The high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30–50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue.We collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC.The multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 − 15 %- versus 6 out of 60 − 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 − 10 %- versus 4 out of 60 − 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity.Our findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %.Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance. Penile carcinoma (PC) is a rare and aggressive disease with an incidence of 0.4 % in the United States and Western Europe, while the incidence is higher in less developed countries, including Africa, Asia and South America, estimated around 6.0 % –3. The mThe PC development can be attributed to two different pathogenic pathways, the chronic inflammation of the lichen sclerosus being the most frequent ; then the infection by the high-risk human papilloma virus (HPV) is relatively less common , 4–6. InThe new World Health Organization (WHO) attributes a great importance to HPV-related cancer, revising the previous classification of PCs based exclusively on the morphology. Thus, PCs are subtyped the into HPV-related and HPV-unrelated tumors , 14.The main HPV-related subtypes are frequently basaloid and warty carcinomas, while the conventional squamous cell carcinoma, the papillary and the verrucous carcinomas are mainly non-HPV-related , 13, 15.The HPV can be detected through direct and indirect different assays, including the polymerase chain reaction (PCR), p16 immunohistochemistry (IHC) and in situ hybridization (ISH) analysis. PCR assay detects HPV DNA identifying both transient and persistent infections. This method is a highly sensitive and it allows the identification of the different genotypes of the virus . ISH anaIHC represents a surrogate method to detect HPV infectious, since it is based on the identification of the p16 expression, being considered as a marker for high-risk HPV-induced transformation. Indeed, since p16 is accumulated as an effect of HPV E7 oncoprotein, its overexpression seems to be a critical early event in carcinogenesis . Thus, iA series of 60 penile carcinomas were included in our study. All cases were collected in our records at the University of Campania “L. Vanvitelli” Hospital, the Istituto Nazionale Tumori, Fondazione G. Pascale, the S.M. delle Grazie Hospital and the University Ribeirão Preto Hospital of São Paulo,. The series included surgical samples and wide biopsies, formalin-fixed paraffin-embedded (FFPE) samples. Sections of 4 μm thickness from each block with a mean of 3 blocks per tumor) were stained with hematoxylin-eosin. All cases were reviewed according to the WHO histopathological classification , 14.p16 IHC was carried out with a proprietary kit CIN tec Histology; MTM laboratories AG) using the clone E6H4 on a Ventana Benchmark autostainer  for the detection of p16INK4a antigen. A cervical carcinoma with high p16 expression was used as a positive control. The primary antibody was omitted from negative controls.In our analysis we identified the subgroups with different p16 IHC staining, as follows: p16 high expression: tumors with staining ≥ 70 % nuclear and cytoplasmic staining; p16 moderate expression: tumors with staining 30–70 % nuclear and cytoplasmic staining; p16 low expression: tumors with staining 10–30 % nuclear and cytoplasmic staining; p16 negative: tumors with staining 1–10 % nuclear and cytoplasmic staining. In addition, also intensity was evaluated as previously reported . The sliSection 4 sitivity .We performed the multiplex HPV RNA ISH /p16 IHC assay according to the protocol previous reported .The protocol utilizes the Diaminobenzidine (DAB) chromogen of the Bond Polymer Refine kit to staining HPV E6/E7 mRNA, the Fast Red chromogen of the Bond Polymer Red Refine kit to staining p16 and hematoxylin to counterstain. Detection of high-risk-HPV E6/E7 mRNA was performed using ready-to-use reagents from RNAscope® 2.5 LS Reagent Kit-BROWN and the HPV-HR18 probe cocktail (Advanced Cell Diagnostics) that were loaded onto the Leica Biosystems’ BOND RX Research Advanced Staining System according to the user manual . The target-specific probes include the E6 and E7 mRNA of 18 h-HPV genotypes HPV . The Ubiquitin C a constitutively expressed endogenous gene was used as positive control to assess the presence adequate RNA quality and avoid a false-negative result. The dapB test was used as negative control to assess non-specific staining, for a comparison in the cases with negative or weakly stained HPV staining.In brief, 4 μm sections were baked and deparaffinized on the instrument, followed by epitope retrieval using Leica Epitope Retrieval Buffer 2 at 95 °C or at 88 °C for 15 min and protease treatment 15 min at 40 °C. Probe hybridization, signal amplification trough different AMP reagent AMP 1–6) and colorimetric detection were subsequently performed. Several washes were performed, subsequently the ready-to-use primary antibody clone E6H4 for the detection of p16INK4a antigen was incubated and colorimetric detection was performed. Finally, a hematoxylin staining was carried out.When the run is completed and the slide trays are removed, the covertiles are carefully lifted upward by the neck to remove. The slides are dehydrated through 2 changes each of 70 %, 95 %, and 100 % alcohol and 2 changes of xylene, before coverslipping. A positive HPV ISH test result was defined as positive if any of the malignant cells showed brown punctate dot-like nuclear and/or cytoplasmatic positivity. p16 IHC was positive if nuclear and cytoplasmic red staining was observed according to the above score. The slides were independently analyzed by three separate observers evaluating simultaneously HPV mRNA and p16 expression.In our series, patients older than 60 years were 28; the tumor site was the glans in 54 cases, the foreskin in 3 and the shaft in 3. Usual squamous cell carcinoma (USCC) was observed in 49, whilest a special histotype in 11 . In USCC histotype, the low grade (G1) was recorded in 15 cases, the moderate grade (G2) in 22, the high grade (G3) in 13. The grade was not applicable in 10 cases since they were special types and in situ carcinomas. Finally, the tumors were staged T1 in 19 cases, T2 in 17, T3 in 11 and T4 in 3, two cases were in situ carcinoma and the stage was not available in 8 cases. The data were reported in Table P16 overexpression was recorded in 28 cases, 19 in USCC; 9 in special histotypes, p16 overexpression was equally distributed in older and younger patients. Particularly, p16 low intensity expression was observed in 5 cases (8.3 %), moderate in 3 cases (5 %) and high intensity in 20 cases (33.3 %).HPV-ISH was observed in 15 cases, 9 in USCC; 6 in special histotypes, HPV-ISH positivity was more frequent in older patients, being observed in 9 cases older than 60 yrs.The data of multiplex approach were fully concordant with single approach methods, also considering the cut-off values and the intensity of p16 overexpression. Notably, all HPV-ISH positive cases overexpress p16 Figs. ; while pThus, when considering only the cut-off without the intensity, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering cut-off combined to high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 % Table .Table 3The role of HPV is well documented in the pathogenesis of several human cancer types, including cervical, oropharyngeal, penile, vaginal, vulvar and anal cancers . Indeed,Although the frequency of the HPV in PC is lower than in cervical cancer, the HPV-related PCs constitute a subgroup with distinct biology and generally associated with a better prognosis and good response to the treatment than HPV-unrelated cancer. The new WHO classification has well identified a subset of PCs whose etiopathogenesis is closely associated with HPV infection. The accurate identification of the HPV-related PCs is necessary for the appropriate classification and management of these tumors.p < 0.000001) was reported between HR-HPV and p16 showing entire and continuous cytoplasmic or nuclear staining in all neoplastic cells, except in the hyperkeratotic or parakeratotic areas when present [Previous findings in PC demonstrated the overall correspondence of p16 IHC overexpression and the HPV presence; a strong association ( present . Althoug present .Martins and colleagues showed that up to 50 % of PC cases positive to HPV molecular assay were negative for p16 IHC expression; in addition, 21 out of 22 cases positive for p16 IHC were confirmed for the presence of HPV . SimilarCDNK2A, the gene encoding for p16, due to the loss of heterozygosity or the promoter hypermethylation [The discordance between HPV PCR assay and p16 expression could be explained by biological reasons, including a non-oncogenic or transient HPV infection, an inactivation of hylation , 24.Beyond biological issues, the p16 IHC positivity in cases defined as HPV negative through PCR methods could be attributed to a detection failure associated with the technical limits, such as the DNA degradation .Although the PCR assay currently represents the gold standard for the HPV detection and genotyping, this method is mainly indicated in fresh-frozen samples while it has a limited yield in FFPE , 31.The RNA ISH is an innovative tool to detect the HPV E6/E7 mRNA transcripts on FFPE samples, providing information not only related to the presence of the virus but also the transcriptionally-active status . The RNAvice versa [In OPSCC, the combination of various assays to detect HPV improves the accuracy compared to the use of a single method, thus diagnostic algorithm including p16 IHC and molecular approaches is used in the clinical practice . The disce versa , 35.Our data showed 13 discordant cases between HPV RNA ISH and p16 IHC results, considering only the cut-off without the intensity of p16 expression. However, when considering the cut-off combined to the expression intensity, there were, the 5 discordant cases. Particularly, when considering only the cut off of 70 % without the intensity of p16 expression, the concordance was recorded in 78.3 % of cases, p16 being more frequently positive, with a sensitivity of 100 %, a specificity of 71 %, a PPV of 54 % and a NPV of 100 %. Furthermore, considering a combined score including the cut off of 70 % and the high intensity of p16 IHC expression, the concordance was recorded in 91.7 % of cases, with a sensitivity of 100 %, a specificity of 89 %, a PPV of 75 % and a NPV of 100 %. Thus, p16 overexpression could be un-related to HPV oncogenic role in PC in at least 8,3 % of cases, also considering that such cases did not show HPV related morphology.In this perspective, as in other district, the multimodal approach based on p16 IHC and HPV detection by molecular assay in PCs leads to a better identification of HPV-related subgroup, reducing the rate of both false-negative and false-positive cases , 24, 30.Multiplex assay in the clinical practice could help to define the HPV-related PC subgroup avoiding the misclassification of these tumors. Our data confirm a high NPV of p16 intense overexpression, but a lower PPV, 8,3 % being the cases overexpressing p16 in absence of HPV-RNA. Thus, as p16 is a parameter potentially, but not exclusively, associated to HPV oncogenic activity, the multiplex approach let a simultaneous study of the HPV presence and its activity, differentiating the HPV-related from HPV-unrelated PCs. In conclusion, the multiplex HPV RNA ISH /p16 IHC assay could solve the diagnosis of HPV, especially in cases with discordant results. This approach has several advantages including the identification of the transcriptionally-active HPV and p16 on the same slide using a single technical approach, high performance in FFPE specimens, the complete not operator-dependent automation and the interpretation in the light field. Overall, the advantages of this new tool may allow wide spreadable for HPV routine testing in PC. Finally, our multiplex assay could help in the clinical practice the definition of HPV-related PC subset avoiding the misclassification of these tumors.


