This cross-sectional electronic online or telephone survey assessed the attitudes and behaviors regarding human papillomavirus (HPV) vaccination and the effect of different factors among a nationally representative random sample of 349 general practitioners (GPs) in Italy. A semi-structured interview was performed between September 2018 and October 2020. Almost all respondents considered the HPV vaccine safe with an overall mean value of 8.8, on a scale ranging from 1 to 10, and 59.9% and 32.6% believed that the vaccination was very effective in preventing the related diseases among 12–26 years’ girls and boys. Multivariate logistic regression analysis showed that GPs who had received information about HPV vaccination from scientific journals were more likely to have positive attitude towards the effectiveness of the vaccine in preventing HPV-related diseases in girls between 12–26 years. A large majority (81.5%) of GPs who provided assistance to girls’ patients aged 11–12 years often or always recommend the HPV vaccine to them, and this behavior was more likely to occur in those who believed that the vaccine was very effective in preventing HPV-related diseases in girls between 12–26 years. GPs were more likely to often or always recommend the HPV vaccine to boys aged 11–12 years if they often or always recommended the vaccine to girls aged 11–12 years, if they believed that the vaccine was very effective in preventing HPV-related diseases in boys between 12–26 years, and if they considered the HPV vaccine very safe. GPs should receive information about the HPV immunization to ensure that they routinely communicate with their patient population in order to achieve better coverage rates. It is well-known that the human papillomavirus (HPV) infection is still one of the most serious public health problems in developed and developing countries, due to the fact that it is one of the leading sexually transmitted infections and is associated with genital warts, cervical, anogenital, and oropharyngeal cancers ,2. AlthoIn spite of the important role that GPs have in HPV prevention, attitudes and behaviors towards HPV vaccination and their predictors among physicians who practice in several health care settings in different geographic areas have received attention in the literature ,8,9,10, The survey lasted from September 2018 till October 2020 and participants were recruited by a nationwide two-stage clustering sample design. The sampling frame included all local health units (LHUs), which provide health care services to the population either directly, through their own facilities, or paying for the services provided by independent public and private structures. In the first stage, 19 LHUs were randomly selected. Initially, the research team contacted the head of each selected LHU with an e-mail explaining the purposes of the study, and asking for their assistance in obtaining names and contact information (e-mail address and phone number) of all GPs working in their LHU. In the second stage, a random sample of GPs was drawn from each LHU of the sample frame.The sample size for this study was calculated using single population proportion formula with 95% confidence level, an expected proportion of 85% of GPs who often or always recommend the HPV vaccination to girls at 11 to 12 years, and relative precision to be 5%. In addition, since a 60% response rate was expected, the total number of enrollment was 327 GPs.Selected GPs received an invitation to participate through an e-mail asking to complete the survey. GPs were provided with a hyperlink to the online electronic survey located on a platform . The e-mail contained a cover letter with the lead investigator’s name and contact details, information about the background of the study, its goals and its voluntary nature, the importance of gaining their perspectives on the issue, the content of the questionnaire, the use and anonymization of the data, and the maximum time needed to complete the questionnaire (10 min). Links were unique to each participant, thereby eliminating the potential for multiple responses being provided by the same individual. Each link was collected and stored independently of survey responses to guarantee the anonymity and to know each participant. A brief introduction of study purpose and participants’ right was showed at the beginning of the questionnaire and participants were also informed that they provided their consent by participating. Participants were also informed that they were free to interrupt the interview without justification at any point during the survey and that when sending back their questionnaires, they were not able to withdraw the data. To maximize the survey response rate, e-mail reminders were sent at seven-day intervals to the GPs if there was no response and subsequently, three follow-up calls were made in different time slots during weekdays and weekends. In the follow-up calls, GPs were asked if they would prefer to participate by responding in a one-to-one telephone interview. If the email of the GP failed to deliver or the phone call was not available, an alternative GP was randomly selected instead from the same LHU. The participants were not compensated with financial or other incentives for their time spent.In a pilot study, the questionnaire was pretested on 25 GPs, and the results were used in order to verify ease of completion and refine the wording of the question, which were excluded afterward from the survey. The instrument, guided by previous published epidemiological surveys conducted by some of us ,11,12,13t-test, as appropriate, explored the relationship between the different outcome variables and the selected variables. A p-value of 0.25 or less at bivariate analysis was considered for variables to be subsequently included in multivariate logistic regression models. In the third step, multivariate logistic regression models for dichotomous outcomes were used to estimate the association between the different covariates and the following outcomes of interest: positive attitude about the effectiveness of vaccine in preventing HPV-related diseases in girls between 12–26 years (Model 1), profile of GPs who often or always recommended the vaccine to girls aged 11–12 years (Model 2), and profile of GPs who often or always recommended the vaccine to boys aged 11–12 years (Model 3). The following covariates were included in the multivariate logistic models: age (continuous), gender , number of years since degree (continuous), number of years in practice (continuous), number of hours worked per week (continuous), source of information related to HPV infection and vaccination , and need of additional information about HPV vaccination . Moreover, the following variables were also included: positive attitude about effectiveness of HPV vaccine in preventing HPV-related diseases among 12–26 years’ girls , and positive attitude about effectiveness of HPV vaccine in preventing HPV-related diseases among 12–26 years’ boys  in Models 2 and 3, and often or always recommend the vaccine to girls aged 11–12 years  in Model 3.All statistical analyses were performed using Stata software v15  . First, p-value were two-sided and the value below or equal 0.05 was considered to be statistically significant.Backward stepwise selection of variables was performed to determine the final models. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to measure the strength of the association between explanatory and outcome variables. All Of the 567 GPs who were selected, 349 completed the interview for a response rate of 61.5%. The survey population’s demographic and professional characteristics are summarized in Regarding the participants’ attitudes, almost two-thirds (59.9%) and one-third (32.6%) exhibited the higher scores on a ten-point Likert-type concerning the effectiveness of the HPV vaccination in preventing the related diseases among 12–26 years’ girls, with a mean value of 9.2, and boys, with a mean value of 8.1. Moreover, almost all (97.1%) considered the HPV vaccine safe with an overall mean value of 8.8, on a scale ranging from 1 to 10. To avoid potential confounders, multivariate logistic regression analysis was performed in order to assess which independent variable would be significantly related to the different outcomes of interest and the results are reported in Among GPs who provide assistance to boys’ patients aged 11–12 years, only one-third (37.7%) often or always recommend the vaccine and 19.3% never do it. The results of the multivariate logistic regression analysis showed that three variables were significantly associated with an increased probability to often or always recommend the HPV vaccine to boys aged 11–12 years. The variable GPs often or always recommending the vaccine to girls aged 11–12 years was found to be the strongest predictors for often or always recommending the HPV vaccine to boys aged 11–12 years since respondents that reported recommending the vaccine to these age-group girls were more than 6 times more likely to recommend the vaccination to boys of the same age . Additionally, GPs who believed that the vaccine was very effective in preventing HPV-related diseases in boys between 12–26 years  and those who considered very safe the vaccine  were also more likely to often or always recommend the HPV vaccine to boys aged 11–12 years , safe (68.1%), and that it prevents HPV-related diseases (21.9%); whereas, the main reasons for not recommending were the lack of information (40.4%), concerns about the efficacy (21.3%), and the safety (17.9%). More than two-thirds of GPs (68.6%) reported that parents refused to vaccinate their sons and the main reasons were concern regarding the side effects (63.9%), lack of knowledge of the infection (30.4%), and because they felt that their boys were not at risk (21.1%). Moreover, only 17.7% of GPs verified that boys aged 13–26 years have been vaccinated and only 12.7% often or always recommended the vaccine to boys not previously vaccinated.The large majority of participants had received information about HPV infection (81.4%) and the related vaccination (73%). The most preferred sources where the GPs got the information about HPV infection and vaccination were scientific journals (71.7%), followed by scientific meetings (42.5%), colleagues (19.2%), and the internet (13.1%). More than two-thirds (68.8%) of participants were willing to acquire more information related to HPV infection and vaccination.This cross-sectional survey aimed to describe the attitudes and behaviors with regard to HPV vaccination among GPs in Italy as one of the priority groups for health-promoting advice and the prophylactic vaccination program. In addition, the study assessed the relationships of several factors with the GPs’ attitudes and decision to vaccinate against HPV.In this study, the majority of the GPs respondents showed a very positive attitude regarding the HPV vaccination in general concerning, for example, the fact that it was considered to be safe and to be effective in preventing the related diseases among 12–26 years’ girls and boys by the vast majority of respondents. The important role of these attitudes was corroborated by the fact that those GPs who considered the vaccine very safe were more likely to often or always recommend the HPV vaccine to boys aged 11–12 years. However, in this study, despite demonstrating this mostly favorable opinion of the respondents regarding the HPV vaccination, one area was detected that may be improved among GPs with continued education. Indeed, unfortunately, it has been observed that the proportion of GPs who often or always recommended the vaccine to the boys’ patients aged 11–12 years was very low with a value of 37.7%. Therefore, targeted health promotion and prevention interventions are needed and must be implemented directed to change this practice in order to reach the target of 95% coverage for this age group. Moreover, based on the multivariate logistic regression model which examined the impact of different predictors on HPV vaccine recommendations, it was found that GPs that recommend the vaccination to girls aged 11–12 years were more likely to recommend often or always the HPV vaccine to boys of the same age. Of interest, the findings from this survey provide further insights into the reasons for not recommending the HPV vaccination to boys. It was surprising that the major impediments to HPV vaccination recommendation reported by respondents were the lack of information, negative perceptions about the efficacy and the safety of the vaccine. While an overwhelming concern in terms of perceived vaccine safety and efficacy was dominant in this survey for the parents for refusing the HPV vaccination for their sons, parents indicated also the lack of knowledge of the infection, and because they did not feel that their sons were at risk. These findings were also consistent with the results from similar previously studies ,17,18,19Sources of information are necessary to gain more HPV-related knowledge. However, one of the most disturbing and surprising findings of the study was that more than one-fourth of the surveyed GPs did not have received information about the HPV immunization. This underscores that it is essential that GPs receive additional information for ensuring that they routinely recommend the vaccine and for the communication dissemination to patients. The important finding that more than half of GPs wanted to know more information related to HPV infection and vaccination supports this. Results of the current study indicated that scientific journals were the most common source of information being identified by the GPs. It is important to highlight that scientific journals have a key and essential role in providing information. Indeed, when it has been measured with multivariate logistic regression analysis the relationship between the sources of information and the different outcomes of interest, it has been found that this source appears to have a significant influential impact on vaccination coverage since those who sought information from scientific journals were more prone to have positive attitude towards the effectiveness of vaccine in preventing HPV-related diseases in girls between 12–26 years compared to those who had not used such source. This result corroborated evidence that has already been proven in a lot of previous published investigations among different groups of the population that these sources are key in improving the level of knowledge, the attitudes, and the recommendation behaviors ,23,24,25As with all similar research, certain limitations of this study should be put into consideration when interpreting the findings and results for the development of broad-scale policy framework. First, the cross-sectional questionnaire-based study design has its own limitations and, therefore, it might be difficulty to draw causative links between the independent variables and the outcomes of interest. Second, the use of the questionnaire with self-reported information may constitute a bias as GPs could be under-reporting or over-reporting their behaviors. Third, the self-reporting nature does not allow for independent validation of the respondents’ answers, especially with regards to HPV behaviors and is also vulnerable to social desirability bias, thus it is possible that respondents may not report truthfully and they tend to answer questions in a manner that will be favorably viewed by others. However, potential bias may have been partially minimized because it has been conducted as an anonymous survey and all GPs were assured that their answers were confidential. Fourth, it is also possible that GPs who may have had an interest in HPV vaccination or who recommend the vaccine were more likely to respond than those who did not share these attitudes and practices. Despite these limitations, the current findings will provide an update about the attitudes and behaviors regarding HPV vaccination in Italy and have important implications for future interventions.In conclusion, importantly, the information acquired from this nationally representative sample of GPs has implications for different policy designs and provides public health perspectives on several fronts for HPV vaccination. First, the positive attitude concerning the effectiveness of the vaccination in preventing the related diseases between girls and boys 12–26 years might result in a higher recommendation of the vaccine mainly among boys. Second, given the low proportion of GPs who often or always recommended the vaccine to the boys aged 11–12 years, it is important to monitor such rate in order to reduce the burden of HPV-related diseases. Finally, GPs should receive information about the HPV immunization for ensuring that they routinely communicate with their patient population for addressing myths regarding the vaccination for increasing adherence rates.


