DdPCR and qPCR data from clinical samples were compared. The assay showed high accuracy, sensitivity and specificity, with low intra-/inter- run variations, in detecting/quantifying HPV16/18/11/45 DNAs. HPV DNA was detected in 51.1% (23/45) CIN DNA samples by ddPCR, whereas 40% (18/45) CIN tested HPV-positive by qPCR. Five CIN, tested positive by ddPCR, were found to be negative by qPCR. In CIN specimens, the mean HPV DNA loads determined by ddPCR were 3.81 copy/cell (range 0.002–51.02 copy/cell), whereas 8.04 copy/cell (range 0.003–78.73 copy/cell) by qPCR. DdPCR and qPCR concordantly detected HPV DNA in SiHa, CaSki and Hela cells, whereas HaCaT tested HPV-negative. The correlation between HPV DNA loads simultaneously detected by ddPCR/qPCR in CINs/cell lines was good . Our data indicate that ddPCR is a valuable technique in quantifying HPV DNA load in CIN specimens and human cell lines, thereby improving clinical applications, such as patient management after primary diagnosis of HPV-related lesions with HPV-type specific assays.Human papillomaviruses (HPVs) are small DNA tumor viruses that mainly infect mucosal epithelia of anogenital and upper respiratory tracts. There has been progressive demand for more analytical assays for HPV DNA quantification. A novel droplet digital PCR (ddPCR) method was developed to simultaneously detect and quantify HPV DNA from different HPV types. DdPCR was initially tested for assay sensitivity, accuracy, specificity as well as intra- and inter-run assay variation employing four recombinant plasmids containing HPV16, HPV18, HPV11, and HPV45 DNAs. The assay was extended to investigate/quantify HPV DNA in Cervical Intraepithelial Neoplasia (CIN,  Human papillomaviruses (HPVs) are small DNA tumor viruses that mainly infect mucosal epithelia of anogenital and upper respiratory tracts . HPVs caThese viruses replicate in the nucleus of the basal layer cells of epithelia. In these cells, the HPV DNA is maintained in an episomal form with a low-copy number . HoweverHPV16 and HPV18 types are the most carcinogenic among HR-HPVs. Indeed, these viruses are linked to about 70 and 60% of all uterine cervical cancers and their pre-malignant lesions, the cervical intraepithelial neoplasia (CIN), respectively . To a leQuantitative real-time PCR (qPCR) has been widely employed due to (i) its use in HPV DNA load quantification, (ii) its broad detection-range of target molecules and (iii) multiplexing potential. However, qPCR has several intrinsic limitations, including: (i) low sensitivity in quantifying the amount of viral DNA when present in a low-copy number ; (ii) laDroplet digital PCR (ddPCR) is an innovative technology, which provides an alternative method for nucleic acids quantification . This tePrevious studies reported on HPV DNA detection/quantification employing ddPCR methods . HoweverThese methodological limitations prompted us to develop a reliable ddPCR method, which allows the simultaneous detection and quantification of DNA sequences from different HPV types. The sensitivity, accuracy, specificity as well as intra- and inter-run assay variation of the ddPCR were initially assessed in HPV DNAs, from HPV16, HPV18, HPV11, and HPV45 types cloned in recombinant plasmids, used as a control . Then, t•NC_001526.4), HPV18 , HPV11 , and HPV52 .pUC19 recombinant plasmid vectors containing the complete genomes of HPV16 .pGEM1 recombinant plasmid vector containing the complete genome of HPV45 . Catalog number: #ER0051.•EcoRI . Catalog number: #ER0271.Restriction enzyme •BamHI-Lsp1109I Buffer (10X) . Catalog number: #B57.•EcoRI Buffer (10X) . Catalog number: #B12.•® HTB-35TM), CaSki (ATCC® CRM-CRL-1550TM), HeLa (ATCC® CCL-2TM) and HaCaT (Elabscience®), Catalog number: #EP-CL-0090.Cell lines: SiHa . Catalog number: #69504.•Salmon-sperm DNA . Catalog number: #AM9680.•TM AutoDG Droplet Digital PCR System-Bio-Rad . Catalog number: #1864100.QX200•TM ddPCRTM EvaGreen Supermix . Catalog number: #1864035.QX200•TM Automated Droplet Generator Cartridges . Catalog number: #1864108.DG32•Automated Droplet Generation Oil for EvaGreen . Catalog number: #1864112.•TM PCR Plate Sealer . Catalog number: #1814000.PX1•TM . Catalog number: #A24811.Thermal cycler SimpliAmp•EIF2C1 gene . Catalog number: #10031258.•TM Real-Time PCR Detection System . Catalog number: #1855196.CFX96 Touch•TM Universal SYBR® Green Supermix, Bio-Rad . Catalog number: #1725271.SsoAdvancedNC_001526.4), HPV18 , and HPV11  along with one pGEM1 recombinant plasmid vector containing the complete genome of HPV45  were employed for the development of the ddPCR assay. These recombinant plasmids were employed as positive controls (X74481) was employed as negative control. The complete HPV16/18/11 genomes were kindly provided by Prof. Pozzi, Department of Biotechnology, University of Siena, Italy and cloned in pUC19 vectors  (9 copy/μL) were serially 10-fold diluted before subsequent ddPCR/qPCR analyses. Linear DNA templates of HPV16/18/11/52 strains were obtained by digestion of the pUC19_HPV16, pUC19_HPV18, pUC19_HPV11, and pUC19_HPV52 recombinant plasmids with Bam H1 , whereas the linearization of HPV45 was obtained by digestion of the pGEM1_HPV45 recombinant plasmid with EcoRI . Restriction enzyme digestions were performed in a total reaction mixture of 20 μL, including 2 μL 10 × enzyme buffer, 1 μL of enzyme, 500 ng plasmid DNA, and ddH2O up to 20 μL. The mixture was incubated for 2 h at 37°C, followed by 20 min at 80°C. for the enzyme inactivation. Digestions were analyzed on 0.8% agarose gel  . The pGErose gel .n = 45) were isolated as described (2O) and a salmon sperm DNA specimen, which is a ready-to-use DNA solution, that is used to verify whether cross-contaminations can occur during DNA extraction and molecular analyses  Heescribed ; (iii) Hescribed . DNA wasescribed . To avoiescribed . In detaanalyses . No crosbin gene .+ 5′-TTTGTTACTGTGGTAGATACTAC-3′ and GP6+ 5′-GAAAAATAAACTGTAAATCATATTC-3′, which amplify the conserved HPV L1 genomic region of different HPV genotypes, including HPV16/18/11/45 were employed  were calculated , SiHa, CaSki, HeLa, and HaCaT cell lines were ddPCR-amplified using the QX200TM Droplet DigitalTM PCR System-Bio-Rad , according to the manufacturer’s instructions . Every sample was partitioned into ∼20,000 stable nano-droplets using an automated droplet generator . After processing, the entire prepared droplet emulsion (40 μL) was transferred into a 96 well PCR plate, covered with pierceable foil, heat-sealed using a PX1rmTM PCR Plate Sealer . Then, the plate was placed in a thermal cycler . The cycling conditions were as follows: heat to 95°C for 5 min, followed by 94°C for 30 s and a range of temperature between 46°C and 60°C for 1 min for a total of 45 cycles (at a heating rate of 2°C/s), followed by 5 min at 90°C, ending at 4°C. After PCR, the 96-well PCR plate was placed in the reader. Data were analyzed using the QuantaSoft analysis tool . Poisson statistics was used to calculate the absolute concentration of HPV DNAs in each sample , SiHa, CaSki, HeLa, and HaCaT cell lines were qPCR-amplified for the detection/quantification of HPV DNA, using the SYBR green chemistry, with the CFX96 TouchTM RT-PCR Detection System . Two different recombinant plasmids pUC19 containing the complete genomes of HPV16 and HPV18 were employed as positive controls. DNA samples and plasmid vectors were analyzed in qPCR by using the GP5+/GP6+ primer set, as reported (2O). Samples were run in triplicate for each qPCR assay. Experiments were run in triplicate by three different operators.DNAs from CIN specimens  was used to assess ddPCR accuracy and consistency between the ddPCR and qPCR for quantitative HPV DNA detection in samples  and negative (gray) controls  diluted