HPV 16 sub-lineages do to not appear to cluster with disease vs asymptomatic carriage or be independently associated with outcomes in anal cancer patients. Further international studies on anal HPV sub-lineage mapping will help to determine whether this is a consistent observation.The incidence of anal cancer is rising worldwide. As identified in cervical cancer management, an improvement in the early detection and management of anal pre-cancer is essential. In other cancers associated with human papillomavirus (HPV), HPV 16 sub-lineages have been shown to be associated with disease status and prognosis. However, in anal cancer, they have been under-explored. A total of 119 HPV 16-positive anal cancer lesions diagnosed between 2009 and 2018 in Scotland and 134 HPV 16-positive residual rectal swabs from asymptomatic men collected in 2016/7 were whole genome sequenced. The association of HPV 16 sub-lineages with underlying disease status (cancer vs. asymptomatic) and overall survival in anal cancer samples was assessed (comparing A1 vs non-A1 sub-lineages). A1 was the dominant sub-lineage present in the anal cancer (76.5%) and the asymptomatic (76.1%) cohorts. A2 was the second most dominant sub-lineage in both groups . We did not observe significant associations of sub-lineage with demographics, clinical variables or survival (A1 vs. non-A1 sub-lineages (HR 0.83, 0.28–2.46  Anal cancer is one of the six cancers shown to have a human papillomavirus (HPV) aetiology . Most HPAdditionally, as with other HPV-driven cancers, anal cancer incidence is increasing worldwide, including in the USA and Europe ,5,6,7.HPVs are formally classified as “types” based on the nucleotide sequence of the open reading frame (ORF) coding for the major capsid protein: L1 . HPV typFor HPV 16, four lineages have been identified , as well as 16 sub-lineages: A, including A1–A3 (previously named European) and A4 (Asian) sub-lineages; B, including B1  and B2 , B3 and B4 sub-lineages; C1 , C2, C3 and C4; and D, including D1 , D2 , D3  and D4 sub-lineages .Although some investigators have assessed the global distribution of sub-lineages, the majority have focused on cervical cancers rather than other HPV-driven cancers. In 2013, Cornet et al. looked at the HPV lineages in cervical cancers and showed that European sub-lineages (A1–A3) were the most common in all regions of the world, except in sub-Saharan Africa and East Asia, whereas the African sub-lineages dominated in the northern sub-Saharan region of Africa, and the Asian variant in East Asia . NicolásIn terms of HPV 16 sub-lineages present in the anus, data is relatively sparce. Volpini et al. (2017) investigated the HPV 16 variants in anal samples collated in Brazil, finding that 70.8% were classified as A1–3 sub-lineages and 29.2% as “other” . A recenIn the context of cervical disease, evidence suggests that sub-lineages and variants may be independently associated with poor clinical outcomes. Mirabello et al. (2015)  revealedWe recently identified that the viral load of HPV16 in anal cancer may be informative for prognosis . Now, duA total of 150 HPV 16-positive anal cancers and 182 DNA extracts from residual rectal swabs obtained from asymptomatic men were selected for HPV 16 sub-lineage identification through whole genome sequencing (WGS).n = 150) sample set as described in detail in Guerendiain et al. (2022) [. Briefly, nucleic acid extract associated with archived formalin-fixed, paraffin-embedded tissue was genotyped using the Seegene Anyplex II 28 , followed by storage at −80 °C. Anal cancer biopsy samples were taken between 2009 and 2018 as part of the management of patients with anal disease from 3 of the 14 territorial health boards in Scotland .For the present work, we used the same anal cancer  at 65 °C overnight. DNA extraction was performed using the Microlab Nimbus IVD  with the StarMAg Universal cartridge Kit (Seegene), following manufacturers’ instructions. Mastermix was prepared with the Nimbus and PCR on the CFX Real-Time PCR instrument .As described in Guerendiain et al. (2022), clinico-demographic information was obtained in January 2020, specifically the patient’s age, sex, stage of cancer (using the American Joint Committee on Cancer (AJCC) TNM system) , responsCases categorized according to the various clinical and demographic variables are summarized in To contextualize the sequences observed in the anal cancers, a disease-free control group of anonymized residual rectal swabs obtained from asymptomatic men attending sexual health clinics were collated for downstream WGS. These samples had previously been genotyped as a consequence of immunization surveillance in Scotland . DNA wasA total of 182 anonymized DNA extracts from residual rectal swabs were collated. Samples had originally been collected in the years 2016–2017 and were included if positive for HPV 16.Use of samples for the present project was approved by the Southeast of Scotland National Research for Scotland Bioresource (NRS) (application reference SR 1283 and SR1364). A favorable ethical opinion to conduct the research was provided by University of St Andrews Teaching and Research Ethics Committee, reference MD 14482.HPV 16 whole genome material was amplified using 47 overlapping amplicons described in Cullen et al.  and optiLibraries were prepared using the Illumina DNA prep kit  following the manufacturer’s instructions, using 450 ng of DNA in 35 μL as input. Sequencing was performed using the Illumina MiSeq instrument and the Illumina MiSeq reagent kit v2 500 cycles (2 × 250 bp). Libraries were normalized to 4 nM in combination with 12.5 pM of PhiX (Illumina).HPV 16-positive (SiHa) and HPV-negative (water) controls were added at the DNA extraction step and carried through the PCR, library preparation and data analysis stages. Individual amplification products were assessed using a Bioanalyzer . Quality control for library preparation included both controlling the library size using an Agilent Tapestation  and determining the DNA concentration using the Qubit dsDNA High-Sensitivity Assay Kit .As a further quality control for analysis of the sequence data generated, a subset of 25 fastq files were sent to the International HPV Reference Laboratory in Karolinska, Sweden for independent bioinformatic analysis and sub-lineage identification.Reads obtained from Illumina were de-multiplexed and converted to fastq files. All fastq files were quality and adaptor trimmed using Trimmomatic (v0.39) . Only hiNew consensus files were aligned using MAFFT (v7.490) with default parameters . A manuaSub-lineages references were obtained from the PAVE for each of the HPV 16 sub-lineages: A1 (K02718.1), A2 (AF536179.1), A3 (HQ644236.1), A4 (AF534061.1), B1 (AF536180.1), B2 (HQ644298.1), B3 (HQ644298.1), B4 (KU053914.1), C1 (AF472509.1), C2 (HQ644244.1), C3 (KU053920.1), C4 (KU053925.1), D1 (HQ644257.1), D2 (AY686579.1), D3 (AF402678.1) and D4 (AF402678.1) A sub-lineage assignment was performed for all specimens excluding those with <100× median depth or low genome coverage (<80% genome coverage).To assess the relationship between HPV sub-lineages and different factors (two or more independent variables), a univariate logistic regression analysis was performed between HPV 16 sub-lineages (HPV 16 A1-positive vs. HPV 16 non-A1-positive), age at diagnosis, collection year and health board of diagnosis. Adjustment was performed for age group , sex, response to treatment, stage of cancer and vital status . Comparison was performed between A1 vs. non-A1 sub-lineages due to the small number of samples identified from the different sub-lineages. The non-A1 group includes the following sub-lineages: A2, A3, A4, B1, B2, B3, B4, C1, C2, C3, C4, D1, D2, D3 and D4.Odds ratios (OR) were calculated to quantify the strength of the association between HPV 16 sub-lineages and the demographic and clinical data. All the statistics were obtained using R-studio macOS, (version 1.2.1335) . The disOverall survival by HPV 16 sub-lineages (HPV 16 A1-positive vs. HPV 16 non-A1-positive) was analyzed using the Kaplan-Meier method. The univariate and multivariate hazard ratios of HPV 16 sub-lineages (HPV 16 A1-positive vs. HPV 16 non-A1-positive) for all-cause death were derived using the cox proportional hazard model. A univariate and multivariate model was derived; age , sex, stage  and response to treatment  were adjusted for. All the statistical analyses were performed using R-studio (version 1.2.1335) . DiffereA total of 182 asymptomatic/control samples and 150 anal cancer samples were subjected to WGS. In the anal cancer cohort, 119/150 (79.3%) samples passed the quality parameters, and in the asymptomatic men cohort, 134/182 (73.6%) were valid. This left a total of 253 samples for inclusion for detailed sequencing/phylogenetic analysis. Twenty-five of these sequences were also analyzed by the International HPV Reference Center as a further quality control for analysis. Results showed 100% agreement.Of the 119 cancer cases with sufficient read depth (>100× median depth and >80% genome), the HPV 16 sub-lineage A1 was identified in 91 anal cancer samples (76.5%), followed by A2, which was identified in 20/119 (16.8%) of samples. A4 was detected in 5/119 samples (4.2%). Two samples were classified as B1 (1.7%), one as A3 (0.8%) and one as D1 (0.8%). Further detail of HPV 16 sub-lineages in anal cancers is described in detail in Of the 134 control samples, most samples were classified (76.1%) as A1, followed by A2, identified in 23/134 (17.2%) of samples. D1 sub-lineage was identified in 4/134 samples (3.0%), and C1 and B1 were identified in two cases each (1.5%). B2 was present in 1/134 (0.7%). No major differences in the proportion of A1 and A2 sub-lineages were observed between the case vs. control cohorts, being 76.4% vs. 76.0% and 16.2% vs. 17.0%, respectively. Whereas the case cohort revealed the presence of the A4 sub-lineage in 4.2% of anal cancers, this sub-lineage was not present in the control cohort. Conversely, the C lineage was present in 1.5% of control specimens and was not detected in cancer cases. Finally, there was a slight increase in the D1 sub-lineage observed in the control vs. case cohort (3.0% vs. 0.8%).From the 119 anal cancers, four samples did not contain vital status information and were not included in the analysis. Due to the dominance of the A1 sub-lineage, the logistic analysis and odds ratio analysis were performed based on the presence or absence of the HPV 16 sub-lineage A1.No significant differences in A1 positivity with sex, age, response to treatment, stage or vital status were observed. This observation was consistent for the adjusted analysis .p = 0.57), For the Kaplan-Meier estimator, overall survival was calculated by classifying HPV 16 sub-lineages into A1 presence or absence. No