P<0.001) were significantly higher in participants with any ISAR following vaccination than in those without an ISAR. Local pain, induration, swelling and systemic fever were significantly correlated with higher GMCs for IgG anti-HPV-16 and/or anti-HPV-18, respectively. Furthermore, the analyses of the immunogenicity bridging study of Cecolin® and the phase III trial of a hepatitis E vaccine yielded similar results. Based on these results, we built a scoring model to quantify the inflammation reactions and found that the high score of ISAR indicates the strong vaccine-induced antibody level. In conclusion, this study suggests inflammation-related adverse reactions following vaccination potentially indicate a stronger immune response.Concerns about vaccine safety are an important reason for vaccine hesitancy, however, limited information is available on whether common adverse reactions following vaccination affect the immune response. Data from three clinical trials of recombinant vaccines were used in this post hoc analysis to assess the correlation between inflammation-related solicited adverse reactions  and immune responses after vaccination. In the phase III trial of the bivalent HPV-16/18 vaccine (Cecolin®), the geometric mean concentrations (GMCs) for IgG anti-HPV-16 and -18 ( Vaccines are widely recognized as one of the greatest public health successes of the last century, significantly reducing morbidity and mortality from a variety of bacterial and viral infections. However, vaccine hesitancy, especially concerns about vaccine safety or adverse reactions, impedes the implementation of vaccines such as the human papillomavirus (HPV) vaccine  and measThe different antigens or adjuvants included in vaccines differ in their mode of action and ability to stimulate the immune system ,9. RegarReactogenicity represents the physical manifestation of the inflammatory response to vaccination and can include injection-site pain, redness, swelling or induration, as well as systemic symptoms such as fever . These sIn this study, we aimed to explore the relationship between adverse reactions that may be caused by inflammation and immune responses following vaccinations on the basis of data from clinical trials of recombinant HPV and hepatitis E virus (HEV) vaccines. Furthermore, we attempted to build a simple model to quantify adverse reactions, which can be an approach to hierarchically assess individual levels of inflammatory responses after vaccination.Escherichia coli-produced bivalent HPV-16/18 vaccine (Cecolin®) involving females aged 18–45 years (NCT01735006)  vs 252.2 IU/ml [95% CI 241.4–263.5], 18 P< 0.001). Also, the incidence of ISAR was higher in women with higher titer of antibodies after vaccination . A relatively larger proportion of women aged 18–26 years had ISARs than women aged 27–45 years  and fever  were the two most common solicited adverse reactions. On covariance analysis , the GMC16 P= 0.047; HPV18: 373.8 IU/ml [95% CI 283.8–492.3] vs 278.0 IU/ml [95% CI 267.8–288.7], 18 P= 0.037). With the increasing number of injections resulting in ISARs, there was an upward trend in GMCs, although a significant difference was only noted in GMCs for anti-HPV-16 (16P = 0.023). When ISAR was subdivided into five symptoms, we found that there was a trend towards higher GMCs in women with any one of the symptoms overall. Significant differences were observed in both HPV types IgG between women with and without pain  or swelling  at the injection site, as well as anti-HPV-16 IgG between women with and without induration at the injection site (16P = 0.047) or fever (16P = 0.001).Higher GMCs for anti-HPV-16 and 18 IgG were recorded in women who experienced grade 3 ISAR, and lower GMCs were recorded in those who experienced grade 1 or 2 ISAR . Men and women with ISARs had significantly higher GMCs for anti-HEV IgG than those without ISARs in the DS of the phase III trial of HEV vaccine . No obvious increasing trend was observed on GMCs in increasing severity or number of injections resulting in ISARs. Significant differences were observed in GMCs for anti-HPV-16 and anti-HEV IgG between subjects with fever and those without fever .Similar findings with significantly higher GMCs in individuals who had ISAR following vaccination were revealed by the analysis of the bridging study of Cecolin®  The effect of the number of injections on the immune response is indeed very small or even negligible, it is due to other unknown confounding factors that cause a significant correlation in HPV-16 specific antibody response of the phase III trial. (2) The quantitative results of immunogenicity induced by different antigens or different reagents are incomparable. The discrimination between different antibody levels induced by the vaccine’s components of HPV-18 might be inferior to that of HPV-16, which could reduce the power of statistical analysis. The significant correlation is hard to show in overall lower antibody levels. (3) The limited sample size of the bridging study of Cecolin® and the phase III trial of HEV vaccine.We also built a scoring model to assess ISARs following vaccination. The parameters of the model are derived from the ratio of GMCs for antibodies, considering five typical symptoms of ISARs and the levels of severity of each ISAR. SI shows a similar exponential relation to GMCs for anti-HPV-16 and 18 IgG in the phase III trial of Cecolin®. This indicates that there are differences in the levels of antibodies between vaccinees with a SI below 6 and those with a SI of 6 or higher. However, the reason for the curve relationship is hard to explain because of the limited sample size of the high SI group in the present study. Notwithstanding that the quantitative model presented here is of limited clinical value, more important is the assumption of a quantifiable inflammatory response following vaccination. Although we know that an effective adaptive immune response requires a certain level of inflammation to be triggered , how to Vaccine-induced immune effectors are essentially antibodies produced by B lymphocytes that can bind specifically to a toxin or a pathogen. The above mentioned improved innate immune system accompanied with more severe ISAR would promote the antigen presentation by antigen presenting cell (APC) and stimulate T cell-dependent B cell differentiate into antibody-secreting cells and memory B cells. From the results in Supplementary Table 3, we also found that the long-term IgG antibody against HPV-18 is significantly correlated with ISAR, which may be largely related to the role of memory B cells.Currently, vaccines have largely been developed empirically, with limited knowledge of how exactly they activate the immune system , let aloThe limitations of this study includes that only data of recombinant, adjuvanted vaccines are analyzed. The extent to which antigens and adjuvants contribute to reactogenicity in addition to other baseline physiological characteritics  needs to be further confirmed and also through similar studies on other types of vaccines (live/nonlive/nonadjuvanted). And both the HPV (Cecolin®) and HEV (Hecolin®) vaccines investigated in this study showed efficacy of 100% in the phase 3 clinical trial against clinical endpoints, thus they are not the proper data sets for analyzing the relationship between ISAR and protection, the correlation between ISARs and the actual efficacy of vaccines is worthy of further study in the future and has practical significance.In conclusion, this study suggests inflammation-related adverse reactions following vaccination potentially indicate a stronger immune response. However, because of incomplete knowledge about the biological mechanisms of vaccine-induced injury and the immune response process, the clinical relevance of these immunological findings warrants further assessment.Click here for additional data file.