This study aimed to evaluate the possible role of human papillomavirus (HPV) and Epstein–Barr virus (EBV) coinfection as an etiological factor for prostate cancer (PCa) development.This case-control study was conducted on 67 patients with PCa and 40 control subjects. The expression levels of cellular and viral factors involved in inflammation, tumor progression, and metastasis were quantified, using the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) assay.n = 10), respectively. No significant relationship was observed between PCa and HPV/EBV coinfection . However, the highest percentage of HPV genome integration was found in the HPV/EBV-coinfected PCa group . Also, the mean expression levels of inflammatory factors , anti-apoptotic mediators (Bcl-2 and survivin), and anti-anoikis factors (Twist and N-cadherin) were significantly higher in the HPV/EBV-coinfected PCa group, compared to the non-coinfected PCa cases. Nevertheless, the tumor-suppressor proteins (p53 and pRb) and E-cadherin (inhibitor of anoikis resistance) showed significant downregulations in the HPV/EBV-coinfected PCa group, compared to the non-coinfected PCa cases.The EBV/HPV coinfection was reported in 14.9% of patients in the case group and 7.5% of the control subjects. The high-risk types of HPV, that is, HPV 16 and HPV 18, were responsible for 50 and 30% of HPV/EBV-coinfected PCa cases (The HPV/EBV coinfection may be an etiological factor for PCa through modulation of cellular behaviors.The online version contains supplementary material available at 10.1186/s12885-021-08658-y. Prostate cancer (PCa) is the second most common male cancer and the fifth leading cause of mortality worldwide . HoweverOf approximately 1400 human pathogens, 220 are related to viral agents. A limited number of these viral agents, which are associated with cancer in various ways, are called oncogenic viruses. A recent theory suggests that virus-induced mechanisms can alter the biology and aggression of many cancers. Also, tumor behaviors alter under the influence of virus-induced mechanisms in cancer and stromal cells in the tumor environment. Some of these changes include the inhibition of cancer cell apoptosis pathways, changes in tumor metabolism, inhibition of anti-tumor immune system, provoked inflammation of the tumor environment, onset of angiogenesis, induction of tumor cell proliferation, increased tumor invasion, and increased metastasis . FollowiApproximately 12% of all cancers are caused by viral infections worldwide, with HPV and EBV accounting for 38% of virus-associated cancers . Both EBIt has been shown that coinfections increase the host’s susceptibility to cancers by affecting the immune responses. They mostly act as cofactors in cancer development and affect cancer behaviors in response to treatment . Howevern = 67) were obtained considering the inclusion and exclusion criteria during the study period in Tehran, Iran. Moreover, healthy tissues (n = 40) dissected from the peripheral area of adenoma removed by surgical procedure were collected as control (in terms of age). At least 24 h prior to surgery, serum samples were prepared from blood by venipuncture and stored at − 80 °C. Additionally, the tumor stage was detected based on the TNM system in accordance with the consultation of a team of experts in cancer including a cancer surgeon, a radiologist and an oncologist. The ethical considerations were in accordance with the Helsinki Declaration, and both verbal and written informed consents were achieved from all research units. Snap-frozen tissue samples in liquid nitrogen were stored at − 80 °C. The clinical profiles of patients included tumor stage, tumor type and age , Tehran, Iran, under the Ethics code of IR.IUMS.REC.1398.642. As well as, the current study was supported by grant no, 15671 from the research deputy of Iran University of Medical Sciences (IUMS).Total DNA was extracted from the tissue samples by the QIAamp DNA Mini Kit based on the kit protocol from the frozen samples of PCa. The quality of extracted DNA was analyzed using a 268-bp fragment amplification of the b-globin gene with the aid of HotStarTaq DNA polymerase  in the presence of G074 (5′-CAACTTCATCCACGTTCACC- 3′) and G073 (5′-GAAGAGCCAAGGACAGGTAC- 3′) primers. The cycling program was 95 ֯C for 9 min, and then 35 cycles at 95C for 30 s, 55 ֯C for 30 s, 72 ֯C for 1 min, and a final extension at 72 ֯C for 10 min .The DNA extracted from 67 and 40 fresh frozen prostate cancer and control samples, respectively were analyzed by PCR for identification of HPV-L1 gene (MY11: 5′-GCMCAGGGWCATAAYAATGG-3′ and MY09: 5′-CGTCCMARRGGAWACTGATC-3′) and for detection of HPV E6/E7 gene (pU-H: 5′- GAGCTGTCGCTTAATTGCTC-3′ and pU-R: 5′-TGCTAATTCGGTGCTACCTG-3′) .For EBV detection nested-PCR method was used by primers that used in Breda et al. study , includiIn EBV positive samples, the EBV typing was performed by the EBNA2 primers . The sizThe samples positive for HPV were genotyped using INNO-LiPA HPV Genotyping v2 test  based on the kit protocol.The expression of cellular gene (SLUG), HPV genes  and EBV genes  was quantified by real-time PCR in the HPV-EBV-positive and HPV-positive PCa samples, respectively. To this end, the extraction of total RNA (1 μg) was performed by QuantiNova Reverse Transcription Kit , followed by constructing the cDNA in a thermal cycler as 27 °C for 10 min, 38 °C for 15 min, 44 °C for 40 min, and 72 °C for 15 min. .The expression level of LMP-1 in EBV-infected PCa tissue was measured by qRT-PCR technique by the probes and primers according to Kubota et al. .Quantitative real-time PCR was carried out in EBV-positive tissues for LMP-2A by QuantiNova SYBR Green PCR kit  which has been described in Busson et al. .The EBER1 and EBER2 levels were measured in EBV-positive PCa tissue using qRT-PCR according to Shannon-Lowe et al. .QuantiNova Reverse Transcription® Kit, one step RT-PCR® kits  and Quantitative SYBR green TaqMan Universal PCR Master Mix®  were respectively applied to recognize the viral E7, E6 and E2 genes. The serial dilutions of E7, E6 and E2 genes cloned in PUC57 vector  were utilized for viral genes, which contained equivalent volumes of these genes from 72 to 865 million copies/reaction, as control.The viral genes of E2 (forward primer: 5′-CTACGAATTCATGGAGACTCTTTGCCAACG-3′ and reverse primer: 5′-GATAGAATTCTCATATAGACATAAATCCAG-3′), E6 (forward primer: 5′-GCAATGTTTCAGGACCCACA-3′ and reverse primer: 5′-ACAGCATATGGATTCCCATCTC-3′) and E7 , were respectively detcted using one step RT-PCR® kits , QuantiNova Reverse Transcription® Kit and Quantitative SYBR green TaqMan Universal PCR Master Mix®  . The serThe SLUG gene was amplified by the qRT-PCR in the presence of specific primers. The real time PCR device  was utilized exploiting the Power SYBR Green PCR Master Mix . The normalization of the relative expression level for the gene was carried out by a GAPDH as housekeeping gene. The sequences of F and R primers to amplify the SLUG gene were as follows: the forward primer sequence was as: 5′-GCCTCCAAAAAGCCAAACTACA-3′, the reverse primer sequence was as: 5′-GAGGATCTCTGGTTGTGGTATGACA-3′ .(−ΔΔCt) exploiting the online data analysis tool of QIAGEN . All reactions were repeated three times, and the internal control was considered to be GAPDH for the normalization of gene expression level  for various specimens.In addition, the expression level was measured by the equation of 2The qPCR technique was used to compute the E2/E6 ratio for the characterization of the HPV-DNA physical status  in the specimens infected with the HPV. The E2/E6 = 0 was defined as a fully integrated status and the E2/E6 ≥ 1 as an episomal status, and the E2/E6 = 0–1 as a mixed status .The taken whole blood was poured into the SST™ serum separation tubes. Each sample had five complete inversions. The blood was left at room temperature for 15–30 min in order to clot. No longer than 2 h after collecting, the separator gel tubes had centrifugation, followed by discarding the clot for 15 min at 1500×g. The serum samples were distributed aseptically at 50-μl volumes and kept − 80 °C for subsequent testing.The serum levels of IL-17, IL-11, IL-8, IL-6 and IL-1 were measured by the related ELISA kit  based on the suggested protocol. Moreover, the serum levels of VEGF, TGF-β and TNF-α were measured by Quantikine Assay Kit (R&D Systems), Human TGF-β Quantikine ELISA® Kit (Minneapolis) and Human TNF-α PicoKine ELISA Kit (Boster), respectively, based on the kit protocols.The survivin (anti-apoptotic mediator) expression level was measured by Survivin Human SimpleStep ELISA® Kit (Abcam) based on the manufacturers’ protocol.In brief, all prostae tissue samples were grounded individually in the liquid nitrogen, and then lysed by BioPlex lysis buffer  inside the microcentrifuge tubes. The tissue lysate was homogenized in a Dounce homogenizer, followed by centrifugation with 13,000 rpm for 10 min at a temperature of 4 °C to obtain a clear supernatant containing the lysate of prostate tissue.The Bcl-2 level was quantified in all tissue lysate samples by the Human Bcl-2 ELISA Kit (Abcam) based on the related kit protocol.The expressin level of anoikis-related proteins including PTPN13, TWIST, N-cadherin and E-cadherin in prostate tissue lysates were measured using Human Tyrosine-Protein Phosphatase Non-Receptor Type 13 (PTPN13) ELISA Kit , TWIST ELISA Kit  and Human E-Cadherin, N-Cadherin ELISA Kit  respectively, based on the related kit protocols.The level of ROS and RNS were determined in tissue lysate samples by the OxiSelect™ Intracellular ROS/RNS Assay Kit , based on the kit protocol.Quantification of NF-kB, Rb and p53 was performed in the tissue lysates by NF-kB p65 Transcription Factor Assay® Kit, Human Retinoblastoma ELISA® Kit  and Abcam’s p53 Simple Step ELISA® Kit , respectively, based on the kit protocols.P < 0.05, adjusted by Bonferroni’s correction test for ANCOVA analysis of data. Benjamini-Hochberg method was applied to correct the false discovery rate for multiple comparisons. Heat maps were plotted by one matrix CIMminer using Ward cluster algorithm and Euclidean distance approachs.Statistical analysis was performed by GraphPad Prism 6 and STATA versions 11.2 software. Kolmogorov–Smirnov test was used to assesse the normality of data distribution. Data were reported as mean ± standard deviation (SD) of parametric variables, and the median and the interquartile range for nonparametric data. In the comparison of central tendency parameters, including the mean for the normal variables and the median for the non-normal variables, two-independent samples t-test or Mann-Whitney non-parametric tests were applied between two groups, as well as one-way ANOVA or kruskal-wallis tests were applied between more than two groups. Fisher’s exact test or Chi-square was utilized to evaluate the associations between the categorical data. Spearman’s rank correlation was measured for obtaining the extent of coexpression between the cellular factors and the viral genes. The statistically sinificance level was considered to be n = 107) are shown in Table P = 0.19). The observed types of PCa tissues included acinar adenocarcinoma , ductal adenocarcinoma  and squamous cell cancer, SCC . In the current study, the lowest and highest cancer stages were respectively IIIA (2.98%) and IVA (17.9%). EBV-DNA was found among 49.3% of PCa samples, HPV DNA in 31.3%, while in control group in 40 and 15%, respectively. Among 67 PCa tissue specimens, HPV mono-infection was detected in 11 (16.4%), EBV mono-infection was detected in 23 (34.3%), and HPV/EBV co-infection was detected in 10 (14.9%). As well as, among 40 control tissue specimens, HPV mono-infection was detected in 3 (7.5%), EBV mono-infection was detected in 17 (42.5%), and HPV/EBV co-infection was detected in 3 (7.5%). There were not significant association between the presence of EBV, HPV and HPV/EBV co-infection with PCa . HPV 16 and EBV 2 were the highest genotypes isolated in both case group  and control group . The most frequent HPV genotypes in HPV/EBV co-infected positive groups were HPV-16 , followed by HPV-18 , HPV-33 , and HPV-6  . No statistically association was reportd between EBV/HPV co-infection and histopathological types of tumor . No significant difference was seen in the frequency distributions of PCa stages between the EBV-positive PCa and the EBV-negative PCa, as well as between the HPV-positive PCa and the HPV-negative PCa (P = 0.17 and 0.54).The demographic profiles of the study participants n = 07 are shP: 0.0009 and 0.002, respectively). It is noteworthy that all integrated form of HPV DNA was found in the HPV/EBV co-infection samples. The HPV 16 DNA integrated type was observed in 5 of 20 (25%) HPV-positive PCa, in 5 of 10 (50%) HPV/EBV-co-infected positive PCa and in 1 of 1(100%) HPV/EBV co-infected positive control groups. The HPV-18 DNA was integrated in 3 of 21 (14.2%) HPV-positive PCa, and in 3 of 10 (30%) HPV/EBV-co-infected positive PCa groups  and IVB  samples, respectively , RNS  and IL-6  in the PCa group positive for HPV compared with the PCa group negative for HPV. The correlation results in Table P < 0.001). The strongest positive correlation was between ROS and E6/E7 , and then between Survivin and E6/E7 .We examined the expression levels of different inflammatory mediators between the PCa samples with the control samples and between EBV/HPV-positive samples with EBV/HPV-negative samples. The result is shown in Tables P = 0.001). Table . Since some of the samples studied were both infected with the EBV and infected with the HPV (HPV/EBV coinfection), the question may now be whether the increased expression of inflammatory factors was the result of the effect of EBV-infection or the presence of HPV-infection and/or effect of HPV/EBV-co-infection? Therefore, as can be seen in Table P > 0.05). Therefore, it can be concluded that an elevated expression level of RNS, ROS, TGF-β, VEGF, TNF-α, IL-17 and IL-8 probably more due to the presence of HPV infection than to EBV infection and also an elevated expression level of IL-11, IL-6, IL-1 and NF-κB factors may be due to the simultaneous presence of HPV and EBV infections. In addition, the level of all inflammatory factors except IL-8 in HPV/EBV co-inffected PCa group was significantly higher than in non-HPV and non-EBV PCa group  and Survivin  (Table P > 0.05). As shown in Table P < 0.001), Bcl-2 , TWIST  and N-cad . Besides, there was a significantly negative correlation between E7 and E6 level and Rb , p53 , E-cad .Following the identification of differences in the expression levels of inflammatory-related mediators between studies groups, we examined the association between the concentrations of antiapoptotic factors (survivin and Bcl-2), tumor suppressor factors p53 and Rb), anoikis-related mediators  and CD44 (as a cell adhesion glycoprotein) in the samples infected with HPV, the samples infected with EBV, the samples coinfected with HPV/EBV, and control groups. In comparison with the control group, the p53 and Rb expression levels were significantly lower and also Bcl-2, Survivin and CD44 levels were higher significantly in the PCa group. Furthermore, among anoikis-related factors, only N-cad and PTPN13 levels significantly were higher and lower, respectively, in the PCa group when comparing with the control group , and also between E7 and E6 levels with the TWIST  and with the N-cad . More information is given in Table According to the results of Table In compared HPV/EBV coinfected PCa group with coinfection negative PCa, mono EBV-positive PCa and non-HPV/non-EBV PCa groups, the E-cad level and the TWIST and N-cad levels had significantly decreased and increased expression, respectively Table . HoweverThe present study aimed to find differences among EBV-positive samples, HPV-positive samples, HPV/EBV-coinfected samples, and control samples concerning the expression levels of EBV and HPV genes, HPV integration physical status, and expression levels of some inflammatory, tumor-suppressor, antiapoptotic, and anoikis-related mediators, including IL-17, IL-11, IL-6, IL-1, IL-8, TNF-α, NF-κB, VEGF, TGF-β, ROS, RNS, Rb, p53, survivin, Bcl-2, CD44, Twist, E-cadherin, N-cadherin, PTPN13, and Slug.Evidence shows that viral infections account for about 12% of all cancers globally , 12. HPVn = 19) [So far, the EBV infection has been reported in prostate tissues. In a previous study, the presence of EBV was reported in 8.8% (31 out of 352) of malignant and benign prostate tissue samples in Sweden , and in n = 19) . Besidesn = 19) . The EBVn = 19) . It seemn = 19) , 44, whiP = 0.0009).Some studies have shown that EBV infection in the cervix accelerates the integration of HPV genomes into the cervical cell genomes , 45, 46.P = 0.35, P = 0.06, and P = 0.34, respectively).Since no study has yet investigated the effect of HPV/EBV coinfection on PCa development, we compared our findings with the results reported for other cancers. The EBV probably acts as a co-factor for HPV to induce the uterine cervix pathology, as shown by Szkaradkiewicz et al.  who repoA previous study showed that EBV LMP1, in combination with HPV16 E6 proteins in transformed mouse embryonic fibroblasts, caused a decrease in the residues of DNA damage response, including p27, pRb, and p53, and led to an increase in the level of checkpoint kinase 1 (Chk1) and Akt, MAPK, and NF-κB signaling . SimilarIn the current study, there was no significant difference between EBV-positive and EBV-negative samples. However, a significant inverse correlation was found between the expression levels of HPV E7 and E6 mRNA and the expression levels of p53 and Rb  and the expression levels of Bcl2/p53 proteins  suggestiIn a study by Castilla et al., PTPN13 gene silencing elevated the expression of invasion-related genes in PCa cells . Also, HIt has been reported that LMP1 downregulates the E-cadherin expression and upregulates Twist and other transcription factors associated with cell motility , 67. An The carcinogenic mechanisms of the HPV and the EBV viruses can be different –81, for One of the limitations of this study is that we had to sample a peripheral area of surgically dissected benign prostatic hyperplasia due to lack of access to normal and healthy prostate samples.n = 10), respectively. The maximum percentage of HPV genome integration was found in the HPV/EBV-coinfected PCa group . Although there was no statistically significant association between the HPV/EBV co-infection and PCa, the expression profile of some cellular factors involved in inflammation, tumor progression, and metastasis were different between the HPV/EBV-coinfected PCa group with both mono-EBV infection and mono-HPV infection. These differences suggest that the co-presence of these viruses alters the expression patterns of cellular factors, compared to mono-infections  were responsible for 50 and 30% of PCa samples coinfected with HPV/EBV (ons Fig. , suggestAdditional file 1: Fig. S1. PCR amplification of the RBV EBER-2 gene. Fig. S2. PCR amplification of EBV genotypes. Fig. S3. Nitrocellulose Strips After Staining.


