respect to age, the majority were employed (69%) and had completed a secondary education (57.4%) . A totalRegarding cytological abnormalities in anal specimens, ASC-US , LSIL  and HSIL  were found in 20.6%, 2.2% and 0.8% of the total samples, respectively. Classifying by gender, abnormal results were significantly more common in males than in females (p = 0.008), and the abnormalities also were also associated with being HPV positive in the male group (p = 0.038). Thirty-three participants were undergoing biopsies with a colposcopic examination of the anal canal. Twenty-nine (18.3%) participants were diagnosed with anal intraepithelial neoplasia (AIN), twenty cases of LSIL, two cases of HSIL and one case of the anal adenocarcinoma. All the cases of HSIL were related to genotypes of HPV 16 or 18 in HIV-positive women.HR-HPV was found in 50.7% (113/223) of the total samples and 74% (113/153) of sample when considering only the HPV-positive cases. HPV16/18 cases were very frequent , in 32% of HR-HPV positive cases in men and 67.4% in women. The frequency of other HPV genotypes is shown . The URRBehavioral data and their association with HPV are shown in . Being yHPV infection and HIV data are shown in . CD4 couThe comparison of behavioral aspects of anal cytology results are shown. . Fifty-tThis study is the first epidemiological study conducted in Espírito Santo state, Brazil, that reports the prevalence rates, the distribution of HPV genotypes and the factors associated with anal HPV infection in HIV patients. The prevalence of anal HPV infection was frequent in this study 68.6%), and it was similar in both genders, even when considering HR-HPV and HPV16/18 infections. In the study population, MSM presented a significantly higher prevalence of HPV, as did having more than 20 partners. This result is similar to results of other studies that were conducted worldwide ,8, and i.6%, and Regarding the HPV genotypes, the tests used were able to identify 39 different types of HPV. The most frequent HR-HPV types were 16, 51 and 52, and the most frequent LR-HPV types were 6, 53 and 70. Some of the types have a low prevalence in the general population but present higher frequency in HIV population ,30. The The results of a global review concluded that 84% of the invasive anal cancer cases contain HPV DNA, most individuals (87% of those who were HPV-positive) were positive for HPV16, and fewer (6%) were positive for HPV18 . In a meIn our study several socio-demographic characteristics had significant differences between men and women; higher education was more frequent in men; the more recent HIV diagnoses (less than 5 years) was more frequent in men, even with an average age over 40 years. Regarding clinical variables, HIV viral load and low CD4 counts were significantly associated with HR-HPV and multiple infections, which could raise the risk of persistent infection and consequently, of malignant development. Our result showed that HAART therapy was protective for multiple HPV infection, and it was previously demonstrated that HR-HPV prevalence was lower in patients in the long-term application of HAART , 40. HowThe prevalence of abnormal anal cytology (25.5%) shows that it is a common finding in HIV-infected patients. Repeated anal cytology testing and referral for HRA could increase the likelihood of AIN diagnosis. The highest positivity of abnormal anal cytology in our study occurred in the MSM population 43.6%), wich is similar to the rate of 53.6% described in a Spanish cohort of MSM that was HIV-infected .6%, wich. In our We found some study limitations, for example, missing data regarding additional tests such as anal cytology due to unsatisfactory samples, although all patients underwent anal cytology. Some patients refused to undergo anoscopy due to complaining of pain or bleeding. We did not have an additional blinded pathologist to perform confirmation of the results. Due to the low frequency of reporting some risk factors in the samples, the number of subjects studied was sufficient enough to find a statistical association between some independent variables and anal HPV infection. The possibility of having been response bias cannot be ruled out due to the general tendency to give socially acceptable answers. However, the limitations do not diminish the importance of this study in providing visibility to a neglected population during preventive actions in assisting HIV-infected individuals.Some aspects of both genital cancers  are shared, as the HPV infection and persistence as a risk factor, HPV16 as the main associated genotype and progression to carcinoma after the emergence of the previous intraepithelial lesions.Since the HR-HPV prevalence and the frequency of the related cancers are strictly associated and have a higher risk in HIV positive individuals, we believe the introduction of HPV DNA testing in this population could have an important role in stratifying the risk of HIV-seropositive patients, as has been proposed by other researchers in the literature .These results reinforce the need to establish an anal cancer screening on high-risk patients, such as HIV-positive women and men. The anus and cervix susceptibility to HPV infection and these HPV-induced malignancies might be explained by the same embryological origins. Anal and cervical cancers have similar tumor biology; hence a similar cancer screening program would appear to be reasonable, considering the success of cervical cancer screening programs around the world . A co-teCurrently, the Centre for Disease Control and Prevention (CDC) in the USA recommends that high-risk populations be vaccinated using the quadrivalent or nonavalent vaccines . In BrazS1 File(DOCX)Click here for additional data file.S2 File(SAV)Click here for additional data file.


Condyloma acuminatum (CA) is a benign epithelium hyperplasia mainly caused by human papillomavirus (HPV), which is now the second most common viral sexually transmitted infection (STI) in China. In total, 90% of CA patients are caused by the low-risk HPV 6 and 11. Aside from low-risk HPV infection there are likely other factors within the local microenvironment that contribute to CA and there has been related research before. In this study, 62 vaginal specimens were analyzed using 16S rRNA gene sequencing. The diversity of the vaginal microbiota was higher and the composition was different with LR-HPV infection. While the relative abundance of dominant Firmicutes was lower, Actinobacteria, Proteobacteria, and Fusobacteria phyla were significantly higher; at the genus level Gardnerella, Bifidobacterium, Sneathia, Hydrogenophilus, Burkholderia, and Atopobium were higher. This study firstly confirmed a more accurate and comprehensive understanding of the relationship between low-risk HPV infection and vaginal microbiota, in order to provide a theoretical basis for further research on the occurrence and development of CA. Condyloma acuminatum (CA) is a benign epithelium hyperplasia mainly caused by human papillomavirus (HPV), which is now the second most common viral sexually transmitted infection (STI) in China , and an HPV-positive CA group . We sought to afford a more accurate and comprehensive understanding of the relationship between low-risk HPV infection and vaginal microbiota in order to provide a theoretical basis for further research on the occurrence and development of CA.Therefore, an accurate understanding of the composition of vaginal microbiota will help to clarify the relationship between vaginal microbiota and the occurrence of CA. Traditional molecular biology techniques cannot fully reflect the structural information of bacterial communities in the whole microenvironment, with the possibility of important information going unnoticed. In this study, we enrolled a total of 62 subjects consisting of a negative control group  and ② two HPV low-risk groups . Exclusion criteria: were persons under the age of 20 and over the age of 50, patients with immunodeficiency diseases; and patients with systemic diseases.A total of 62 individuals who underwent physical examination and/or diagnosis and treatment in the Jinan Central Hospital Affiliated to Shandong University were enrolled in this study. The samples were divided into three groups according to the results of LR-HPV screening: ① HPV negative control  following the manufacturer's instructions. The extracts were kept at −20°C.2O. Amplicons were extracted from 2% agarose gels and purified using the Axy Prep DNA Gel Extraction Kit  according to the manufacturer's instructions and quantified using Qubit®2.0 . After preparation of the library, these tags were sequenced on a MiSeq platform  for paired end reads of 250 bp, which were overlapped on their three ends for concatenation into original longer tags. DNA extraction, library construction and sequencing were conducted at Realbio Genomics Institute .The V3-V4 region of the bacteria 16S ribosomal RNA genes was amplified by PCR  using primers 341F 5′-CCTACGGGRSGCAGCAG)-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3.′ The KAPA HiFi Hot Start Ready Mix PCR kit  was used for high-fidelity amplification. PCR reactions were performed in a 30 μL mixture containing 15 μL of 2 × KAPA Library Amplification Ready Mix, 1 μL of each primer (10 μM), 50 ng of template DNA and ddHhttp://drive5.com/uparse/), and chimeric sequences were identified and removed using Userach  using a confidence threshold of 0.8 . A P-value of < 0.05 was considered statistically significant.In addition to the statistical analyses described above, the statistical analysis of quantitative data was conducted by Student's A total of 62 genital tract samples were obtained from the study population for sequencing . After pR = 0.228, p = 0.001) indicate that the differences between groups were greater than those within groups with great statistical significance , which also included Gardnerella, Prevotella, Pseudomonas, Atopobium, and other bacteria. The community structure of the vaginal microbiota in the HPV-LR group was significantly altered at the genus level, with the most prominent manifestation being the decrease in Lactobacillus content, the increase of Gardnerella (up to 3 times), Bifidobacterium (up to 146 times), Sneathia (up to 110 times), Hydrogenophilus , Burkholderia (up to 177 times), and Fusobacterium (up to 122 times), which together constituted the dominant bacteria of the vaginal microbiota with decreased Lactobacillus. According to the species annotation results, a corresponding histogram of species profiling was made for each group at the classification levels of phylum, class, order, family, and genus. The histogram showing the average relative abundance of species visualizes the species and proportion of each group with higher relative abundance at different classification levels . The anaP < 0.05, the linear discriminant analysis LDA value > 2). We found 69 differentially abundant taxa (19 taxa were higher in the NC group and 50 taxa were higher in the HPV-LR group) among the two groups , while partial CA can be transformed into VIN or squamous cell carcinoma and further lead to vaginal and cervical epithelium neoplasia and canceration by means of sexual transmission. In fact, most patients can clear HPV infection through their own immune response. The median clearance time for female cervical HPV infection is 9.4 months, while the clearance time for male genital HPV infection is 7.5 months. Within 1 year after diagnosis of CA, 80% of female patients can be cured after treatment  can decompose vaginal epithelial glycogen to produce lactic acid, maintain a weakly acidic environment in the vagina and help maintain the balance between normal vaginal microbiota and microecology  was higher in CA patients with LR-HPV infection, which is consistent with the existing conjecture. Another kind of anaerobic bacterium Bifidobacteria may enhance the antitumor immunity and efficacy of immunotherapy , but many studies of human microecology have found that the changes in the abundance of many low-abundance species have an important influence on human health and disease progress. Lee suggested that anaerobic bacteria Lactobacillus and Gardnerella appear to be higher in the LR group in P < 0.05, the linear discriminant analysis LDA value > 2). Due to the higher P-value of Lactobacillus and Gardnerella, they cannot be shown in the LEFSe diagram. We added P-value for all species). Although the Wilcoxon rank-sum test is a commonly used method for LEfSe difference analysis in the study of flora, it may not apply to the difference analysis of Lactobacillus and Gardnerella in this study. So, we added a Welch's t-test on these two bacteria and found that the difference between the two groups was significant . The results are displayed in In addition, we found there are some inconsistencies between Mycoplasma, Chlamydia and other microorganisms were higher in CA patients with HPV LR-6/11 infection. The accompanied infection of CA patients with other sexually transmitted diseases  is an important factors that led to the recurrence and a worse prognosis of CA (Mutoh et al., The recurrence rate of CA is as high as 59% and the time span for recurrence of CA varies from a few weeks to a few years. In this study, we also found Taken