To assess the prevalence and distribution of HPV genotypes among Chinese Han women, and to explore the risk of high-grade cervical lesions associated with individual hr-HPV genotypes.Genotyping and histopathology data from the Chinese Multi-Center Screening Trial (CHIMUST) and its pilot screening trial, from 6 regions across mainland China, were re-analyzed. The data from physician- and self-collected samples from 10,867 Chinese Han women (ages 30–69) were used to determine the prevalence and distribution of hr-HPV and to explore the risk association between hr-HPV genotypes and precancerous lesions.9.2% of the study population tested hr-HPV positive in physician-collected sample. The prevalence varied regionally from the lowest in Guangdong (6.3%) to the highest in Inner Mongolia (13.0%). The most prevalent genotypes found were HPV-52 (21.7%), HPV-16 (19.2%), HPV-58 (15.0%), HPV-39 (8.9%), and HPV-51 (8.2%). The overall odds ratios for CIN2+ and CIN3+ for the presence of HPV-16 was 58.6 (95% CI 39.2–87.5) and, 91.6 (95%CI 54.3–154.6), respectively and remained the highest odds ratio for CIN3+ in all 6 regions.Geographical variation exists in the prevalence and distribution of hr-HPV in mainland China. HPV-16/52/58 were the most prevalent genotypes, and HPV-16 had the highest risk for high-grade cervical lesions.ChiCTR-EOC-16008456. Registered 11 May 2016.CHIMUST, Registration number:  Cervical cancer is ranked as the fourth most common cancer in women, with an estimated of 570,000 newly diagnosed cases and over 310,000 deaths in 2018 worldwide, more than three quarter of these are from low-income and middle-income countries (LMICs) [Persistent high-risk human papillomavirus (hr-HPV) infection by certain types is the main cause of cervical cancer and cervical intraepithelial neoplasia . HPV is This Chinese Multi-Center Screening Trial (CHIMUST) (Registration number: ChiCTR-EOC-16008456) is a multi-center, cross-sectional, population-based cervical cancer screening trial conducted between Aug 2016 and Jan 2018 in 5 regions in mainland China: Inner Mongolia (Northern China), Hebei province (Northern China), Hubei province , Jiangxi province  and Guangdong province (Southern China). Women who were non-pregnant, sex exposed, no prior pelvic radiation, no hysterectomy, and no cervical cancer screening in the past 3 years were eligible to be enrolled. The protocol of this trial was approved by the Institutional Review Board (IRB) of Peking University Shenzhen Hospital (IRB: PUSH2016001) and Cleveland Clinic Institutional Review Board (IRB:15–1549). Before CHIMUST, in 2014 our team conducted a Pilot Screening Trial in Shenzhen city using the same protocol. A total of 12,897 women between 30 and 69 years of age were enrolled in these two screening trials, but only 10,867 Han ethnicity women were included in this study. Of these 10,867 Han ethnicity women, 8891 were from CHIMUST while the other 1976 were from Shenzhen Pilot Screening Trial. All participants signed an informed consent document before enrollment.All participants provided a self-collected vaginal sample and a physician-collected endocervical sample. The physician-sampling was performed following self-sampling. The physician placed a vaginal speculum to expose the cervix, then obtained a cervical exfoliated cell sample at the squamocolumnar junction of the cervix. All samples were tested with the PCR-based high-risk HPV assays: Cobas and SeqHPV . The physician-collected samples were also processed and interpreted by PUSH cyto-pathologists using the Hologic I2 Imager (computer assisted cytology). Women testing HPV positive by either Cobas or SeqHPV (self or direct) were referred for colposcopy, using the quadrant-based POI  protocol of directed and random biopsies plus ECC . The impFor this manuscript, we merged the data from the Shenzhen Pilot Screening Trial and CHIMUST, and made the following revisions: a. Shenzhen is listed as a new first-tier city as its population is dominated by immigrants; b. The Mentougou District of Beijing is actually located in the administrative area of Hebei Province, so these two parts of data were merged. Finally, listing 6 screening centers: 1. Inner Mongolia; 2. Hebei province; 3. Hubei province; 4. Jiangxi province; 5. Guangdong province; 6. Shenzhen city. Of these 6 screening centers, Inner Mongolia and Hebei province located in northern mainland China, Hubei province and Jiangxi province located in central mainland China, Guangdong province and Shenzhen city located in southern mainland China. The data referring to HPV genotyping of physician-collected and self-collected samples with SeqHPV and the associated histologic diagnoses among Chinese Han women were reanalyzed for this manuscript . It was developed by BGI Shenzhen, P.R. China. This NGS assay uses index-multiplex-PCR techniques to amplify the HPV L1 gene of cervical exfoliated cells in the sample using a “double index system”. It is a high-throughput technology that can identify 14 h-HPV genotypes (HPV-16、-18、-31、-33、-35、-39、-45、-51、-52、-56、-58、-59、-66 and − 68) . All proWomen testing HPV positive for any HPV sample (self or direct on either assay) were called back for colposcopy. The colposcopy protocol used in this trial was done by quadrant. We used directed biopsies for all visible lesions, and random biopsies at the squamocolumnar junction in normal quadrants. All colposcopy patients had an endocervical curettage (ECC) . Study cSPSS v.24.0 software  was used for all data analysis in this study. Odds ratios and 95% confidence intervals (95%CI) were calculated by univariate logistic regression modelling to assess the risk of CIN2+/CIN3+ associated with hr-HPV genotypes. McNemar’s Chi-square was performed to calculate differences between paired proportions at a probability level of 0.05. Agreement between physician- and self-collected sample was measured by absolute agreement and Kappa statistics (Cohen’s Kappa).A total of 12,897 women from 6 regions were recruited in CHIMUST and its pilot screening trial. Of these, 1754 of non-Han ethnicity, 40 Seq HPV testing failures and 236 Seq HPV positives who did not return for colposcopy were excluded. Therefore a total of 10,867 Chinese Han women with mean age 44.5 ± 7.6 years had complete data are included in this analysis. Among the 10,867 Chinese Han women, 10,508 (96.7%) were pathologically normal, 242 (2.2%) had CIN1, 116 (1.1%) had CIN2+ and 62 (0.6%) had CIN3 + .P < 0.001).A total of 995(9.2%) women were positive for Seq HPV with specific HPV genotypes in physician-collected sample while 1030(9.5%)in self-collected sample. And 912 women tested positive for Seq HPV in both two sampling methods. The concordance rates of HPV positive between physician-collected and self-collected sample was 81.9%(912/1113). The agreement in Seq HPV detection between physician- and self-collected sample was very good, Jiangxi (10.4%), Hebei (8.7%), Hubei (8.6%), Shenzhen (7.1%) and Guangdong (6.3%) in descending order. The type-specific prevalence of hr-HPV by regions is presented in Table 3.0%, JiaThe odds ratios for the various hr-HPV genotypes according to regions is shown in Figs. In May 2018, the Director-General of World Health Organization (WHO), Dr. Tedros Adhanom Ghebreyesus, made a global call to take action to eliminate cervical cancer which is considered one of the greatest threats to women’s lives worldwide . To achiMany studies have shown significant geographic variation in the prevalence of HPV. The estimated HPV prevalence in Sub-Saharan was 24.0%, Latin America and Caribbean 16.1%, Eastern Europe 14.2%, and Southeastern Asia 14.0% according to a comprehensive meta-analysis involving over 1 million women from 5 continents , Shanxi Of the 6 regions we studied, Inner Mongolia showed the highest prevalence (13.0%), similar to that reported by Wang et al. , who comThe lowest prevalence in our study was in Guangdong (6.3%), which was less than a half of reported by Zhao et al. in northeastern Guangdong (15.7%) , but jusIn an investigation in 11 cities of Jiangxi province , the pre2 = 2.816, p > 0.05) while the provincial capital, Wuhan, has a higher prevalence (13.9%) reported by Xiang et al. [Hubei is in the central mainland China similar to Jiangxi province, the estimated prevalence of hr-HPV was 8.6% in our study, showed no significant difference with Jiangxi province almost equal to that in Hubei province. In the northern city of Tianjin, which borders on Hebei, hr-HPV infection was reported as (13.5%) . GeneralAn estimation of population attributable fraction (PAF) of HPV attributable cancer from GLOBOCAN 2012 data showed that HPV-16/18 are responsible globally for 71% of the cervical cancer . By usinAnother meta-analysis on prevalence and attribution of HPV-52 and HPV-58 in cervical neoplasia worldwide indicated that these two genotypes shared a higher prevalence and attribution among cervical intraepithelial neoplasia in Eastern Asia; and the attribution of HPV-58 to invasive cervical cancer was nearly 2-fold higher than that of HPV-52 . In our Our present study has strengths and limitations. This screening trial was a multi-center, cross-sectional, population-based study, that enrolled over 10,000 women from northern, central and southern mainland China. Therefore, it allowed a relatively robust analyses of HPV prevalence and distribution under strictly comparable protocols. The colposcopy protocol used in this trial was directed biopsies for all visible lesions, and random biopsies at the squamocolumnar junction in normal quadrants. All colposcopy patients had an endocervical curettage (ECC). We believe this protocol can improve detection of prevalent precancers . AlthougIn conclusion, geographical variation existed in the prevalence and distribution of hr-HPV in mainland China. HPV-16/52/58 were the most prevalent genotypes. The present study provided guidance for the refinement of cervical cancer screening strategies and vaccine implementation in China.