differences in overall survival were found between both sub-lineage groups (p = 0.751). Variables associated with worse overall survival in the univariate model were stage IV vs. stage I with HR of 15.7 (3.38–72.8), p < 0.001 and response to treatment vs. no response to treatment with HR of 0.11 (0.05–0.25) p < 0.001. After adjustment for age, gender, stage and response to treatment, non-A1 sub-lineages did not significantly influence the overall survival compared to A1, with a HR 0.83 .p = 0.575).If only A1 and A2 samples were considered, no significant differences in HR were found when using A1 as the reference  of the anal cancer samples. This absence indicates the potential integration of the HPV16 in the human genome. The E2 gene was the most frequently missing region, followed by E4, E5 and L2 E1 and L1 .In the control group of asymptomatic men, a similar prevalence of A1 and A2 was observed. Differences identified were the presence of A4 in the anal cancers (4.7%), which was absent in the control group; presence of the C lineage only detected in the control group; and the presence of the sub-lineage D1 in the control group (3%), which had a lower prevalence of 0.81% in the cancers. This higher prevalence of sub-lineages A1 and A2 is consistent with previously published studies in European cohorts. Gonçalves et al. (2022) found a higher prevalence of the A lineage in the anal canal of asymptomatic men, mainly A1 , and NicThe data collated in the study add to the limited information on the pattern and implications of HPV sub-lineages in the anus. Though we did not see significant associations with demographic and underlying disease status, these observations need to be confirmed or refuted by future studies with larger sample sizes.To our knowledge, no other studies have investigated the association of HPV 16 sub-lineages in anal cancer and overall survival. We did not observe that A1 vs. non -A1 sub-lineages influenced overall survival in the univariate and adjusted analysis. Interestingly, a recent study was reported by Lang Kuhs et al. (2022) in which the authors looked into the genetic variation of HPV 16 and its association with clinical outcomes in HPV 16-positive oropharyngeal cancer patients . They inAlthough we identified potential integration of the HPV16 genome , due to the small number, we did not perform any further analysis, including in relation to implications for survival. Given the relative lack of information on the extent and implications of integration in anal cancer, we would assert that this is an area that would benefit from further study.n = 253), particularly given that the Scottish European age-standardized rate (EASR)  was 2.6 in 2017, we appreciate that detecting rarer sub-lineages with precision can take large sample sizes.We acknowledge this study has limitations; the asymptomatic population were all men, whereas the cancer population had a majority of female (75.63%) samples compared to males (24.37%); this was due to pragmatic reasons relating to available material. However, data did not show differences in the distribution of HPV 16 sub-lineages between women and men in the anal cancer group. Additionally, as discussed earlier, we believe the observations made in the present work would benefit from validation in a larger sample of cases and controls and would hope this study serves as a primer for such. Though the number of cases of cancers was not trivial  reported that HPV 16 lineage variants B, C and D exhibited slightly (<two-fold) reduced sensitivity to nonavalent vaccine sera compared to lineage A .Therefore, the high prevalence of lineage A in the samples included in this study could be interpreted as positive for vaccine efficacy, particularly given that gender-neutral vaccination is now a part of core policy in the UK and several other countries.This study has demonstrated the technical feasibility of detecting HPV 16 sub-lineages in anal cancer samples and residual material from rectal swabs. Though some differences in the presence of non-A sub-lineages were detectable between the cancer and asymptomatic population, the consistency, magnitude and implications of these would benefit from further study. The domination of lineage A is consistent with existing European data and suggests that sub-lineage identification in itself may not be informative for prognostication.


Currently, cervical cancer prevention is undergoing comprehensive development regarding human papillomavirus (HPV) vaccination and cervical cancer screening. In Sweden and many other countries, high coverage vaccinated cohorts are entering screening within the next few years. This entails demands for baseline HPV genotype data across the screening age range for surveillance and a basis for screening program adjustment. In 2016, Örebro County, Sweden, changed to primary HPV screening using HPV mRNA testing followed by cytology triage. An alternative triage method to cytology could allow for a fully molecular screening algorithm and be implemented in a screening program where self-sampling is included. Hypermethylation analysis of the human genes FAM19A4/miR124-2 has been suggested as a promising triage method. HPV mRNA-positive screening samples (n = 529) were included and subjected to genotyping targeting a broad range of both low-risk and high-risk genotypes in addition to hypermethylation analysis of the two human genes FAM19A4/miR124-2. Data were connected to cytological and histological status and age. The most commonly detected genotypes were HPV31, 16, and 52. In addition, HPV18 was one of the most common genotypes in high-grade squamous intraepithelial lesions (HSILs) samples. In relation to available vaccines, 26% of the women with histological HSIL or cancer (≥HSIL) tested positive for only hrHPV included in the quadrivalent vaccine and 77% of the genotypes in the nonavalent vaccine. According to these figures, a relatively large proportion of the HSILs will probably remain, even after age cohorts vaccinated with the quadrivalent vaccine enter the screening program. Hypermethylation positivity was associated with increasing age, but no HPV-related independently predictive factors were found. Accordingly, age needs to be considered in development of future screening algorithms including triage with hypermethylation methodology. HPV infections are the underlying cause of cervical cancer (CC) and its precursors . More thInternationally, CC is a major health issue with over 500,000 new cases yearly, and in some regions CC is the leading cause of cancer related death in women, much due to lack of prevention and access to healthcare . In SwedIn Sweden, all women are invited to participate in cervical cancer screening from the age of 23 years. In August 2016, new guidelines were implemented in Örebro County whereby women over 30 years of age receive primary HPV screening followed by triage with cytology . The scrBetween October 2016 and April 2017, 7673 women over age 30 years (30–58 years) participated in cervical cancer screening in Örebro, Sweden. Within the screening program in Örebro County, professional sampling by liquid-based cytology (LBC) method  is followed by primary HPV analysis with Aptima HPV assay (Hologic) detecting E6/E7 mRNA of the hrHPV genotypes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 for women over age 30 years. Results are given as hrHPV positive or negative but without specific genotype information. Positive HPV mRNA screening results are followed by cytological triage according to Bethesda classification . Women wDNA from HPV mRNA–positive samples was extracted in two steps; first, directly from the Aptima transfer tube for DNA genotyping, and later from biobanked LBC samples for FAM19A4/miR124-2 analysis and for samples extracted from Aptima transfer tubes that were HPV negative.Following the HPV screening analysis, 200 μl of liquid specimen from the Aptima transfer tube was used for DNA extraction in the QIA symphony system  using the QIAamp DNA Minikit and the Tissue LC 200 V7 DSP protocol with elution in 50 μl, according to the manufacturer’s protocol.For 504/529 study participants, LBC samples from a biobank (Örebro biobank) were available for the second DNA extraction. A cell pellet from 100 μl of biobanked LBC samples was incubated with 180 μl ALT and 20 μl proteinase K at 56˚C on a thermoshaker for 2 h. In cell-abundant samples, extraction was performed with the QIA symphony system as described above. For samples with fewer cells, DNA was extracted in the QIA cube system (QIAGEN) with the QIAamp DNA FFPE Tissue Kit and tissue program with 50 μl elution volume according to protocol from the company.HPV genotyping on mRNA–positive samples was performed first on DNA extracted from specimen transport tubes. Second, analysis on all HPV DNA–negative samples was repeated with DNA extracted from LBC samples. Anyplex™ II HPV28 , which targets the viral L1 gene of 28 genotypes  together with the human gene Beta-globin (HBB), was used for genotyping. The PCR was manually prepared and performed after manufacturer’s recommendations with approximately 50 ng DNA input into both multiplex reaction mixes per sample and was run on a CFX96TM Real-Time PCR System . The assay includes human internal control for every sample, genotype-specific plasmid controls for all genotypes, and non-template control in every run. Results were analyzed in the Seegene Viewer software (version 2.0). Melting curve analysis after 30, 40, and 50 cycles gives a semi-quantitative indication of the positive results.Extracted DNA from LBC samples (≤200 ng) was subjected to bisulfite treatment using the EZ DNA Methylation-Gold Kit  according to protocol.Approximately 50 ng of bisulfite-converted DNA was added into the methylation analysis. QIAsure (QIAGEN), a multiplex-methylation–specific rtPCR method, was used to evaluate the hypermethylation status of two human host cell genes (FAM19A4 and miR-124-2), including one internal control gene (ACTB). Cycle threshold values of the two targets in each sample were reported in relation to the internal control and a low copy number plasmid control, the calibrator. The assay was run on the Rotor-Gene Q MDx 5plex HRM (QIAGEN) system and results were automatically analyzed with the Rotor-Gene Assay Manager software.In addition to an internal control validation for every sample, for each run a low copy number plasmid control, for example, the calibrator, and a non-template control, was included. Cycle threshold values of the two targets in the samples are reported in relation to the internal control and the calibrator. Detected hypermethylation in any of the two targets resulted in a positive test result.Data calculations and table and figure preparation were carried out in Microsoft Excel 2010 , and for statistical analyses IBM