linearized pUC19/pGEM1 plasmids containing the complete genomes of HPV16/18/11/45 types . Each dilution was tested by ddPCR in triplicate. The theoretical/detected values were converted into logarithms and compared. Results indicate a good degree of linear correlation with the dilutions of all tested plasmids, which met the requirements of the ddPCR accuracy. In detail, the ddPCR method presented high accuracy for detecting genomes belonging to HPV16/18/11, with R2 of 1 and P = 0.0001, whereas HPV45 DNA was detected with good accuracy, with R2 of 0.9999 and p = 0.0001 , SiHa, CaSki, HeLa, and HaCaT cell lines. HPV DNA was detected in 40% (18/45) CIN with both assays, whereas an additional 5 CIN, which resulted positive for ddPCR, tested negative in qPCR (The presence of HPV DNA was investigated by ddPCR and qPCR in CIN specimens ( in qPCR . Specifi in qPCR . In celln = 18) and with ddPCR alone (n = 23), respectively. Quantitative results obtained by qPCR in CIN (n = 18) indicate that the mean HPV DNA load was 8.04 copy/cell (range 0.003–78.73 copy/cell). The mean HPV DNA load detected in SiHa and CaSKi cell lines by ddPCR was 3 and 493.9 copy/cell, respectively, whereas HeLa cell line harbored 30 HPV copy/cell. QPCR data indicate that SiHa and CaSki cell lines harbored 4.66 and 424.17 HPV copy/cell, whereas 46.64 HPV copy/cell were detected in HeLa cell line. Quantitative results obtained by ddPCR/qPCR in HPV-positive samples (n = 21), including CIN (n = 18) and cell lines (n = 3) were then compared between the two PCR assays  and 3.81 copy/cell (range 0.002–51.02 copy/cell) in CIN found to be HPV-positive with respective methods  three HR-HPVs, including HPV16 and HPV18, which are considered the most common oncogenic HR-HPVs; (ii) HPV45, which has been frequently found in cervical carcinomas and others malignancies of the anogenital/upper-respiratory tract  ory tract . Notablyry tract .+/GP6+ can be applied to ddPCR-based assays. The ddPCR was then tested on circular and restriction enzyme-digested linearized plasmids carrying the HPV16 genome . These qualitative/quantitative data are in agreement with previous studies on CIN lesions (Following ddPCR set up, as first approach on biological/clinical samples, we extended the assay on restriction enzyme-undigested DNA isolated from 45 CIN tissues and 4 human cells lines, including 3 HPV-positive cervical cancer cell lines. To this end, we considered a Cancer Genome Atlas study showing that HPV integration occurs in >80% of HPV-positive cervical cancers. Of these, 76% of HPV16-positive samples have integrated HPV, whereas integration is evident in all HPV18-positive samples . During  for HPV , whereas for HPV . When co lesions  and cerv lesions , thereby lesions . However lesions . In our  lesions . These r lesions . In summ lesions .The ddPCR method described in this pilot study may be potentially helpful for clinical purposes. Indeed, our ddPCR method might be of interest for patient management since it will allow, employing only one ddPCR run, to detect few copies of HPV DNA in clinical samples. Our primary aim of this investigation was the set up of this assay, which increases the sensitive HPV-related cancer-recognition methods. Indeed, undetectable low-copy HPV DNA could be an indication of integration into the human genome, which can be one of the carcinogenesis steps . On thisIn conclusion, in this study, the ddPCR assay exhibited high sensitivity, accuracy and specificity in quantifying HPV DNA sequences. In our experimental conditions, the method is repeatable and reproducible. The robustness of the ddPCR was also demonstrated through assay validation in CIN specimens and human cell lines. This ddPCR assay is a promising method that offers the potential to simultaneously quantify viral DNAs from different HPV types with a high analytical approach, thereby improving clinical applications, such as patient management after primary diagnosis of HPV-related lesions with HPV-type specific assays.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the University Hospital of Ferrara Ethical Committee. The patients/participants provided their written informed consent to participate in this study.JR and FM: conceptualization. JR, CM, LO-G, and CL: data curation, formal analysis, software, validation, and visualization. JR, MT, and FM: funding acquisition. JR, CM, LO-G, CL, and MI: investigation. JR and LO-G: methodology and roles, writing – original draft. MT and FM: project administration, resources, supervision, and writing – review and editing the final version. All authors contributed to the study conception and design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Archival tissue samples collected longitudinally from a patient who died from HPV16-induced high-grade anal intraepithelial squamous cell carcinoma with vertebral HPV16–positive metastasis were retrospectively analyzed by the Capture-HPV method (Capt-HPV) followed by Next-Generation Sequencing (NGS). Full length nucleotide sequences of the same HPV16 were identified from the initial and second anal biopsy samples, from plasma sample and from vertebral metastasis biopsy. Remarkably, HPV was episomal in each sample. The HPV genome sequence was closest to the HPV16 Qv18158E variant subtype (A1 lineage) exhibiting base substitutions and deletions in 7 and 2 HPV loci, respectively. In conclusion, the powerful Capt-HPV followed by NGS allows evidencing the detailed cartography of tumoral and circulating HPV DNA, giving rise to a unique and unexpected episomal virus molecular status in a context of aggressive carcinoma, underlying the importance of HPV status and its association with clinical features for further prospective studies. Although the natural history of human papillomavirus (HPV) infection in ASCC remains unclear, it has been shown that high-risk HPV16 was by far the most carcinogenic genotype for ASCC1.Despite being a rare cancer accounting for approximately 20,000 new cases each year globally, the incidence of anal squamous cell carcinoma (ASCC) has been increasing dramatically over the past decade in HIV-positive patientsversus integrated) as well as the integration sites in both HPV and human genomes2.New tools and biomarkers to assess the prognosis and to improve patient stratification as well as identifying therapeutic targets are needed. Recently, sensitive double capture-HPV method (Capt-HPV) followed by Next-Generation Sequencing (NGS) allowed the capture of 208 variants from 88 HPV genotypes and thus determining at once HPV genotype and nucleotide sequence, the molecular status , the patient hospitalized at Hôpital Européen Georges Pompidou (HEGP), Paris, presented vertebral HPV16–positive metastasis, associated with a paravertebral collection. The patient received first line palliative chemotherapy with docetaxel, cisplatin, and 5FU (DCF), for 6 months, leading to stability according to RECIST criteria4. Due to grade 3 asthenia, a second line chemotherapy with weekly paclitaxel and carboplatin was started. After 2 months, CT-scan imagery showed progression according to RECIST criteria, and the patient presented with an ECOG performance status of 3, leading to discontinuation of chemotherapy. He received supportive care for 4 additional months and died 12 months after the first round of chemotherapy. Witnessing this very aggressive and unexpected clinical presentation prompted us to evaluate the molecular status of the HPV16 detected in the collected samples from different origins using Capt-HPV and NGS, including the initial anal biopsy that diagnosed HGAIN, a 2-year post-diagnosis follow-up anal biopsy showing HGAIN recurrence, the vertebral metastasis biopsy, and the plasmatic HPV16 ctDNA.We previously