Randomised controlled trials (RCTs) provide valuable information and inform the development of harm profiles of new treatments. Harms are typically assessed through the collection of adverse events (AEs). Despite AEs being routine outcomes collected in trials, analysis and reporting of AEs in journal articles are continually shown to be suboptimal. One key challenge is the large volume of AEs, which can make evaluation and communication problematic. Prominent practice is to report frequency tables of AEs by arm. Visual displays offer an effective solution to assess and communicate complex information; however, they are rarely used and there is a lack of practical guidance on what and how to visually display complex AE data.In this article, we demonstrate the use of two plots identified to be beneficial for wide use in RCTs, since both can display multiple AEs and are suitable to display point estimates for binary, count, or time-to-event AE data: the volcano and dot plots. We compare and contrast the use of data visualisations against traditional frequency table reporting, using published AE information in two placebo-controlled trials, of remdesivir for COVID-19 and GDNF for Parkinson disease. We introduce statistical programmes for implementation in Stata.Visualisations of AEs in the COVID-19 trial communicated a risk profile for remdesivir which differed from the main message in the published authors’ conclusion. In the Parkinson’s disease trial of GDNF, the visualisation provided immediate communication of harm signals, which had otherwise been contained within lengthy descriptive text and tables. Asymmetry in the volcano plot helped flag extreme events that were less obvious from review of the frequency table and dot plot. The dot plot allowed a more comprehensive representation by means of a more detailed summary.Visualisations can better support investigators to assimilate large volumes of data and enable improved informal between-arm comparisons compared to tables. We endorse increased uptake for use in trial publications. Care in construction of visual displays needs to be taken as there can be potential to overemphasise treatment effects in some circumstances.The online version contains supplementary material available at 10.1186/s13063-020-04903-0. When evaluating a new treatment in a randomised controlled trial (RCT), it is well accepted that potential harms should be monitored alongside any potential benefit. Harms are assessed through the collection of adverse events (AEs) that occur during the trial period. Information on AEs is collected from several sources within an RCT and typically include spontaneous reports from participants and clinical observations . AEs areSome of these AEs will have occurred to the participants regardless of their involvement in the trial and are therefore unrelated to the treatment, and those that are related to treatment are termed adverse reactions (ARs) , 3. Tryip value < 0.05 as a way to identify an AR [How to analyse, summarise, and present large numbers of AEs collected in a trial is a primary challenge. Prominent ‘analysis’ practice in published RCT articles is the use of contingency tables to present frequencies by arm , 6. The fy an AR . Hypothefy an AR , 9.It can be problematic to evaluate (often lengthy) lists of AEs in tables. Visual representation of this information can provide an immediate and informal way to effectively communicate the same information, and facilitate the identification of harm signals. In 2016, a collaboration of industry experts and journal editors undertook a consensus and published recommendations to improve AE reporting in trial publications . The recIn this article, we critically appraise the presentation and description of AEs reported in two recently published RCTs of drug interventions and illustrate how the same information can be presented graphically using two newly develop statistical commands. We contrast the two plots (volcano and dot) with the originally published tables to demonstrate the strength and limitations of each way to display the information.We include two plots that were identified in a recent AE methodology review . They wvolcano plot proposed by Zink et al. in 2013 [x-axis, against the log10-transformed p value from a test of the researcher’s choice on the y-axis. The summary statistic plotted can be the absolute risk difference, or alternatively a relative measure such as the risk ratio, odds ratio, or the incidence rate ratio. This plot can be produced in Stata using the aevolcano [The first is the  in 2013 . The volevolcano  or aevolssc install aevolcanoThe relevant Stata command and associated help files can be freely installed by typing into the Stata command line:https://ideas.repec.org/c/boc/bocode/s458736.html.or it can be downloaded at p value from the Fisher’s exact test or the chi-squared test as requested by the user. The command will incorporate a correction when fitting odds ratios or risk ratios if there are zero events in one of the treatment arms by adding half an event to each arm (numerator and denominator). This is a commonly used continuity correction but has been shown to be inferior when used for meta-analysis models on rare events as it will affect the variance and therefore its weight in the model [p values [For binary AE data, the Stata command calculates the he model . While tp values . The FDRUnder a null hypothesis of no difference, the expected shape of the plot would be symmetrical around the value of no difference for the summary statistic used. The volcano plot can provide a way to immediately identify outlying events for further investigation by means of an asymmetrical plot. Any asymmetry should not be overinterpreted as an ADR as in practice, when there are low numbers of adverse events included or when there are differing incidence rates, it will not be uncommon to obtain an asymmetrical plot. The plot should be used as a means to identify ‘signals’ of ADRs taking into consideration the role that the number of AEs included and the heterogeneity in incidence rates have in this visual impression.dot plot, first proposed by Amit et al. for AE data in 2008 [y-axis) in each treatment arm on the left hand side of the plot and a relative measure, such as the relative risk, with corresponding 95% confidence interval (CI), on the right hand side. An indication of the precision around estimates is particularly important when the event rate is low. We propose an adaption to the original plot proposed by Amit et al. [The second plot is the  in 2008 . The dott et al.  and inclt et al.  dependenssc install aedotThe relevant Stata command and associated files can be installed using the Stata ssc install command, typing into the Stata command line:https://ideas.repec.org/c/boc/bocode/s458735.html.or downloaded at If zero events are experienced in either treatment arm, the Stata command adds half an event to each arm (numerator and denominator) to calculate the relative risk, standard error, and 95% CI. This does not affect the percentages presented or calculation of the risk difference. The calculation of the 95% CI uses the normal approximation.The dot plot is a useful, space-efficient, visual alternative to the traditional two-by-two table AE data is typically displayed in, providing a clear and concise summary of the overall burden of harm with a measure of precision for the relative point estimate.In this article, two recently published high-profile drug intervention trials are used to demonstrate the implementation of these plots , 23. We n ≈ 453 participants) in a 2:1 ratio. The trial was stopped early by the Data Monitoring Committee after recruiting 237 patients . The decision was stated to be due to a higher proportion of participants discontinuing from the study treatment due to AEs (12% versus 5%), alongside consideration of the improved control of the COVID-19 outbreak in Wuhan.Wang et al. undertook a randomised, multicentre, placebo-controlled trial of remdesivir in adults admitted to hospital with severe COVID-19 infections in Wuhan, China . RemdesiIn the study, there was daily monitoring of AEs and screening of AEs using clinical laboratory tests on days 1, 3, 7, and 10; 12-lead electrocardiogram on days 1 and 14; and daily vital signs.Participants experiencing at least one AE were reported in a total of 66% and 64% of remdesivir and placebo participants respectively. The original AE frequency table reported in the publication can be seen in Fig. p value. In the absence of any disproportionality in harm between arms, we would expect to see a symmetrical curve, with a random scatter of AEs (not related to the drug) centred around the null value, in this case the RD of zero. Compared to the table, the strength of the volcano plot is its provision of an overarching and immediate representation of the AE profile that allows the user to draw attention to a particular signal of interest, but it does not allow a detailed review of all AEs.Figure increased aspartate aminotransferase which occurs more frequently in the placebo arm (9 (12%) vs 7 (5%)). The greatest harm signal in the remdesivir arm is for rash (2 (3%) vs 11 (7%)). Rash was not reported by authors in the text, but increased aspartate aminotransferase was one of the most common events in the placebo arm. Not reporting rash in the text for the remdesivir arm demonstrates the subjectivity in describing AEs by arm which may make it a less transparent way to communicate results compared to a visual tool. Often common events are mixed up with potentially important differences indicative of ARs.In Fig. The same data are presented in Fig. increased aspartate aminotransferase in the placebo arm, and the upper 95% CI excludes the value of no difference. The corresponding absolute risks can be read from the left hand side of the graph with the number of AEs by arm included in the right hand side of the plot. This detailed information on absolute values can be especially useful in situations such as when the event rate is low. In contrast to the volcano plot, the dot plot provides information that requires more assimilation but contains more data.In Fig. p value and therefore cannot be uniquely seen. The volcano plot does display a highly asymmetrical pattern and highlights a signal for a higher risk in the placebo arm of cardiopulmonary failure (7 (9%) vs 8 (5%)) and multiple organ dysfunction syndrome (2 (3%) vs 1(1%)). The prominence in the volcano plot of multiple organ dysfunction syndrome is arguably overemphasised in the plot considering the extremely low event rate. This provides an example where the use of the volcano plot alone to draw inference in the absence of other information could provide a misleading impression.The volcano and dot plots for SAEs reported in Fig. The dot plot  patients versus four (5%) patients who stopped placebo early.’ The volcano plot for discontinuations due to AEs in Supp. Figure A% patientsecondary infections (7 (9%) vs 4 (3%)), which is not mentioned in the authors’ summary, and while respiratory failure is notable in the plot, the strength of evidence is less than for serious secondary infections. However, it should be noted that the plot can only display the numerical difference and does not convey whether secondary infections are less of a concern than respiratory failure and therefore a smaller difference in respiratory failure may be of more importance than a greater one in secondary infections. The dot plot  vs 7 (5%)).The volcano plot highlights a signal for harm in the placebo arm for serious Finally, the authors provided a descriptive summary of AEs in the text. The authors selected to list the names of the ‘most common’ events by arm. The definition of ‘most common’ was not provided. They reported the six most common AEs in the remdesivir arm and the seven most common AEs in the placebo arm. The usefulness of highlighting the most common events, listing them by arm, is unclear as they may not be clinically important or numerically imbalanced between arms. In this instance, five of those listed were the same in both arms, and without any further qualification, interpretation is difficult. Picking out the most commonly occurring AEs by arm means the AEs could just relate to the underlying infection or participant comorbidity when they are the same across both arms. If they are different between arms, it could indicate a drug effect.Whone et al. performed a single-centre, randomised, double-blind, placebo-controlled trial of glial cell line-derived neurotrophic factor (GDNF) for patients with Parkinson’s disease delivered using intermittent intraputamenal convection-enhanced delivery via a skull-mounted transcutaneous port . GDNF haAll participants regardless of treatment arm assignment had to undergo robot-assisted surgery for implantation of a skull-mounted port to allow administration of the drug. This necessary surgery to deliver the active drug or placebo added an additional layer of complexity to the reporting of AEs in the trial.Dyskinesia, falls, adverse changes in mood, and impulsivity were pre-specified as AEs of special interest.AEs, routine laboratory tests, and anti-GDNF serum antibodies were used to assess safety. Treatment-emergent adverse events (TEAEs) were defined as any event starting on or after the date of the first dose of study medication, and analysis included participants from both the pilot and primary phase. In the publication, the authors selected to report all treatment-emergent adverse events (TEAEs) experienced by at least three participants in either treatment arm , placebo 2 (10%)), Lhermitte’s sign (GDNF 8 (38%), placebo 0 (0%)), and ON/OFF phenomena (GDNF 7 (33%), placebo 2 (10%)).In the abstract, the authors reported that ‘GDNF appeared to be well tolerated and safe, and no drug-related serious adverse events were reported.’ This message was reiterated in the discussion. On inspection of the AE table in Fig. Lhermitte’s sign is immediately communicated via the volcano plot, and it can be seen that the risk is greater in the GDNF arm compared to placebo. There are also three other events of note: two in the GDNF arm (paraesthesia and ON/OFF phenomena) and impulsive behaviour in the placebo arm. We selected these to be labelled in the plot as removing these events would leave a symmetrical curve. These events are less apparent from a review of the article’s frequency table. The authors did comment on paraesthesia, Lhermitte’s sign, and ON/OFF phenomena within the text, but they did not report on the increased risk of impulsive behaviour due to the rules used to select events to report.Figure Lhermitte’s sign, paraesthesia, and ON/OFF phenomena in the GDNF arm.The same data is presented in Fig. We can see that summarising the same AE data in a graphical display allows for both the full picture and a comprehendible summary, helping communicate this information. The author’s use of ad hoc rule-based thresholds (e.g. ≥ 3), either as a minimum number of events for inclusion in the table or as a between-arm difference to highlight a signal, can result in important AE information being omitted and not communicated.As well as data in the table, the authors included a commendable comprehensive descriptive summary of these in the text. The authors clarified that no participants had a TEAE that led to discontinuation of the study medication. The authors listed out some of the TEAEs where there was a between-arm difference of ≥ 3 participants experiencing an event in the GDNF arm compared to the placebo arm. It was not clear why the threshold of a difference in ≥ 3 participants in the GDNF arm was used, why they selected only some AEs to highlight in the text, and why they did not highlight TEAEs that occurred more frequently in the placebo arm. It is possible authors selected a subset they subjectively assessed to be most relevant that met their original ad hoc rules as a means to provide a comprehendible summary to readers.adverse changes in mood; this may be because no events were reported, but it is not clear. In the publication, text information on serious AEs by treatment arm (GDNF 5 (24%), placebo 0 (0%)) is reported, with authors noting that none of these events was related to study medication.In the publication text, the authors also reported a balance of the overall frequencies for the pre-specified AEs of special interest in each treatment arm (GDNF 13 (62%), placebo 11 (55%)). Event-specific information is included in Fig. port site infections and local hypertrophic scarring, noting that many of these events occurred post-surgery but pre-treatment. These post-surgery, pre-treatment events do not meet the definition of TEAEs, and therefore, a summary of AEs associated to surgery and the device was omitted from the article. This information may have been helpful for real-world application as patients will require that a port is fitted in order to receive the drug.The authors also reported on device-related adverse events being dominated by An additional example is provided in the supplementary material tables Aa and b tThere have been many different graphical displays proposed , 13, 15,We have used data from randomised trials in COVID-19  and ParkContemporary trial statisticians require statistical knowledge across many different domains to undertake the best statistical practice. As the field is advancing in each domain year-on-year, it can be challenging for applied statisticians to learn and programme new methods at the rate of emerging progress. Supporting applied statisticians with accessible software tools can enable them to undertake the best practice and enable early adoption of emerging statistical methods. We have demonstrated and provided accessible software code to produce graphical tools that would otherwise require lengthy complex coding with the aim to accelerate the translation of better AE analysis methods into practice.The volcano plot had the benefit in immediate communication of extreme differences in AE rates through fast and simple evaluation of asymmetry in the plot. It provided a means to assess the ‘profile’ of AEs but without the detail, suggesting the approach as a highly effective way to communicate signals for ARs. Users are able to highlight AEs of concern by adding labels to the required events in the plot. The volcano plot was found to be less effective when there were either few AEs in total, or where several AEs shared the same frequency, which occurs most often when the event rates are low. As a result, the volcano plot will be most useful when there are around 10 events or more and are also not dominated by low frequency counts, e.g. 0 and 1. The advantage of the volcano plot is also what brings its disadvantage, i.e. its effective ability to communicate a signal for an AR does not transparently highlight the precision of the estimate, and we found it was possible to give a misleading impression in some scenarios when the event numbers were small. Researchers need to be cautious when constructing and interpreting a volcano plot and check if they assess the communication to be a fair representation of the raw data and message delivered.The dot plot provided a very different way to communicate results compared to the volcano plot and allows more detailed information to be included. The dot plot required more involved appraisal, but arguably shorter than that required when reviewing a frequency table. The dot plot could be viewed as providing a substitute to an AE frequency table, whereas the use of a volcano plot will likely still require presentation of more detailed information in a table. While the dot plot laudably contains both the absolute risk by arm and relative between-arm comparison, we found the inclusion of 95% CIs for the relative statistic strongly encourages the temptation to interpret the 95% CI as a test of no statistical difference—which in turn may encourage false flagging as well as missed signals.The volcano plot encourages review of the risk profile as a whole, as events are not all labelled, whereas the dot plot allows for more scrutiny by clinical investigators who may want to mentally group similar body system terms in their interpretation, e.g. noting the direction and strength of effect across multiple cardiovascular terms rather than viewing each as individual events.This study was limited to the data selected for inclusion in the original articles and has only demonstrated use in two example trials. Trials of differing sample size, event rates, and AE complexity may highlight further advantages and disadvantages for visual displays.Visual displays of AE data provide an effective way to communicate harm and support different perspectives on interpretation and aid communication. Visualisations can aid investigators to assimilate large volumes of data and also encourage informal between-arm comparisons in contrast to tables. Care in construction of visual displays is needed as they can overemphasise treatment effects in some circumstances. The use of tables, dot plots, and volcano plots can encourage differing interpretations. Volcano plots bring high focus attention to the most extreme events, and the dot plot provides a more detailed summary of all events. We recommend trialists examine both crude numbers along with graphical display to help draw inferences.Additional file 1. Supplementary file


However, no vaccine has yet been licensed for therapeutic use. Several studies have suggested that administration of prophylactic vaccines immediately after surgical treatment of CIN2 cervical lesions can be considered as an adjuvant to prevent reactivation or reinfection, and other studies have described the relevance of prophylactic vaccines in the management of genital warts. This review summarizes the leading features of therapeutic vaccines, which mainly target the early oncoproteins E6 and E7, and prophylactic vaccines, which are based on the L1 capsid protein. Through an analysis of the specific immunogenic properties of these two types of vaccines, we discuss why and how prophylactic vaccines can be effective in the treatment of HPV-related lesions and relapse.Human papillomavirus (HPV) is the most common sexually transmitted virus. The high-risk HPV types  are considered to be the main etiological agents of genital tract cancers, such as cervical, vulvar, vaginal, penile, and anal cancers, and of a subset of head and neck cancers. Three prophylactic HPV vaccines are available that are bivalent , tetravalent , and non-avalent . All of these vaccines are based on recombinant DNA technology, and they are prepared from the purified L1 protein that self-assembles to form the HPV type-specific empty shells . These vaccines are highly immunogenic and induce specific antibodies. Therapeutic vaccines differ from prophylactic vaccines, as they are designed to generate cell-mediated immunity against transformed cells, rather than neutralizing antibodies. Among the HPV proteins, the E6 and E7 oncoproteins are considered almost ideal as targets for immunotherapy of cervical cancer, as they are essential for the onset and evolution of malignancy and are constitutively expressed in both premalignant and invasive lesions. Several strategies have been investigated for HPV therapeutic vaccines designed to enhance CD4 Despite the introduction of prophylactic vaccines, the incidence of human papillomavirus (HPV)-related tumors remains high , particuSeveral studies have suggested that administration of HPV prophylactic vaccines after surgical treatment of high-grade cervical intraepithelial neoplasia (CIN2-3) can be considered as an adjuvant to prevent HPV reactivation or reinfection. The relevance of prophylactic vaccines has also been demonstrated in the management of genital warts, although clinical studies have not delivered univocal results to date.In this review, the HPV-related tumors and the life cycle of HPV are described, to better understand the characteristics of the different viral proteins that are targeted by prophylactic and therapeutic vaccines. Then, we summarize the leading features of prophylactic and therapeutic vaccines that target the L1 and E6, E7 oncoproteins, respectively. Finally, through an analysis of the specific immunogenic properties of these two types of vaccines, we discuss how prophylactic vaccines can be effective for the treatment of HPV-related lesions, and for prevention of HPV-related relapse.,Human papillomavirus is the main agent of sexually transmitted diseases, and it can cause cancer in different anatomical districts with different prevalences . The higThere are 15 high-risk (HR)-HPV genotypes that can lead to cancers of the cervix, anus, penis, vagina, vulva, and oropharynx   represents the precursor of invasive anal cancers, where 65% are cervical squamous cell carcinomas. For both sexes, 88–94% of these cancerous lesions are positive for HPV DNA, with HPV16 as the most commonly detected (~87% of HPV-positive tumors), while only 9% of these anal cancers harbors HR-HPV18Annually, about 18,000 women are diagnosed with anal cancer worldwide, and this cancer is more frequent in women than in men . FurtherIn men, the risk of anal cancer development is strictly related to sexual behavior and HIV immune status .Approximately 50% of penile cancers can be attributed to HPV infections, although HPV also infects healthy subjects without progressing to neoplasia. In a British study carried out among 43 couples, 69% of the men were HPV-positive in the uro-genital tract . AnotherIt is estimated that 40–50% of vulvar cancers are associated with HPV. Overall, vulvar cancers represented 3% of gynecologic cancers in 2002 , and 60%Cancer of the vagina is rare, and it accounts for only 2% of gynecological neoplasia. Nevertheless, from 2000 to 2015 there was an increase in vaginal cancer, which corresponded to 0.4% of vulvar carcinomas in the USAThe most common head and neck cancers are squamous cell carcinomas (HNC) and they include neoplasia of the oral cavity, tongue, tonsils, oropharynx, hypopharynx, and larynx. The HR-HPV types most frequently detected in head and neck cancers are HPV16, followed by HPV18*1301-HLA-DQA1*0103-HLA-DQB1*0603)  . This prresponse .Different annual incidences of respiratory papillomatosis have been reported in different countries. For example, in Denmark, similar incidence has been reported in children (juvenile onset)  and in adults  , while iHPV6 and HPV11 are the main genotypes detected in respiratory papillomatosis. As spontaneous regression is rarely observed, surgical treatment is necessary to prevent progression of the lesions. Moreover, recurrence of papillomatosis is often observed, and retreatment is needed in most cases, which comes at a high economic burden . AlthougHuman papillomavirus infection can not only cause cancer, but also benign genital warts. These are very diffuse in the young and in adults, with prevalence from 4 to 11% –43. TreaCondylomas were classically considered a benign lesion, with the exception of Buscke-Lowenstein tumors. This large tumor can undergo local invasion and can transform into anal cervical squamous cell carcinoma .Human papillomaviruses are non-enveloped icosahedral viruses that consist of 72 capsomers and are 55 nm in diameter. The viral genome is a circular double stranded DNA of ~8,000 bp in length.cis-acting regulatory sequences that are involved in the control of viral replication and post-transcriptional phases  the early region that encodes the E1, E2, E4, E5, E6, E7, E8 viral proteins that have regulatory functions in infected epithelial cells; (ii) the late region that encodes the two viral capsid proteins L1 and L2; and (iii) the long control regions  that contain l phases .Different viral proteins are expressed in different layers of the epithelium. The early proteins are expressed in basal epithelial cells, while the late proteins are expressed in the granular layer that includes more differentiated cells, and from where the virus is assembled and released. E1 is an ATPase helicase that is involved with E2 in viral replication and transcription regulation. The E1 and E2 complex interacts with the long control region ori site, which is considered to be the origin of HPV DNA replication –53. In tE6 and E7 are small proteins of about 150 and 100 amino acids, respectively. The E6 oncoprotein acts through its PDZ-binding