SPSS Statistics version 25  was used. Pearson Chi2 test was used for comparisons of proportions and binary logistic regression was used for analysis of predictive factors for hypermethylation . In analyses where comparisons between proportions were made with stratification, Fisher’s exact test was used. For the statistical analysis, p < 0.05 was considered statistically significant. Due to rounding, percentages in text and tables may not always add up to 100%.The study was approved by the regional ethical committee board in Uppsala, Sweden (D-nr. 2017/297). The ethics committee waived the need for consent, since data were pseudonymized for analysis.Of all 529 women who tested positive for HPV mRNA in the cervical cancer screening, 495 (94%) tested HPV DNA–positive in this study . In 34 samples (6%), HPV DNA from the targeted 28 genotypes was not detected. In 59% (292/495) of the positive samples, a single HPV genotype was detected, and multiple genotypes, counting both hrHPV and lrHPV, were found in the remaining samples . Between two and seven genotypes were detected together . Regarding hrHPV, 87% (429/495) of the samples harbored HPV from the IARC1 hrHPV class. Most of these samples included one IARC1 genotype  and a minority  more than one . Out of 495 HPV-positive samples, 13% (n = 66) tested positive for solely non-IARC1 genotypes. They were predominantly from the IARC risk groups 2A and 2B, and most often HPV66 and or 68 (n = 44), which are also targeted by the Aptima mRNA assay. However, in 22 samples none of the genotypes targeted in the Aptima test could be found and were instead from the IARC2B group , lrHPV, or unclassified .The most detected genotypes were HPV31 (11%), HPV16 (9%), HPV52 (7%), and HPV68 (6%). In single infected samples, HPV31, 16, and 52 were also most abundant, with HPV18 placing fourth, presenting the proportions of 20%, 12%, 10%, and 7%, respectively. The most common genotypes in multi-HPV samples were HPV16 followed by HPV31 and HPV42 .The majority of the study group, 52% (n = 274), were aged 30–39 years, 33% (n = 172) were 40–49 years, and 16% (n = 83) were 50–58 years. In total, 517 out of 529 women had complete follow-up. Of these, 70 cases were defined as ≥HSIL . In the control group with no evidence of disease (n = 447), there were 299 women with normal cytology, 61 with ≤LSIL cytology, 54 with LSIL histology, and 33 with normal histology. Of the 12 excluded women, six had a screening sample with non-assessable cytology result and six had no follow-up.Regarding specific genotypes, as in the total group, HPV16 and 31 were the two most common genotypes . HPV18 wIn the present cohort, the probability of having a histologically confirmed abnormality of ≥HSIL decreased with age . Also, women in the younger age group had a higher proportion of HPV IARC1 compared to the other age groups . In analysis by screening outcome strata, this association was, however, only statistically significant in samples with no evidence of disease.All confirmed HSIL cases were HPV DNA positive . The proMost ≥HSIL samples were positive for genotypes covered by available HPV vaccines, and 83% (58/70) contained at least one hrHPV vaccine genotype . Fewer samples, 77% (54/70), were exclusively positive for vaccine genotypes. Concerning only HPV16/18, 39% (27/70) of ≥HSIL samples were positive for one or both, and 26% (18/70) contained solely HPV16 or 18.LBC samples were available for hypermethylation analysis in 504 cases, and data could be obtained in 487 samples. Analytical results were positive for hypermethylation in one or both of the targets in 32% (158/487) and negative in 68% (329/487).Of samples with a hypermethylation result, loss of follow-up was evident in 11 cases, five had a screening sample with non-assessable cytology result and six had no follow-up. Of the 476, 61 cases were defined as ≥HSIL , and 415 as control group with no evidence of disease.The proportion of methylation positivity increased by severity of screening outcome and presented with 67% in ≥HSIL compared to 28% in samples from women with no evidence of disease  . Among hThe probability of a positive methylation result increased with increasing age of the woman. In the age group 50–58 years, almost half of the samples were positive (48%). There was a statistically significant difference in hypermethylation-positive proportion between the three age groups  compared to hypermethylation-negative samples . Also in samples with no evidence of disease, the hypermethylated samples were numerically more often positive for multiple genotypes  compared to hypermethylation-negative samples .Genotype-specific hypermethylation patterns in samples with no evidence of disease showed generally low frequencies of methylation in samples positive for all genotypes. In IARC1 genotype–positive samples, frequencies were between 18% and 38%. In ≥HSIL samples, the frequencies of methylation were generally higher, notwithstanding ≥HSIL samples positive for HPV51, where only 1/5 (20%) was hypermethylated and 4/5 (80%) unmethylated. In ≥HSIL IARC1–positive samples, the methylation frequencies were between 50% and 89%, with genotypes 52, 33, and 45 showing the highest methylation frequencies .DNA negative samples (n = 34) presented invalid methylation results in two samples. The methylation rate in this group was 31% (10/32). In the group testing positive for non-Aptima targeting genotypes (n = 22), 18% of the samples tested hypermethylation positive.The study cohort included women screened with primary HPV in the beginning of conversion to the new screening algorithm with primary HPV testing in Örebro, Sweden. Messenger RNA HPV–positive cervical screening samples were included in the study for genotyping, while previous studies have largely been based on cytological abnormalities. In this set-up, we are able to identify all active hrHPV infections in the screening population and provide information on the genotype distribution in the screening population.The concordance in detection of HPV between the mRNA screening method and DNA genotyping was not complete, which is known from other studies ,15. A miThe most common genotypes in the present study were HPV31, 16, and 52 both in total and in only single infected samples ; in ≥HSIWith implementation of vaccine programs, a considerable decrease in hrHPV in the screening population is expected. Data have already been reported on population level showing a decrease in HPV positivity and in precancerous lesions . In the Hypermethylation of the human genes FAM19A4 and miR124-2 has shown to detect cervical cancer with high sensitivity . A negatIn addition, the results show that the hypermethylated samples were numerically more often positive for multiple genotypes. Multiple genotypes in one sample could potentially cumulatively increase the oncoprotein effects, thereby driving hypermethylation and transformation . At the Women with a confirmed histological HSIL are most often treated due to the risk of progression to cancer in this group. Even so, it is known that HSIL is a heterogeneous group, where many lesions would spontaneously regress . In thisIn the present study, hypermethylation of the targeted genes was associated with age, where older women were more likely to present with a positive test result independent of screening outcome . AssociaThis study presents the genotype distribution, with a broad range of targeted genotypes, both lr- and hrHPV and hypermethylation status of two human genes, in mRNA hrHPV–positive women participating in cervical cancer screening. The study population is a well-screened and essentially unvaccinated cohort, which makes it suitable as a baseline comparison in surveillance regarding future developments in screening populations. Future perspectives include linking genotyping and methylation data to data from upcoming screening rounds for these women. Further studies are also of importance to explore whether methylation and genotyping could be sufficient on self-collected samples.HPV genotyping in this study shows evidence that a relatively large proportion of histological ≥HSILs will remain, even after age cohorts vaccinated with the quadrivalent, as well as the nonavalent, vaccine enter screening. Except for age, no HPV-related independently predictive factors for hypermethylation were found. Accordingly, age needs to be considered in development of future screening algorithms, if including triage with hypermethylation and HPV genotyping.


Studies have shown the low uptake of the human papillomavirus (HPV) vaccine among university students. Knowledge about the HPV virus and the vaccine can be a factor that may sway an individual’s decision to receive the vaccine. This study aimed to investigate HPV vaccine uptake and knowledge about HPV vaccination among Croatian university students.This cross-sectional questionnaire study was conducted from February to May 2021 period. A validated, anonymous questionnaire that contained questions regarding demographic data, data about HPV vaccine uptake, and data regarding knowledge about the HPV virus and the vaccination was self-administered to a cross-faculty representative student sample of the University of Osijek in Eastern Croatia.The study sample included 840 subjects with, median age of 20 years (interquartile range 20-21), 45.8% males, and 54.2% females. The prevalence of vaccination uptake in the studied population was 20.8%. The study revealed that there were 25.6% of students with a low level of knowledge and 74.4% of students with satisfactory levels of knowledge about the HPV virus and the vaccination. The excellent knowledge about the HPV virus and the vaccination was more frequently shown by students who studied in biomedicine and health area of science (p < 0.001) and students with an excellent average grade of study (p < 0.001). HPV vaccination uptake was higher among females (p < 0.001), students who studied within biomedicine and health area of science (p < 0.001), students with an excellent average grade of study (p < 0.001), and students who showed excellent knowledge about the HPV virus and the vaccination (p < 0.001).The majority of Croatian university students had a satisfactory level of knowledge about the HPV virus and the vaccination but the vaccination uptake is still very modest. Additional efforts are needed to organize more appropriate education and promotion of vaccine uptake in the studied population.• The HPV vaccination uptake among Croatian university students is very modest although the majority of students have a satisfactory level of knowledge about the HPV virus and the vaccination.• Continuous examination of HPV knowledge gaps and identification of factors influencing vaccine uptake is key to increasing vaccination rates in the Croatian university student population.