together, our study found CA patients with LR-HPV infection are often accompanied by vaginal microecological imbalance and systematically demonstrated the diversity and community structure of vaginal microorganisms between CA patients with LR-HPV infection and healthy people. It may be that the microbiota of LR subjects changed upon infection or upon development of CA or it may be that the LR subjects had a different microbiota and were more susceptible to infection or CA development. We hope this study will provide implications for the development of rational interventions against LR-HPV infection and CA, and will also provide a theoretical basis for vaginal microecological therapy.https://www.ncbi.nlm.nih.gov/sra/PRJNA548879.Publicly available datasets were analyzed in this study. This data can be found here: This study was undertaken with the approval of the Jinan Central Hospital Affiliated to Shandong University Ethics Service Committee. All experiments were carried out in accordance with the approved guidelines. Informed consent was obtained from all the participants prior to sampling.YZ and YW conceived and directed the study. LW, FP, and MJ analyzed the data. FZ and YS made the clinical diagnoses. QZ, YH, XW, and JT collected and extracted the DNA. YZ was the major contributor in writing the manuscript. All authors read and approved the final manuscript.The author YZ is currently a postdoctoral fellow jointly trained by Shandong University and Shandong LaiBo Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To estimate long‐term cervical intraepithelial neoplasia grade 3 (CIN3) risks associated with different triage strategies for human papillomavirus positive (HPV+) women with a view to reducing unnecessary referrals.The ARTISTIC trial cohort was recruited in Manchester in 2001–03 and was followed up for CIN3 and cancer notification through national registration until December 2015.The 10‐year cumulative risk of CIN3+ was much higher for women with HPV16/18 infection  than for those with other HPV types . Among the 379 women with normal to low‐grade cytology and new HPV infection, the 10‐year cumulative CIN3+ risk was 2.9% (95% CI 1.6–5.2%).The CIN3 risk is confined to women with persistent type‐specific HPV so partial genotyping test assays identifying HPV16/18 as a minimum are essential for efficient risk stratification. Immediate referral to colposcopy for HPV+ women with borderline or low‐grade cytology and referral after a year if still HPV+ with normal cytology may be unnecessary. Low‐grade lesions can safely be retested to identify those with persistent HPV. Recall intervals of 1 year for HPV16/18 and 2 years for other high‐risk HPVs are justified for women with normal cytology and might also be considered for women with borderline/low‐grade cytology. The minimal risk of invasive cancer that has progressed beyond stage 1A must be weighed against the advantages for patients and the NHS of reducing the number of referrals to colposcopy.Cervical screening would be better for women and cheaper for the NHS if women with HPV and normal to low‐grade cytology were retested after a year or two when many infections will have cleared. Cervical screening would be better for women and cheaper for the NHS if women with HPV and normal to low‐grade cytology were retested after a year or two when many infections will have cleared. Primary human papillomavirus (HPV) testing with cytology triage of HPV‐positive (HPV+) women has been piloted at several sites in England following publication of the ARTISTIC Trial results over three rounds of HPV screeningIn England, women are invited for screening 3‐yearly from age 25 to age 49 years then 5‐yearly from age 50 to 64 years. Women who are HPV+ with borderline or worse cytology are referred to colposcopy.The NHSCSP must strike an acceptable balance between cancer risk and the cost and patient inconvenience of excessive screening and unnecessary treatment. The current policy of reflex cytology for HPV+ samples and immediate colposcopy for high‐grade cytology provides effective management for these women. Those with high‐grade cytology at entry to ARTISTIC included all ten prevalent cancers.Women were recruited to the ARTISTIC trial after attending routine cervical screening in Greater Manchester and randomly allocated in a ratio of 3:1 to have the HPV result revealed and acted upon, or concealed. Liquid‐based cytology samples were collected for cytology and HPV testing, and followed for histopathology between 2001 and 2009. Management of women with abnormal cytology was identical in both arms. Women in the revealed arm with normal cytology who tested HPV+ were invited for repeat HPV testing at 12 months and if still positive chose between immediate colposcopy or repeat HPV testing at 24 months. The study was extended to a third round where women on both arms were managed according to national guidelines. HPV testing was performed with Hybrid Capture 2  with a cut‐off of relative light units/control of 1 pg/µl. Three HPV‐typing assays were used to genotype HC2‐positive samples over various periods of the trial. Any high‐risk HPV type detected by any of the assays was included in the analysis.All participants in the study have been traced through the NHS Central Register to December 2015 for mortality and cancer registration, including CIN3. The cohort was linked to the NHSCSP call–recall database to obtain lifetime cervical screening records, including reasons for ceasing screening .No patients were involved in the development of the research. The work was funded by the NIHR‐HTA and Cancer Research UK.Women were classified hierarchically into mutually exclusive groups: HPV16 or HPV18, any other HPV without HPV16 or HPV18 , and HPV‐negative (HC2+ with untyped HPV or HC2‐negative). We have shown that the 10‐year CIN3+ risk in women with normal cytology is higher among those with HPV16  than those with HPV18  but these types were grouped because in the UK 80% of invasive cervical cancers are caused by them.Infections detected in the first HPV sample taken after entry were classified as new or persistent by comparing HPV genotypes with those identified at entry. Women with both new and persistent infections were classified as persistent. This analysis was stratified by time from entry to next test, which varied by cytology and randomisation arm: those with normal cytology were invited for a repeat HPV test after 1 year in the revealed arm, whereas in the concealed arm they were invited for routine follow‐up cytology after 3 years. All women with borderline or low‐grade cytology were invited for repeat cytology after 6 months.Cumulative CIN3+ risks were estimated by Kaplan–Meier methods modified to allow for interval‐censoring. Cumulative risks were calculated from entry (round 1) and also from round 2 (defined as the first HPV test taken 30–48 months after entry). Women were censored at date of last smear before hysterectomy except 608 women who were censored at last smear within the trial as they were not successfully linked to call–recall data. All analyses were censored on 30 April 2015 to allow for late cancer registration. CIN3+ histology was backdated to the beginning of follow up (round 1 or round 2) where the histology/registration occurred within a year of the beginning of follow up. These cases at time zero give an initial step in the Kaplan–Meier curve showing prevalence. Later CIN3+ histology/registration dates were backdated to the first test in the preceding year, then further backdated to the midpoint of the interval between that test and the preceding test to estimate the approximate date when they became screen‐detectable. The cumulative risks therefore include CIN3s which would be diagnosed if screened at that point in time. Cervical cancers are shown in brackets in the tables by time to cancer registration, including those preceded by CIN3 diagnosis. All analyses were programmed in STATA 15.1 .The analysis includes the 24 496 women who were successfully flagged for cancer incidence and mortality. From enrolment to the end of follow up 482 CIN3 and 23 invasive cervical cancers were diagnosed from national registrations or from local histopathology data. Of the 13 591 women who had an HPV test at round 2 approximately 3 years after entry, 75 were diagnosed with CIN3 and seven with invasive cervical cancer before April 2015. Average follow up was 12 years.P = 0.2) but a much lower 10‐year CIN3+ rate  at age 45 years or over (P < 0.0001 compared with women aged under 45 years). Among women with high‐grade cytology at entry there was no age difference . In women who were HPV+ at entry the cumulative CIN3 and cervical cancer risks showed opposite effects in relation to age. Respective cumulative risks (average follow up 12 years) for ages at entry 20–29, 30–44 and 45–64 years were 16.0% (236/1475), 16.0% (165/1032) and 7.0% (19/273) for CIN3 and 0.2% (3/1475), 1.0% (10/1032) and 1.5% (4/273) for cervical cancer. The ten prevalent invasive cancers were all diagnosed within 7 months of entry, and all presented with high‐grade cytology. In contrast, only two of the 13 incident cervical cancers diagnosed 4 or more years later had abnormal cytology at entry .There were 2383 high‐risk HPV+ women with normal, borderline or low‐grade cytology at entry Table . The majTable P = 0.001). Among women with borderline cytology at entry, 80% (140/174) of HPV16 infections persisted until the next test 6 months later compared with 63% (239/382) of non‐HPV16 infections (P < 0.001).HPV16 was more likely to persist than other high‐risk HPV types. In HPV+ women with normal cytology at entry retested at 1 year, 55% (74/135) of HPV16 infections persisted compared with 39% (195/494) of non‐HPV16 infections . The continuing clinical burden of repeated HPV testing, cytology and colposcopy will therefore be largely confined to women who are HPV+ at their first test, some of whom will have long‐standing pre‐cancer missed by previous cytology.Reflex cytology provides a good triage strategy for women undergoing their first HPV test as well as for previously screened women. However, CIN3+ rates were significantly lower in women with a previous negative HPV test  was unrelated to age but was lower (7.0%) in HPV+ women aged ≥45 years. These findings are similar to those reported from the UK pilot (4.8% in HPV+ women aged ≥50 years versus 9.1% in HPV+ women aged 30–49 years).Immediate referral of all HPV+ women with borderline or low‐grade cytology as recommended for the roll‐out of primary HPV screening will not be cost‐effective, particularly in women who have tested HPV‐negative in the previous round of screening. We suggest that low‐grade lesions can safely be retested to identify those with persistent HPV. Recall intervals of 1 year for HPV16/18 and 2 years for the remainder have been piloted in some regions for women with normal cytology and might also be considered for women with borderline/low‐grade cytology. The CIN3+ risk is confined to women with a persisting type‐specific HPV so partial genotyping test assays identifying HPV16/18 as a minimum provide more efficient risk stratification. The minimal risk of invasive cancer that has progressed beyond stage 1A must be weighed against the advantages for patients and the NHS of reducing the number of referrals to colposcopy.The major unresolved issue, which will become increasingly important, is management of women with persistent HPV infection and normal cytology after two rounds of triage. A study should be conducted to evaluate whether random biopsy  is substantially more sensitive than colposcopically directed biopsy for diagnosing disease in women with type‐specific HPV persistence and normal or low‐grade cytology.No additional data are available, in compliance with the ethical and governance regulations under which this research was undertaken.CG received speaker fees from Roche paid to employer and a grant from NIHR‐HTA to fund this project. AS attended meetings with HPV assay manufacturers; speaker fees from Roche; travel and accommodation from Roche and Abbott for training and user group meetings; Roche, Abbott, Hologic, Becton Dickinson and Cepheid provided kits for assay validation purposes; PHE provided funding to support the NHS screening laboratory activity for the pilot. JP received a grant from NIHR‐HTA to fund this project. Completed disclosure of interest forms are available to view online as supporting information.CG conducted all statistical analysis and drafted the report. AS conducted all HPV testing for the ARTISTIC trial. JP was the epidemiological PI of the ARTISTIC trial and contributed to the design of the trial, initiated the follow‐up study, and contributed to the statistical analysis and writing of the report.Ethical approval of the study was obtained from the South East Coast NRES Committee (14/LO/0627) on 13 May 2014 and Section 251 support to process confidential patient information without consent was given by UK Health Research Authority Confidentiality Advisory Group.The work was funded by the NIHR‐HTA and Cancer Research UK.Table S1. Cumulative CIN3+ risks from round 2 by HPV partial genotype and cytology. Invasive cervical cancers (ICC) are also shown in brackets.Click here for additional data file.