reported the contribution of digital droplet polymerase chain reaction for early detection of HPV-induced aggressive metastatic anal cancer in an HIV-positive MSM Fig. 3. In bri2, but the present study is the first one to describe episomal HPV16 ctDNA in peripheral blood.We identified similar HPV16 sequences with full genome coverage in the initial and second anal lesions, the plasma sample, and the metastasis biopsy. This HPV sequence was classified into the A1 lineage. The closest variant in our reference data base was HPV16 Qv18158E (GenBank accession number: AY686583.1). The virus clearly appeared episomal in each sample, without any chimeric HPV-human genome reads potentially resulting from HPV integration in the human genome. De novo assembly of the NGS reads allowed circularizing the genome, confirming its episomal status  and 5140 (L2 gene) were silent at the protein level while the nucleotide deletions at positions 4184 and 4215 were located in the same intergenic region. No mutations in the E7 and E4 genes nor in the long control region were identified.Compared to the closest HPV16 Qv18158E genome reference, the present HPV genome analysis revealed 7 mutations. Namely, 2 base substitutions at nucleotide positions 176 and 350 were located in the Our observations report the comprehensive longitudinal molecular characterization of HPV DNA sequences from initial aggressive anal tumor to ctDNA extracted from plasma and deadly metastasis. The present study is the first one to describe episomal HPV16 ctDNA in peripheral blood. Indeed, and unexpectedly, the HPV status of each full length HPV16 sequence evidenced in tumor tissue samples as well as plasma ctDNA from study patient was episomal.. showed that the HPV16 E7 protein leading to cervical cancer was invariant5. Accordingly, we did not find any of the 32 mutations they identified in the E7 ORF from their control subjects. However, the E6-G350T found in our patient was associated with good prognosis in cervical cancer6, and none of the other mutations we found were previously described, underlying the need to aggregate HPV complete sequences associated with clinical features in order to correlate the presence of specific mutations with prognosis.Mirabello et al7. Using Capt-HPV, we also recently showed that 2 out of 4 patients with metastasis after an initial ASCC presented episomal HPV in their initial tumor, but no data were available regarding ctDNA nor metastasis, and de novo assembly was not performed to confirm the episomal status neither8. Our observation that episomal HPV can lead to metastatic progression challenges the widespread claim that tumors with integrated HPV are of worse prognosis than the ones with episomal HPV. Furthermore, HPV variants remained unchanged over time in this very aggressive metastatic ASCC, raising the central issue to which extent the cancer-associated HPV virus was functionally active during the advance of the disease and the therapeutic treatments or remained passive once the oncogenic process had started.Capt-HPV was used previously to confirm the metastatic nature of a tongue carcinoma in a patient but no ctDNA data were provided and the same signature for HPV integration was found in both the tumor and the metastasisThe present case report highlights that the availability of robust, affordable and reliable technologies such as Capt-HPV followed by NGS should facilitate the design of prospective studies where HPV status and its complete DNA sequence at baseline and/or during the follow-up would be associated with clinical data, especially to anticipate overall and progression-free survival and to offer the best adapted personalized treatment.9. Capt-HPV was performed using the SeqCap EZ library reagents (Roche NimbleGen)2. Illumina MiSeq systems were used to sequence the post capture libraries (150 bp paired-end reads). NGS data were automatically analyzed using a specific pipeline developed by the HEGP biomedical informatics team. HPV genotyping, HPV full length sequence, HPV molecular status, and positions of HPV-human junctions when present were obtained. To confirm the assumption that HPV was episomal when no integrations sites were found, we performed de novo assembly of the virus using the software Geneious . This software was also able to circularize the HPV genome if episomal, confirming its structure. The assembled genome was aligned against our 208 reference HPV sequences in order to identify the closest variant. The closest variant lineage was assessed by building a phylogenetic tree including the assembled genomes and 10 HPV16 A, B, C and D variant lineage reference sequences10.DNA extracts were obtained from formalin-fixed and paraffin-embedded anal and vertebral biopsies as well as from HPV16 circulating tumoural positive plasma, as describedhttps://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000032719520&categorieLien=id).Our report is an observational study, in which the biological analyses were carried out with the patient approval before his death. Their post-mortem interpretation and publication are not in the scope of an ethical committee, according to the French Jardé’s law on biomedical research (No animal was done for the study.


Human papillomavirus (HPV) is responsible for the rise in the incidence of cancer in the oropharynx, tonsils, and base of the tongue . HPV triggered the changes in the epidemiology of oropharyngeal and oral cavity cancer (OPC/OCC) in Asia, Europe, North America, and Oceania. Hence, the incidence of cancer in HPV-related subsites is augmenting, while that in other HPV-unrelated subsites is decreasing. In South America, although the incidence of HPV-positive tumors has gradually increased, there is an atypically low prevalence of HPV in people with OPC/OCC. To clarify whether this dramatic shift in incidence trends also occurred in this population, we estimated the burden of HPV on the incidence trends of OPCs/OCCs in São Paulo city in Brazil. In this population-based study, we categorized OPCs/OCCs by HPV-related and HPV-unrelated subsites. We used Poisson regression to assess the age-standardized incidence rates (ASRs) stratified by sex and age groups, as well as to examine the age-period-cohort effects. There were 15,391 cases of OPCs/OCCs diagnosed in HPV-related  and HPV-unrelated  subsites. Overall, the ASRs decreased for most subsites, for both sexes and for all age groups, except for HPV-related OPC/OCC in young males and females, which increased by 3.8% and 8.6% per year, respectively. In the birth-cohort-effect analysis, we identified an increasing risk for HPV-related OPC/OCC in both sexes in recent birth cohorts; however, this risk was sharply decreased in HPV-unrelated subsites. Our data demonstrate an emerging risk for HPV-related OPC/OCC in young people, which supports prophylactic HPV vaccination in this group. Human papillomavirus (HPV) is an oncogenic virus that is sexually transmitted, and it is the cause of an estimated 630,000 new cancers each year worldwide . There iIn the mid-2000s, there was a striking rise, from 40.5% to 72.2%, in the overall HPV prevalence of oropharyngeal tumors , OceaniaTo the best of our knowledge, no population-based study has focused on the burden of HPV associated with OPC/OCC incidence in Brazil. Prophylactic HPV vaccinations were introduced in 2014 in an effort to hasten a decrease in HPV prevalence, but its need in oral cavity and oropharyngeal subsites at the population level was unknown. For these reasons, we assessed the incidence trends of OPC/OCC in HPV-related and HPV-unrelated subsites over the 15 years prior to large-scale HPV immunization in São Paulo city.The Research Ethics Committee (nº 83218318.8.0000.5421) and the Technical Advisory