Overcalling of abnormalities has been a concern for using cytology triage after positive high‐risk human papillomavirus (HPV) tests in cervical screening.The authors studied the detection of cytological and histological abnormalities at age 24 to 64 years, using data from the English HPV pilot. The pilot compared routine implementation of primary cervical screening based on cytology , where HPV test results were not available before cytology reporting, with that based on HPV testing , where cytology was only required after positive HPV tests.adj], 1.16; 95% confidence interval [CI], 1.14‐1.18). Laboratories with higher direct referral referred fewer persistently HPV‐positive women after early recall. The detection of high‐grade cervical intraepithelial neoplasia (CIN2+) after direct referral increased with an ORadj of 1.17  for informed versus uninformed cytology. Generally, the positive predictive value (PPV) of colposcopy for CIN2+ remained comparable under both conditions of interpreting cytology. In women 50 to 64 years old with high‐grade dyskaryosis, however, the PPV increased from 71% to 83% after revealing HPV positivity .Revealed HPV positivity was associated with a higher direct referral to colposcopy after any abnormality  test can contribute to a higher overall detection of high‐grade cervical intraepithelial neoplasia without a concomitant increase in overreferral of low‐risk women. The practices used in England, where the national cervical screening program is quality‐assured using rigorous national standards, may serve as a model for screening settings where revealing HPV positivity has led to overgrading of cytology. Women who are high‐risk human papillomavirus (HPV)‐positive in a cervical screening context are generally triaged given the transience of most HPV infections. One of the most common triage tests internationally is cytology. In randomized trials, where cytology slides were prepared for all women and interpreted without knowledge of the HPV test result, cytology triage showed both safety and efficiency in preventing unnecessary colposcopy referral and biopsies.In a number of studies, cytological abnormalities were reported more frequently after HPV positivity had been revealed,The English HPV screening pilot was undertaken within the quality‐controlled cervical screening program (CSP).Using these data, we studied how cytology reporting and the downstream consequences thereof were affected according to whether a woman's positive HPV test was unknown (LBC screening) or revealed (HPV screening).The CSP routinely recalls women 25 to 49 years old every 3 years and those 50 to 64 years old every 5 years. The first invitation is sent at 24.5 years. Abnormal cytology in squamous or endocervical cells is defined as borderline changes or worse  and ThinPrep  LBC systems .We first studied whether revealing the positive HPV test result affected cytology interpretation. For both HPV‐based and LBC screening, we calculated age‐specific proportions of women with abnormal cytology requiring direct colposcopy referral after the baseline sample Fig. . BecauseThereafter, the data were stratified by laboratory, and, within each laboratory, into consecutive 3‐month periods . For each laboratory and calendar quarter, we calculated the ratio of the proportions with a direct colposcopy referral after informed versus uninformed cytology. These ratios were standardized for age (<30 and ≥30 years) and IMD decile (deciles 1‐5 vs deciles 6‐10) to represent the female population of England in 2013.We then investigated the downstream consequences of revealing the HPV infection. First, we studied the detection of CIN2+ after direct colposcopy referral for informed versus uninformed cytology and the associated PPV for CIN2+ of those colposcopies. ORs were calculated and adjusted as described above. For women with positive primary HPV tests, we also stratified these data by LBC system (ThinPrep and SurePath). Here, we estimated the sensitivity, specificity, PPV, and the negative predictive value (NPV) for CIN2+ of baseline triage cytology with exact binomial 95% confidence intervals (CI). CIN2+ were counted until the end of the episode, including both early recalls for women with initially negative triage cytology; cases diagnosed at early recall were considered missed by baseline triage cytology. ORs for the observed accuracy measures comparing ThinPrep with SurePath were adjusted for age, IMD decile, and HPV test type used in the laboratory . Second, we studied the relationship between direct referral and early recall referral. For each laboratory and calendar quarter, the proportions of women with direct referral were calculated as described above, using the numbers with a positive HPV test as the denominator. The proportions of women with a positive HPV test, screened in the same laboratory and calendar quarter, who satisfied the criteria for colposcopy referral at early recall were calculated separately. Total referral was defined as the sum of direct and early recall referral out of all women with a positive HPV test in that laboratory and calendar quarter. All proportions were standardized to the English population as described above. R version 3.6.1 was used for analyses.adj for informed vs uninformed cytology, 1.16; 95% CI, 1.14‐1.18). Across all age groups, the increase in referrals associated with informed reading was highest for borderline changes . The increase in low‐grade dyskaryosis was smaller and limited to women younger than 50 (with ORadj consistently approximately 1.15). Although the reporting of high‐grade dyskaryosis with informed reading only showed a very small increase in women younger than 30, it was significantly less frequent among women older than 50 .The study included 931,539 women screened with LBC and 403,269 women screened with HPV testing  .Overall, informed reading was associated with a higher detection of CIN2+ at direct referral for all abnormal cytology grades combined . For all ages combined, 4.5 women with informed borderline cytology needed to be referred to detect one CIN2+. This varied between 4.0 among women younger than 30 to 9.1 among women older than 50. Among women with low‐grade abnormalities detected through informed cytology, 5.5 needed to be referred overall to detect 1 CIN2+, varying between 4.8 and 12.5 depending on the women's age. Among those with high‐grade abnormalities, 1.1 women needed to be referred to detect 1 CIN2+, varying between 1.1 and 1.2 depending on the women's age.Informed cytology was associated with a slightly higher PPV of baseline colposcopies for CIN2+, but this varied by age group , they had similar proportions of positive HPV tests followed by abnormal cytology triage . In ThinPrep sites, baseline triage cytology after a positive HPV test detected 69% (1842/2686) of all CIN2+, whereas in SurePath sites this was 73% (4787/6556)   reported no differences in the detection of CIN2+ between the SurePath and ThinPrep systems in primary LBC screening.In conclusion, implementation of HPV‐based primary screening with cytology triage in a controlled manner, supported by rigorous cytology training and performance monitoring, does not lead to inappropriate overgrading of cytology. Rather, an earlier recognition of cytological abnormalities appears to partly explain why the detection of CIN2+ is increased in HPV‐based screening compared with LBC screening.Public Health England supported the epidemiological evaluation of the HPV pilot (ODR1718_428). Matejka Rebolj and Christopher Mathews were supported by Cancer Research UK (C8162/A27047). Public Health England had a role in designing the pilot and in the collection of the data and commented on the manuscript. Cancer Research UK had no role in designing the study, in the collection of the data, or in the writing of the manuscript.Christopher S. Mathews held an honorary appointment at Public Health England to process the data for the pilot. Karin Denton reports personal fees from Public Health England during the conduct of the study and travel support from Hologic outside the submitted work; chairs the Public Health England Laboratory Clinical Professional Group, the HPV Development Group, and several groups related to the evaluation of self‐sampling; was a consultant to the Scally Review of cervical screening in Ireland and the Royal College of Obstetricians and Gynaecologists review of cervical cancer audit in Ireland (both completed in 2019); and has prepared expert medicolegal reports for claimants and defendants, including cases of cervical cancer. Matejka Rebolj reports grants from Public Health England during the conduct of the study and lecture fees from Hologic outside the submitted work, is a member of the Public Health England Laboratory Technology Group and HPV Self‐Sampling Operational Steering Group and Project Board, and has attended meetings with various human papillomavirus assay manufacturers.Matejka Rebolj: Conceptualization, methodology, formal analysis, writing–original draft, and writing–review and editing. Christopher S. Mathews: Data management and writing–review and editing. Karin Denton: Conceptualization, methodology, and writing–review and editing. The Pilot Steering Committee was responsible for the study design of the pilot.Supplementary MaterialClick here for additional data file.