The aim of this study was to assess the bedside brain function monitoring of color density spectral array (CDSA) for early prognostic evaluation of coma patients in pediatric intensive care unit (PICU).Forty-two consecutive pediatric coma patients were enrolled. The individual conscious state was evaluated according to the Glasgow coma scale (GCS). CDSA parameters including CDSA pattern (CDSAP), sleep–wake cycle (SWC), sleep stage (SS), and drug-induced fast wave activity (DIFWA) were recorded. Three months later, prognosis was evaluated according to pediatric cerebral performance category (PCPC) score, based on which the patients were divided into FP-group (favorable prognosis) and PP-group (poor prognosis).P = .83) and that of SWC was 0.12 . Furthermore, the absence of SWC was correlated with poor prognosis in nonintracranial infection patients.The changeable type of CDSAP, appearance of SWC, SS, and DIFWA were significantly correlated with favorable prognosis. Both GCS and SWC were significantly correlated with the prognosis. However, there was substantial overlap in GCS between FP-group and PP-group. Although the absence of SWC was statistically an independent risk factor for poor prognosis but with a high false positive rate (0.143), a linear logistic regression showed the odds ratio of GCS for predicting prognosis was 0.93 (95% confidence interval: 0.48–1.80; Our study found that several CDSA factors are associated with prognosis of coma patients in PICU. SWC may be a potential indicator for evaluating the prognosis of coma patients in PICU. Currently, the Glasgow coma scale (GCS) is the most widely used tool for evaluating the conscious state and brain functions. The 15-point scales include 3 aspects including eye opening response, verbal response, and motor response. A lower GCS score indicates a more severe condition and predicts a poor prognosis. However, the GCS scoring system has some inherent limitations: drug use or low blood oxygen can alter the patient's level of consciousness, leading to an inaccurate score on the GCS; tracheal intubation or tracheotomy and severe eye swelling may make it impossible to test the verbal responses and eye opening; and the assessment of these responses has a certain of subjectivity. Thus, an objective evaluation method is needed for bedside brain function monitoring and prognosis prediction. However, conventional continuous EEG monitoring is information-heavy, time-consuming, and hard to analyze, and thus, it is rarely used bedside in PICU. Color density spectral array (CDSA) is a quantitative and simplified EEG method that uses Fourier transformation to reflect EEG signals by color and frequency (y-axis) over time (x-axis). The interpretation of CDSA is much more intuitionistic and easier, and this technique has been applied in critically ill patients.,4 Nevertheless, the prognostic value of CDSA remains unclear. In this study, we aimed to investigate the clinical value of CDSA for early evaluation of prognosis in patients with coma in PICU.Electroencephalography (EEG) is a noninvasive electrophysiological monitoring method for recording the spontaneous electrical activity of the brain, which can sensitively reflect brain injury and the metabolism of cerebral neuronal cells. The amplitude in EEG decreases when the neurons are injured or suppressed, and abnormal metabolism or reduced conduction velocity in nerve fibers can lead to a reduced frequency.22.1 The inclusion criteria were coma within 3 days; GCS ≤8; and age ranging from 1 to 14 years. The exclusion criteria included coma induced by antiepileptic drugs or sedative drugs; other neurological or psychological disorders that may affect the brain function assessment; or significant artifact in EEG.A total of 42 consecutive pediatric patients with coma in PICU in the First Hospital of Jilin University were enrolled between March 2016 and March 2017. This study was approved by the local Institutional Review Board and Ethics Committee. Written informed consent was obtained from the guardians of each pediatric patient. Coma was determined according to the GCS.2.2 Long-term (≥16 hours) EEG monitoring was performed using a digital EEG monitor . The electrodes were placed according to the International 10–20 system, and CDSA of EEG parameters was recorded. The EEG signals in 4 leads  were transformed into four-channel CDSA patterns (CDSAPs); the spectrum diagram is a two-dimensional image onto which the three-dimensional  images were projected. The x-axis represented the time, the y-axis represented the frequency, and the color represented the EEG power . The CDSAPs were classified into 3 types according to change of the spectrograms: changeable, which is characterized by a constant and predominant activity in the low-frequency band with variously pronounced and organized peaks in the alpha- or high-frequency band , and the poor-prognosis group .Three months after the initial examination, individual prognosis was evaluated according to the pediatric cerebral performance category (PCPC) score Table ,[7] whic2.3t test. Chi-squared or Fisher exact test was used to screen the potential risk factors. Wilcoxon rank-sum test was performed to assess the prognostic value of GCS, and Fisher exact test was performed to assess the prognostic value of SWC. Then logistic regression analyses were performed to identify the risk factors for prognosis and to evaluate the prognostic value of combining GCS and SWC. A two-tailed P value <.05 was considered indicative of a statistically significant difference.Statistical analysis was performed using SPSS software package . Continuous variables are presented as mean ± standard deviation (SD); comparison between groups was performed using two-sample 33.1P > .05). The demographic data, the causes of coma, and the etiological comparisons between the FP-group and the PP-group were summarized in Table The study population included 21 females and 21 males, with an average age of 89.81 ± 46.39 months . According to the PCPC score, the prognosis was favorable in 21 cases (FP-group) and poor in 21 cases (PP-group), yielding a poor prognostic rate of 50%. There were no significant differences in gender, age, and cause of coma between the FP-group and the PP-group . However, there was substantial overlap in GCS between the FP-group and the PP-group , Fisher exact tests showed GCS was significantly correlated with the prognosis , SS , DIFWA (present or absent), and CDSAP . Fisher exact tests showed that changeable type of CDSAP, appearance of SWC, SS, and DIFWA were significantly correlated with favorable prognosis , and that of SWC was 0.12 .As we observed that GCS and SWC might be significantly associated with the prognosis, we further performed a linear logistic regression. The OR of GCS for predicting prognosis was 0.93  had a favorable prognosis. In addition, among the 5 children with slow-monotonous type, 3 died and 2 remained unchanged CDSAP. Two children with slow-monotonous type of CDSAP had a poor prognosis. All the 5 children with nonspectrum type died.4 In current clinical practice, GCS is the most widely used scale for the assessment of conscious status. In the present study, we also noted the correlation between GCS and prognosis, and we found that a lower GCS score predicted a poor prognosis. However, we evaluated the prediction value of GCS, and observed that there was substantial overlap in GCS between FP-group and PP-group, indicating that GCS alone cannot serve as a robust indicator of prognosis in our cohort. The GCS scoring system has some inherent limitations for predicting the prognosis of coma patients in PICU.Brain function monitoring can provide important information for clinical evaluation and prognosis prediction in patients with coma. Recently, CDSA has been widely used in the clinical evaluation of patients with sleep disorders and epilepsy. However, the application value of CDSA in brain function monitoring has not yet been well elucidated. Here, we examined the reliability of CDSA to predict the prognosis of pediatric patients with coma.Although conventional EEG recording can promote the assessment of conscious status and coma severity, the time-consuming and high analytical skill requirements limit its usage in PICU. CDSA uses Fourier transformation to process long-term EEG signals and converts EEG parameters  into visualized images, making it possible to evaluate the dynamic EEG changes in different-frequency bands. We speculate that the brain may still reserve certain adjustment function in the early period of coma, manifesting as diffuse slow waves on EEG, whereas by 3 to 5 days after coma onset, the brain function may deteriorate due to abnormal circulation and metabolism. Furthermore, the follow-up data showed that slow-monotonous type and nonspectrum type of CDSAP were associated with a poor prognosis. Changeable type may indicate that a child's brain function impairment is still reversible, and early effective treatment may help the function recovery.In this study, according to EEG signals, CDSA parameters including CDSAP, SWC, SS, and DIFWA were analyzed. We classified the spectrograms into 3 types: changeable, slow-monotonous, and nonspectrum. On the initial examination, 32 children (76.1%) exhibited changeable type, of which 21 had a favorable prognosis. Among the other 11 children with poor prognosis, only 4 children had significant SWC (36.4%), and these 11 children did not show DIFWA. Five children showed slow-monotonous type and 5 children showed nonspectrum type. CDSA of these 10 children had no SWC, SS, or DIFWA, and 8 of these children died . These findings indicate that changeable type of CDSAP may predict a better prognosis than slow-monotonous type and nonspectrum type, which is consistent with the findings reported by Bricolo et al. Thus, the preservation of physiological sleep structure may be closely associated with a favorable prognosis. Dario et al studied 27 patients with subacute disturbance of consciousness and found that the presence of SS can predict a favorable prognosis. Karnaze et al noted that an alternating CDSA pattern was significantly associated with survival in patients with head injuries. Pineda et al found that the sleep structure on EEG was correlated with early prognosis of acute encephalopathy and the presence of SS could predict favorable outcomes. In the present study, we used spindle waves to identify the SS. In this study, we used a much larger sample size which supported their speculation.There were 22 children had clear SWC, all of which showed changeable type, and 18 (81.8%) children among them had a favorable prognosis. Notably, these 18 children had power variation or a fast wave spectrum in the 12 to 14 Hz frequency