Background: HPV vaccination of both girls and boys can protect against infection and eliminate the risk for HPV-associated cancer. Due to a common misconception that the virus only poses risks to women, vaccine coverage is suboptimal among men in many countries. It is urgent to identify barriers to vaccination of boys and men. Methods: We conducted a narrative review of publications examining attitudes and beliefs regarding HPV vaccination for boys and young men. The electronic databases searched were PubMed, PsychInfo and Scopus . A total of 103 original articles were included in the final analysis. Results: The central barriers against vaccination of boys and men are: (1) lack of knowledge, (2) vaccine hesitancy in general, (3) lack of recommendation from and/or discussions with healthcare providers, (4) cost and logistics, and (5) the idea that HPV vaccination may promote promiscuity. Men who have sex with men and families belonging to ethnic minorities express a need for information tailored to their situation. Conclusions: Boys should be included in national immunization programs and men should also be offered catch-up vaccinations. Future studies should focus on addressing vaccine hesitancy and developing interventions to promote pan-gender HPV vaccination. Human papillomavirus (HPV) infection is a substantial contributor to the global cancer burden. The virus is transmitted through sexual contact and infection is extremely common both in women and men; the estimated lifetime probability of being infected is above 80% . High-riVaccine hesitancy in society is a growing challenge and was in 2019 listed by the World Health Organization (WHO) as one of the top 10 global public health threats. The stated WHO goal is to achieve a worldwide HPV vaccine coverage of ≥90%. However, misconceptions and fake news can disrupt national vaccination programs ,5,6.National HPV vaccination programs are implemented in at least 107 countries worldwide . Pan-genThe aim of this review is to provide a concise update on the latest progress made about HPV vaccination in boys and men, and the barriers against vaccine coverage of this half of humanity.This narrative review was performed in line with Onwuegbuzie and Frels (2016), and Polit and Beck (2016) ,12. A coBased on the material found, and our personal expertise, we conducted a narrative review ,12 to prBoys have, in general, lower knowledge about HPV compared with girls .There is a broad variation in boys’ opinions about HPV vaccination, and whether they intend to be vaccinated or not. Only 29% were willing to be vaccinated among boys in the USA and 37% in France, while a Swedish study found that boys seem to be in favor of being vaccinated ,15,16.According to a Dutch study, fear of unknown side effects is a cause behind the boys’ vaccine hesitancy . UnsurprSchoolboys believe that the school health service is an adequate arena for information about HPV and HPV vaccination. Information from healthcare providers increases the chances that adolescents in low-income families will become vaccinated; adolescents who recalled that their provider had given them written information were more likely to complete the vaccine series . Boys alThe parental perspective mirrors that of the boys themselves: knowledge is poor ,22,23,24The intention among parents to have their sons vaccinated varies considerably between countries and sociodemographic groups. Parents in Brazil seem to be in favor with an acceptance of 86% , whereasAs was the case with the boys themselves, increased parental knowledge predicts acceptance of the vaccine ,34. SeveFinally, perceived support from others in the decision is a facilitator for vaccine completion , and recMen are much less HPV vaccinated than women ,46,47. AAs with boys and parents, there are great differences between countries/regions, as an example: the willingness to vaccinate is high in California but low in Hong Kong ,56.Concerns that vaccination would lead to promiscuous behavior as well as the perception that one will only have one partner, i.e., not be sexually active until marriage, are other reasons for unwillingness to vaccinate against HPV . ArguablAs usual, knowledge—especially knowledge regarding men’s risk to be affected by HPV—increases vaccine acceptance ,60. FurtObviously, HCPs play a key role in the success of HPV vaccination programs, since the attitudes and confidence of these professionals affect vaccine acceptance ,62,63,64HCPs face challenges such as parental vaccine hesitancy and lack of time to provide adequate information to parents and children ,66,70. TThere is a broad variation in knowledge and awareness about HPV and HPV vaccination among MSM. An Irish study on young MSM found that only half (54.9%) of them were aware of HPV , and simDespite low awareness among many men ,82, MSM In spite of these favorable beliefs and attitudes, only a minority have discussed the vaccine with HCPs . MSM alsAs in the other groups, MSM consider the cost a barrier against vaccination ,89, but There are differences in attitudes and beliefs about HPV vaccination for boys and young men between countries and cultural groups. The acceptability of vaccinating young boys against HPV is high (83%) in Brazil  while itCultural norms and religious and ethnic customs definitively play a role as determinants of vaccine acceptance .Several barriers are found among Latinos in the USA who have low knowledge about HPV, have low confidence in vaccine efficacy, and perceive low vaccine benefits compared to other groups ,24,92. FAlthough minorities are often vaccinated to a lower extent, there are exceptions. For instance, in the Arabic community in Israel, boys and young men have high vaccine uptake ,94. AlsoImportantly, minority groups request targeted information about HPV and HPV vaccination adjusted to their cultural and ethnic group .Finally, religion has an impact on beliefs and knowledge about HPV vaccination, and the decision-making process to vaccinate or not ,96. One As should be clear from the above sections, low HPV and HPV vaccine knowledge is a major barrier against vaccination. In particular, the link between HPV and oral and anal cancer is not sufficiently well known ,87. KnowThe perception that HPV vaccination is associated with increased risk of promiscuity is widespread among both parents, sons, and youth in general ,98. ThisThis assumption is not supported by science. HPV-vaccinated Swedish girls in the catch-up program had less sexual risk-taking compared with girls who were not vaccinated . Sexual Fear of side-effects is a common barrier against parental acceptance of HPV vaccination ,101. We Obviously, to have to pay for the vaccine is an essential barrier  especialAn important and related factor is whether the vaccine is included in nationwide school-based vaccination programs, or if it is up to the parents or the young man himself to take the initiative . In the As has been highlighted from several perspectives in this review, knowledge is crucial. The more one knows about HPV and the HPV vaccine, the greater the likelihood is that one will become vaccinated or have one’s sons vaccinated . But howMass media can have a positive effect and act as a vector for vaccine awareness ,108 but Healthcare, and especially HCPs, play an undisputed role in informing about HPV and addressing misconceptions and unfounded fear ,111,112.Only a handful of researchers have addressed the question of how to best disseminate knowledge and influence attitudes among adolescents of all sexes. There have been a few prospective school-based information interventions ,114,115.all boys and before sexual debut. As should be clear from this review we are still a long way from reaching this goal.Vaccination against HPV may lead to the eradication not only of cervical cancer in women but also of cancers in the head and neck region, anal cancers and genital tumors in both men and women. But to achieve this goal vaccination needs to be given to boys as well, to (1)HPV vaccination is only relevant for girls and women;(2)HPV vaccination leads to increased promiscuity or sexual risk-taking;(3)HPV vaccination is unsafe.Vaccine coverage of boys, not just girls, has been included in nationwide school-based vaccination programs across the globe, but uptake is patchy, knowledge is deficient and misconceptions linger. Central misconceptions that lead to diminished vaccine acceptancy are the following:It is not surprising that parents of boys are more unsure about HPV vaccination compared to parents of daughters . For a lThe nature of HPV transmission poses a challenge to parents that other infections do not: they have to discuss sexual health behavior with their sons. The parental opinion that HPV vaccination should be delayed since (they believe) their child is not sexually active  may partVaccine hesitancy is one of the greatest health challenges facing the world today . This isThe role of HCPs in the success of national HPV immunization cannot be overstated ,119,120.School-based HPV vaccination programs are linked to high vaccine coverage and contribute to sociodemographic equality ,125,126.Besides, school-based vaccination is an opportunity for HCPs to inform children about the virus and discuss sexual health in general, before the children have become sexually active. After all, children have the right to be informed about actions that can promote their future health. This is also in line with the Convention on the Rights of the Child .So what can be done? Boys and young men seem to be in favor of being vaccinated against HPV ,87,120. This narrative review provides a concise update on the latest progress made about HPV vaccination in boys and men, and the barriers against them becoming vaccinated. Although the included studies provide a broad spectrum of the stakeholders’ perspective—i.e., boys and their parents, men, MSM and HCPs—and were undertaken in different countries and sociodemographic areas, it was impossible to include all relevant studies. We still believe that the crucial points have been made.Much needs to be done if full HPV vaccination of all boys and young men is to be reached. The main barrier is a lack of knowledge and misconceptions regarding the vaccine’s safety and effectiveness. However, most parents, boys and young men will accept vaccination if they are given reliable information, and also have the opportunity to discuss HPV with healthcare providers. The goal can be reached.