To validate the HPV viral loads that are reflected by the cycle threshold values of Cobas4800 as the viral load indicators by verifying the consistency of the viral loads per unit  from the BMRT assay.The analysis is based on data from the Chinese Multi-Center Screening Trial (CHIMUST). The cases included in the analysis are all positive for physician-collected hrHPV on SeqHPV and/or Cobas4800 or negative for hrHPV but abnormal in cytology (≥LSIL), and some cases selected by nested case-control randomization from those negative for physician-collected hrHPV and cytology. With HPV testing results and relevant Ct values from Cobas4800 available, we tested the entire sample set with the BMRT HPV testing assay and analyzed their agreement with Cobas4800, followed by a comparison of the CtV from Cobas4800 and viral loads (lg) from BMRT by lesion grade.HPV16 = −0.641, P < 0.001; r 12−HPVpool = −0.343, P = 0.109; In CIN2+, r HPV16 = −0.754, P < 0.001; r 12−HPVpool = −0.429, P < 0.001).We included 4,485 women (mean age: 45.4 years) in the study, and 4,290 had complete data. The consistency of genotypes from Cobas4800 and BMRT for hrHPV, HPV-16, HPV-18, and 12-HPV pools was 94.9% , 99.1% , 99.6% , and 95.3% , respectively. Further analysis shows that any inconsistency between the two assays is likely among samples with comparatively lower viral loads. When analyzing per lesions of CIN2+ and CIN3+, the CtV from Cobas4800 and VL (lg) from BMRT are highly correlated inversely and follow the linear regression for HPV16 and 12-HPV pool (Pearson's or Spearman's correlation coefficient (r): In CIN3+, r The CtV from Cobas4800 and the viral loads (lg) of per unit cells from the BMRT are well correlated for lesion grading when tested on physician-collected samples. Cobas-CtV is worthy of further study for clinical application. Cervical cancer is the fourth most common cancer in women. In 2020, an estimated 604,000 new cases of cervical cancer were found, and there were 342,000 deaths worldwide . The incCurrently, the coverage of cervical cancer screening in China is low and needs to be increased with effective technologies and efficient implementation. Compared with cytology, which is highly resource-dependent, has relatively low sensitivity, and is very skill-dependent, HPV testing for primary screening is an objective technology, higher in sensitivity and negative predictive value (NPV), and is very reproducible . ConsequHPV testing has a significant disadvantage: specificity for detecting CIN2/3. Because most HPV infections are non-neoplastic, this creates challenges for risk stratification and the need for secondary screening . CytologCurrently, there are about 250 HPV tests worldwide, most of them PCR-based assays in China. PCR-based assays can report the cycle threshold value (CtV). However, only a small number of assays have been clinically validated . The CobThe Bioperfectus Multiplex Real-Time (BMRT) HPV assay is the only PCR-based HPV testing assay that reports the genotype-specific viral load of 21 HPV genotypes per unit  . We haveThis study used physician-collected cervical samples from the Chinese Multi-center Screening Trial (CHIMUST). CHIMUST was a population-based cross-sectional study  conducted collaboratively from August 2016 to January 2018 by six hospitals in six provinces in northern, middle, eastern, and southern China . A totalThe physician-collected samples were tested by the Cobas4800  and SeqHPV  assays. Colposcopy-directed and random biopsies by quadrant were performed on all cases positive on either of the two primary testing assays. Using a nested case-control method, we performed BMRT HPV testing on the part of the stored physician-collected specimens from CHIMUST and compared the results with the Cobas4800. We evaluated the sensitivity and specificity for CIN2+, the concordance of CtV from Cobas4800 (CtV-Cobas), and the genotype-specific viral load reported by BMRT (VL-BMRT). We also analyzed the trends of the CtV-Cobas and the VL-BMRT by the pathologic grade of the confirmed cervical lesions. This CHIMUST sub-study was approved by the Ethics Committee of Peking University Shenzhen Hospital (IRB: PUSH2019044).Non-pregnant women aged 30 to 59 with an intact uterus, no history of pelvic radiation or damaging cervical treatment, and no cervical cancer screening for 3 years were enrolled in CHIMUST. All participants provided informed consent to participate in the trial and for the future use of their samples and related personal de-identified information.® solution  for HPV testing.At the first participation visit, each participant obtained a vaginal sample by herself (self-collected sample). Then, the participant was escorted to another room for physician sampling following the standardized sampling procedures  to obtain exfoliated cervical cells from the transformation zone and the squamo-columnar junction of the cervix. The physician-collected cervical sample was placed into a vial containing 20 ml of PreservCytBoth the self- and physician-collected samples were tested on the Cobas4800 and SeqHPV, while the physician-collected samples were also processed for cytology using ThinPrep by senior cytopathologists from PUSH . Participants who were positive for hrHPV on one or both of the testing assays (self- and/or physician-collected samples) were referred for colposcopy and biopsy plus endo-cervical curettage (ECC) .This study was conducted based on 8,856 physician-collected samples from the screening sites in Beijing, Hebei, Hubei, Jiangxi, and Shanghai. Among those samples, 1,434 were hrHPV positive on SeqHPV and/or Cobas4800, and 61 samples were HPV negative but with cytology ≥LSIL. Therefore, a total of 1,495 samples were considered to be “positive samples.” We included the cases abnormal by cytology only for fair validation of BMRT's effectiveness in detecting CIN2+ cases. The nested case-control sample included all the positive samples and doubled that number for the matched negative samples by women's ages, sampling site, and sampling time. A total of 2,990 samples negative for HPV and cytology (the negative samples) were randomly picked, making a total of 4,485 samples to be the “sample set” for BMRT testing .Cobas4800 is an HPV test based on real-time fluorescent PCR amplification technology. The specially provided equipment can automatically run the testing procedures, including sample preparation, DNA extraction, PCR amplification, and DNA detection for 14 hrHPV genotypes. It refers human β-globin gene in each run to monitor cell adequacy and to provide an internal control for positive and negative results. With four configured channels, Cobas4800 reports HPV-16, HPV-18, 12 types of hrHPV in pool , and β-globin. At the same time, it can report the CtV of each channel, which reflects the number of cycles of DNA amplification required in the PCR process to detect HPV DNA. The CtV is inversely correlated with the logarithm of the target DNA in the testing sample , 18. Thevia analyzing the linear relationship between the single-copy gene and the number of cells. The BMRT HPV test reports the viral loads for each of the 21 HPV subtypes. In this study, we refer to the viral loads of 14 hrHPV genotypes from BMRT to compare them with the CtVs of the same hrHPV genotypes from Cobas4800. The viral copy in 10,000 cells is reported together with the testing results of BMRT by genotype.The BMRT HPV test is also based on real-time fluorescent PCR amplification technology. It detects 21 HPV genotypes, including 14 high-risk HPV genotypes , 4 mid-risk HPV genotypes  and 3 low-risk HPV genotypes . It performs eight reactions in eight tubes simultaneously per sample, in which HPV-16/-18/-31, HPV-59/-66/-53, HPV-33/-58/-45, HPV-56/-52/-35, HPV-68/-51/-39, HPV-73/-26/-82, and HPV-6/-11/-81 are tested in groups in tubes A, B, C, D, E, F, and G, respectively, leaving tube H to detect human topoisomerase III (human TOP3), a single-copy gene within a single human cell. Analytic software is applied to calculate the number of viral copies per 10,000 cells in the HPV-positive samples  All patients who returned for diagnosis had colposcopy and biopsies. We followed a POI protocol: multiple biopsies were performed directly at the lesion site (s) plus randomly at the squamocolumnar junction in other quadrants without visible lesions and endocervical curettage (ECC) for all patients . Peking t-test was used to compare the two assays for the mean CtV-Cobas and VL (lg)-BMRT of the 14 hrHPV, HPV-16, HPV-18, and 12-HPV pool. Pearson's or Spearman's correlation coefficient (r) was applied to analyze CtV-Cobas and VL (lg)-BMRT relative to the histology diagnosis, and linear regression lines were fitted in the scatterplot. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.SPSS 25.0 software was used for all analyses. The consistency of BMRT and Cobas4800 for detecting hrHPV , HPV-16 (referring to cases positive of HPV-16 singly or multiple types including HPV-16), HPV-18 (referring to cases positive of HPV-18 singly or multiple types including HPV-18), and the other 12 types of hrHPV in the pool (12-HPV pool) (referring to cases positive of another type (s) singly or multiply) was tested by Kappa coefficients. Student's The average age of the 4,485 women included in this study is 45.4±7.2 years, with no significant difference from that of all the participants of CHIMUST. After excluding 189 cases that were positive for Cobas4800 and/or BMRT who did not return for colposcopy, two tests failed for Cobas4800, one test failed for cytology, and three had missing Cobas4800 data from the 1,495 HPV positives. A total of 1,300 positive cases were included in the analysis, with 2,990 negative cases, totaling 4,290 cases with complete data. Pathology diagnostics were performed on 1,258 cases, including 987 normal, 149 CIN 1, 74 CIN 2, 43 CIN 3, and 5 cervical cancers.Among the 4,290 cases, 807 were positive for Cobas4800 HPV testing, with a rate of 18.8% (807/4290), and 745 were positive for BMRT HPV testing, with a rate of 17.4% . Of the 807 Cobas4800 positives, 185 were positive for HPV-16 and/or−18, and 682 were positive for the 12-HPV pool. Among the 745 BMRT positives, 140 were positive for HPV-16 and/or−18, and 645 were positive for the 12-HPV pool. The consistency of genotypes from Cobas4800 and BMRT for hrHPV overall, HPV-16, HPV-18, and 12-HPV pool was 94.9% , 99.1% , 99.6% , and 95.3% , respectively, indicating good consistency of the two assays.n = 35), HPV-18 (n = 16), and 12-HPV pool (n = 119) are significantly higher, respectively. The mean VL (lg) of the cases negative for Cobas4800 but positive for BMRT for HPV-16 (n = 4), HPV-18 (n = 1), and 12-HPV pool (n = 82) is significantly lower, respectively. This indicates that the inconsistencies between the two assays are likely associated with the samples having comparatively lower viral loads.Further analysis of all the cases with inconsistent testing results between the two assays  showed tCobas4800 and BMRT detected 100% of the 48 CIN3+. Of the 122 CIN2+ cases, Cobas4800 and BMRT detected 115 (94.3% and 115/122) and 113 (92.6% and 113/122) cases, respectively, while among the 1,136 ≤CIN 1 cases, Cobas4800 detected 692  and BMRT detected 632 (55.6% and 632/1136) .HPV16 = −0.641, P < 0.001; r 12−HPVpool = −0.343, P = 0.109; In CIN2+, r HPV16 = −0.754, P < 0.001; r 12−HPVpool = −0.429, P < 0.001). Correlation analyses were not conducted for CIN2+ cases positive for HPV-18 because we have only 1 CIN3+ and 3 CIN2+ cases positive for HPV-18  from BMRT are highly correlated and follow linear regression. (Pearson's or Spearman's correlation coefficient (r): In CIN3+, r r HPV-18 .Our study shows that the Cobas4800 and BMRT assays consistently detect the viral load of hrHPV overall, HPV-16, and 12-HPV pool from the physician-collected samples. A possible reason for the good consistency might be that the two assays are both based on real-time fluorescent PCR amplification technology, which needs to be demonstrated further. There were only a few cases with inconsistent results between the two assays, of which 3  in the 141 Cobas (+) /BMRT (-) cases and 1 in the 79  Cobas (-) /BMRT (+) cases were diagnosed as CIN2. All those CIN2 cases were reported positive in the 12-HPV pool with low viral loads. This fact indicates that most CIN2+ cases and all the CIN3+ cases are related to a medium or high viral load level in the specimens. We need further follow-up to demonstrate whether CIN2 with low viral load may become potentially regressive. Supportive to those indications are the known facts that HPV DNA integrating into the human genome is a vital event in cervical carcinogenesis ; HPV-16 Inconsistent measurement of viral loads in previous studies has been a barrier to its clinical application for a referral. Many investigators believe that it is difficult to make the per-sample number of cells similar. Since the number of cells in a sample could influence the circulation time of DNA amplification to give a quantification of viral load on a semi-quantitative HPV testing , homogenWith the samples collected by physicians using a standardized sampling technology, our team demonstrated that CtV from Cobas4800 was diversely correlated to the grading of cervical lesions caused by HPV-16 and 12-HPV pools and published relevant papers  per unit cell from the BMRT were well correlated. CtV-Cobas merits additional clinical promotion and validation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee from Peking University Shenzhen Hospital and Cleveland Clinic Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.RW, LW, and JB conceived and designed the study. QY, HD, and XQ analyzed, interpreted the patient data and drafted the manuscript. WD, LG, CL, CW, CG, and YZ contributed to the data collection and quality control. All authors contributed to the article and approved the submitted version.The study was supported by the Shenzhen High-level Hospital Construction Fund (YBH2019-260), the Shenzhen Key Medical Discipline Construction Fund (Grant No. SZXK027), and the Sanming Project of Medicine in Shenzhen (Grant No. SZSM202011016).Author JB was employed by Preventive Oncology International, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-vaccinated women had a higher education (p = 0.0068) and were more frequently in a committed relationship or married (p = 0.01). The most trusted source of medical and vaccination information was healthcare providers (55.2%). The main reasons for vaccine acceptance were protection against HPV-related disease (93.4%), severity of preventable diseases (82.7%), HPV vaccine safety (66.8%), free HPV vaccine availability (62.8%), and the existence of vaccination recommendations (55.5%). The main reasons for refusing vaccination were the need for additional vaccine-related information (31.4%) and vaccine safety concerns (29.4%). To increase vaccine coverage, information about the benefits and safety of HPV vaccination must be widely disseminated to all health professionals and the general public. We are convinced that the knowledge obtained in this study can be reliably applied to other countries in the region that lack such information and have a very high cervical cancer burden.HPV immunization programs are mainly focused on girls and boys, but adult women and men could also benefit from vaccination. A multinational CoheaHr-WP4 study investigated the acceptability of HPV vaccination among 25–45 years old women. A total of 607 women from Slovenia participated in the study, and 49.6% (301/607) agreed with HPV vaccination, with a significant difference ( Cervical cancer, which is the fourth most common cancer in women worldwide and led to more than 340,000 deaths in 2020, is caused by persistent human papillomavirus (HPV) infection . Two revThe first HPV vaccine—the quadrivalent HPV6/11/16/18 Gardasil vaccine —was licensed in Europe in 2006, followed by the bivalent HPV16/18 Cervarix vaccine  in 2007, and finally by the nonavalent HPV6/11/16/18/31/33/45/52/58 Gardasil 9 vaccine  in 2015. Since 2006, the great majority of European countries have implemented HPV vaccination in their national vaccination programs  and laryIn Slovenia, a central European country with a population of 2.1 million, the crude incidence rate of cervical cancer was between 8.3 and 12.4 per 100,000 women between 2017 and 2020, and it has significantly decreased from 20.7 per 100,000 women in 2003 when the National Cervical Cancer Screening Program (NP ZORA) was implemented . This orIn addition to girls and boys targeted in the great majority of national vaccination programs worldwide, adult women and men could also benefit from HPV vaccination . Women aThe COHEAHR-WP4 study was part of the COHEAHR project funded under the European Commission FP7 Framework Health 2013 Innovation 1 initiative . Participating countries obtained national ethical and regulatory approvals. In Slovenia, the study was approved by the National Medical Ethics Committee (consent no. 0120-657/2015-2-KME 81/12/15) and by the Agency for Medicinal Products and Medical Devices of the Republic of Slovenia (consent no. 1050-17/2016-3).For the Slovenian part of the COHEAHR-WP4 study, two gynecologists from different regions—Litija (Center 1) and Nova Gorica (Center 2)—recruited women to participate in the study between August 2016 and October 2018. Both gynecologists consequently invited women 25 to 45 years old with no previous history of HPV vaccine administration that attended the routine organized national screening program until they reached the target number of participants. As in other countries participating in the COHEAHR-WP4 study, the same core protocol and data collection forms were used. Before the COHEAHR-WP4 study launch, the questionnaire was validated in a small pilot study. The women were given a brochure with information about cervical cancer, HPV, the HPV vaccine, and the aims of the study, as well as a detailed summary of the study protocol, and they were invited to participate in the first part of the study  targeting nine HPV genotypes: HPV6/11/16/18/31/33/45/52/58. Exclusion criteria for vaccination were the following: known history of severe allergic reactions or hypersensitivity to any of the vaccine components; known history of immune-related disease; current acute severe febrile illness; administration of immunoglobulin or blood-derived products within six months before the planned first dose of HPV vaccine; pregnancy or planning to become pregnant in the following 12 months; and women that had undergone hysterectomy.Study data were collected and managed using REDCap (Research Electronic Data Capture), a secure web-based application designed to support data collection for research studies. It provides (i) an intuitive interface for validated data entry, (ii) audit trails for tracking data manipulation and export procedures, (iii) automated export procedures for seamless data downloads into common statistical packages, and (iv) procedures for importing data from external sources .t-test for continuous data available in the open-source program OpenEpi 3.01 (http://www.openepi.com/Menu/OE_Menu.htm (accessed on 13 May 2019). Values p ≤ 0.05 were considered statistically significant.Statistical significance tests were performed using the mid-P exact test for categorical data and the A total of 607 women—363 (59.8%) at Center 1 and 244 (40.2%) at Center 2—met the inclusion criteria and decided to participate in the study. Of these, 306 (50.4%) participated only in the first part of the study (the anonymous questionnaire) and 301 (49.6%) participated in both study parts  was found in the educational level between the vaccinated and non-vaccinated, with the non-vaccinated having a higher educational level. A statistically significant difference was also found in marital status, with more non-vaccinated women being in a committed relationship or married. Of all participants, 80.6% (489/607) were in a committed relationship or married, whereas 17.1% (104/607) were single or divorced, and 0.5% (3/607) were widowed. According to the questionnaire data, 3.3% (20/607) of participants had already had anogenital warts. The great majority  of the participants enrolled had previously attended cervical cancer screening, 76.4% (464/607) attended screening regularly, and 72.2% (438/607) had had more than two cervical cancer screenings in their lifetime. Significantly more non-vaccinated individuals had previously participated in cervical cancer screening (p = 0.0077). Reasons for not attending cervical cancer screening regularly included lack of time , discomfort with the procedure , misinformation or lack of information on the need for periodic cervical screening , economic reasons (including work leave and travel costs) , and other reasons . Only 1.6% (9/575) of participants had a history of medical problems linked with any previous (non-HPV) vaccinations, with no significant differences observed between HPV vaccinated versus HPV non-vaccinated in this study. Of all participants, 78.4% (476/607) of participants have correct knowledge that cervical cytology or a Pap test is the main tool used in cervical cancer screening in Slovenia, and 27.5% (167/607) wrongly thought that HPV testing is the main screening tool in Slovenia. Interestingly, significantly more HPV vaccinated versus HPV non-vaccinated in this study thought that HPV is used as the main cervical cancer screening tool in Slovenia (p = 0.0001).As shown in p = 0.0071) between HPV vaccinated and HPV-non-vaccinated participants, with HPV-vaccinated participants more likely to have heard of the HPV vaccine previously. Participants’ knowledge and opinions about the HPV vaccine were assessed using several questions in an anonymous questionnaire. Most participants that answered the question “Before reading the information provided earlier, had you ever heard about HPV vaccines?”  