band, yielding identifiable SS. Of 22 children with SWC, 4 had a poor prognosis . Of 14 children without clear SWC, 13 (92.9%) had poor prognosis and 1 (7.1%) remained functionally disabled. Ayas et al found that sleep deprivation may adversely affect the metabolic, immune, and circulatory functions, and they also proposed that sleep disorder can interfere the nerve regeneration.Considering the presence of the SWC is a prerequisite of SS, we suggest that SWC may be used as a practical and intuitionistic prognostic predictor. Fisher exact test showed that SWC had statistical significance for prognosis prediction. Although logistic regression analysis showed that the absence of SWC was an independent risk factor predicting poor prognosis with a high OR (43.751), SWC was still not a robust indicator to serve as an independent factor to predict prognosis in our cohort, due to the high false positive rate (0.143). As GCS and SWC might be associated with the prognosis, we further performed a linear logistic regression, which indicated that the combination of GCS and SWC could not significantly improve the predictive ability when compared with SWC alone. Therefore, we speculate the prognostic value of SWC in comatose children may be better than that of GCS. Further analysis revealed that the absence of SWC might be a useful predictor for poor prognosis in coma patients without intracranial infection Table . HoweverThe present study has some noticeable limitations. We did not perform repeated monitoring to evaluate the dynamic changes on CDSAP. In addition, the sample size was small, and definitive conclusions still require further studies involving a much larger cohort.5The CDSA diameters may be a useful bedside tool for PICU physicians to evaluate the status of critical patients. Our study presents some interesting discoveries and found several CDSA factors that may be associated prognosis of coma patients in PICU. Both GCS and SWC were informative but had limitation. The predictive value of SWC is superior to that of GCS, and SWC may be a potential indicator for evaluating the prognosis of coma patients in PICU.We gratefully acknowledge the valuable cooperation of Prof. Yumei Li, Dr. Cuiying Wang, and the staff who helped us in this study. This work was supported by the National Nature and Science Foundation of China (No. 81771396); the Nature and Science Foundation from the Science and Technology Department of Jilin Province (No. 20180101159JC); and the Special Fund Project of Industrial innovation of Jilin provincial (No. 2017C029-1).Data curation: Jiangtao Wang, Xiaosheng Hao, Ge Qu, Chunnv Li.Formal analysis: Yanfeng Zhang, Yinbo Chen, Jianmin Liang.Methodology: Ge Qu.Resources: Chunnv Li.Validation: Yanfeng Zhang, Chunnv Li.Writing – original draft: Jiangtao Wang.Writing – review and editing: Xiaosheng Hao, Guiling Liu, Yinbo Chen, Jianmin Liang.


It bound to the positively charged region in the HPV L1 protein, suggesting that 3HP-β-LG binds to HPV L1 protein through the interaction between the negatively charged region in 3HP-β-LG and the positively charged region in HPV L1 protein, thus competitively blocking the binding of HPV to the receptor on the basement membrane in vaginal mucosa. Although 3HP-modified chicken ovalbumin (3HP-OVA) also carries high net negative charges, it exhibited no anti-HPV activity, suggesting that the interaction between 3HP-modified protein and HPV L1 protein relies on both electrostatic and matchable conformation of the binding sites in both proteins. When topically applied, 3HP-β-LG did not enter the host cell or blood circulation. These findings suggest that 3HP-β-LG targets HPV L1 protein and blocks HPV entry into the host cell, thus being safe and effective for topical application in the treatment of HPV infection.We have previously demonstrated that 3-hydroxyphthalic anhydride (3HP)-modified bovine beta-lactoglobulin (3HP-β-LG) is highly effective in inhibiting entry of pseudovirus (PsV) of high- and low-risk human papillomavirus (HPV) into the target cell. Intravaginally applied 3HP-β-LG-containing vaginal gel could significantly inhibit HPV infection and reduce viral load in the cervical region. However, we still do not understand the underlying molecular mechanism by which 3HP-β-LG is able to inhibit HPV infection. Here, though, we showed that 3HP-β-LG did not inactivate HPV PsV, but rather blocked entry of HPV PsV into the target cell  Cervical cancer is the second most common cancer among women in China . Almost T = 7 HPV virus-like particle (VLP) reveals that 72 L1 pentamers form the icosahedral shell of HPV , 2,4,6-trinitrobenzenesulfonic acid (TNBS), human serum albumin (HSA), β-lactoglobulin (β-LG), chicken ovalbumin (OVA), and bovine serum albumin (BSA) were purchased from Sigma. Horseradish peroxidase (HRP)-conjugated goat anti-human IgA and IgE antibodies were purchased from Abcam (UK). HRP-conjugated rabbit anti-mouse IgG antibody was purchased from Dako (Denmark). The HPV16- and HPV-58-L1/L2-expressing plasmids and Luciferase pCLucf plasmid were kindly provided by Dr. John Schiller at the Laboratory of Cellular Oncology, National Cancer Institute, NIH, MD, USA.Anhydride-modified proteins were produced as previously described . Briefly6 cells) at 16 h before transfection, were transfected with a mixture of HPV16-L1/L2-expressing plasmid (p16sheLL) and pCLucf plasmid for HPV16 PsV, or a mixture of HPV58-L1/L2-expressing plasmid (p58sheLL) and pCLucf plasmid for HPV58 PsV, using VigoFect (Vigorous Biotechnology Corp.). The cells were suspended in 0.5 ml of lysis buffer and incubated for 24 h at 37°C with slow rotation. The lysate was cooled on ice for 5 min, mixed with 5 M NaCl solutions to adjust the concentration of NaCl to 0.85 M, and further kept on ice for 10 min. Then, the lysate was centrifuged at 5,000 × g for 10 min at 4°C, and quantified for the concentration of L1/L2 capsid protein levels. To remove aggregates, the pseudovirion stock was filtered before use for the neutralization assay. The inhibitory activity of 3HP-β-LG against HPV PsV entry into HeLa or HaCaT cells was detected as previously described (4 cells in 100 μl of 10% FBS DMEM (DMEM-10) per well in a 96-well plate, followed by a culture at 37°C overnight. 3HP-β-LG or β-LG was serially diluted in DMEM and incubated with 100 μl of HPV PsV . Mixtures were added to HeLa or HaCaT cells and incubated at 37°C for 16 h. After replacement of the culture supernatants with free medium and culture for additional 72 h, cells were lysed for measurement of luciferase activity, according to the manufacturer’s manual .The HPV pseudovirus was produced as previously describedescribed . Brieflyin vivo safety and efficacy of the intravaginally applied 3HP-β-LG-containing vaginal gels (Registry No.: ChiCTR-TRC-12002016)  at 4°C overnight. After blocking with protein-free blocking buffer , HPV L1 (type16 or 18) protein at 1 μg/ml was added into the wells for 50 μl/well, and the plate was incubated at 37°C for 1 h. After washing with PBST three times, mouse anti-HPV L1 (type16 or 18) antibody  was added at a 1: 1,000 dilution. After incubation at 37°C for 1 h and washing again, HRP-conjugated rabbit anti-mouse IgG antibody  was added for 1 h. After washing and adding tetramethylbenzidine , the absorbance was measured at 450 nm. Similar protocols were used for detection of 3HP-β-LG in the serum of rhesus monkeys and 3HP-β-LG-specific IgA and IgE in the vaginal swab eluates, which were leftover samples from the clinical trials for evaluating the 2002016) ,b.Two positively charged peptides of HPV L1 protein, L1-I (residues 474–488: LKAKPKFTLGKRKAT) and L1-II (residues 493–505: STSTTAKRKKRKL), were synthesized by a standard solid-phase Fmoc method as previously reported . The pep5/well) overnight. Then, cells were incubated with 3HP-β-LG at a final concentration of 15 μg/ml at 0.5 h before or 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after addition of HPV PsV , followed by an incubation at 37°C for 16 h. After replacement of culture supernatant with fresh medium and an incubation for additional 72 h, cells were lysed to determine the entry inhibition ratio, according to the manufacturer’s manual (Promega).HeLa cells were plated in a 96-well plate (for 1.0 × 10HeLa cells in 96-well plates were preincubated with 100 μl of 3HP-β-LG (5 μM) or β-LG (5 μM) at 37°C for 1 h and then washed with DMEM three times before addition of HPV PsV. For the control, 3HP-β-LG- or β-LG-pretreated HeLa cells were not washed before HPV PsV was added. HPV PsV entry into HeLa cells was detected as described above.The plated HeLa cells and 3HP-β-LG mixture were both prechilled at 4°C for 30 min. Then, the cells were incubated with the 3HP-β-LG mixture at 4°C for 12 h. After washing with cold PBS buffer twice, the cells were further incubated at 37°C for 72 h. Then the cells were lysed to determine luciferase activity, according to the luciferase assay system manual .The experiment on rhesus macaques was conducted under ethical guidelines and approved by the Ethics Committee of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College . In brief, the rhesus macaques were randomly divided into treatment group and control group. In the treatment group, vaginal gel containing 3HP-β-LG (1.8 mg per dose) was dispersed into carbomer gel and administered intravaginally or rectally. In the control group, only carbomer gel was applied. Then, 2 ml of blood was collected before or after injection at −1 h, 0 h, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h. The serum was separated after condensation.5) were seeded onto coverslips in a 6-well plate. Then, 3HP-β-LG (0.5 μg/ml) was added for 12 h. Then, the supernatant was replaced with DMEM containing 2% FBS. After 24 days, the cells were fixed by 4% paraformaldehyde , perforated by 0.2% Triton X-100, and blocked with 3% BSA . Next, cells were incubated overnight with anti-3HP-β-LG mAb or anti-β-actin mAb  at 4°C. After five washes, the cells were incubated with Alexa Fluor 488-labeled donkey anti-mouse IgG  at room temperature for 1h. After five washes, the coverslips were sealed with Prolong Gold Antifade reagent with 4,6-diamidino-2-phenylindole (Thermo Fisher Scientific) and scanned with the Leica SP8 confocal microscope.To determine whether 3HP-β-LG could enter into HeLa or 293 T cells, cells  of 0.59 μg/ml, which is more potent than that of 3HP-HSA (IC50: 2.28 μg/ml). 