Objective: According to the 2019 American Society for Colposcopy and Cervical Pathology (ASCCP) recommendations, women with a positive high-risk human papillomavirus (HR-HPV) diagnosis and low-grade cervical intraepithelial lesion (LSIL) cytology result should be referred for further colposcopy examination. However, this strategy results in over-treatment in several cases. In this study, we assessed the performance of extended HR-HPV genotyping in women with a simultaneous positive HR-HPV and LSIL diagnosis with the aim of improving the current triage strategy.Methods: This study was an observational analysis of women from the Fujian Province Cervical Lesion Screening Cohorts (FCLSCs). Women who were HR-HPV-positive and had a cytological examination of LSIL, which were followed up with colposcopy and biopsy, from 2015 to 2018 were included. The study endpoint was defined as the detection of histological cervical intraepithelial neoplasia grade 2 or worse (CIN2+). We combined HR-HPV genotypes according to the prevalence rate in histological CIN2+ and ranked them from high to low to establish HR-HPV genotyping models. Outcomes were assessed with respect to sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and colposcopy referral rate.Results: Overall, 56,788 women undergoing preliminary screening for HR-HPV genotyping were included in this study. Among them, 10,499 women positive for HR-HPV underwent a cytology examination, and 902 women with LSIL cytology diagnosed and subsequent biopsy results were included in the final evaluation. Among these patients, 25.1% (226/902) were found to have CIN2+ in histology. HPV-16, -58, -52, -18, -33, and -31 infections were the most common genotypes, and HPV-16, -18, -58, -33, and -31  were associated with the potential for histological CIN2+, from the highest to lowest. In the detection of CIN2+ lesions in HR-HPV-positive LSIL women of different HR-HPV genotyping models, the extended HPV 16/18/31/33/52/58 genotyping model was found to have better efficacy with higher sensitivity (92.9%) and NPV (93.0%), but a significantly lower colposcopy referral rate (74.7%) than the ASCCP-recommended HR-HPV non-genotyping model.Conclusion: For HR-HPV-positive women with LSIL, the HPV 16/18/31/33/52/58 genotyping model can serve as an alternative approach to the ASCCP recommendations, potentially reducing the unnecessary colposcopy referral burden in China. Cervical cancer is the fourth most frequent cause of death in women Since the publication of the 2015 American Society for Colposcopy and Cervical Pathology (ASCCP) interim guidance, HPV testing has been used as the primary screening strategy for cervical cancer in situ (AIS), or cancer (CIN2+) with less resource waste are needed in HPV-positive patients with LSIL. However, most of the related articles published to date have mainly investigated HPV-positive patients with LSIL using the cytology as a primary screening method but not the HPV testing Low-grade squamous intraepithelial lesion (LSIL), as defined by the Bethesda system, is the second most common cervical abnormality in cytological results Extensive evidence indicates that the persistent infection of HPV promotes the progression of cervical cancer The purpose of this retrospective study was to assess the potential of extended HR-HPV genotyping in identifying underlying histological CIN2+ in women with a positive HR-HPV and LSIL finding. Furthermore, we aimed to assess the efficacy of different HR-HPV genotyping models to optimize the 2019 ASCCP risk-based management consensus guidelines for these patients to achieve a reduction in the unnecessary use of colposcopy resources.The study population was selected from the Fujian Province Cervical Lesion Screening Cohorts (FCLSCs) with more than 200,000 cases, including a provincial-level hospital, nine municipal-level hospitals, and more than 500 community health service centers Prior to registration, each woman provided informed consent. An experienced doctor conducted a confidential interview with a questionnaire to collect basic information, including the patient's history of medication, cervix-related diseases and treatments, other malignancies, education background, smoking and drinking habits, and reproduction history. All eligible individuals underwent gynecological examinations. Exfoliated cervical cells from the ecto- or endo-cervical canals were obtained with a cytobrush. For HPV genotyping, specimens were stored using a preservation solution in 2 ml vials at -20 °C. For cytology, samples were stored using ThinPrep® PreservCyt®  in 20 ml vials at 4 °C. Samples were subsequently transferred to the laboratory and cytological site where HR-HPV genotyping and cytology were conducted.Polymerase chain reaction-reverse dot blot (PCR-RDB) was used for the analysis of HR-HPV, including 14 HR-HPV genotypes , in cervical exfoliated cells . All detection procedures were conducted according to the manufacturer's instructions An auto-image system  was used for cervical cytological examinations. All slides prepared for the cytological examinations were analyzed independently by two experienced cytopathologists and were diagnosed according to the Bethesda system HR-HPV-infected participants who were also diagnosed with LSIL were subsequently referred for colposcopy and/or biopsy within 10 weeks. Colposcopy results that were deemed normal had no requirement for a biopsy. In contrast, participants with abnormal colposcopy results received an immediate biopsy of visible lesions. The results were interpreted in accordance with the CIN system The referral rate was determined by dividing the number of HR-HPV-infected LSIL patients by the overall number of LSIL patients. The mean and standard deviation (SD) of the classified variables were assessed. The values and percentages were calculated. The study endpoint was defined as the detection of histological CIN2+ A total of 56,788 women with HR-HPV genotyping results who also underwent cervical cytology were recruited for this study. Among these, 10,499 (18.5%) women with positive HR-HPV genotyping results underwent cytology examination, and 356 (3.4%) HR-HPV-positive women without cytology results were excluded. Among the remaining 10,143 (17.9%) HR-HPV-positive women with cytology results, 1,201 (2.1%) women were HR-HPV-positive with both HR-HPV genotyping and LSIL cytology results. After exclusions, 1,036 (1.8%) women were eligible for the study. 134 (12.9%) of whom without colposcopy and/or biopsy results were lost to follow-up and excluded, and the remaining 902 women (87.1%) with colposcopy biopsies were included for final evaluation. The baseline features of the participants are described in Table Among all HR-HPV-positive participants with LSIL, the most prevalent HR-HPV genotype was HPV-52, accounting for 24.9% (225/902), followed by HPV-16 , HPV-58 , HPV-51 , and HPV-56  Figure .Figure Table The sensitivities and NPVs were higher in HR-HPV genotyping models that combined more HR-HPV genotypes, with a decrease in the specificity and PPV. The sensitivity and NPV rates for the extended HPV 16/18/31/33/52/58 genotyping model were 92.9% and 93.0%, respectively, and represented the highest values observed for the detection of underlying CIN2+ pathology, followed by the extended HPV 16/18/33/52/58, HPV 16/18/52/58, HPV 16/18/58, and HPV 16/18 genotyping models. In addition, the colposcopy referral rate of women with the extended HPV 16/18/31/33/52/58 genotyping model was only 74.7%  for histological CIN2+.Women with positive HPV results and LSIL face a significant risk of developing cervical precancerous lesions A potential limitation of this study included the small number of participants with a CIN3+ biopsy. Using histological CIN3+ as the main study outcome can result in an unstable and imprecise sensitivity result. Therefore, our analysis was restricted to CIN2+ as the study endpoint. A second limitation is the enrollment of participants from a single region. In addition, the study lacked large-scale follow-up, which will be conducted in future work.In conclusion, with a high sensitivity and NPV, but lower referral rates, the extended HPV 16/18/31/33/52/58 genotyping model can provide an alternative to current triaging management of HR-HPV-positive women with LSIL, based on ASCCP guidelines, resulting in a significant reduction in unnecessary referrals for subsequent testing. Future work should focus on the evaluation and validation of this model in large-scale follow-up and different populations.Supplementary tables.Click here for additional data file.


Lactobacillus (L.), the dominant bacteria in maintaining vaginal microecological balance, was decreased gradually. In contrast, the abundance of Actinobacteria in the four disease groups was significantly higher than that in normal group. Furthermore L. iners may be related to the serious progression of cervical cancer. After analyzing the whole process, we found that Gardnerella(G.), Atopobium(A.) and Dialister(D.) have important effects on both persistent HR-HPV infection and the pathogenesis of cervical cancer. In addition, PICRUSt2 and KEGG results showed that the KEGG pathways enriched by the predicted genes of vaginal microbiota in cancer group included metabolic diseases, endocrine system and immune systems when compared with that in normal group. These findings may provide insights into the pathogenesis of cervical cancer, and help to improve the early detection and prevention of cervical precancerous lesions.The process from high-risk human papillomavirus (HR-HPV) infection to cervical cancer is a continuous and long-term process, but the pathogenesis of the whole process is not completely clear. Here, 59 Chinese women were engaged in this study, and divided into five groups: normal healthy group, HR-HPV infections group, low-grade intraepithelial neoplasia (LSIL) group, high-SIL(HSIL) group, and cervical cancer group. With the occurrence of HR-HPV infection and the development of cervical lesions, the diversity of vaginal microbiota species was increased, and the relative abundance of  Lactobacillus (L.), which is known to produce lactic acid to maintain low vaginal pH (< 4.5), and this is the first line of defense against pathogenic agents  than others. And it is associated with microbiological disorders, and increases the risk of cervical and other female genital tract diseases  Subjects having immunosuppressive diseases; 2) Subjects using antibiotic within the last 7 days; 3) Subjects currently having a sexually transmitted infection or vaginal infection; 4) Subjects using suppositories, vaginal applied medications and douching substances within 48 hours prior to the visit; 5) Subjects having had sexual intercourse less than 48 hours prior to the visit; 6) Subjects having a previous history of cervical treatment.The total of 59 subjects were divided into five groups according to the HPV infection status and Histology of colposcopy-directed biopsy samples, including normal controls , HR-HPV infection (HPV), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and cervical cancer (cancer). HPV status was determined by the Linear Array HPV Genotyping Tests , and divided into two groups: HPV-positive (including HPV 16/18 genotypes) and HPV-negative controls . This study was approved by the Ethics Committee of International Peace Maternity and Child Health Hospital . Vaginal swabs were collected by clinicians according to the clinical standards, and patient information and experiments were performed according to approval standards. All samples were sent to the laboratory immediately after collection and saved at −80 °C for subsequent DNA extraction.Total DNA of samples were extracted according to the Fast DNA SPIN extraction