motif, which promotes its interactions with PDZ domains in multidomain proteins, to alter their functionality. These PDZ domains are present in many multidomain proteins that regulate key steps in the cellular processes of apoptosis, adhesion, and polarity –43, 58. The main targets of E7 are the pRB, p107, and p130 proteins, which are components of a complex that can repress the E2F transcription factor , 60. WheThe immune response has an important role in clearing most HPV infections, although sometime the virus cannot be eliminated and can persist for several years, which represents a risk factor for neoplasia development . HPV-ass+ T-cell responses that are specific for HPV16 E6, E7, and E2 have been frequently detected in peripheral blood mononuclear cells of healthy individuals   . At vari regress . Also, ll cancer . Taken tThe humoral immune response to HPV infection is mainly directed against conformational epitopes in the variable regions of the major coat protein L1 . This deIgG and IgA are the most abundant isotypes in sera from natural infections. Other HPV antigens  do not commonly induce antibody responses in patients with acute or persistent HPV infections.Studies that have investigated whether naturally acquired HPV antibodies can protect against subsequent HPV infections have reported mixed results –82. MoreThree prophylactic vaccines for prevention of HPV infection are available at present: a bivalent vaccine against HPV16 and HPV18 (Cervarix) that was approved in 2007; a tetravalent vaccine against HPV6, 11, 16, and 18 (Gardasil) that was approved in 2006; and a nonavalent vaccine against HPV6, 11, 18, 31, 33, 45, 52, 58 (Gardasil 9) that was approved in 2016. However, the non-avalent vaccine is the only HPV vaccine that is currently available in the USA, and it was approved for males and females from 9 to 45 years by the US Food and Drug Administration in late 2018.Initially, the administration of the HPV vaccines was in three doses, with the more recent change to a two-dose schedule driven by evaluation of girls aged 9–13 years who had received either two or three doses. The antibody responses of the young women (aged 16–26 years) who had followed a two-dose schedule were similar to those who received all three doses . TherefoAll three of these vaccines use recombinant DNA technology and are prepared from the purified L1 protein, which self-assembles to form HPV type-specific empty shells . Only intact VLPs can generate protective antibodies, which supports the evidence that conformational epitopes of L1 are required to generate neutralizing antibodies .The evidence that HPV VLP vaccines protect against high viral challenges through induction of neutralizing anti-L1 antibodies was obtained in preclinical studies in animals, which thus provided the strong rationale for development of VLP-based vaccines. In particular, in a canine model of experimentally induced oral papillomas, it was demonstrated that dogs vaccinated with the major capsid protein, L1, of canine oral papillomavirus developed antibodies against canine oral papillomavirus and became completely resistant to the viral challenge . SimilarIn humans, analysis of vaccine-induced antibody responses measured by several methods has demonstrated that almost 100% of vaccinated individuals generate a strong type-restricted serum antibody response to L1 VLP. These methods have included conventional enzyme-linked immunosorbent assays, competitive radioimmunoassays, competitive Luminex-based immunoassays, and pseudovirion-based neutralization assays.Initial and follow-up studies that assessed the immunogenicity of the HPV 16/18 AS04-adjuvant vaccine in 15- to 25-year-old women showed that after vaccination, anti-HPV16 and anti-HPV18 total IgG antibodies peaked at month 7, reached a plateau between months 18 and 24, and remained constant for up to 76 months . MeasureComparisons of the immunogenicities of the HPV16/18 and HPV6/11/16/18 vaccines in healthy women aged 18–45 years revealed that 7 months after vaccination, the serum neutralizing antibody responses elicited by the bivalent vaccine were significantly higher than those for the HPV6/11/16/18 vaccine. The differences in these responses for HPV16 and HPV18 were maintained at month 24, and also up to month 60 in women aged 18–45 years.in situ for HR-HPV–naïve women. In particular, the non-avalent vaccine showed the highest efficacy for prevention of onset of CIN1 , CIN2 (97.1%), and CIN3 (100%) neoplasia  caused by the HPV genotypes included in the vaccine, HPV-related lesions remain a public problem worldwide for several reasons: (i) only 8% of low and middle income countries have introduced HPV vaccination programsCurrently, the treatment of high-grade disease (CIN2-3) includes electrosurgical excision of the transformation zone, with carbon dioxide lasers or knives used to perform conization, where the entire transformation zone is removed , 102 Ta. Incomplin-vivo virus-specific T-cell responses against established HPV infections and lesions. For therapeutic vaccination to deliver unequivocal clinical benefits, improvements must be achieved at two levels: by maximizing the induction of T-cell responses with optimal amplitude, specificity and effector profile; and by ensuring that vaccine-induced T-cells can reach the tumor site and perform their functions without restraint , and protein-based, peptide-based or dendritic-cell-based vaccines. Among the bacterial vectors, live attenuated Listeria monocytogenes has been used to generate a promising HPV therapeutic vaccine. L. monocytogenes is considered a potent vaccine vector because it enters professional antigen-presenting cells and induces antigen-presenting cell maturation, and strong innate and adaptive immunity  was demonstrated in a phase I clinical study that was conducted with 15 patients with late-stage metastatic cervical cancer  in a prospective phase II clinical trial as second-line and third line for patients with recurrent metastatic cervical cancer showed that 12-month overall survival was 38%, which exceeded the historical overall survival of such patients, of 25%. A phase III clinical trial of Lm-LLO-E7 for high-grade cervical cancer is being conducted at the time of writing (see NCT02853604).Several strategies have been investigated for HPV therapeutic vaccines designed to enhance CD4immunity . In addibacteria . The safl cancer . Evaluatin-situ production of the antigen(s) as a result of the transfection of antigen-presenting cells and non-antigen-presenting cells, with the presentation of the expressed antigen(s) by both MHC class I and class II molecules. Furthermore, this results in activation of all three arms of the adaptive immune response .Encouraging data have also been obtained in clinical studies that have tested DNA-based vaccines. DNA vaccination consists of direct introduction into tissues of a plasmid that contains the DNA sequence that encodes the antigen(s) against which an immune response is sought. This relies on + and CD8+ T cells as well as humoral immune responses in small animal models, clinical data from multiple studies have demonstrated that they induce poor T-cell responses . This study showed that 78% of the patients developed CD8+ T-cell responses, and 100% showed antibody positivity to at least two vaccine antigens . NotablyPost-hoc immunological analysis here demonstrated that VGX-3100 elicited significantly increased frequency of T-cell responses against HPV16/18 E6 and E7, and that the magnitude of the T-cell response against E6 was associated with clinical outcome. Humoral immune responses were also lower in placebo recipients than in VGX-3100 recipients, and the antibody responses against HPV16, HPV18, and E7 were significantly higher in the patients who had concomitant histopathological regression and viral clearance, compared to those who did not . However, despite this significantly improved HPV viral clearance with this vaccine, it has still not been licensed for clinical use because of the modest efficacy  can be used to express the E2, E6, and E7 oncoproteins, and they can stimulate CD4efficacy .Finally, a vaccine designed on recombinant MVA that contained the bovine papillomavirus E2 protein (MVA E2) was used to treat HPV-induced ano-genital intraepithelial lesions. A phase III study showed that 90% of female patients had complete elimination of lesions after treatment with MVA E2, with 100% seen for men. All of these patients treated with MVA E2 developed antibodies against the MVA E2 vaccine and generated a specific cytotoxic response against the papilloma-transformed cells .Interestingly, novel vaccination strategies aimed to maximize systemic as well as genital resident memory T cell responses to treat sexually transmitted infections and human papilloma virus neoplasia are being developed. In this context several studies have investigated the effect of either the topical delivery of host and pathogen derived immunomodulatory molecules or the delivery route of immunization in the induction of cervicovaginal long lived CD8+ T cell responses .The treatment of premalignant lesions  by LLEP or conization sometimes fails to prevent lesion recurrence –118. ThiA systematic review of studies of the treatment of high-grade lesions (HSIL/ CIN2-3) reported that a median of 28% of the women remained positive for oncogenic HPV types 3 months after treatment. A decrease in this HPV persistence was seen during follow-up, as it fell to 21% after 6 months . Also, hAs no vaccine has yet been licensed for therapeutic use, the prophylactic vaccines have been tested in several trials to determine their effectiveness for prevention of HPV disease recurrence or reinfection after CIN2-3 treatment. The recurrence for MSM who undergo treatment for high-grade anal intraepithelial neoplasia (HGAIN) is particularly high, as 50% show recurrence within 1 year . This maIn 2013, Kang et al. investigated the effectiveness of the tetravalent HPV vaccine to prevent recurrence of CIN2-3 in patients with high-grade CIN treated by the loop electrosurgical excision procedure . RecurreThe data obtained on the efficacy of the tetravalent HPV vaccine for the prevention of anal condylomas are, however, more encouraging . Three hAltogether, these data suggest that there is possibility that prophylactic vaccines reduce the risk of HSIL recurrence in previously infected patients, although the exact protective mechanisms in infected individuals is not understood. The high risk of recurring infections is consistent with either auto-inoculation across anatomic sites or new inoculation or episodic reactivation of latent infection. As HPV vaccines prevent infection by induction of L1-specific antibodies that block viral entry, and L1 is not generally expressed during the oncogenic process, it is expected that these vaccines will be effective in the prevention of auto-inoculation or new infections.The greater effectiveness obtained with prophylactic vaccines in the prevention and regression of genital warts might be related to the integration state of the HPV genome. In genital warts, the virus is not generally integrated into the host genome, and therefore viral particles are produced. In this case, the prophylactic vaccines that block viral entry through induction of L1-specific antibodies can prevent reinfections, which will favor the elimination of the virus. Conversely, in high-grade lesions, the virus genome is often integrated into the host genome, and infected cells do not express L1 and do not produce viral particles. Thus, as transformed cells are frequently in the basal layer of the derma, they will not be recognized by vaccine-induced antibodies, which are ineffective in the control of the disease course.Furthermore, there are some cases in the treatment of HPV-related cancers where the use of prophylactic vaccines might not be recommended:(1) Anal and cervical cancers that are not attributable to the HPV types that are included in the non-avalent HPV vaccine. Several studies have demonstrated that half of the HPV infections in MSM are caused by HPV types that are not included in the non-avalent HPV vaccine , 133 Ta. Here, o(2) HPV DNA-negative cervical tumors. Over recent decades, several studies have reported that some cervical cancers are HPV-negative –139. OftThe data reported in this review highlight the significant efforts that have been carried out to set-up therapeutic vaccines against HPV-related malignancies. Although several approaches to produce an effective vaccine have been attempted, including the use of proteins, synthetic peptides, and viral proteins expressed in different vectors, and although some of the data appear encouraging, no therapeutic vaccines have been licensed in clinical practice yet. Recently, prophylactic vaccines have been used for treatment of recurrent forms or reinfections in subjects who have previously undergone surgical resection. However, the trials here have offered conflicting results, and vaccination did not guarantee 100% effectiveness. This is probably due to a residual burden of transformed cells that can persist after the surgical treatment, and that are not targeted by the humoral L1-specific immune response induced by the prophylactic vaccines. Although it cannot be excluded that the therapeutic potential of prophylactic vaccines could be improved by using different adjuvants or route of immunization, an additional limit in using prophylactic vaccines for therapeutic purposes is seen by the evidence that the non-avalent vaccine does not include all of the HR-HPV types. As the real extent of protection given by the non-avalent vaccine against other HPV types is not known, its use in the treatment of tumors related to these other HR-HPV types is questionable. Furthermore, for endometrial adenocarcinomas, such as gastric-type adenocarcinomas, which are HPV DNA-negative, careful virological and histological diagnosis must be made before administration of HPV prophylactic vaccines to treat HPV recurrence or reinfection.AG and PD wrote the original version and edited versions. MC edited versions. DL and CS prepared figures and tables.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.