3HP-OVA and all three unmodified proteins at the concentration as high as 10 μg/ml showed no significant inhibitory activity against HPV16 PsV entry into the target cell. These results confirm that not all, just some, proteins, such as β-LG and HSA, modified by 3-hydroxyphthalic anhydride gain the ability to inhibit HPV infection.Our previous studies have shown that 3-hydroxyphthalic anhydride-modified bovine beta-lactoglobulin (3HP-β-LG) is a promising HPV entry inhibitor against entry of PsV of both high-risk and low-risk HPV types . This in50 of 0.28 μg/ml), confirming that 3HP-β-LG has broad-spectrum anti-HPV activity.In our previous studies, we proved the antiviral activity of 3HP-β-LG against entry of the high-risk HPV types 16 and 18 and low-risk HPV type 6 into the target cell . Aside fConsidering that the HaCaT cells express abundant integrin and heparan sulfate proteoglycan (HSPG), the receptors for HPV  and act As we proposed before, 3HP-β-LG inhibited HPV PsV entry into the target cell possibly through the binding of the negatively charged residues in 3HP-β-LG with the positively charged residues in a protein on the surface of the viral particle, thereby blocking the interaction between viral protein and receptor on the target cell . Howeverα value equals 2.31 × 105/M) and L1-II . These results suggested that 3HP-β-LG may bind to the positively charged sites in the C-terminal region of L1 protein on the HPV surface to block the interaction between the viral particle and cell receptor.The C-terminal positively charged region of HPV L1 was proved to regulate the binding of HPV to the receptor . This reTo determine whether 3HP-β-LG could inactivate HPV PsV, we incubated HPV PsV with 3HP-β-LG at room temperature for 2 h and then removed 3HP-β-LG by PEG-8000 precipitation. As shown in HPV entry into the host cell takes more than 10 h, a relatively slow process compared to other viruses , which pTo confirm the functional stage of 3HP-β-LG in blocking viral entry, we used a temperature shift assay to slow down the viral infection process. At 4°C, HPV PsV could bind to cell receptors, but could not fully enter the cytoplasm because the speeds of membrane fusion and cytoskeletal transformation are largely reduced at such low temperature. We incubated HPV PsV and HeLa cells at 4°C for 12 h so that HPV could bind to the cell surface. Then we washed away the unbound HPV before shifting the cells to 37°C for further 72 h of incubation. When 3HP-β-LG was added into the cells at the beginning and then removed by washing, no viral entry occurred . This prTo determine whether intravaginally applied 3HP-β-LG could enter into epithelial cells, we incubated HeLa cells and 293 T cells with 3HP-β-LG for 24 h and then washed the cells three times using PBS. The cells were stained with mouse anti-3HP-β-LG antibody or anti-β-actin antibody, as a control, and Alexa Fluor 488-labeled donkey anti-mouse IgG. Different from the anti-β-actin antibody-treated cells that showed strong fluorescence signals, anti-3HP-β-LG antibody-treated cells displayed no significant fluorescence signals inside or outside the cells . These rTo investigate whether the topically applied 3HP-β-LG enters the blood circulation, a carbomer gel with or without 3HP-β-LG was topically administered in the vagina or anus of rhesus macaques. Their blood samples were collected at different time points before and after gel application. The concentration of 3HP-β-LG in the blood samples and that in the gel (as positive control) were quantified with ELISA using anti-3HP-β-LG mAb. As shown in About 10–20% of acute HPV infections become persistent, leading to various forms of cancer , such asViral entry inhibitors have been proven effective and safe for the treatment of viral infections . We havei.e., the net negative charges on 3HP-β-LG   and HSV Based on the results obtained from this study, we proposed a mechanistic model of 3HP-β-LG for inhibiting HPV infection in the cervical mucosa . The new3HP-β-LG that is topically applied in the vagina can bind to the L1 protein of the newly invaded or the newly released HPV and block the attachment of HPV to the basement membrane, thereby inhibiting the entry of HPV into the host cells for replication. After treatment with 3HP-β-LG for a certain period of time , the newly produced HPV-free epithelial cells in the lower part of the mucosal epithelium are expected to move upward and push the HPV-infected cells away from the vaginal mucosa , making all layers of mucosal epithelium free of HPV infection.Considering that the presence of micro lesions or breaks in the cervical epithelium, possibly caused by sexual activity, is the requirement for HPV to access the HSPG receptor on basement membrane , combinaThe results from the time-of-addition assay, cell washout assay, and virus inactivation indicate that 3HP-β-LG could interact with the virions, rather than the cells, to effectively block the entry of HPV PsV into the target cell, but could not inactivate the virions . These bAll datasets generated for this study are included in the manuscript/supplementary files.The animal study was reviewed and approved by Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College .SJ, LSu, and YZ conceived and designed the experiments. CH, CW, LSi, and QW conducted the experiments. YZ performed the computer modeling and analysis of the interaction between 3HP-β-LG and HPV L1 protein. CH, YZ, LSu, and SJ analyzed the data and wrote the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although routine vaccination of females before sexual debut against human papillomavirus (HPV) has been found to be cost-effective around the world, its cost-benefit has rarely been examined. We evaluate both the cost-effectiveness and cost-benefit of routine female adolescent nonavalent HPV vaccination in Hong Kong to guide its policy, and by extension that of mainland China, on HPV vaccination. One major obstacle is the lack of data on assortativity of sexual mixing. Such difficulty could be overcome by inferring sexual mixing parameters from HPV epidemiologic data.We use an age-structured transmission model coupled with stochastic individual-based simulations to estimate the health and economic impact of routine nonavalent HPV vaccination for girls at age 12 on cervical cancer burden and consider vaccine uptake at 25%, 50%, and 75% with at least 20 years of vaccine protection. Bayesian inference was employed to parameterize the model using local data on HPV prevalence and cervical cancer incidence. We use the human capital approach in the cost-benefit analysis (CBA) and GDP per capita as the indicative willingness-to-pay threshold in the cost-effectiveness analysis (CEA). Finally, we estimate the threshold vaccine cost (TVC), which is the maximum cost for fully vaccinating one girl at which routine female adolescent nonavalent HPV vaccination is cost-beneficial or cost-effective.As vaccine uptake increased, TVC decreased  in the CBA but increased in the CEA. When vaccine uptake was 75% and the vaccine provided only 20 years of protection, the TVC was US$444 ($373–506) and $689 ($646–734) in the CBA and CEA, respectively, increasing by approximately 2–4% if vaccine protection was assumed lifelong. TVC is likely to be far higher when non-cervical diseases are included. The inferred sexual mixing parameters suggest that sexual mixing in Hong Kong is highly assortative by both age and sexual activity level.Routine HPV vaccination of 12-year-old females is highly likely to be cost-beneficial and cost-effective in Hong Kong. Inference of sexual mixing parameters from epidemiologic data of prevalent sexually transmitted diseases  is a potentially fruitful but largely untapped methodology for understanding sexual behaviors in the population.The online version of this article (10.1186/s12916-018-1118-3) contains supplementary material, which is available to authorized users. The cost-effectiveness of routine female adolescent human papillomavirus (HPV) vaccination and other strategies  has been extensively studied for many high-income countries  as well as middle- and low-income countries  . The conDespite the recent advent of a second-generation HPV vaccine , Hong KA key challenge in a rigorous evaluation of HPV vaccination programs is to adequately parameterize the HPV transmission model. To robustly evaluate the health impact of HPV vaccination, evidence-based HPV transmission models are needed to estimate the herd immunity effect . ExtensiHerein, we briefly describe the model structure. Please see the Additional file Our model comprises (1) a deterministic age-structured compartmental dynamic model for simulating heterosexual transmission of high-risk HPV (HR-HPV) and (2) a stochastic individual-based cohort model for simulating the development of cervical cancer over the lifetime of each female. This hybrid approach has been used in previous studies of HPV vaccination . We groEach individual enters the model at age 10 without any HPV infection , 76 1-year age groups , and three sexual activity levels . Let Ng, a, u(t) be the number of individuals in stratum  at time t, and cg, a, u be the rate at which these individuals form new sexual partnerships. The age-specific distribution of individuals with different sexual activity levels are based on the sexuality study results published by the Family Planning Association of Hong Kong (FPAHK) [g, a, u) forms a sexual partnership at time t, the probability that their partner belongs to stratum ,  g ≠ g', isδuv has value 1 when u = v and 0 otherwise, and Φ(⋅) is the Gaussian kernel. We use the Gaussian kernel to model age assortativity because its shape conforms with intuition as well as the patterns empirically observed in sexual activity surveys from the UK, Australia, and the US [εA and σg whereas risk assortativity is controlled by εS. For simplicity, we assume that σg is the same for males and females.We stratify the population into two sexes ( (FPAHK) ; see Add (FPAHK) . Specifid the US –18. In tIg, a, u, h(t) be the prevalence of HPV class h among individuals in stratum  at time t. The force of infection from HPV class h for individuals in stratum  at time t isβh is the class-specific baseline probability of transmission per sexual partnership, g, a, u) and   for individuals aged below W1; (2) linearly decreases from 1 to μ as individuals age from W1 to W2; and (3) remains at αa = μ for individuals aged above W2. The parameters μ, W1, and W2 are inferred during model parameterization.for modeling the effect of age on susceptibility and infectiousness ; (4) assortativity of sexual mixing ; and (5) age-specific susceptibility and infectiousness .We infer the following model parameters by fitting the model to these data using Markov chain Monte Carlo methods with non-informative flat priors : (1) claWe compare routine vaccination for girls at age 12 to opportunistic vaccination with status quo vaccine uptake (12% ). We assWe assume that vaccination does not affect screening behavior. The Cervical Screening Programme (CSP) in Hong Kong recommends women aged between 25 and 65 years to follow the 1-, 1-, 3-yearly cycle of cervical screening  . Based oIn the CBA, we used the human capital approach to monetize health and life-year loss into productivity loss based on average personal income    and the For each vaccination uptake and protection scenario, we considered 10,000 probabilistic sensitivity analysis scenarios which comprise all combinations of (i) 100 sets of transmission and natural history parameters randomly generated from their posterior distributions obtained from model parameterization ; σg = 2.1 (0.2 – 0.49)) and sexual activity level (εS = 0.98 (0.89 – 0.99)) ) Fig. . The infThe prevalence of HPV-16, HPV-18, and HPV-OV decrease monotonically over time and reached a steady state 50–60 years after routine female vaccination began Fig. . This agTVCCBA decreased but TVCCEA increased with TVCCBA > TVCCEA across all scenarios considered: TVCCBA was lower than TVCCEA by approximately 13%, 30%, and 36% when vaccine uptake was 25%, 50%, and 75%, respectively , the total cost for fully vaccinating one girl would need to be less than US$444 ($373–506) and $689 ($646–734) for routine HPV vaccination to be cost-beneficial and cost-effective, respectively. Compared to this scenario, TVCCBA (TVCCEA) increased by 2.3% (3.9%) if vaccine protection duration increased to 30 years or longer. If vaccine uptake decreased from 75% to 50%, TVCCBA increased by 6.6% whereas TVCCEA decreased by 2.3%. For TVCCBA and TVCCEA to be similar, the willingness-to-pay threshold would need to be approximately US$30,000 for vaccine uptake at 25% and US$20,000 for vaccine uptake at 75%  as the duration of vaccine protection decreased Fig. . As vaccely Fig. . When th75% Fig. .Fig. 4CoWe have evaluated the cost-benefit and cost-effectiveness of routine female adolescent nonavalent HPV vaccination for reducing cervical cancer burden in Hong Kong. Our results suggest that, at a vaccine uptake of between 25% and 75%, routine vaccination for 12-year-old girls  as part of a centrally funded program represents good value for money if the cost of fully vaccinating one girl is no greater than US$444 ($373–506) and $689 $646–734), respectively. The current market price of fully vaccinating one girl at age 12 at the FPAHK (as of November 2017) is $284, and the tender prices for bulk purchases in Italy, Norway, South Africa, and Spain were 66–77% lower than market prices [–734, resFurthermore, because of the lack of local data, our analysis did not examine many of the benefits of HPV vaccination such as protection against anogenital warts, recurrent respiratory papillomatoses, or vulvar, vaginal, penile, anal, and oropharyngeal cancers. The value of protection against these non-cervical cancers has been estimated to be almost as great as the value of protecting against cervical cancers in some scenarios . FurtherTVCCBA/TVCCEA < 1. The generalizability of this finding to other populations hinges on two other factors. The first factor is that CBAs in Hong Kong consider only changes in economic productivity . Intuitively, TVCCBA/TVCCEA increases with the ratio of average personal income to willingness-to-pay threshold. The second factor is the age distribution of cervical cancer in relation to retirement age and life expectancy. HPV vaccination will be (1) more cost-beneficial if the gap between the average age of cervical cancer cases and retirement age increases; and (2) more cost-effective if the gap between the average age of cervical cancer and life expectancy increases. Consider an illustrative comparison among Hong Kong, the US , and the UK . The ratio of average personal income to willingness-to-pay threshold is approximately 0.5 for Hong Kong, 0.72 for the US, and > 0.7 for the UK. On the other hand, the median age of cervical cancer diagnosis is 45 in the UK, 49 in the US, and 52 in Hong Kong, whereas the female life expectancy is 82, 79, and 87, respectively. The retirement age in these populations are similar. Applying the rationale above, the difference between TVCCBA and TVCCEA is likely to be significantly smaller for the US and UK compared to Hong Kong, and thus CBA and CEA will likely result in similar conclusions on the health economics of HPV vaccination in the US and UK.To our knowledge, our study is the first to compare the CBA and CEA implications of HPV vaccination. For Hong Kong, the CBA threshold vaccine cost is always lower  than its CEA counterpart, i.e., A recently published review of cost-effectiveness studies of HPV vaccination , togetheDespite the different model scenarios, some results from previous studies can be compared with ours. We estimate that the female-only organized HPV vaccination with 75% vaccine uptake and lifelong vaccine protection would reduce cervical cancer incidence by 85% compared to 12% opportunistic vaccine uptake. In the CEAs for high-income countries, the additional reduction in cervical cancer for routine vaccination compared to no vaccination (which is the most similar scenario to the opportunistic vaccination scenario in our study) ranged between 70% and 92%, depending on vaccine uptake of females and males , 52, 54.Although not explicitly stated, the TVC of nonavalent HPV vaccines for routine vaccination compared to no vaccination can be estimated from two previous studies where sufficient detail about the overall cost of vaccination is reported , 53. In Our study has several other important limitations. First, we assumed that the duration and transmissibility of HPV infection are the same for males and females. While some evidence from other settings suggests that this is not generally true , Hong KoA major strength of our study is that, as we evaluate the health economics of HPV vaccination, we simultaneously characterize sexual mixing in Hong Kong by fitting the transmission model to epidemiological data . The resth 5-year plan (2016–2020) of China aims to link the cities of Hong Kong, Macau, Guangzhou, Shenzhen, Zhuhai, Foshan, Zhongshan, Dongguan, Huizhou, Jiangmen, and Zhaoqing into an integrated economic, business, and technology hub that constitutes an area of 56,000 km2, a combined population of 68 million, and an economy of $1.51 trillion. Given the low uptake of HPV vaccination and cervical cancer screening and the high burden of cervical cancer in these cities, prevention of cervical cancer will likely be a top-priority public health issue in the GBA Initiative. Over the next decade, demographics, sexual mixing, and disease transmission in these heterogeneous cities will be substantially impacted by the massive increase in short- and long-term human mobility and interaction brought about by the GBA Initiative. A recent study showed that interstate migration has a strong impact on the population-level benefit of HPV vaccination in the US because of herd immunity and the long duration between HPV infection and resultant cervical cancer [The Greater Bay Area (GBA) Initiative in the 13l cancer . As suchRoutine HPV vaccination of 12-year-old females is highly likely to be cost-beneficial and cost-effective in Hong Kong. Inference of sexual mixing parameters from epidemiologic data of prevalent sexually transmitted diseases  is a potentially fruitful but largely untapped methodology for understanding sexual behaviors in the population.Additional file 1:Table S1. The distribution of individuals with no, low, and high level of sexual activity in each age group. Table S2. Posterior distributions of inferred parameters. Table S3a. Probability distributions of cervical cytology testing. Table S3b. Probability of cytology results given true health states. Table S4. Probability distributions of cost parameters. Table S5. Probability distribution of QALY weights for different health outcomes. Figure S1. Schematic of the natural history model for HR-HPV infection and cervical cancer among females. Figure S2. Trace plots and the posterior distributions of the fitted parameters. (PDF 544 kb)


Human papillomavirus (HPV) contributes to the development of cervical and oropharyngeal tumors. The increased incidence of HPV associated oropharyngeal tumors is lately being observed also in Polish population. The worldwide distribution of HPV varies and the studies rarely combine analysis of virus genotypes in both: genital and oropharyngeal locations. Therefore, in our study, we investigated HPV distribution in both anatomical sites of females with previous history of cervical cancer or with pre-cancerous lesion and their partners to establish the dominant types in Polish couples in genital and oropharyngeal areas, as they can be easily sexually transmitted.The study group consisted of 197 females and their partners. Each female had current or previous cervical pathology and HPV detected in gynecological swab with the use of Anyplex™ II HPV28 Detection system. This system is based on multiplexed real time PCR and enables detection of 19 high-risk and 9 low-risk HPVs.p = 0.035). Moreover, regular presence of low risk type 42 was noticeable in both sexes, in both kind of swabs. There was a trend observed towards its prevalence as single infectious agent in women with previous history of cervical cancer (p = 0.069).Beside women, the virus was found in 114/197 of men in their foreskin swabs. Additionally, HPV was detected in oropharyngeal swabs of 39/197 female and 56/197 male participants. HPV 16/31/42/39/54 dominated in female and HPV 66/42/16/31/53 in male genital locations. The incidence of HPV in oropharynx was lower, top five genotypes included: HPV 6/39/42/35/16 in women compared to HPV 39/6/42/40/33 in men. HPV16 was the most frequently detected virus type, found in 70/197 examined cervical swabs. It was significantly more prevalent as single infection in females, previously treated for the cervical cancer  contains supplementary material, which is available to authorized users. In the last decades, there has been an increase in data concerning the incidence of HPV infections in humans. The most significant premise from these data is that HPV is the most important risk factor for the development of cervical cancer , 2. AccoHPV is small (50–55 nm in diameter) double stranded virus that exhibits tropism to epithelial cells (skin and mucosa). The virus genome length is around 8 kb and is divided into three regions: E (Early), L (Late) and LCR (Long Control Region). In general, HPVs constitute the heterogeneous group of viruses. So far, more than 200 mucosal and cutaneous subtypes were identified , 8–10.The worldwide distribution of given genotypes varies among different localizations , 5, 11. HPV genotyping is essential for preventing cervical cancer and may also be useful for simultaneous screening of HPV in the