had heard of the HPV vaccine previously, with a statistically significant difference  and 50.8% (124/244) agreed at Centers 1 and 2, respectively . Fewer pThe most important vaccine-related issues relevant to improving current HPV vaccination coverage in Slovenia’s general population were the following: recommendation by the primary care physician or gynecologist, need for more information or studies on vaccine safety and health benefits, additional information before making a decision, better vaccine efficacy, and inclusion in the national vaccination program or public funding for vaccination. Less relevant were lower cost per dose, two instead of three doses of vaccine, and potential reduction in the number of future cervical cancer screening visits after HPV vaccination .Participants were also asked to indicate the reason(s) for accepting or refusing HPV vaccination. Most women accepted HPV vaccination in this study because they believed that the HPV vaccine protects against cervical cancer or genital warts , because cervical cancer is a serious disease , because the HPV vaccine is safe  or offered for free , and finally because it is recommended to get the vaccine ; an additional 3.3% (10/301) stated other reasons for accepting HPV vaccination. On the other hand, the reasons for refusing HPV vaccination in this study were a need for more information , concerns about HPV vaccine safety or side effects , a need to consult other people or concerns about whether the HPV vaccine works and how long protection lasts , dislike of vaccines in general , a feel that they might not benefit from HPV vaccine protection , and lack of time or personal advice not to get vaccinated . Nearly 6%  refused to answer the question, and 3.6% (11/306) stated other reasons for refusal.The clinical part of the questionnaire completed by gynecologists recorded the cervical cytology, colposcopy, histology, and HPV results obtained during the last previous cervical cancer screening visit by the HPV-vaccinated women. Of 301 HPV-vaccinated women, 264 (87.7%) had normal cytology results, 22 (7.3%) had atypical squamous cervical cells of undetermined significance (ASC-US), six (2.0%) had a high-grade squamous intraepithelial lesion (HSIL), four (1.3%) had a low-grade squamous intraepithelial lesion (LSIL), two (0.7%) had atypical squamous cells for which HSIL (ASC-H) could not be excluded, and one (0.3%) had glandular abnormalities. In two women (0.7%), the cytology result was not obtained.Adverse events after HPV vaccination were reported by 13.3% (40/301) of women vaccinated in this study, and a total of 45 events were reported. The most adverse events were of low intensity , mainly pain, swelling and redness at the injection site, headache, and fatigue, lasting from a few days to a month after vaccination. There were two reported cases of moderate adverse events , but neither the women affected required drug treatment or hospitalization.Vaccination against HPV as a tool for primary prevention of cervical cancer has dramatically expanded the world’s potential for the elimination of cervical cancer and most probably other HPV-related neoplasms in the anogenital and head and neck areas. In the 16 years since its introduction, HPV vaccination has seen many positive developments such as a reduced number of doses and more flexible schedules, which have reduced costs significantly and facilitated program implementation and gradual program expansion to multiple-age cohorts and both sexes . All thrThis study assessed the acceptability of HPV vaccination by adult women in Slovenia and identified determinants of uptake and completion of the vaccination schedule for the first time. Although it was performed in a relatively small country, we are persuaded that knowledge gained in this study can be reliably applied to other similar countries in central and eastern Europe, some of which have the highest burden of cervical cancer in Europe, in terms of both incidence and mortality. Despite the fact that (i) only 1.6% of participants had a history of medical problems linked with previous (non-HPV) vaccinations, (ii) more than 90% of participants thought that cervical cancer is a serious medical condition, and (iii) we offered the HPV vaccine at no cost, more than half of eligible women refused to be vaccinated against HPV. Because the market price for three-course vaccination in Slovenia  ranges from EUR 180 to 300, we deemed that offering vaccination at no cost would be an important incentive for vaccine uptake, which did not turn out to be the case. However, as expected, the acceptance rate of HPV vaccination among adult women (49.6%) observed in this study mirrored the coverage rates Slovenia achieved in the national HPV vaccination program for girls attending the sixth grade of primary school in 2016–2018 (46.4%–49.5%) . AlthougOne of the main factors when deciding to accept the vaccine or not is informed knowledge about the vaccine, which was also shown in this study because the great majority of vaccinated participants had previously heard about the HPV vaccine and many of those that refused vaccination stated that they needed more information and counseling. Interestingly, although up to 80% of women believed that the HPV vaccine prevents cervical cancer, significantly fewer at both centers were confident that the nonavalent HPV vaccine prevents genital warts. Many of the participants were also unaware that adult women and men can also be vaccinated against HPV, although the efficacy of the HPV vaccine in these cohorts has been demonstrated in large clinical trials ,30,31. TIn this study, women that refused vaccination had significantly higher educational attainment, similar to other countries participating in the COHEAHR-WP4 study as well as in other studies from France, Italy, and Norway ,33,34. TIn addition to the lack of reliable and trusted information, the second most important factor influencing the decision to refuse vaccination in this study was safety concerns. Approximately half of the participants challenged the safety of the HPV vaccine. In addition, approximately one-tenth of participants refused vaccination because they disliked vaccines in general, although only 1.6% of participants reported that they had a history of medical problems linked with previous (non-HPV) vaccination(s). General vaccine aversion and low confidence in the safety of the HPV vaccine specifically have also been previously identified as major factors in the low uptake of the HPV vaccine . HoweverAlthough this study was not sufficiently powered to appropriately address potential adverse events of the HPV vaccine, most of them were mild, with pain, redness, and swelling at the injection site most frequently reported. In addition, there were two reported moderate adverse events, but neither of the women required any medical treatment or hospitalization.This study has some limitations: (i) it was conducted in only two selected centers, which may not be representative of the entire country; (ii) the recruitment of study participants in the healthcare centers may have overrepresented women with higher concerns for their health and/or women interested in free HPV vaccination may have been referred by other study participants; (iii) the limited study size.This study has shown that, despite offering the HPV vaccine at no cost due to various reasons (the major being safety concerns), more than half of Slovenian women 25 to 45 years old refused to be vaccinated against HPV. To improve vaccination coverage among all people that would benefit from HPV vaccination, including adult women, it is necessary to disseminate information about the benefits and safety of HPV vaccination to the widest possible range of health professionals and to ensure that their knowledge is continuously updated. Concurrently, trusted information about HPVs, HPV-related diseases, HPV vaccines, the proven safety and efficacy of these vaccines, and the opportunity to protect adult women and men through HPV vaccination should be widely disseminated to the general public using well-planned educational campaigns and all available communication channels.


The Chinese government has taken action to prevent cervical cancer by implementing the National Cervical Cancer Screening Programme in Rural Areas (NACCSPRA), which was launched in 2009. Numerous studies have demonstrated that long-term cervical cancer screening alters human papillomavirus (HPV) infection rates and cervical disease detection. Nearly 80 million women have been screened over 10 years, representing <30% of the target population; however, in some rural areas, such as Ordos City of Inner Mongolia Autonomous Region, Xiangyuan County of Shanxi Province, and Jinyun County, and Jingning County of Zhejiang Province, programs for prevention and treatment of cervical cancer have been implemented. Numerous studies have demonstrated that long-term cervical cancer screening alters rates of human papillomavirus (HPV) infection and cervical disease detection. In this study, we aimed to determine the infection rates of high-risk HPV (hrHPV) and the detection rate of cervical lesions; and changes in factors associated with cervical cancer, to provide scientific data to inform efforts to eliminate cervical cancer in rural areas.This was a cross-sectional, population-based, and multi-center survey. Populations from three rural areas of China  were selected and 9,332 women aged 20–64 years old were invited to participate in cervical cancer screening by both cytology and HPV testing. The outcomes assessed were: infection rates with hrHPV, HPV16, 18, 16/18, and other 12 hrHPV types ; detection rates of cytological and histological lesions; and factors associated with HPV infection.P < 0.05). Infection rates with hrHPV, 16, 18, 16/18, and the other 12 hrHPV types showed a single peak infection mode, with a peak age of 56–65 years old. Age, marital status, number of live births, education level, reproductive disease history, and a history of alcohol consumption were risk factors for hrHPV infection. The detection rate of cytological abnormalities was 12.98% in the study and was higher in women older than 56 years old. The detection rates of cervical intraepithelial neoplasia CIN2+ and CIN3+ in the population were 1.45% and 0.77%, respectively. The highest incidence rates of CIN2+ and CIN3+ were 32.12% and 17.51%, respectively, in the 41–45 years old group.A total of 9,217 women aged 45.62 ± 8.02 years were included in this study. Infection rates with hrHPV, HPV 16, 18, 16/18, and other 12 hrHPV types were 16.3%, 3.0%, 1.5%, 4.3%, and 13.6%, respectively. There were significant differences among the age-specific HPV infection rates (Infection rates with hrHPV, HPV16, and cervical lesions among our screening population were lower than the mean level in rural areas of China. Infection rates with hrHPV, HPV16, 18, and 16/18 showed a single-peak infection pattern, with the peak age of infection being 56-65 years old. Risk factors for hrHPV infection were age, history of alcohol consumption, marital status, reproductive diseases, education level, and the number of live births. Based on these data, we recommend that cervical cancer screening be offered to women older than 30 years in rural areas, particularly those aged 41–45 years. Cervical cancer is ranked third among gynecological malignancies in terms of both estimated new cases and deaths of women worldwide. An estimated 604,000 new cervical cancer cases and 342,000 deaths were reported globally in 2020 . In ChinThere has been heavy investment in cervical cancer prevention and control in China in recent years; however, the goal of eliminating cervical cancer, especially in rural areas, remains some way from being achieved . The ChiHPV infection rates, particularly the HPV 16 infection rate, were altered by the implementation of cervical cancer screening measures after ten years follow-up . The preThis was a multicenter, population-based, and cross-sectional study conducted in rural areas of China from 2016 