kits  instruction. DNA concentration and purity were detected by NanoDrop2000, and DNA quality was tested using 1% agarose gel electrophoresis. DNA was stored at -20 °C before further analysis.Pfu Buffer, 0.4 μL of Fast Pfu Polymerase, 0.8 μL of each primer (5 μM), and 10 ng of template DNA. PCR amplified procedure was as follows: 95 °C for 3 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and a final extension at 72 °C for 10 min (PCR instrument: ABI GeneAmp9700).PCR amplification of the V3-V4 regions of the bacterial 16S rRNA gene was performed using 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) primers. The PCR reagent system contained 2 μL of 2.5 mM dNTPs, 4 μL of 5× Fast *300 library was constructed from purified amplified fragments according to Illumina MiSeq platform  standard operating procedures. Steps for library construction: (1) Connecting “Y” connector; (2) Using magnetic beads to remove self-connecting segments; (3) Enriching library templates by PCR amplification; (4) Producing single stranded DNA fragments by sodium hydroxide denatures. And the Illumina MiSeq PE300 platform  was used for sequencing.PCR products were separated with 2% agarose gel, and purified using an AxyPrep DNA Gel Extraction Kit . Quanti Fluor - ST  was used for quantitative detection. PE 2in vitro polymerase chain reaction (PCR) assay was used to identify HPV-16, -18 and 12 other HR HPV subtypes . HPV 16/18 genotyping was performed using the Roche Linear Array HPV Genotyping Test  according to manufacturer’s guidelines. A clinically validated The original sequencing was controlled by Trimmomatic software  and split-test (p < 0.05)  software and R packages were used to analyze the sequence information. Bacterial diversity was assessed using the alpha diversity: Shannon and Simpson diversity index. The difference of alpha diversity index between different groups was analyzed using Student’s  < 0.05) . The R l < 0.05) . By calc < 0.05) . In the current study, 59 eligible women were recruited and classified into five groups: normal controls , HR-HPV infection , low-grade squamous intraepithelial lesion , high-grade squamous intraepithelial lesion  and cervical cancer   . In totFirmicutes, Bacteroidetes, Fusobacteria, Actinobacteria, Proteobacteria and Tenericutes. Of these, the number of L. spp. belonging to Firmicutes was the most abundant (78.04%) in all five groups. In addition, the species diversity of vaginal microbiota in the four disease groups  was increased compared with that in normal group. Among them, the species diversity of vaginal microbiota in the cancer group was further increased, and the most significant change at the genus level was the large increase in the relative abundance of low-abundance bacteria or even unknown bacteria, indicating that the community structure of vaginal microbiota was disorganized.In this study, the compositions of vaginal microbiota in the five groups were investigated . The majL. spp. abundance in the HR-HPV infection, LSIL, HSIL and cervical cancer group was significantly decreased compared with the normal group (Actinobacteria (mainly G. and A.) in the four disease groups was higher than that in the normal group, and cancer group had the highest abundance (Bacteroidetes (mainly Prevotella) and Fusobacteria (mainly Sneathia) was also positively correlated with disease progression, but the increase was not as significant as that of Actinobacteria.The al group . In contbundance . The inct-test indicated significant differences between the normal control group and disease group; the diversity became more significant with the severity of the disease , but there were no statistically significant differences between the disease groups  in this study. In addition, L. jensenii were identified in 4.3% of subjects, and L. gasseri is was even rarer, with only 0.9%. Heat map analysis of the sequence data using nearest neighbor linkage at the species level identified four major vaginal microbiota community state types (CSTs) . Among aL. crispatus-dominant) decreased with increasing disease severity . Conversely, the rate of CST II (L. iners-dominated) was associated with an increase in HSIL and cancer group compared with the normal group . None of normal group samples were found in CST III (L.-depleted), and the cancer group accounted for the largest proportion . Due to the small sample size, two of the CST IV samples were LSIL, which could not explain the relationship between CST IV and disease. The frequency of CST I  of species sequence data was used to evaluate the vaginal community structure in the context of disease grade  . CompareThen, we used Partial Least Squares Discriminant Analysis (PLS-DA) to analysis the high-dimensional data . The bacD., Sneathia, and Senegalimassilia. The Acetobacter taxa were remarkably prevalent in the controls  effect size (LEfSe) model was used to identify the differences in microbiota composition, which may be associated with increased disease severity. At the genus level, three taxa exhibited significantly higher abundances in the HPV group than in the normal group, including controls .Faecalibaculum, Rhodococcus, Roseburia, L., Faecalibacterium (p<0.01) and Alloprevotella (p<0.04). In the LSIL group, Streptococcus was significantly overrepresented, and in the HSIL group, the highest abundance was Ralstonia (p<0.01) and Anaerococcus (p<0.03). G. (p<0.01), A. and D. (p<0.03) were the predominant genera in the cancer groups. These bacteria with significant differences between groups can be suggested as potential biomarkers for disease diagnosis   .PICRUSt was used to infer the bacterial functional metagenomic content of each sample and to classify the inferred genes into the level 2 KEGG pathways. The vaginal microbiota of the normal group was characterized by signal transduction, over representation of membrane transport, excretory system, genetic information translation and processing, all of which may be related to the normal physiological metabolism of bacteria and cells. In contrast, compared with the normal group, the cancer group showed highlighted metabolic diseases, endocrine system, immune system, cellular processes, cell growth and death, cell motility, transport and catabolism as well as “folding, sorting and degradation” .HR-HPV infection is a recognized risk factor for squamous intraepithelial lesions (SILs) and cervical cancer occurrence. In addition, there are many other factors contributing to cervical cancer development. In this study, we compared the structure and diversity of vaginal microbiota in normal, HR-HPV infection, LSIL, HSIL and cervical cancer groups, and analyzed the differences in bacterial composition and gene function during the development of cervical cancer. L. spp., the dominant bacteria in maintaining vaginal microecological balance, was decreased gradually with the occurrence of HR-HPV infection and the development of cervical lesions. Among them, the change was most pronounced in the cancer group. In contrast, the abundance of Actinobacteria (mainly G. and A.) in the four disease groups was significantly higher than that in normal group. Among them, the change was also most pronounced in the cancer group. With the development of cervical lesions, the relative abundance of low-abundance bacteria and even unknown bacteria in vaginal microbiota was increased, indicating that the imbalance of vaginal microflora became more and more serious.We found that the species diversity and community structure of vaginal microbiota were different among normal, HR-HPV groups, and groups with different grades of cervical lesions. With the occurrence of HR-HPV infection and the development of cervical lesions, the diversity of vaginal microbiota species was increased, and the community structure became more complex and disordered. In addition, the relative abundance of L., which is known to produce lactic acid to maintain low vaginal pH (< 4.5), and this is the first line of defense against pathogenic agents  was increased during the disease progression. In this study, L. spp. in the HSIL group was slightly higher than that in the LSIL group, but the proportion of L. iners in L. spp. in the HSIL group was significantly higher than that in the LSIL group. The L. spp. content in the cancer group was reduced, while the proportion of L. iners continued to be increased, accounting for 80.44% of the L. spp.. The role of L. iners in the healthy cervix is unclear, since it was detected both in normal and disease groups  was increased in HSIL and cancer group compared with that in normal group. L. iners may also related to the serious progression of cervical cancer. In addition, the analysis of PCA and PLS-DA further confirmed the significant difference between the vaginal microbiota in disease group and the normal group. As a result, vaginal bacterial dysbiosis may have existed in the development of cervical cancer. This study further found that the distribution of CSTs in normal group was different from the other disease groups. The abundance of e groups . L. inernfection . Some sttic acid . Most L.L. iners , indicatD., Sneathia and Senegalimassilia were significantly overrepresented in HPV group. Streptococcus were the most representative in LSIL. Whereas Anaerococcus and Ralstonia were significantly enriched in HSIL samples. Three other BV-associated bacteria, G., D. and A., were also observed to be markers of cancer. Previous studies have identified Sneathia spp. as a microbiological marker for HPV infection, cervical neoplasm and increased HIV acquisition (Sneathia spp. was more abundant in HR-HPV infection women than in patients with cervical cancer. To this extent, we confirmed that cervical disease was correlated with distinct bacterial combinations, which also changed with disease status. In addition, these specific bacteria were present in HR-HPV infection and CIN severity. We can assume that they contribute an important coordinating role in clearing HR-HPV infection or facilitating the transition from normal cytology to cervical squamous intraepithelial lesion status. Based on LEfSe analysis, some potential biomarkers related to different disease stages were found. uisition . In our Based on the 16S rRNA sequence information, PICRUSt was used to infer the function of the bacterial community, and further analyzed the differences between normal control and cervical cancer groups. The results showed that changes in bacterial abundance and composition could lead to significant changes in gene functional expression, especially in metabolic diseases, endocrine system, and immune system, which might be the factors of the development of cervical lesions.A major strength of our study is that we have analyzed the vaginal microbiota of Chinese women from HR-HPV infection to precancerous lesions and further to cervical cancer. There are still some limitations, including a relatively modest sample size and uncollected pH data. In summary, our work provides evidences for elucidating the complex relationship between vaginal microbiota composition, HR-HPV infection and cervical intraepithelial neoplasia. We also identified bacterial biomarkers to distinguish HR-HPV infection, LSIL, HSIL and cervical cancer. This may improve our understanding of the role of the bacterial microenvironment in HPV persistence, CIN development and cancer progression. The relationship between vaginal microbiota and functional prediction may provide insights into the pathogenesis of cervical cancer. These specific bacteria will improve women’s health status while helping to improve the early detection and prevention of cervical precancerous lesions. In future studies, we will further study the relationship between vaginal bacteria and the tumor microenvironment.