oropharynx, as tumors of this region of head and neck seems to be most frequently associated with infection of these viruses. Therefore, in this study, we took an attempt to track the distribution of HPV genotypes in the genital and oral swabs of females with current or previous cervical pathology  and their partners, to determine which HPV types dominate in Polish, unvaccinated population and to find whether this distribution is similar in couples in both anatomical sites. Such report will enrich the knowledge concerning the distribution of HPV genotypes in female patients suffering from genital pathology and their asymptomatic partners.The examined group consisted of 197 Polish female participants and their matched partners. Women were qualified to the group based on two premises: current or previous pathology of the cervix (including cancer) and HPV detected in the smear taken during gynecological examination. If a woman fulfilled these criteria, then her partner was included automatically to the study group. Women were diagnosed during routine cervical cytology or in the framework of preventive screening programs. The examinations were performed by specialists (gynecologists and oncologists) in “Boramed” Medical Center in Warsaw and two Clinics of Poznan University of Medical Sciences: Department of Gynecology and Department of Gynecological Oncology in Poznan, between years 2014–2016. The brush smears were taken with the use of Cervex-Brush®  from the endocervix, ectocervix or cervical transformation-zone from women with CIN I–II or from the vaginal vault in patients after hysterectomy, performed previously to treat cervical cancer. From each patient’s partner the swab from the foreskin was taken with the use of FLOQSwabs . Ultimately, 71 women with previous history of cervical cancer were included, out of which 18 had cancer detected in the year of material collection and 53 had cancer detected in the past . All of them were successfully treated for cervical cancer and during the study they were free of disease, with no symptoms of either recurrence or other malignancy. The remaining, 126 women had CIN I or CIN II detected . Each sample was designated for HPV genotyping with the same method. The experiments were performed in accordance with relevant guidelines and regulations, approved by the Ethics Review Board of Poznan University of Medical Sciences (decisions no. 21/13 and 75/15) and the written, informed consent was obtained from all participants.DNA was isolated from 1 ml of hypercellular suspension containing gynecological or laryngological smear collected on ThinPrep liquid medium. The suspension was first centrifuged for 15 min at 13.000 rpm in order to separate cellular material from the brush or swab. Then, the supernatant was discarded and the pellet was suspended in 200 μl of 1 x PBS. Further, cell lysis, DNA precipitation, binding to the membrane and elution were performed according to procedure of NucleoSpin® Blood Kit .HBB) gene - was simultaneously amplified to ensure that the DNA amount is sufficient and that the PCR reaction is efficient. The positive (for each HPV type) and negative controls were included during each PCR run. The results were analyzed using the Seegene Viewer v. 2.0 program and were considered as valid, when internal control was present in each sample. A positive result (+++/++/+) indicated the presence of HPV DNA, while the negative result (−) indicated the absence of the HPV virus. The detection limit of the applied test is 50 copies of HPV per reaction.The HPV genotyping was performed with the use of Anyplex™ II HPV28 Detection system , which detects simultaneously 19 high-risk HPVs  and 9 low-risk HPVs  in a single specimen. This system, targeting L1 gene, is based on multiplex real-time PCR with high sensitivity and specificity due to the utilization of DPO™  and TOCE™ (Tagging Oligonucleotide Cleavage and Extension) technologies. The reaction mixture of 20 μl included 1 x HPV28 primer mix A or B and 1 x Anyplex Master Mix. The real time PCR reaction was performed on a CFX96 Real-Time PCR thermocycler  with the following steps: 50 °C/4 min., 95 °C/15 min. and 50 cycles of 95 °C/30 s., 60 °C/60 s., 72 °C/30 s. Melting curve analysis was performed from 55 to 85 °C. The internal control - Beta-Globin  and HPV detected in the gynecological location as well as of their male partners. Each female patient was infected with at least one out of 28 types of the virus, detectable by the test , in which both partners were infected in the genital area. In this group of couples, 33/114 pairs (28.9%) were infected with at least one concordant virus type. The stable relationship with one partner during the period of at least a year before the examination was declared by 22/33 pairs. In the remaining 81 couples (71.1%), the infection with distinct HPV types was determined  included as follows: HPV 31, 16, 18, 66 and 42 (Table The infection with only one (any) HPV type predominated, however depending on the examined anatomical site and sex, the infection with more than two HPV types was also observed Table .p = 0.002; Table HPV16 was the most frequently detected virus type, found in 70 out of 197 examined cervical swabs 35.5%). Within the group of HPV 16 positive females, 40 (57.1%) had been previously treated for the cervical cancer, while 30 (42.9%) presented low grade CINs at the time of study. The study shows that HPV 16 was significantly more prevalent in patients with the cervical tumor history in comparison to those, that had not developed tumor of this location , HPV 42 in 20 women (10.2%), HPV 39 in 18 women (9.1%) and HPV 54 in 16 women . It is noteworthy, that 9 out of 15 women with previously treated CC had HPV 42 detected as single infection. On the contrary, in none of the women with CINs (0/5) HPV 42 was detected as single one. Among 11 women who had HPV 42 and coinfection with other HPVs, high risk types dominated  and in men . In women, HPV 6 and HPV 39 were detected more frequently , while in men HPV 39 was most common , followed by HPV 6 .HPV infection contributes to the cancer development in the mucosa of two distinctly distant anatomical sites: genital tract and upper aerodigestive tract. It is an indispensable factor of cervical cancer development but it may also affect other parts of female and male genitals. In the last decade, HPV detection rates have been increasing in the oropharyngeal cancers , 4. ThisThe genotyping allows to establish the profile of particular HPVs in clinically involved tissues in different populations. It is often difficult to compare the results of HPV genotyping derived from different studies, as diverse methodology is used and various materials are analyzed. This may translate into false negative results , 11. AddThe genotypes in the individual couples overlapped partially: the concordant types were detected in 33/114 of couples in genital swabs and in 9/11 of couples in oropharyngeal swabs. In these groups, 22/33 and 6/9 couples, respectively, declared a stable relationship lasting at least a year. In the remaining couples we cannot be certain if the virus was transmitted between partners of a given pair. However, our result - 28.9% is concordant with the available data showing, that 13 to 63.2% of couples share the same HPV type in the genital area . NeverthIt should be highlighted, that neither male nor female participants had pathology in the oropharynx during laryngological examination. Similarly, none of the men reported any complaints from the penile. However, beside low risk types, the detected genotypes encompassed also high risk types, potentially causing cancer, therefore there is a constant need for these persons to perform regular check-ups. Especially in cases of oropharyngeal swabs, the positive HPV status is particularly disturbing in the light of recent results from Rosenthal et al., who found that normal, healthy controls lack HPV infection in their oral rinses . The speTo conclude, our results shed lights on the distribution of HPV genotypes in Polish couples, in which female partner suffered from previously diagnosed cervical cancer or from cervical intraepithelial neoplasia grade I or II. We have pointed out the common infection with HPV 42 in women but also in their partners, in two anatomical sites: in the genital area as well as in the oropharynx. Importantly, HPV 42 was detected as the single infectious agent more often in females with previously diagnosed cervical cancer than in those with CIN I or II. Despite this, we cannot infer that HPV 42 should be reconsidered as high risk type in the general Polish population. Our findings should be verified on a larger group of Polish participants, including healthy controls, followed regularly in 6 months intervals, in oral as well as genital areas.Additional file 1:Table S1. The characteristics of patients and their partners data. The file contains information concerning clinical data of the study participants, together with the results of HPV genotyping. (XLSX 29 kb)


The prevalence of some human papillomavirus (HPV) genotypes has been shown to change with age. So, also the distribution of HPV genotypes included in the nonavalent vaccine may not be the same at all ages, and this could mean that vaccine protection against cervical cancer may be affected by age. The present study aimed to evaluate whether there are age-related changes in the fraction of high-grade cervical intraepithelial neoplasia (CIN) attributable to HPV genotypes included in the nonavalent vaccine.  Two hundred four consecutive women undergoing conization with a histological diagnosis of CIN3 were retrospectively analyzed. All included women had a preconization HPV genotyping (HPV Sign® Genotyping Test). The women were divided into three groups according to age: <35, 35–44, and ≥45 years of age. Based on HPV genotypes detected in cervical lesions, the age-related changes in the expected vaccine protection were evaluated by the Cochran–Armitage test for trend. p=0.006). The rate of HPV-16 and HPV-33, included in the vaccine, showed a negative trend with age . Among HPV genotypes not covered by the vaccine, the rate of non-high-risk HPVs (genotypes: 53-54-70-73-82-85-87) showed a significant positive trend with increasing age (p=0.018).  