Human papillomavirus (HPV)-45 genotype circulates in high percentage in Bandung area - Indonesia, after HPV-16 and HPV-18. The aim of this study was to analyse variations of major capsid (L1) HPV-45 and its phylogeny. Furthermore in silico protein structure and epitope prediction was explored. L1 gene of HPV-45 was amplified, sequenced and aligned. Phylogenetic tree had been built and compared with a complete L1 HPV-45 sequence. Structure and epitope prediction of L1 protein were then developed in silico. Of 5 L1 HPV-45 sequences collected, we have detected one variant of sub lineage A2 which was considered as a new variant, and two variants of B2. Superimposition of structure of these two variants with reference showed very similar structure. Furthermore, seven amino acid substitutions were found within these L1 variants of which two substitutions might change the polarity of corresponding amino acid I329T and S383G. The S383G occurred in surface loop (HI-Loop) of new L1 HPV-45 variant. Similar structure of Indonesian variants indicates that amino acids variations do not affect the L1 structure. However, one substitution with altered amino acid polarity found within the area of surface loop suggests a potential impact in antibody recognition and neutralization.  Human papillomavirus (HPV) is a small, circular, double-stranded DNA virus with almost 8 kb genome size. Despite its small genome, the virus has diverse genetic classification with more than 170 HPV types have been identified . Of these, twelve HPV types which comprises of HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59 are classified as high-risk to cause cancer . Persistent infection with any of these high-risk HPV type is correlated with cervical cancer development . The classification of HPVs is based on open reading frame (ORF) of major capsid protein (L1), which is the most conserved gene within the genome. The difference within this gene with its closest isolate by more than 10% is defined as HPV genotype, between 2%-10% as HPV subtype and if the difference is less than 2% is defined as HPV variant . Although HPV variants show highly similar sequence, the biological behaviours may distinct, for example, the difference in viral infectivity, persistence, antibody recognition and progression of cervical dysplasia to cervical cancer . The L1 protein is the major structural protein which encapsidate the HPV genome, and has a major role in infectious entry to the host. The protein form 72 pentameric capsomers and can self-assembled to construct an icosahedral capsid. The icosahedral structure has made L1 protein displayed evenly on the surface of the capsid, therefore, this L1 protein is highly immunogenic . The surface loop of the L1 protein may contain the sites of sequence variation among HPV types and also the location of dominant surface epitopes .HPV-45 is one of the high-risk HPVs that contribute for about 5% of cervical cancer cases worldwide . Compare to other HPV types, HPV-45 together with the infection of HPV-16 and HPV-18 are suggested to have a relatively more carcinogenic potential than other high-risk HPV types . To date, it has been known that HPV-45 is classified into two major variants lineage A and B, and within this major lineage, the virus is grouped further into five variants of sub lineage A1, A2, A3, B1, and B2 . Some studies suggest that different HPV-45 variant may have been linked to different risk for cervical cancer development as well as protection from currently available HPV vaccine . Unlike HPV-16 and HPV-18, the geographic distribution of HPV-45 variant lineage varies around the world. Information about HPV-45 variant distribution may have beneficial impact for cervical cancer prevention strategies. HPV-45 is the third most prevalent HPV found in cervical cancer after HPV-16 and HPV-18 in Bandung, West Java province, Indonesia . To our knowledge, studies describing on genetic variation and lineage of HPV-45 from Indonesia are limited. The purpose of this study was to analyse nucleotide variations of complete HPV-45 major capsid protein (L1) from Indonesia and its phylogeny. Furthermore, in silico protein structure and epitope prediction was explored.Sample CollectionDNA analysed in this study were obtained from cervical cancer tissue, and further underwent HPV Genotyping Test . Only samples with confirmed HPV-45 were included in this study. The study protocol was approved by Ethical Committee of Faculty of Medicine, Universitas Padjadjaran no. 289/UN6.C2.1.2/KEPK/PN/2013. PCR amplification and sequencingAmplification of L1 HPV-45 ORF (±1600 bp) was performed using sense primer 5’-GTGGCCTAGTACATCTCCTA-3’ and antisense primer 5’-TCATACATACACGCACGC-3’. 10mMol of each sense and antisense primer were added into the PCR mixture containing of 12.5 µl PCR mix , 20ng template DNA, and PCR grade water added for a total reaction volume of 25 µl. The cycling condition was as followed: initial denaturation at 94°C for 3 minutes; amplification phase for 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds; followed by 1 cycle of 72°C for 10 minutes . PCR products were confirmed by 1% agarose electrophoresis and visualized under UV light . All confirmed PCR products were sent for purification and sequencing, of which samples were sequenced in both direction sense and antisense . Sequence analysis and phylogenetic tree constructionThe L1 HPV-45 sequences were edited to exclude PCR primer binding sites and corrected manually (BioEdit 7.2), and the obtained sequences were further aligned (MEGA 7.0.) . Nucleotide variations of L1 HPV-45 from this this study were then compared to reference sequence published in GenBank (accession number X74479.1). Any nucleotide change in L1 HPV-45 sequence was explored for its implication. To construct phylogenetic tree, additional open reading frame (ORF) of L1 HPV-45 sequence was downloaded (http://www.ncbi.nlm.nih.gov/), and the referenced source list sequences used in this study was provided in Suppl. 1. The phylogenetic tree was further constructed (MEGA version 7.0) , using maximum likelihood method and fit-substitution model. Tamura three-parameter and the non-uniformity of evolutionary rates among sites were modelled by a discrete Gamma distribution (T92+G). A 1,000 bootstrap replicates were performed to assess the confidence of branching pattern of the tree. Homology ModellingThe template structure for homology modelling was determined by using BLAST-x (blast.ncbi.nlm.nih.gov). The best template structures were then downloaded from Protein Data Bank (RCSB.org). The nucleotide of target sequences were translated to amino acid sequences by using ExPASy Translate Tool (web.expasy.org/translate/). The target and template sequences were aligned using Biovia Discovery Studio, by using structure oriented alignment . The alignment sequences were used as the input file of the Modeller 9.19 software . Twenty homology models were generated from each target sequences, and ranked by their DOPE score. The best models were further refined and energy minimized by steepest descent method using Modeller 9.19. Furthermore, the refined models were assessed by Rampage Ramachandran plot analysis  and DOPE graph profile . Epitope PredictionsStructure-based epitope prediction had been made using Ellipro (http://tools.immuneepitope.org/tools/ElliPro) . The predicted epitopes were mapped on the pentamer protein structure and visualized by using Biovia Discovery Studio .L1 HPV-45 Sequence VariationsIn total, 5 cervical cancer tissues infected by HPV-45 in single or multiple infection were included. The histopathological diagnosis and the distribution of HPV infection among isolates was shown in Phylogenetic analysisThe HPV 45 L1 from Indonesian isolates were distributed into two groups in phylogenetic tree  that corHomology ModellingThree crystal structures were found at the top ranking templates in terms of percentage identity and query cover. Crystal structure of L1 papillomavirus type 18 (PDB ID 2R5I) was selected as the best template structure with 82% identity and 84% query cover. Crystal structure of L1 papillomavirus type 59 (PDB ID 5J6R) showed 80% identity and 88% query cover, and crystal structure of L1 papillomavirus type 16 (PDB ID 1DZL) showed only 67% identity and 89% query cover. Twenty homology models from each sequence had been constructed. Protein model with the best DOPE score was further refined and optimized. Stereochemical quality of the models in term of Phi-Psi plots (Ramachandran Plot) showed that 99.1% residues fall in the favorable and allowed region and the rest of 0.9 % residues in the disallowed region. The structure of all three variation sequences of HPV-45 L1 were modelled. In general, these structures were geometrically similar, as shown in Structural Epitope Prediction and Amino Acid MutationEpitope prediction produced ten putative linear B-cell epitopes, which comprise of 9 - 36 amino acid residues (Suppl. 