to 2019. Three rural areas were chosen based on their high incidence rates of cervical cancer, including the Ordos, Inner Mongolia (Hang jin banner and Yi jinholo banner), Shanxi Province (Xiangyuan Country), and Zhejiang Province (Jinyun County and Jingning County). A total of three tertiary hospitals and five maternal and child health hospitals were selected. Ethics approval was obtained from the Cancer Hospital, Chinese Academy of Medical Sciences (No. 16-013/1092), and the study was approved by all institutional review boards of the participating hospitals.In the initial stage of the study, 9,332 women participated, while 9,217 eligible women aged 20 to 64 years who lived in villages or sub-districts participated in the questionnaire survey, gynecological examination, and laboratory testing. All eligible women provided informed consent before enrolment. The investigators carried out procedures, inspecting whether the women complete the questionnaires or met the inclusion and exclusion criteria .The survey was conducted face-to-face by trained interviewers. If patients had difficulty reading and completing the scales, trained interviewers helped them read and explain, or family members helped them answer questions. Information collected included: demographic ; and other factors, including a history of disease, pregnancy, reproduction, lifestyle , contraceptive measures, menopausal status, and sexual behavior. A strict quality control scheme was adhered to throughout the entire investigation process, including data collection, filing, entry, and checking, revision, and data security. The trained interviewers checked the questionnaires immediately on completion to avoid missing items and logical errors. If the questionnaires had missing items or obvious logical mistakes (such as missing items and errors), the trained interviewers called the patient to amend them and check the information. All procedures were performed by trained local physicians, while the materials for and results of cytology and hrHPV analyses were provided by central hospitals.All women were tested by cytology and for hrHPV using Thin Prep medium and the Cobas 4,800 test (Roche Diagnostics). Women positive for either HPV 16/18 or other HPV genotypes with positive cytology results were deemed to have screened positive; colposcopy and biopsy of suspicious lesions were performed if necessary. Women positive for other HPV genotypes and negative on cytology, and those negative for HPV genotypes, were deemed to have screened negative. The results of cytology show atypical squamous cells of undetermined significance (ASCUS) or higher , low-grade intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and atypical glandular cell (AGC), among others) indicated the need to undergo colposcopy and biopsy of the suspicious lesions. Women negative for intraepithelial lesion or malignancy (NILM) were screened negative, with follow-up observation to be carried out in 3 years.According to CIN terminology, CIN was diagnosed as one of four stages: NILM, CIN grade 1 (CIN1), CIN2, CIN3; and cervical cancer as micro-invasive carcinoma, invasive carcinoma, and others.Precancerous lesions diagnosed by cytology were classified into four stages: NILM, ASCUS, LSIL, and HSIL+.2 test was performed to compare proportions in subjects with specific characteristics and incidence rates of hrHPV, HPV16, 18, 16/18, and another 12 high-risk HPV types  and cytology. Linear trend tests were used to compare infection rates with total hrHPV, HPV16, 18, 16/18, and the 12 other hrHPV genotypes; detection rates of abnormal cells; and detection rates of cervical precancerous lesions, according to age group.A database was established using Microsoft Access 2007 software. Statistical analyses were performed in SPSS, version 28.0. A χP < 0.001) , 41–45 years old (19.5%), and 56–60 years old (9.4%), respectively. There were significant differences in age distribution, marital status, and educational level (< 0.001) .P < 0.05), with the peak age of infection at 56–65 years old (P < 0.05).There was no significant difference in infection rate of HPV 18 among the selected areas. Comparing the age groups, the infection rates of hrHPV and HPV 16/18 were statistically different among age groups in Inner Mongolia Autonomous Region and Shanxi Province (P < 0.05), except for the infection rates of HPV16. While infection rates of HPV16, 18, 16/18, and other types in the Zhejiang Province were statistically significant (P < 0.05), with the peak age of infection at 61–65 years old.Among the women screened for cervical cancer, the positive rates for the hrHPV types, 16, 18, 16/18, and others (12 high-risk types) were 16.3%, 3.0%, 1.5%, 4.3%, and 13.6%, respectively. Further, the infection rates of HPV16, 18, 16/18, and other types in the total population differed significantly among age groups (ears old . The infP > 0.05). However, the hrHPV infection rate differed significantly according to age at first pregnancy, age at first delivery, number of births and live births, method of contraception, menopause status, and the number of sexual partners (P < 0.05). Infection rates were higher in women who were younger at first pregnancy and delivery, had given birth 3–4 times, had 3–4 live births, had disease history, had ≥ 2 sexual partners, and used other method of contraception   .P < 0.0001], education level , number of births , marital status , number of live births , age at first delivery , and alcohol consumption history .Seven factors were candidate predictors that were associated with hrHPV infection on univariate analyses. HrHPV infection rates differed significantly according to age [odds ratio (OR) = 1.124, Multivariate analysis confirmed that five variables were independently associated with hrHPV infection, including age, marital status, number of live births, education level, and alcohol consumption history .n = 8,020), inconclusive (n = 45), ASCUS , LSIL , HSIL , AGC , ASC-H , and cervical cancer . ASCUS or above was not detected in the 21–25 year-old age group, and the detection rate of abnormal cells differed significantly among age groups (P < 0.05), with the highest in women aged > 56 years  , 66 cases of CIN2 , 68 cases of CIN3 , 3 cases of cervical cancer, and 3 cases of vulvar intraepithelial neoplasia among those women with abnormal histological findings. The overall CIN2+ detection rate was 1.45%, of which 0.04%, 0.33%, 0.48%, 0.24%, 0.22%, and 0.17% were in the 21–30, 31–40, 41–45, 46–50, 51–55, and > 56 years age groups, respectively; there was an overall CIN3+ detection rate of 0.77%, with 0.02%, 0.12%, 0.26%, 0.26%, 0.26%, and 0.12% in each age group, respectively. CIN 2/3+ was not detected in women aged 21–25 years, and the highest CIN 2/3+ detection rates were found in the 41–45 years age group, accounting for 32.12% and 17.51% of all cases respectively. The detection rate of abnormal cervical histology differed significantly among the different age groups ( < 0.05) .HrHPV infection is closely associated with genital warts and penile, anal, oropharyngeal, and cervical cancers. The elimination of HPV-related cancers, particularly cervical cancer, has attracted global attention as a public health problem . The WHOIn our study, we found that the hrHPV infection rate was 16.3% in the screened population, which was consistent with the findings of Zhao et al. regarding the national screened population but lower than rates reported in other parts of the world, such as Africa 20.9%–23.4%) , 18. Com.9%–23.4%P < 0.05), with the peak age of infection at 56–65-years-old in the total mount, Inner Mongolia, Shanxi Province and Zhejiang Province. These findings are inconsistent with the double peak HPV infection rate phenomenon previously reported in rural areas of China   and the We found three cases of vulvar intraepithelial neoplasia, which were not analyzed according to age group; however, rates of abnormal cervical tissue differed among age groups. The detection rates of CIN2+ and CIN3+ were 1.45 and 0.77%, respectively, which were lower than those previously published in the literature for rural areas , 41. TheThis study has some strengths. The study population was large and a multicenter design was applied, using HPV genotype testing for cervical cancer screening in minority or rural areas of mainland China. To confirm the effects of HPV infection, we chose doctors at local hospitals to perform all screenings, permitting assessment by a superior hospital. Cervical cancer screening has been implemented in rural areas of China for more than ten years; however, the associated risk factors, HPV infection rates, rates of abnormal cervical cytology, and rates of abnormal histology and precancerous lesions in these areas are unclear, and elimination of cervical cancer by screening and prevention programs is expected. Second, our data provide a reference for real-world assessment of the effects of prevention and treatment programs for cervical cancer in rural areas of China.The study also has some limitations. There was an inevitable loss to follow-up, with a rate of 18.74% in this study; however, analysis of the basic characteristics, main risk factors, and main outcome indicators of the lost population demonstrated that it is unlikely to have influenced our analyses. Furthermore, differences in the cytology, histology, and diagnosis findings of gynecologists in different regions may have had certain impacts on our results.The rates of hrHPV and HPV16 infections and cervical lesions in the screened population included in this study were lower than the mean rates in rural China. The infection rates of hrHPV, HPV16, 18, and 16/18 showed a single-peak infection pattern, with a peak infection age of 56–65 years. Risk factors associated with hrHPV infection were: age, history of alcohol consumption, marital status, reproductive diseases, education level, and number of live births. Cervical cancer screening is not recommended for women aged 21–25 years in rural areas, while women aged 26–30 years may decide to undergo screening, according to their economic status. Older women (>30 years old), particularly those aged 41–45 years, are recommended to undergo cervical cancer screening.The original contributions presented in the study are included in the article/Central Ethics Approval was obtained from the Cancer Hospital, Chinese Academy of Medical Sciences (No. 16-013/1092). The patients/participants provided their written informed consent to participate in this study.Y-QY, M-YJ, R-MF, MB, WC, and Y-LQ participated in study design, data analysis and visualization, validation of the entire study, and preparation of the manuscript. Y-QY and M-YJ conducted data collection and supervision. Y-QY analyzed the data and write the article. All authors read and approved the final manuscript.The study was funded by Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Nos. 2017-I2M-B&R-03 and 2021-I2M-1-004). The authors declare that this study received funding from Roche company. Roche was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.Y-LQ and WC received grants from the Ministry of Science and Technology of the People's Republic of China during the conduct of the study, and personal fees and non-financial support from Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.