The fraction of CIN3 attributable to HPV genotypes included in the nonavalent vaccine showed a significant negative trend with increasing age, with potential vaccine protection of 82% after the age of 45 ( Although the fraction of CIN3 attributable to genotypes included in the nonavalent HPV vaccine was high even after age 45, older women appeared to be more at risk of high-grade CIN related to HPV genotypes not included in the vaccine. Interestingly, older women showed a higher rate of precancerous cervical lesions associated with non-high-risk HPV. The present findings seem to raise the question about the management of cervical pathology at a later age in a future postvaccination era. Cervical cancer (CC) is one of the most common neoplasms among women worldwide, especially in developing countries . CurrentConcerning primary prevention, there are three types of HPV vaccines with a well-established safety profile . The bivAlthough the vaccine protection is very high, it should be taken into account that the distribution of HPV genotypes is age-dependent . Some HPBased on the question mentioned above, the present study aimed to evaluate whether there are age-related changes in the fraction of high-grade CIN related to HPV genotypes included in the nonavalent HPV vaccine.The present retrospective observational study included women with CIN3 diagnosis on cone specimens at the Cervical Cancer Screening Centre of Reggio Emilia and University Hospital of Ancona, Italy, between September 2011 and November 2014. The present study was approved by the Local Ethical Committee  with the following protocol number: N 2015 0486OR. All patients provided written informed consent for the use of their data for research purposes before any diagnostic or therapeutic procedure.All included women were sent to colposcopy for an abnormal Pap smear and had a preconization diagnosis of CIN2+ on cervical biopsy or persistent low-grade CIN for at least two years. They had a presurgery HPV genotyping performed on the same day of conization, as a routine procedure, because our local protocols provided a different time interval between conization and the first subsequent colposcopic evaluation according to preconization HPV genotype. All included women had a histological diagnosis of CIN3 on cone specimen . Women with an anti-HPV vaccination, previous conization, immunological disease, concomitant malignancies , or pregnancy were excluded. All data were retrieved from computerized software used in our Colposcopic Clinics.Cervical conizations were performed in an outpatient setting with local anesthesia and under colposcopic guidance. Based on previous studies , HPV genThe women were divided into three categories according to age: <35 years of age, 35–44 years of age, and ≥45 years of age. The age-related changes in the fraction of CIN3 related to HPV genotypes included in the nonavalent HPV vaccine were evaluated by comparing the rate and trend between groups based on HPV genotypes detected in cervical lesions. Furthermore, the possible age-related changes of each HPV genotype included in the vaccine were evaluated (including its presence in both single and multiple infections). Finally, the rate and trend between age groups of HPV genotypes not included in the vaccine were assessed (hr-HPV genotypes and non-hr-HPV genotypes). All results from the three different age groups were expressed as numbers and percentages.Given that HPV genotype distribution may be affected by age, ethnicity, and sample collection year, an univariate analysis was performed to assess the impact of these confounding variables.DNA was extracted from 204 human cervical samples and analyzed using the HPV Sign® Genotyping Test . All samples were collected with an endocervical swab and Thin Prep (TP)  the same day of conization before surgery. Twelve of these samples showed the absence of HPV DNA with a confirmed lesion on cone specimen. The latter samples underwent further molecular testing by cone biopsy.μL of proteinase K in 180 μL of lysis buffer (buffer ATL) at 56°C for one hour and then incubated in 200 μL of lysis solution (buffer AL) at 70°C for 10 min. After that, 200 μL of ethanol (96–100%) was added to each sample, followed by vortexing for 15 s. After briefly centrifuging the samples, they were washed repeatedly, and the DNA was eluted in 60 μL of buffer and extracted with the QIAmp DNA Mini Kit  following the manufacturer's instructions.For cytological samples, 4 mL of each specimen collected by TP was digested with 20 For tissue samples (cone specimens), DNA was extracted after deparaffinization as described for the cytologic samples. DNA was stored at −20°C until use.β-globin gene was used as the internal control, followed by pyrosequencing with multiple sequencing primers. Melting curve analysis detected specific peaks for HPV sequences and the β-globin gene, allowing the semiquantitative determination of the presence or absence of HPV DNA. Only HPV-positive samples were further analyzed by pyrosequencing using the Pyromark Q24 System  and four specific sequencing primers to identify the viral genotypes. These sequencing primers allowed the synthesis of genotype-specific sequences of 30 bases. Furthermore, they had a high discriminatory power for identifying the HPV genotype of each sample. IdentiFireTM software  was used to compare the obtained sequences with genotype-specific sequences in the HPV Sign® Q24 library. The assay was performed following the manufacturer's instructions.As previously described , this asThe Kolmogorov–Smirnov test was used to assess the distribution of continuous variables (age). Given that our data originated from ordered categories, to test the relationship between two classification factors , we used the chi-squared test for trend (or the Cochran–Armitage test for trend), which is more potent than the unordered independence test when a classification table has two columns and three or more rows (or two rows and three or more columns) .https://www.medcalc.org; 2019). A p value <0.05 was considered statistically significant.All statistical analyses were performed using MedCalc Statistical Software version 19.0.3 , 35–44 years of age (75 women), and ≥45 years of age (50 women). Preconization hr-HPV genotypes were detected in 196/204 (96.1%) women (with at least one hr-HPV type). Preconization non-hr-HPV genotypes were detected in 8/204 (3.9%) women. Single infections were found in 181 (88.7%) women. Multiple infections were found in 23 women (11.3%). Potential HPV vaccine protection was 91.7%. Patient characteristics are shown in HPV genotype distribution in women with CIN3 is shown in p=0.047); HPV-33 showed a similar significant negative trend with increasing age (p=0.044). HPV-18, -31, -52, -45, and -58 showed no significant trend with age   . There wp=0.006) .p=0.153). The distribution of non-hr-HPV genotypes showed a significant positive trend with increasing age: 1.3% in women <35 years of age, 2.7% in women of 35–44 years of age, and 10.0% in women ≥45 years of age (p=0.018) . Hr-HPV genotypes: 3 women with HPV-59, 2 women with HPV-56, 2 women with HPV-35, 1 woman with HPV-39, and 1 woman with a multiple infection including HPV-66 + HPV-53; non-hr-HPV genotypes: 4 women with HPV-73, 1 woman with HPV-53, -87, and -82, and 1 woman with a multiple infection including HPV-70 + HPV-85. hr-HPV genotypes not included in the vaccine showed a positive but not significant trend with increasing age: 2.5% in women <35 years of age, 4.0% in women of 35–44 years of age, and 8.0% in women ≥45 years of age (p=0.018) .The present study showed that the fraction of CIN3 lesions attributable to HPV genotypes included in the nonavalent vaccine decreased with increasing age. The same significant trend was observed for HPV genotypes 16 and 33, included in the vaccine. Conversely, among HPV genotypes not covered by the vaccine, non-hr-HPVs showed a significant positive trend with increasing age.Nonavalent HPV vaccine was initially approved for women aged 9–26 years, and in October 2018, the Food and Drug Administration extended its use to men and women aged 27–45 years . It has The inclusion of specific HPV genotypes in the nonavalent vaccine has led to very high protection against precancerous and cancerous cervical lesions. However, several studies in the literature have shown that the prevalence of HPV genotypes, and therefore of those HPV-related cervical pathologies, varies with age , 16. TheIn a fascinating paper, Guardado–Estrada et al. showed that almost half of CCs in older women were due to non-hr-HPV . Other sSo, it would seem that older women are more at risk of high-grade cervical lesions due to genotypes not included in the nonavalent vaccine. These results may be a matter to be investigated for the future management of cervical cancer screening programs. There are studies in the literature that used simulation models to assess the appropriate screening intensity in vaccinated women. They showed that for women undergoing bivalent vaccine, including HPV-16 and 18 genotypes, three lifetime screens would be cost-effective; while for women subjected to the nonavalent vaccine, only two lifetime screens would be needed . These rA further result of the present study was that non-hr-HPV genotypes showed a significant positive trend with increasing age in CIN3 lesions. Although they represented a small percentage of cases, in this group of women, there was an increase of high-grade cervical lesions unrelated to hr-HPV. Interestingly, these women would have been negative at the primary HPV screening test that includes only hr-HPV genotypes. Currently, in Italy, only the HPV test is used in cervical screening program from 30 years of age. Based on these results, it would be interesting to ask the question about the need to use a cotesting (Pap smear + HPV test) at a given age cutoff. In a recent paper, Sun et al. showed that about 40% of older women with CIN2+ and carcinoma had a negative hr-HPV test, and these cases would have been missed without using the Pap test .As reported previously, these age-related changes in HPV genotype distribution may be due to the woman's immunological status . Older wThe present study has the limitation of being retrospective. Furthermore, although the group of women not covered by the vaccine provided significant results, it included a small sample of subjects. Not last, it must be taken into account that the hierarchical attribution of HPV genotypes may have led to an overestimation of vaccination protection. Conversely, it should be emphasized that we only included women with CIN3 that represent the real precancer cervical lesion. Furthermore, our histological reference standard was represented by cone specimens and not by cervical biopsies. Finally, HPV genotype sampling was carried out on the same day of conization, making the data very reliable.To conclude, although the expected HPV vaccine protection against CIN3 was high even after age 45, the present results seem to raise the question about the management of cervical pathology at a later age in a future postvaccination era. Further studies with even larger sample sizes would be needed to confirm these results.