Committee of the Population-based Cancer Registry in the city of São Paulo (RCBP-SP) approved our investigation. We accessed data on the RCBP-SP repository in July 2018 . All datThis population-based study included new cases of OPC/OCC diagnosed between 1997 and 2013 in the RCBP-SP database. In São Paulo city, the RCBP-SP records cancer data of more than 12 million residents from 301 sources, including hospitals, clinics, and death investigation services; this database is one of the oldest and largest cancer registries in Latin America .To investigate the potential role of HPV in the burden of OPC/OCC, we classified anatomical codes as a proxy for HPV exposure based on robust scientific evidence ,8,24–27 The incidence rates were further age-standardized per 100,000 persons using the direct method, the World Health Organization (WHO) standard population , and datFor the incidence trend analysis, we adjusted Poisson regression models by sex. The dependent variable was the annual incidence, and the explanatory variables were the HPV group (HPV-related or HPV-unrelated subsites), age group , and time (years). We used as offset the population size according to the age group and year. With the models, we estimated the incidence rates and the annual percent change  for each segment . To assess whether the trends were similar in different segments, we assessed contrasts under the general linear hypotheses .Additionally, we investigated the effects of age, calendar year (period), and birth year (cohort) on incidence rates with Poisson regression to measure the relative risk ,34. We cOver the entire study period, there were 15,391 new cases of OPC/OCC in São Paulo city. Of these cases, 5,898 occurred in HPV-related subsites (38.3%), and 9,493 occurred in HPV-unrelated subsites (61.7%). There was a marked male dominance in both HPV-related and HPV-unrelated subsites and all anatomical site groups . NeverthIn the temporal analysis, incidence trends decreased or remained stable for all anatomical sites, sexes, and age groups . For theWhen we compared the inclines (β1) with the contrasts under the general linear hypotheses, we found statistically significant differences among HPV-related and HPV-unrelated subsites for young females (p≈ 0.010) and males (p = 0.033). Additionally, the incidence trends were different between individuals aged ≤39 years and older age groups in both sexes for HPV-related subsites, which indicates a distinct pattern for HPV-related and HPV-unrelated subsites in the young population.In the age-period-cohort analysis, the incidence rates of HPV-related OPC/OCC increased in the youngest birth cohorts in both sexes, while it decreased in HPV-unrelated subsites . For HPVUnderstanding the HPV pattern over time is critical to support prophylactic interventions focused on diminishing its harmful impacts on populations. For this reason, we performed this study to determine the burden of HPV in OPC/OCC in São Paulo city, which is the largest city in Latin America. Overall, the incidence rates decreased for most OPCs/OCCs in both sexes in all age groups, and particularly in those with HPV-unrelated subsites. However, we identified a dramatically increasing incidence trend for HPV-related OPC/OCC in young females, emphasizing the highest risk in recent birth cohorts.Our study revealed a striking reduction in the overall incidence trends of OPC/OCC and an age-period-cohort effect in HPV-unrelated subsites. Since the 1990s, the Brazilian tobacco control policy has led to a decrease in smoking  and, conThe lung cancer incidence is decreasing worldwide, while the OPC incidence is increasing, which suggests that HPV infection is a reason for this growth . In thisA possible cause for the increased incidence of cancer at HPV-related subsites is the changing sexual behaviors . People Another potential reason for our findings is the prevalence of alcohol and tobacco use by the individuals with cancer in HPV-related subsites. Previous studies have reported an HPV infection rate of 6.2% in a healthy Brazilian population , and theAlthough our results are not sufficiently robust to indicate a viral epidemic in São Paulo city, they highlight the need to monitor the burden of these cancers in young populations. Considering that cancer is a time-dependent disease, we expected stable trends for individuals aged ≤ 39 years, but we found an upward trend in HPV-related OPC/OCC in young individuals. In addition, the remarkable decrease in smoking may be masking the increasing incidence in the HPV-related subsites, as this tumor classification occasionally includes tobacco-related cancers. For this reason, our data suggest that a dramatic increase in HPV-related OPC/OCC in its initial phase is occurring in the city of São Paulo. Indeed, this is a public health concern due to high survival rates and its potential magnitude, which impairs patients’ quality of life and the healthcare and social security systems, particularly in developing countries, such as Brazil.Our study had limitations that are important to its interpretation. As in other countries ,46, cancThis population-based study had strengths including its generalizability and limited selection bias, which allowed us to demonstrate the emerging risk of HPV-related OPC/OCC in young males and females, whereas the risk in HPV-unrelated subsites sharply decreased. For these reasons, our data support the HPV vaccination program and the continuity of the Brazilian tobacco control policy, which has had positive effects on OPC/OCC incidence in São Paulo city. Furthermore, it is crucial to continue to specifically monitor the incidence of HPV-related OPC/OCC and to investigate vaccine effectiveness in the oropharyngeal and oral cavity subsites in the long term. Moreover, cancer control programs should broaden screening coverage in young subjects to prevent sexually transmitted infections and HPV-related cancers to minimize the increasing risk.S1 Fig1; [E]: gums; [F]: hard palate; [G]: mouth; and [H]: other parts of the tongue.a,b APC: annual percent change; *: statistically significant APC (95% CI). a For better graph visualization, we applied the simple moving average of 5 years. b We analyzed these data with joinpoint regression models. 1 As there were 11 cases of cancer in Waldeyer's ring, they were combined with the cases of cancer in the soft palate and uvula.[A]: Base of the tongue; [B]: oropharynx; [C]: tonsils; [D]: Waldeyer’s ring, soft palate and uvula(TIF)Click here for additional data file.S2 Fig1 by age groups; [E]: gums by age groups; [F]: hard palate by age groups; [G]: mouth by age groups; and [H]: other parts of the tongue by age groups.a,b APC: annual percent change; *: statistically significant APC (95% CI). a For better graph visualization, we applied the simple moving average of 5 years. b We analyzed these data with joinpoint regression models. 1 As there were 11 cases of cancer in Waldeyer's ring, these cases were combined with the cases of cancer in the soft palate and uvula. # There were insufficient cases for analysis.[A]: Base of the tongue by age groups; [B]: oropharynx by age groups; [C]: tonsils by age groups; [D]: Waldeyer’s ring, soft palate and uvula(TIF)Click here for additional data file.S3 Figa,b APC: annual percent change; *: statistically significant APC (95% CI). a For better graph visualization, we applied a simple moving average of 5 years. b We analyzed these data with joinpoint regression models.[A]: Incidence trends for OCC/OPC according to HPV groups; [B]: incidence trends for OCC/OPC by sex and HPV groups; [C]: incidence trends for HPV-related OCC/OPC by age groups; and [D]: incidence trends for HPV-unrelated OCC/OPC by age groups.(TIF)Click here for additional data file.