Cervical cancer incidence is high in Kenya due to HIV and limited access to cancer prevention services. Human papillomavirus (HPV) has been shown to increase HIV acquisition; however, the potential impact of HPV vaccination on HIV is unknown. We modeled the health impact of HPV vaccination in the context of the HIV epidemiology in Kenya.Using a validated compartmental transmission model of HIV and HPV set in Kenya, we evaluated five scenarios of nonavalent HPV vaccination: single-age-vaccination of 10-year-old girls at 90% coverage; multi-age-cohort (MAC) vaccination of 10-14-year-old girls at 90% coverage; MAC plus moderate-coverage (50%) catch-up vaccination of 15-24-year-old women; MAC plus high-coverage (80%) catch-up of 15-24-year-old women; and MAC plus catch-up of 15-44-year-old women at 80% coverage (HPV-FASTER). We compared cervical cancer incidence, HIV prevalence, and cumulative cervical cancer and HIV cases averted after 50 years to a baseline scenario without vaccination. In all scenarios, we assumed the UNAIDS 90-90-90 goal for HIV treatment is attained by 2030.In 2021, model-estimated cervical cancer incidence is 44/100,000 and HIV prevalence among women is 6·5%. In 2070, projected cancer incidence declines to 27/100,000 and HIV prevalence reaches 0·3% without vaccination. With single-age-vaccination, cancer incidence in 2070 is reduced by 68%, averting 64,529 cumulative cancer cases. MAC vaccination reduces cancer incidence by 75%, averting 206,115 cancer cases. Moderate and high-coverage catch-up and HPV-FASTER reduce cancer incidence by 80%, 82%, and 84%, averting 254,930, 278,690, and 326,968 cancer cases, respectively. In all scenarios, HIV prevalence in 2070 is reduced by a relative 8-11%, with 15,609-34,981 HIV cases averted after 50 years.HPV vaccination can substantially reduce cervical cancer incidence in Kenya in the next 50 years, particularly if women up to age 24 are vaccinated. HIV treatment scale-up can also alleviate cervical cancer burden. However, HPV vaccination has modest additional impact on HIV when antiretroviral therapy coverage is high.National Institutes of Health, Bill and Melinda Gates Foundation Cervical cancer is the leading cancer among women in Kenya, due to the high burden of HIV and low coverage of vaccination against human papillomavirus (HPV) infection and other cervical cancer prevention services. We searched PubMed on September 5, 2021 for modeling studies evaluating HPV vaccination impact in Kenya without language or date restrictions using the terms: (“model* OR “simulation”) AND (“HPV” OR “human papillomavirus”) AND (“vaccin*”) AND (“Kenya”). None of the resulting studies evaluated high-coverage vaccination strategies, such as catch-up vaccination for older adolescents and young women or accounted for the impact of HIV on cervical cancer. In addition, modifying our search to remove restrictions to Kenya and adding the terms: , we found no studies evaluating the impact of HPV vaccination on HIV burden.Our results suggest that HPV vaccination strategies that include young and mid-adult women have the largest impact in reducing cervical cancer burden. In addition, HIV prevention interventions can reduce cervical cancer incidence in the absence of HPV vaccination. Although HPV vaccination has a modest impact on HIV burden in Kenya, vaccination may have a larger impact in settings with higher HIV incidence.Our findings support the widespread implementation of HPV vaccination to prevent cervical cancer. In addition to a high-coverage routine vaccination program, temporarily providing catch-up vaccination for older adolescents and young women can lead to significant reductions in cervical cancer incidence in a country with limited access to cervical cancer screening and treatment.Alt-text: Unlabelled box,With advances in primary and secondary cervical cancer prevention in high-income countries (HICs), cervical cancer is increasingly an indicator of global health disparities. Approximately 90% of the estimated 604,000 new cervical cancer cases and 342,000 deaths due to cervical cancer in 2020 occurred in low- and middle-income countries (LMIC).Cervical cancer is one of the most common cancers and cause of cancer deaths among Kenyan women.,HPV vaccines are safe, durable, and highly efficacious in preventing infection with several oncogenic HPV types and the sequelae associated with these infections.A crucial component to the success of the cervical cancer elimination initiative is increasing HPV vaccination coverage in LMICs.We adapted a previously published compartmental, dynamic model of heterosexual HPV and HIV transmission to the Kenyan setting.Susceptible individuals in the model acquired HIV through heterosexual contact or, to a lesser extent, through mother-to-child transmission. We assumed women with an HPV infection had higher probability of HIV acquisition compared to those without HPV infection .,We modeled oncogenic HPV infection in two groupings: infection with an HPV type targeted by the nonavalent HPV vaccine (HPV 16/18/31/33/45/52/58) and infection with other oncogenic HPV types (HPV 35/39/51/56/59/68) . Acquisition and progression of these two groups of infection were independent of each other and could occur simultaneously. While men could only be HPV-infected or susceptible, women in the model had additional HPV-associated states of cervical intraepithelial neoplasia (CIN) grades 1 to 3 and cervical cancer. Further, women who cleared HPV infection develop natural immunity against reinfection with the same HPV group that wanes over time. Women with HIV had a higher probability of HPV acquisition, persistence, and progression to CIN, and reduced regression from CIN states.We calibrated the model by fitting epidemiological and clinical parameters, including HIV and HPV transmission probabilities and CIN progression rates, to reproduce HIV, HPV, and cervical cancer epidemiology. Specifically, we calibrated the model to HIV prevalence from nationally representative surveys , S6-7 an1No vaccination. This is the baseline for evaluating the impact of vaccination strategies.2Single-age-cohort vaccination. 90% of girls vaccinated by age 10. This scenario reflects Kenya's vaccination strategy in 2019.3Multi-age-cohort (MAC) vaccination. Girls aged 10-14 are vaccinated at 90% coverage. This scenario represents the vaccination strategy proposed by the WHO to eliminate cervical cancer.4MAC plus moderate-coverage catch-up vaccination. Vaccinating girls aged 10-14 at 90% coverage, with one year of catch-up vaccination for women aged 15-24 years at 50% coverage.5MAC plus high-coverage catch-up vaccination. Vaccinating girls aged 10-14 at 90% coverage, with one year of catch-up vaccination for women aged 15-24 years at 80% coverage. Scenarios 4 and 5 reflect the strategies adopted by several high-income countries.6HPV-FASTER. Vaccinating girls aged 10-14 at 90% coverage, with one year of vaccination for women aged 15-44 at 80% coverage. This scenario is similar to the strategy proposed by Bosch et al based on clinical trial data showing high vaccine efficacy among mid-adult women up to aged 45.We modeled the impact of nonavalent HPV vaccination on cervical cancer incidence over 50 years, with vaccination beginning in 2021. Specific scenarios are as follows:We assumed the nonavalent vaccine provided lifelong protection and is 100% efficacious against the HPV types targeted by the vaccine regardless of the recipients’ HIV status. Only girls and women who were not currently infected with a vaccine-targeted HPV type received protection from vaccination, consistent with data showing low vaccine efficacy in recipients infected with a vaccine-targeted HPV type at the time of vaccination.To evaluate vaccination impact on cervical cancer, we estimated annual cervical cancer incidence rates from 2021 to 2070, which were age-standardized using the WHO 2000-2025 Standard Population distribution. We calculated the impact of vaccination by dividing incidence rates from scenarios with vaccination by the rates from the baseline no vaccination scenario in the same year. We also estimated the cumulative number of cervical cancer cases averted 30 and 50 years after vaccine introduction relative to no vaccination. To assess the impact of HPV vaccination on HIV burden, we estimated yearly relative percent reductions in HIV prevalence compared to no vaccination. We also estimated the cumulative number of HIV cases averted after 30 and 50 years of vaccination. For all outcome estimates, we report the median and the interquartile range (IQR) of the 100 iterations.Study funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. GL had full access to all of the data and the final responsibility to submit for publication.The model-estimated cervical cancer incidence in 2021 was 44 per 100,000 women  of cervical cancer over 30 years b. With MBy 2070, relative to no vaccination, cervical cancer incidence was reduced by 68% with single-age-cohort vaccination, 75% with MAC vaccination, 80% with moderate catch-up, 82% with high-coverage catch-up, and 84% with HPV-FASTER b. Over 5HIV prevalence was projected to be 6·5% (IQR 4·4-7·5) among women and 2·9% (IQR 2·1-3·6) among men in 2021. Assuming the UNAIDS 90-90-90 goals are achieved by 2030, prevalence among women and men in the baseline scenario were predicted to decrease to 1·0% (IQR 0·7-1·3%) and 0·5% (IQR 0·3-0·7%) in 2050 and to 0·3% (IQR 0·2-0·4%) and 0·2% (IQR 0·1-0·2%) in 2070 a. In 205Relative to no vaccination, HIV prevalence among women in 2070 was reduced by 7·6-11·0% in the vaccination scenarios, with similar relative reductions in men . However, the absolute changes in HIV prevalence due to vaccination were <0·1% . The cumThe impact of HPV vaccination on cervical cancer and HIV burden is slightly modified by ART coverage levels among people living with HIV (Table S19-S20). Findings from our sensitivity analyses show that HPV vaccination led to a greater reduction in cervical cancer incidence (Table S19) and HIV prevalence (Table S20) when ART coverage is lower.HIV prevention and HPV vaccination have the potential to substantially decrease cervical cancer burden in Kenya in the next 50 years, particularly if young women up to age 24 are vaccinated. Compared to no vaccination, cervical cancer risk in 2070 was three times lower with single-age-cohort vaccination of 10-year-old girls and was up to 5·7 times lower with catch-up vaccination of women aged 15-24. Catch-up vaccination could prevent 90,401 to 114,161 cases of cervical cancer in addition to the 164,529 cases prevented through single-age-cohort vaccination. The total cumulative cervical cancer cases averted by 2070 was almost doubled with HPV-FASTER compared to single-age-cohort vaccination. In addition, HIV prevention can also reduce cervical cancer incidence on the population-level, as shown by the results from the no vaccination scenario and the sensitivity analyses.Since catch-up vaccination and HPV-FASTER lead to higher vaccination coverage in older age groups, the impact of vaccination on cervical cancer incidence will be realized earlier. With catch-up vaccination and HPV-FASTER, 23-30% of the projected cumulative cases of cervical cancers by 2050 were prevented in our simulations, compared to 13% with single-age-cohort vaccination. These findings are consistent with post-vaccination HPV surveillance data from European and North American countries, which show that population-level HPV prevalence declined more rapidly and to a greater extent with catch-up vaccination (up to age 19 or 26) than with single-age-cohort vaccination.While the WHO strongly recommends routine vaccination of girls 9-14 years old, its recommendation for vaccinating young women 15 and older is more tentative, leaving countries to decide whether to vaccinate young women.We modelled bidirectional interactions between HIV and HPV infections, such that HIV infection increased the risk of HPV acquisition and progression, and HPV infection increased HIV acquisition. Kenya has made marked progress in HIV prevention, leading to a decline in HIV prevalence in the last two decades.,Of note, we found that even in the highest coverage scenario (90% of 9-14-year-old girls and 80% of 15-44-year-old women vaccinated), cervical cancer elimination, defined as incidence of ≤4 per 100,000, was not achieved in Kenya by 2070. This is consistent with other modeling studies showing that cervical cancer elimination in LMICs may not be achievable even in the next century with vaccination alone.A strength of our model is that HIV and HPV transmission occurred dynamically, allowing the model to capture herd effects from interventions. This enabled us to estimate the total, population-level effect of vaccination on cervical cancer in Kenya. In addition, we explicitly modelled the biological interactions between HIV and HPV infections, making it possible to isolate the effect of HPV vaccination on cervical cancer from the effect of HIV prevention on cervical cancer. Accurate projection of vaccination impact will be important as Kenya plans to meet the challenge of cervical cancer elimination amidst changing HIV epidemiology. Further, while the convergence of evidence that HPV infection increased the risk of HIV acquisition prompted questions regarding the effectiveness of HPV vaccination for HIV prevention, clinical trials to investigate these questions were not recommended due to ethical and financial concerns.,We acknowledge the study limitations and their impact on the findings. Sexual behavior patterns, including condom use and mixing, are highly influential on disease trends. For simplicity, we assumed these parameters remained constant. However, the accuracy of our projections of HIV and cervical cancer burden would be affected if changes in future sexual behavior patterns occur. Similarly, our predictions regarding the benefit of catch-up vaccination among young women aged 15-24 could be biased if sexual behaviors in this population change in the future. Due to a lack of national-level cervical cancer incidence data in Kenya, we calibrated our model to the cervical cancer incidence rates estimated by GLOBOCAN.In conclusion, cervical cancer burden in Kenya can be substantially reduced with HPV vaccination and HIV prevention. The reduction in cancer incidence is greater and occurs earlier when multiple age cohorts are included in the vaccination strategy. While the elimination threshold is not projected to be reached, substantial cervical cancer-associated morbidity and mortality can be averted in high HIV prevalence settings with more equitable HPV vaccine coverage. In addition, concurrent scale-up of cervical precancer screening and treatment will be needed to eliminate cervical cancer as a public health problem in Kenya.Conceptualization and design: GL, CJB, DRW, and RVBModel adaption, calibration, and validation: GL, CJB, and DRWData review for parameterization and scenario definition: GL, NRM, CJB, DRW, and RVBScenario definition and analysis planning: all authorsResults interpretation and critical review: all authorsReview and approve final manuscript: all authorsWe did not collect participant data for this study, however, data used to parameterize, calibrate, and validate the model are available in the Supplemental Appendix.NRM is the recipient of an investigator-initiated award from Merck for a HPV vaccine study. RVB declares support from the National Institutes of Health and the Bill and Melinda Gates Foundation; and from Regeneron Pharmaceuticals for manuscript writing and abstract submission outside the submitted work. All other authors declare no competing interests.