2). There were four variable epitopes, in which two of them were located on the surface area . Seven aHPV 45 variant lineagesOur recent study has shown that there were three nucleotide variations obtained of which one of these variations (sample BDG-163) is considered as a new L1 variant, since there is no HPV-45 L1 sequence that 100% identical with this isolate. The closest isolate with HPV-45 L1 sequence is Qv34178 from Costa Rica (GenBank accession number: EF202159.1) with 99.9% similarity. According to the HPV classification, a difference less than 2% within ORF of L1 with its closest isolate can be classified as HPV variant , however, other study has recommended to classify HPV variants based on its complete genome. With the definition of HPV variant, it should share >90% nucleotide identities, while intratype variant lineage should differ by 1-10% and intratype sublineage variant should differ by 0.5-1.0% .Despite the discrepancy in classifying variant of HPV type, this minute finding in HPV-45 L1 nucleotide variation from Indonesia may indicate another variation in different region of the genome, since variation of nucleotide within a region in HPV genome may describe nucleotide variation in another region within its genome. HPVs are known to evolve through nucleotide changes, thus, nucleotide change in one region is highly correlate and attached to another changes in different regions within genomes from the same lineages . Moreover, from the phylogenetic tree, it appears that isolate BDG-163 has formed its own branch . FurtherBased on previous studies on HPV 16 and 18, it is suggested that the variant of HPV-16 and HPV-18 are correlated with geographic region from which they are isolated . However, the correlation between specific variant lineage and geographical region for other HPV genotype, including HPV-45, remains unanswered . It is well known that the variation within HPV genotype will have difference in viral persistence and cervical cancer development, for example, non-European HPV-18 variants has higher risk to develop cervical cancer,  and similarly non-European HPV-16 variants also correlate with higher risk in progression to cervical cancer . The variants of HPV genotype other than HPV-16 and HPV-18 may also contribute to various risks in cancer development, including HPV-45 as this genotype is the most closely related with HPV-18 . Hence, the geographical distribution of HPV-45 variants is diverse . Currently, the taxonomy of HPV-45 has been determined according to study by Chen et al., which classify HPV 45 into two main lineages A and B, and five sublineages A1, A2, A3, B1 and B2 . From this study, we found that four out of five HPV-45 from Indonesian isolates were grouped in a sublineage B2, which are known to be associated with higher risk of cervical cancer . Although in this study we used data of L1 HPV-45 sequence only from cervical cancer isolates , and canHomology modelling of HPV-45 L1 and epitopes predictionThe L1 protein is a major structural protein which forms a HPV capsid. On the surface of the capsid, there are 5 loops designated as BC, DE, EF, FG, and HI loop . These surface exposed loops are known to be important for neutralizing antibody recognition,  and sequence variation within the loops may affect efficiency of antibody neutralization . In this study, we have observed that the amino acid variation of HPV-45 L1 from Indonesian isolates are not showing major different in protein structure . SeveralIn conclusion, the HPV-45 from Indonesian isolates in this study are grouped into sub-lineage A2 and B2, with most of the isolates are clustered in B2 sub-lineage, which known to correlate with higher risk in developing cancer. Mutation in HI loop of new HPV-45 L1 variant results in an altered amino acid polarity found within the area of surface loop, suggesting a potential impact in antibody recognition and neutralization. Further study with more HPV-45 samples and exploration using molecular dynamics simulation are needed to reinforce these findings.


Mild local hyperthermia at 44°C has been proven efficacious in the treatment of cutaneous warts induced by human papillomavirus (HPV), while its effect on cervical intraepithelial neoplasia (CIN) caused by high risk type of HPVs has not been reported.are reported in this study.Three patients with low grade CIN and positive high risk HPV types  The diagnosis was based on identification of HPV types and abnormal cytological findings.The 3 patients were treated with local hyperthermia from ceramic heating  to cervix. The treatment was delivered once a day for 3 consecutive days, plus two similar treatments 10 ± 3 days later, with each session lasting 30 minutes. HPV and cytology test were performed 3 months thereafter.All the 3 patients recovered to normal cytological findings. Two of the patients were negative for HPV, the remaining patient with pre-treatment HPV 56 and 58 positivity changed to HPV58 positive alone.This pilot observation inspires that mild local hyperthermia be recommended as a new method in the treatment of CIN patients with persistent HPV infection, once validated by qualified RCT. Currently, preventative vaccines, a bivalent, a quadrivalent and a 9-valent vaccine are available to protect against HPV infection, however there still remains quite a number of high-risk HPV types without vaccines. Previously infected women by high-risk HPVs could not benefit from the available vaccine and their chance to develop malignant transformation is unlikely to change. High-risk HPV infection remains a major therapeutic challenge nowadays.Cervical cancer is the fourth most commonly diagnosed female cancer worldwide, accounting for 7.5% of all female cancer deaths, especially in the less developed or developing regions, where more than 87% cancer deaths were recorded. Our previous study demonstrated that hyperthermia at 44°C attained about 50% cure rate in the treatment of benign skin neoplasia caused by low risk HPVs, while this non-destructive method has not yet been applied in conditions by high risk HPVs. Herein, we presented 3 cases with cervical low grade squamous intraepithelial lesion (LSIL) with presence of high-risk HPVs, who responded favourably to local hyperthermia.Artificial elevated local temperature (exogeneous hyperthermia) has been used to treat infectious diseases and neoplasms, with varied efficacy.2Three patients underwent a routine uterine cervical cancer screening with no other discomfort, and cytosmear indicated the existence of high-risk HPV and atypical squamous epithelial cells, but colposcopic examination showed no obvious abnormity. They were referred for treatment in our institution. Informed consent was obtained for publication of the case. This study was approved by the Ethical Committee of China Medical University ([2017]2016-207-2).2.1We used a patented local hyperthermia device , which was approved by the Ethics Committee of China Medical University. The heat was generated from an energy source of ceramic heating element, which was delivered through a metal disk (2–3 cm in diameter) to the whole uterine cervix with direct contact, irrespective of the lesion location  test was negative and the cytology examination was normal.2.1.2A 49-year-old Chinese woman was diagnosed with LSIL 1 year and a half ago. Her cervical cervical cytosmear was positive for HPV31 and 56, tested by flow-through hybridization method. The patient received a combination of topical application of recombinant interferon (IFN) а-2b and subcutaneous injection of 1.6 mg thymalfasin, twice a week for 1 year, when she recovered with normal cytology and negative HPV test. Unfortunately, she was reinfected by HPV51 and 58, and was diagnosed with LSIL by cervical cytosmear examination 1 month ago. She also had a 4-year history of hepatic adipose infiltration. 3-month after the treatment, HPV (Scraping) test and the cytology examination displayed normal.2.1.3A 33-year-old married woman was diagnosed cervical intraepithelial neoplasia at grade 1 by pathological examination for a year. At the time, her cervical cytosmear was positive for HPV52, 58 and 16, and cyto-immunohistochemical staining presented positive p16 and the percentage of Ki-67 positive cells was greater than 20%. The patient was treated with topical recombinant IFNа-2b accompanied with injection of 20 mg thymopentin every other day for half a year. She was identified as HPV52 and 58 positive with a cervical cytological result of LSIL 1 month ago.Three months after completion of hyperthermia treatment, she remained HPV58 positive and the cytology examination revealed normal Table .All patients did not experience any adverse effects during and after the treatment.3 Eradiation of HPV in the early state of the disease is pivotal in prevention of the progression of cervical neoplasia. In our cases, the patients were completely or partly resolved of high-risk HPV and reversed intraepithelial neoplasia by local hyperthermia treatment. Hyperthermia treatment has shown its effectiveness in HPV infected skin lesions via several mechanisms.–6 Depending on our experience, we adopted the same temperature, the same therapeutic course and the same parameters in the eradication of HPV, irrespective of HPV types. HPV may inhibit the activation of cytotoxic T cells, which is critical for eradicating HPV-infected cells. Hyperthermia may promote the release of cytokines and expression of cell surface molecules in dendritic cells, resulting in stimulation of antigen-specific cytotoxic T lymphocytes. Furthermore, hyperthermia may elevate phagocytosis and antigen uptake by macrophages and dendritic cells, promote the migratory maturation of Langerhans cells in HPV-infected skin.,9 Regression of HPV-infected lesions after hyperthermia treatment displayed dense infiltration of CD4+ and CD8+ T lymphocytes. In addition, hyperthermia increased the percentage of apoptotic keratinocytes infected with HPV and cervical cancer cells; induced mutation of HPV DNA sequence and inhibited the virus or virus replication; reduced the copy number of HPV16 E6 in cervical cancer tissue; decreased viral load and viral integration degree.–13 It is widely known that the majority of females will harbor HPV at some time in their lives, but in about 2 thirds of women the infection becomes persistent, and the more prolonged the HPV infection, the greater risk of the progression of cervical carcinogenesis.,15Early clearance of HPV relieved the heavy mental burden of the patients. Currently, immunotherapy and other target therapies against HPV infection is emerging. Our cases support the feasibility and acceptability of the use of hyperthermia on the cervix infected with HPV.With the changing of sexual culture, the incidence rate of uterine cervical neoplasia has been increasing among young women.There were no serious adverse effects associated with hyperthermia such as heavy vaginal bleeding or burning during treatment and follow-up. Despite incomplete understanding of the mechanism, we consider that the present study may prove beneficial to patients and may be another option to clear HPV when patients fail to respond to other therapies or do not apply other therapies. A randomized controlled trial is required to consolidate the efficacy of thermotherapy on early stage cervical neoplasias with positive high risk HPV.Data curation: Ruiqun Qi, Xiaodong LiMethodology: Wei Huo, Ruiqun QiSupervision: Xin Wu, Hongduo ChenValidation: Hongduo Chen, Xinghua GaoWriting- original draft: Yang Yang, Lan ZhangWriting- review & editing: XingHua Gao