Objective. This study investigated whether women with a history of human papillomavirus (HPV) infection have an increased risk of infertility. Material and Methods. All patients with an HPV infection  in Taiwan’s National Health Insurance Research Database (2000–2012) were propensity score matched with control subjects  without an HPV infection by age, sex, index year, and relevant co-morbidities. Both groups were tracked until a diagnosis of infertility was recorded. The Chi-square test was used to analyze the distribution of demographic characteristics in the HPV group and non-HPV group. A Cox proportional hazards regression was used to estimate the hazard ratios (HRs) for the development of infertility, adjusting for age, sex, and co-morbidities. The Kaplan–Meier method was used to plot the cumulative incidence curves. We also performed negative controls to test for possible unmeasured confounding. Results. The HPV cohort had a higher risk of infertility. The adjusted HR (aHR) was found to be 1.39 (95% CI = 1.19–1.63) after adjusting for demographic characteristics and relevant co-morbidities. In the age subgroup analysis, patients with an HPV infection had an increased risk of infertility compared to the non-HPV cohort in the group aged 26 to 35 years . As we used propensity score matching to treat measurable confounders and negative controls to access unmeasured confounders, the findings of the study are robust. Conclusions. Among females of reproductive age, HPV infection is a potential risk factor that predisposes individuals to subsequent infertility. Chlamydia trachomatis and Neisseria gonorrhoeae are pathogens that have been shown to cause cervical, tubal, and peritoneal damage to the host, causing infertility [Infertility is defined as the inability to conceive after one year of regular intercourse without contraceptive measures involving both a male and a female partner . It is aertility .Human papillomaviruses (HPVs) are double-stranded DNA viruses that only infect humans and are considered some of the most common sexually transmitted viruses . HPV infIn this study, we hypothesized that HPV infection might be a risk factor for female infertility. We conducted this original longitudinal nationwide cohort study to explore this important issue.Data source: The Taiwan’s National Health Insurance Research Database (NHIRD) contains a large amount of data about the health of Taiwan’s residents. The NHIRD was established in 1995 when a National Health Insurance (NHI) program was launched by the Taiwanese government. In this retrospective cohort study, we utilized a sub dataset of the NHIRD, the Longitudinal Health Insurance Database (LHID), which contains the medical claim data of one million insured people from 1996 to 2013. The disease codes in the LHID were determined according to the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). The Institutional Review Board of the China Medical University in Taiwan approved this study (CMUH-104-REC2-115). Study population: To ensure the accuracy of the participants’ data, patients with missing demographic information were excluded from the analysis before the index date. All diagnosis codes in the NHIRD were recorded based upon the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). For this cohort study, we recruited patients who had been diagnosed with human papillomavirus (HPV)  between 2000 and 2012 as the study cohort and patients without a history of HPV as the control cohort. To ascertain that the enrollees had accurate diagnoses, the subjects in the study cohort were only confirmed if they had undergone HPV-related examinations and procedures such as colposcopy, cervical conization, or cryotherapy in the three months prior to or after the diagnosis of HPV. The index date was defined as the first date of HPV diagnosis for the case patients, and the controls were assigned a date between 2000 and 2012. To exclude the dated diagnosis issue, for each patient, the diagnosis of HPV was required to be after 2000, and the diagnosis of infertility had to be after the index date. We excluded patients who were under fifteen or above forty-five years of age, had received a hysterectomy, had been diagnosed with cancer, or had been diagnosed with infertility before the index date. Each control patient was matched with an HPV patient according to their propensity score. Patients who were not matched in the HPV cohort were excluded. The matched non-HPV patients had the same index dates and distribution of relevant co-morbidities as their paired HPV individuals. We identified the main outcome from the index date to the end of our study (31 December 2013).The primary focus of this study was infertility (ICD-9-CM code 628). All participants were followed up until December 2013, unless they were diagnosed with infertility, withdrew from the NHI program, or died. Co-morbidities associated with a risk of infertility include endometriosis (ICD-9-CM code 617), polycystic ovarian syndrome (PCOS) (ICD-9-CM code 256.4), benign neoplasm of the ovary (ICD-9-CM code 220), pelvic inflammatory disease (PID) (ICD-9-CM code 614), uterine leiomyoma (ICD-9-CM code 218), and infertility-associated operations, such as myomectomy (ICD-9-CM procedure code 68) and tuboplasty (ICD-9-CM procedure code V26.0). Information on co-morbid medical disorders was obtained by tracing all of the ambulatory medical care and inpatient records in the NHIRD within the two years prior to the index date.First, the demographic characteristics, including the distributions of categorical age, sex, and co-morbidities between the HPV cohort and non-HPV comparison cohort were analyzed by chi-square tests. The incidence density of infertility per 1000 person-years was calculated in both cohorts.Second, to investigate the possible effect of HPV, a Cox proportional hazards regression analysis was conducted to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) after the adjustment of covariates. The covariates included in the multivariable models included age, sex, co-morbidity of endometriosis, polycystic ovarian syndrome, benign neoplasm of the ovary, pelvic inflammatory disease, uterine leiomyoma, myomectomy, and tuboplasty. Propensity score matching was estimated using logistic regression to minimize the measurable confounders and potential selection bias in terms of previously mentioned variables.Third, the Kaplan–Meier method was used to describe the cumulative incidence of infertility in the two cohorts. The difference between the two cohorts was examined by a log-rank test. The incidence of infertility was estimated by dividing the number of infertility events by the follow-up person-years for both cohorts. p value for the interaction test was statistically significant, this indicated an observable subgroup effect on the incidence rate of infertility.Fourth, to examine the effects of sex, age, and each mentioned co-morbidity on the incidence of infertility among patients with an HPV infection, we used the multivariable Cox regression model adjusted for age, sex, and co-morbidities. The HR adjusted for covariates was calculated for male and female patients and for the following age groups: 15–25, 26–35, and 36–45 years. We conducted a test to assess interactions. If the Last, because lifestyle and behaviors are also important risk factors for infertility, we used negative controls to test for the possibility of such unmeasured confounding. We chose lung cancer  and hepatocellular carcinoma . The literature review showed that there seems to be no relationship between HPV infection and lung or hepatocellular carcinomas. However, if the result showed a significant association between HPV infection and lung cancer using the model in this study, it would indicate the presence of an unmeasured confounder (such as tobacco use) from this point of view. Again, if the result showed a positive association between HPV infection and hepatocellular carcinoma, it would indicate the presence of an unmeasured confounder  from HPV to infertility. In contrast, if the results of the negative controls showed insignificant associations between HPV infection and both malignancies, the finding of HPV being a potential risk factor for infertility would be more reliable.This study included 11,198 patients with HPV and the same number of controls. As shown in Between 2000 and 2012, we identified 13,953 female patients from the LHID with an HPV infection. In the analysis with propensity score matching, we performed multivariable-adjusted analyses using the age-, sex-, and index date-matched controls to estimate the HRs of lung cancer and hepatocellular carcinoma in patients with an HPV infection in comparison with the non-HPV cohort. The negative control outcome showed non-significant associations between HPV infection and lung cancer  and between HPV and hepatocellular carcinoma .We adopted this method to show that the potential magnitude of any uncontrolled confounder is minimal. However, we did not completely control for confounders in our study.This first retrospective cohort study using 13 years of nationwide population-based data suggests that people with HPV have a nearly 1.4-fold increased risk of developing female infertility compared with the general population. HPV is one of the most common STIs  and the p = 0.0008) but surmised that HR-HPV is more likely a trigger factor rather than an independent cause [Though studies have established an association between HPV infection and both male infertility ,25,26,27nt cause . Shannonnt cause . Other snt cause ,33. BV ant cause ,35.Chlamydia trachomatis infection and endometriosis [HPV infection often co-exists with other conditions that are related to infertility, such as etriosis ,37. We petriosis . With thThe strength of our study was the use of nationwide population-based data and PSM to evaluate infertility risk in patients with an HPV infection . The advThis population-based cohort study demonstrates a higher risk of female infertility in patients with previous HPV infection, especially those aged 26–35 years. Further studies are needed to clarify the underlying biological mechanisms of these associations.