Since 2007, the Standing Committee on Vaccination (STIKO) has recommended that all girls receive vaccinations against the human papillomavirus (HPV) in order to reduce the disease burden of cervical cancer. Persistent infections with high-risk HPV subtypes increase a woman’s risk of developing cancer. In the second wave of the German Health Interview and Examination Survey for Children and Adolescents , 42% of 2,958 11- to 17-year-old girls reported that they had received at least one HPV vaccination, and 31.4% reported a full HPV vaccination. 45.3% of 14- to 17-year-old girls reported a complete series of HPV vaccinations. Compared to the figures reported in KiGGS Wave 1 five years ago, HPV vaccination coverage has therefore remained stable. A vaccination coverage below 50% in girls is too low to exploit the potential of HPV vaccination to reduce cervical cancer rates in Germany. The human papillomavirus (HPV) is sexually transmitted. Without vaccination, over two thirds of all women will contract HPV during the course of their lives . HPV is Factors such as a high virus burden and the carcinogenicity of a specific virus type further increase the risk of cancer. Smoking or an HIV infection increase the risk of cancer as well. In most cases, however, an HPV infection is cleared without further consequences. The infection persists only in about one in ten women for one to two years and increases the risk of developing cancer . In 2014Since 2007, the Standing Committee on Vaccination (STIKO) has recommended HPV vaccination for all girls as a means of reducing the burden of cervical cancer. Until 2014, a series of three vaccinations was generally recommended for 12- to 17-year-old girls. In August 2014, the STIKO lowered the recommended age for vaccination of girls to 9- to 14-years and advised two instead of three vaccinations for this age group for full immunisation . Catch-uSecond follow-up to the German Health Interview and Examination Survey for Children and AdolescentsData owner: Robert Koch InstituteAim: Providing reliable information on health status, health-related behaviour, living conditions, protective and risk factors, and health care among children, adolescents and young adults living in Germany, with the possibility of trend and longitudinal analysesStudy design: Combined cross-sectional and cohort studyCross-sectional study in KiGGS Wave 2Age range: 0-17 yearsPopulation: Children and adolescents with permanent residence in GermanySampling: Samples from official residency registries - randomly selected children and adolescents from the 167 cities and municipalities covered by the KiGGS baseline studySample size: 15,023 participantsKiGGS cohort study in KiGGS Wave 2Age range: 10-31 yearsSampling: Re-invitation of everyone who took part in the KiGGS baseline study and who was willing to participate in a follow-upSample size: 10,853 participantsKiGGS survey waves► KiGGS baseline study (2003-2006), examination and interview survey► KiGGS Wave 1 (2009-2012), interview survey► KiGGS Wave 2 (2014-2017), examination and interview surveywww.kiggs-studie.de/englishMore information is available at The second wave of the German Health Interview and Examination Survey for Children and Adolescents  now provides data on HPV vaccination coverage based on a population-based representative sample. These data complement the calculation of vaccination coverage conducted by the Robert Koch Institute (RKI) in cooperation with the Associations of Statutory Health Insurance Physicians (ASHIPs) which is based on the analysis of health insurance claims data (ASHIP vaccination monitoring project) and permit the inclusion of privately insured girls as well as an analysis of further indicators for differences in vaccination uptake. A comparison with data collected five years ago in KiGGS Wave 1 allows for a trend analysis in HPV vaccination coverage and their determinants.New data for action. Data collection for KiGGS Wave 2 has been completed in issue S3/2017 as well as in KiGGS Wave 2 cross-sectional study – participant acquisition, response rates and representativeness in issue 1/2018 of the Journal of Health Monitoring [KiGGS is part of the health monitoring system at the RKI and includes repeated cross-sectional surveys of children and adolescents aged 0 to 17 years  that are representative for Germany. The KiGGS baseline study (2003-2006) was conducted as an examination and interview survey, the first follow-up study  as a telephone-based interview survey and KiGGS Wave 2 (2014-2017) as an examination and interview survey. A detailed description of the methodology used in KiGGS Wave 2 can be found in HPV vaccination coverage was surveyed in KiGGS Wave 2 through the answers of 11- to 17-year-old girls to a written questionnaire. It contained the question: ‘Have you been vaccinated against human papillomavirus (HPV) to protect yourself from cervical cancer?’ Respondents answering ‘yes’ were asked how many vaccinations they had received (‘How many HPV vaccinations have you received so far?’) and how old they had been when they received their initial vaccination (‘At what age were you first vaccinated against HPV?’).The analyses are based on data from 2,958 girls aged 11 to 17 with valid information on HPV vaccination. The proportion of missing values on HPV vaccination in the group of 11- to 12-year-old girls is high (20.3%) compared with the group of 13- to 17-year-old girls 10.6%). The analyses on the completed series of HPV vaccinations is based on the data collected from 2,916 girls (98.6%) because 42 girls did not provide answers to the respective subquestions. Results are presented as prevalences (frequencies) for at least one vaccination and for the completed vaccination series according to current STIKO recommendations. A completed series of vaccinations is defined as two vaccinations if started until the age of 14 years or three vaccinations if started at 15 years or older. The analysis did not consider whether there was a minimum interval between vaccinations of six months between the two vaccinations . The tables show prevalences stratified according to age, residence in East or West German federal states and socio economic status (SES) [.6%. The The calculations were carried out using a weighting factor that corrects deviations within the sample from the population structure with regard to regional structure , age (in years), gender, federal state (as of 31 December 2015), German citizenship (as of 31 December 2014) and the parents’ level of education (Microcensus 2013 ). P-valuIn KiGGS Wave 2, 42.0% of 11- to 17-year-old girls reported having received at least one HPV vaccination. The proportion of girls who have received at least one HPV vaccination increases with age. Whereas in the group of 11- to 13-year-old girls, 20.6% had received at least one vaccination, this proportion rises to 55.5% among 14- to 17-year-olds. Less than one third of 11- to 17-year-old girls has received the complete vaccination series. The proportion of girls with complete HPV vaccination also increases with age. Only 9.3% of 11- to 13-year-old girls, but 45.3% of 14- to 17-year-old girls have received a complete HPV vaccination series . Figure Compared to the girls interviewed by telephone five years ago for KiGGS Wave 1, there are no apparent differences in vaccination coverage for 14- to 17-year-old girls. The proportion of fully vaccinated adolescent girls was 43.7% in KiGGS Wave 1 and therefore only slightly lower than the currently surveyed prevalences 45.3%) . The sli.3% . TheWhen comparing the HPV vaccination coverage surveyed in KiGGS Wave 2 between 2014 and 2017 with the vaccination coverage calculated for 15-year-old girls in 2015 based on the ASHIP vaccination monitoring project , a higheIn the years following the HPV vaccination recommendation for girls, use and risk profile of HPV vaccination has been discussed widely and critically in the general public , leadingHPV vaccination coverage surveyed in KiGGS indicates that only about half of all girls or their parents/legal guardians, respectively, decide for a vaccination to protect them against HPV infections. The preventive potential of HPV vaccination against cervical cancer is so far not being fully exploited. In addition to the direct, individual protection, with a sufficiently high vaccination coverage of around 80% protection through herd immunity can be observed , also al45.3% of 14- to 17-year-old girls have received a complete series of HPV vaccinations.Vaccination coverage of girls in East German federal states is significantly higher than in the West German federal states.12- to 17-year-old girls received their first HPV vaccination on average at the age of 13 years and nine months which is five months earlier than in KiGGS Wave 1 five years ago.