The prevalence of multiple types was significantly higher in HIV-positive than HIV-negative women (p = 0.034). The frequently detected genotypes were HPV35 (23.9%), HPV58 (23.9%), HPV45 (19.6%), and HPV16 (17.3%) in CIN2 cases, while in CIN3, HPV35 (22.5%), HPV16 (21.8%), HPV33 (15.6%), and HPV58 (14.3%) were the most common identified HPV types, independent of HIV status. The prevalence of HPV types targeted by the nonavalent HPV vaccine was 60.9% and 68.7% among women with CIN2 and CIN3, respectively, indicating that vaccination would have an impact both in HIV-negative and HIV-positive South African women, although it will not provide full protection in preventing HPV infection and cervical cancer lesions.South African women have a high rate of cervical cancer cases, but there are limited data on human papillomavirus (HPV) genotypes in cervical intraepithelial neoplasia (CIN) in the Eastern Cape province, South Africa. A total of 193 cervical specimens with confirmed CIN from women aged 18 years or older, recruited from a referral hospital, were tested for HPV infection. The cervical specimens, smeared onto FTA cards, were screened for 36 HPV types using an HPV direct flow kit. HPV prevalence was 93.5% (43/46) in CIN2 and 96.6% (142/147) in CIN3. HIV-positive women had a significantly higher HPV prevalence than HIV-negative women (98.0% vs. 89.1%,  Human immunodeficiency virus (HIV) infection is highly predominant in Africa. Globally, approximately 25.7 million people live with HIV infection, of which 80% (20.7 million) reside in Eastern and Southern Africa, as reported in 2019 . HIV preAn interaction between HIV infection and specific cancers has been established. Cervical cancer is one of the three cancers established as AIDS-defining cancers . South APersistent infection with HPV16, HPV18, and other HR-HPV genotypes is the most significant risk factor for developing cervical lesions and cervical cancer . HPV16/1Molecular HPV DNA testing has been implemented as the alternative to non-molecular testing for cervical cancer, particularly cytology testing . HPV tesThere is limited information on the epidemiology of HPV types in women with preinvasive cervical lesions from the Eastern Cape province. It is essential to investigate the prevalence of HPV genotypes and their distribution among women with different immune statuses and confirmed CIN histology results in this region. Therefore, these data will help achieve better understanding of the HR-HPV types involved in cervical lesions and cervical cancer cases. Furthermore, this information will contribute to discussions about implementing strategies for cervical screening and monitoring HPV types that are not present in the current vaccines to reduce cervical cancer disease in this population. Our study aims at investigating the distribution of HPV genotypes among HIV-positive and HIV-negative women with cervical intraepithelial lesions from Eastern Cape, South Africa.The study obtained ethical approval from the Human Ethics Committees of the University of Cape Town , Walter Sisulu University (016/2017), and Eastern Cape Department of Health (EC reference 2017RPO_484). The recruitment procedure for this study was reported previously . BrieflyDNA elution of cervical specimens from FTA cards was done following the procedure previously described ,25. Fourp-value was < 0.05. All data and statistical analyses were done using GraphPad Prism version 6 . The chi-squared test was used to determine a statistical difference between HPV infection and variables. A variable was considered significant if the The median age of women was 40 (IQR: 33–48) years. A high number of women were HIV-positive (76.2%) and never smoked (93.3%), and half of the women had their first sexual experience at the age of 16–18 years (53.9%) . Women wp = 0.012)  with CIN2 and 96.6% with CIN3 had an HPV infection. HIV-positive women had a significantly higher prevalence of any HPV infection compared to HIV-negative women  . HIV-negWomen with CIN2 were more likely to be infected with two HPV types A. The moWomen with CIN3 were more likely to be infected with two HPV types B. Their p = 1.000 and p = 0.823, respectively)  (p = 0.157). However, CIN3/HIV-negative women had a similar prevalence of nonavalent HPV types (HPV6/11/16/18/31/33/45/52/58) to CIN3/HIV-positive women   increased from 20.0% of CIN2/HIV-negative to 25.0% of CIN2/HIV-positive and from 27.8% of CIN3/HIV-negative to 29.7% of CIN3/HIV-positive, with no statistical significance (ctively) . For quactively) . CIN2/HI= 0.761) .This study investigated the prevalence and distribution of HPV types among HIV-positive and HIV-negative women with high-grade precancerous cervical lesions. A high number of women in this study were HIV-positive (76.2%). A significantly high overall prevalence of any HPV infection (98% vs. 89%) and multiple infections (65% vs. 49%) was observed among HIV-positive compared to HIV-negative women with cervical intraepithelial lesions. This higher prevalence of multiple types of HPV infection among women with HIV infection agrees with other cohorts of women with high-grade lesions from Botswana and South Africa ,28,29. HA high prevalence of HPV in women with CIN2 (93.5%) and CIN3 (96.6%) was observed in this study, which is expected as these women had abnormal cytology from the referral clinic. Similarly, a high HPV prevalence was reported in a global meta-analysis study, whereby HPV prevalence ranged from 86% to 93% in women with high-grade lesions (CIN2/3) . In the Previously, cervical histological lesions were associated with one HPV type . HoweverInterestingly, in our study, HPV16 was not the predominant HPV type, as it ranked fourth in CIN2 and second in CIN3 cases in the present cohort. A low prevalence of HPV16 has been observed in other sub-Saharan studies, while a high prevalence was observed in European studies ,46,47. ThpVIR real-time PCR [hpVIR real-time PCR for HR-HPV infection (HPV59) but had a low HPV copy number (11.3 copies) and viral titre (0.052). The observed negative results of HPV infection in women with high-grade lesions may suggest that it could result from sample storage, inadequate sampling, or low viral load. Alternatively, there may be novel HPV types causing the cancers that are not detected by the test used.Persistent HR-HPV infection is regarded as a significant factor in the development of cervical cancer lesions. However, in the current study, 4.2% (8/193) of women with CIN2/3 were negative for any HPV type, and seven of these samples were also negative when typed with time PCR . One speThe currently available HPV vaccines are estimated to prevent 70–90% of cervical cancer cases . The nonVaccines have been found to provide cross-protection against specific vaccine HPV types as well as some types that are not present in the vaccine. Numerous trial studies have reported that the bivalent vaccine offers a wider extent of cross-protection against nonvalent specific types (31/33/45/52/58), with less extensive cross-protection by Gardasil-9 ,54,55. SWe observed a significantly higher prevalence of HPV and multiple HPV infections in HIV-positive compared to HIV-negative women with cervical intraepithelial lesions. The distribution of HPV genotypes was similar between CIN2 and CIN3 cases independently of HIV status. The HPV nonavalent vaccine would have an impact on South African women, although it will not provide full protection in preventing HPV infection and cervical cancer lesions. Therefore, the high prevalence of the non-vaccine type (HPV35) underscores the need to incorporate this HPV type into the next HPV vaccines. This study also highlights the importance of introducing cervical cancer screening strategies to monitor non-vaccine HPV types.


P < 0·001). The HPV types included in the quadrivalent vaccine were detected in 1002 (14.8%) specimens, with a different pattern of HPV infection between sexes. Characteristics associated with overall HPV detection included female gender, self-declaration of race as brown/pardo, lower socioeconomic class, single or dating, current smoking and having 2 or more sex partners in the past year. We found a high prevalence of HPV, with significant differences between regions. Our data provide information that may be considered when developing HPV prevention policies and constitute a baseline against which the impact of the HPV immunization program in Brazil can be measured in future years.For Brazil, there are no nationwide data on HPV prevalence against which the impact of the HPV immunization program can be measured in the future. Therefore, we aim to evaluate the prevalence of genital HPV infection among adolescents and young adults in Brazil. A cross-sectional, multicentric, nationwide survey was conducted between September 2016 and November 2017. Sexually active unvaccinated women and men aged 16 to 25 years old were recruited from 119 public primary care units, including all 26 state capitals and the Federal District. All participants answered a face-to-face interview and provided biological samples for genital HPV analysis. We used an automated DNA extraction method and HPV genotyping was performed using the Linear Array genotyping test (Roche). Of 7,694 participants, 53.6% (95% CI 51.4–55.8) were positive for any HPV type. The prevalence of high-risk HPV types was significantly higher in women (38.6% vs. 29.2%,  HPV is one of the main causes of mortality among women in underdeveloped countries5. Although the prevalence of HPV has already been evaluated in some specific groups and regions, there are no data on HPV prevalence in young general populations of the different regions of Brazil6.Human papillomavirus (HPV) is globally the most common sexually transmitted infection1. In addition, the prevalence and type-specific HPV frequency can change according to race, with a higher incidence in indigenous7 and black populations8.The prevalence and types of circulating HPV vary widely both among different populations and among age groups within populations. All regions of the world have shown an overall decline in prevalence according to age, except Latin America and the Caribbean, where the prevalence increases later in life, presenting a bi-modal distribution9. Although vaccine efficacy in decrease cervical intraepithelial neoplasia has been demonstrated in clinical trials12, measuring the impact of vaccination on HPV prevalence in young populations is important to evaluate the effectiveness of immunization against HPV in different scenarios under pressure of the vaccines. The impact of national HPV vaccination programs is being evaluated with different strategies, including data obtained from residual cervical screening18, including women 25 years or older, that are dependent on screening rates and the population profile of each specific country. Few studies have had the opportunity to use data from national surveys19 and evaluate young populations.The introduction of HPV vaccination is an opportunity to prevent infection and associated lesions, thus changing the patterns of mortality by cervical cancer. Brazil introduced an HPV immunization program using a quadrivalent vaccine in 2014 for children 9 to 14 years old and is currently adopting a 2-dose vaccination schedule (0–6 months)This study reports prevalence data on type-specific genital HPV in a nationwide sample of adolescents and young adults who use the Public Health System in Brazil. These data allow comparison between different geographical regions and provide important baseline estimates to evaluate the impact of the HPV vaccination program.20.In this cross-sectional study, we evaluated the prevalence of HPV types in a convenience sample of sexually active women and men, aged 16 to 25 years from public primary health care units of the 26 state capitals and the Federal District of Brazil, from September 2016 to November 2017. Participants were recruited through different approaches as individuals who came to the unit for any reason, community health agents extended personal invitations, and nurses in school-based health promotion programs. The study sample of 7935 was calculated to detect a prevalence of at least 30% with an 80% power and to detect a difference of at least 5.2% between the regions of the country. Males were included (M:F ratio of 1:6) to allow subsequent assessment of herd immunity as a result of the HPV vaccination program in future years. This research was approved by the Moinhos de Vento Hospital human subjects research board, and written informed consent was obtained from all participants . We excluded pregnant women, those who had undergone hysterectomy or trachelectomy, and participants who had ever had cervical intraepithelial neoplasia grade 2 or higher. The study protocol was published previously21. Race/skin color was self-reported. We also asked about current and past cigarette smoking, educational level, marital status, number of sex partners in the last five years and last 12 months, age at first sexual intercourse, and condom use.All participants were interviewed by primary care professionals trained specifically for the study using structured questionnaires on risk factors for HPV infection and demographic and behavioral data. Socioeconomic status distribution is a composite score calculated based on the number of household assets, degree of education of the household head and presence of monthly paid housekeeperFor women, the cervical sample was obtained using the Qiagen HC2 DNA collection device according to the manufacturer’s instructions. Men were instructed to self-collect samples from the entire surface of the scrotum, glans penis/coronal sulcus and penile shaft using a saline-wetted Dacron swab, under supervision of a health professional. The cervical and genital swabs were placed in a tube containing 1 mL of Digene Specimen Transport Medium (STM), stored at controlled room temperature (15–25 °C) and shipped to a central laboratory weekly where the samples were aliquoted and stored at −80 °C until processing.DNA was extracted from 0.5 mL of specimen transport medium (STM) using magnetic beads for isolation and purification on a robotic system  according to the manufacturer’s extraction instructions and the DNA concentration in the extract was determined using the NanoDrop 2000 (Thermo Scientific™).22. The assay incorporated β-globin as an internal control for sample amplification. To ensure reproducibility of LA, an automated AutoBlot instrument (Fujirebio) was used for the hybridization and wash steps23. As the HPV 52 probe cross-reacts with types 33, 35 and 58, additional analyses were performed, if needed, using a type-specific real-time PCR assay25. Samples negative for β-globin and HPV  were considered inadequate and excluded from analysis. Six hundred eighty-seven (8.93%) men and women were considered nonresponders because they used an incorrect sampling technique or sample DNA was less than 5 ng/µl. We chose not to genotype these samples to avoid potential bias in the results. From the analyzed samples, eight (0.13%) were considered inadequate after genotyping.HPV genotyping was performed using the Roche Linear Array® (LA) genotyping test which amplifies a 450 bp fragment in the L1 gene. Per reaction, 25 μl working master mix was combined with DNA extract (between 100–150 ng) diluted in 25 μl of ultrapure DNase/RNase-Free water. Polymerase chain reaction cycling conditions and hybridization were performed as recommended by the manufacturer. Probes could detect 37 types of HPV simultaneouslyHPV results were grouped as follows: positive for any HPV type, positive for 13 high-risk HPV , positive for other HPV , positive for vaccine types , and multiple types (more than one HPV type).26.The variable number of partners in the past year was categorized as ≤1 or >1 and the variable number of sexual partners in the last five years was categorized as ≤1, 2–3 and ≥4, according to the variable distribution. Social class was categorized according with the Associação Brasileira de Empresas de Pesquisa (ABEP – Brazilian Association of Survey Companies b) who uses a system based on the purchasing power and the level of education of the families to identify social and economic status, ranging from the highest (Class A) to the lowest (Class E)27.We used the response rate definition from the American Association for Public Opinion Research, which defines the response rate as the number of participants divided by the number of persons that were ever contacted and screened as eligible28. SAS software , version 9·4, was used to conduct the analyses, and a 2-sided confidence level of 5% was determined to be statistically significant. Data with a normal distribution (Anderson-Darling test) were analyzed with Student’s t test or ANOVA, and the Chi square test was used for categorical variables.Sampling weights were applied to the data from our convenience sample in each capital to estimate HPV prevalence among the male and female population aged 16–25 years according to the Brazilian CensusP < 0.20 in univariate analysis were included hierarchically  in a multivariate model. The multivariate model was controlled by other variables included in the model, using the Hosmer-Lemeshow procedure29.The association between demographic and behavioral characteristics and the prevalence of HPV  was evaluated through Poisson regression with robust variance for binary outcomes. Variables with For each region, a univariate model was used to evaluate the prevalence ratio of characteristics in relation to HPV. Additionally, a multivariate model  was used to evaluate the prevalence ratio among each of the five regions of Brazil, using the South region as reference . Interaction terms between variables were examined to evaluate effect modification. The vaccine status was investigated, and 810 participants who reported having been vaccinated were excluded from the analysis.No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for recruitment, design, or implementation of the study. No patients were asked to advise on interpretation or writing up of results. The results from the present study will be disseminated through institutional websites, written communication, events and conferences, networks, and social media.All procedures performed in studies involving human participants were in accordance with the ethical standards of the Moinhos de Vento Hospital research board  and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.27, defined as the number of eligible people who agreed to participate in the study, was 99.1%.Of the 7694 recruited participants, 6388 (83.0%) provided valid samples for HPV analysis (5268 women and 1120 men). There were no significant differences in race/skin color, educational level or family income in the population that was able to provide a valid sample (Supplement 1). Higher social class, younger age and the Southeast region had higher levels of inadequate sample (Supplement 1). The overall response rateThe majority declared themselves as brown/pardo n = 3671/ 57.0%), 3817 were not engaged in a formal relationship (63.5%), 3402 were socioeconomic status C (55.5%), and 3571 were attending or had finished secondary school (55.9%) , with a higher prevalence of HPV infection in the 16–21 year old group  than in the 22–25 year old group . Detection of both overall HPV and HR-HPV prevalence varied by age, being highest among 18–19-year-olds and decreasing in older ages. The prevalence of HR-HPV types was 35.2% , with significant differences between sexes, with a higher frequency in women  than men  , with no differences between women  and men . When stratified by age group (16–21 and 22–25), there was a significant difference only among women  specimens [27.9% (95%CI 25.5 to 30.4) of those with any HPV detected], with no significant differences between men and women (data not shown). Nonavalent HPV types were detected in 1842  specimens being 51.6% (95%CI 48.7 to 54.6) of all HPV positive samples.P < 0.02).Although most individuals who tested positive for HPV had only one HPV type , multiple types were detected in 2036  participants: 883  had 2 types, 484  had 3 types, and 669  had 4 types or more. There was a statistically significant difference in the proportion of multiple types between sexes , HPV 16 , HPV 62 , HPV 89 , and HPV 61 . Overall, 247  participants were positive for HPV 18, and 757  were positive for both HPV 16 and 18. The prevalence of HPV 6 was 5.6% , and 74 participants  were positive for HPV 11. The pattern of HPV infection differed widely between sexes. While HPV 16  and 52  were the most prevalent HR-HPV types in women, HPV 59  and 52  were the most frequent in men , followed by the Midwest , North , Southeast , and South .The geographical regions also varied in the socio-behavioral characteristics that associated with HPV detection Table . Dating This study with over 6000 participants is the first to provide nationwide data on genital HPV prevalence in Brazil. Furthermore, to our knowledge, this is one of only a few large studies enrolling women and men simultaneously, allowing a direct comparison of HPV rates between genders. We found a high prevalence of any HPV (53.5%) and HR-HPV (35.2%) in a nationwide unvaccinated sample of 16–25-year-olds. While the overall HPV prevalence is similar between sexes, women have higher rates of HR-HPV and multiple infection. The type-specific detection of HPV infection varied widely between sexes. The geographical regions of Brazil differed in socio-behavioral characteristics that were associated with HPV detection.30 up to 62.3%31, depending on the population evaluated and method of detection. Compared to global rates32, the prevalence in this study is higher than the ones reported for the same age range in South America (18.1%)6 and similar to the US HPV detection rate (53.8%)18 for 20–24 year old.Approximately half of the studied women had HPV detected. Previous small studies in the Brazilian population have shown HPV rates from as low as 6.7%33 except for the HIM Study (men aged 18–70 years), which found higher rates of HPV (72.3%)34. In the United States (NHANES) and England  surveys have also compared the prevalence of HPV between sexes. Although the overall prevalence of HPV was higher among men in the US, this varied by age, with younger women (age 14–24 years) having higher prevalence of any type and HR-HPV35, similar to our results. Although we used similar genotyping methods, there were differences compared to our study. In the United States, the number of sexual partners was associated with an increase in HR-HPV prevalence in men35. In contrast, our study found that the number of partners increased the HR-HPV prevalence in women  but only in those who initiated sexual activity after age 15 (data not shown).The majority of studies on HPV prevalence are focused on sex-specific populations, preventing a direct comparison between genders. In the present study, the prevalence of overall genital HPV in men was similar to women and higher than previous Brazilian studies36. In women, the most prevalent HR type was HPV 16. These data emphasize the importance of screening considering that approximately 68.2% of invasive cervical cancers are attributed to HPV 16 or 1832.Although men and women share the main HPV types, the prevalence varies between sexes. In men, the most prevalent HR type was HPV 59, an oncogenic type related to HPV 1833. Although there are differences in prevalence according to region, race/color, social class and educational level were generally not significantly associated with HPV detection. Similar results have been reported in previous research studies in American and British populations38. Studies showing an effect of race and social class on HPV prevalence are often not comparable due to differences in the included population41, which is generally limited to specific groups. Behavioral characteristics, such as smoking and number of partners, may explain differences in prevalence rates according to region. Current and prior cigarette smoking was associated with HPV prevalence in all but the South and Southeast regions, possibly due to the higher prevalence of smokers in these regions . Cigarette smoking contributes to cervical cancer32, and it has been associated with HPV prevalence43, incidence44, and persistence45.Brazil covers a large portion of the South American continent and has high socioeconomic and cultural diversity. Therefore, it was reasonable to assume that the Brazilian population might have different risk factors associated with HPV infection from region to region. The majority of studies available were performed in women in the Southeast region and no reports were found that included all geographic regions of Brazil46.This study has some limitations that must be noted. This multicentric community-based study was not population-based; instead, it was performed in convenience primary health care units spread throughout Brazil. We were able to weight our estimates for the age structure, but all people included in the survey were sexually active and therefore likely to overestimate the true population prevalence. Furthermore, we do not have data to weight for differences in sexual behavior between people living in capitals and other regions of the state, adding uncertainty around estimates extrapolated to the wider population. Although we have a large sample size, when we presented the prevalence stratified by sex or region, some estimates may be unstable due to the small sample size in specific categories. Additionally, males had a higher rate of invalid samples because they had lower levels of extracted DNA, similar to other studiesIn summary, we report a high prevalence of any HPV type and HR-HPV types in a nationwide sample of men and women aged 16–25 years who were unvaccinated for HPV, with a higher prevalence of HR-HPV types observed in women. There were some differences in HPV prevalence across regions in Brazil that might be partially explained by behavioral variables. Our data provide information to be used in HPV prevention policies and constitute a baseline against which the impact of the HPV immunization program in Brazil can be measured in future years.Supplement 1.


The Delta Regional Authority (DRA) consists of 252 counties and parishes in 8 states in the US Mississippi Delta region. DRA areas have high rates of disease, including cancers related to the human papillomavirus (HPV). HPV vaccination coverage in the DRA region has not been documented.We analyzed data for 63,299 adolescents aged 13 to 17 years in the National Immunization Survey-Teen, 2015–2017. We compared HPV vaccination initiation coverage estimates (≥1 dose) in the DRA region with coverage estimates in areas in the 8 Delta states outside the DRA region and non-Delta states. We examined correlates of HPV vaccination coverage initiation and reasons parents did not intend to vaccinate adolescents.Vaccination rates in the DRA region  and in Delta areas outside the DRA region  were similar, but these rates were significantly lower than rates in non-Delta states . Inside the DRA region, reasons for parents’ vaccine hesitancy or refusal were similar to those expressed by parents in the Delta areas outside the DRA region. Some parents believed that the vaccine was not necessary or had concerns about vaccine safety.HPV vaccination coverage in the DRA region is similar to coverage in other Delta counties and parishes, but it is significantly lower than in non-Delta states. Activities to address parental concerns and improve provider recommendations for the vaccine in the DRA region are needed to increase HPV vaccination rates. Routine human papillomavirus (HPV) vaccination is recommended for children aged 11 or 12 years to prevent HPV and associated cancers.Geographic disparities in HPV vaccine coverage exist in the Mississippi Delta Regional Authority (DRA) counties and other counties in Delta states, compared with states outside the Delta region.Efforts to improve coverage are needed, particularly in the DRA region and other counties in Delta states. Providing parents and guardians with information and strong, compelling recommendations can improve HPV vaccination coverage.The Delta Regional Authority (DRA) was established in 2000 by the US Congress to support economic development and improve living standards for approximately 10 million residents in 252 designated counties and parishes in 8 Mississippi Delta states: Alabama, Arkansas, Illinois, Kentucky, Louisiana, Mississippi, Missouri, and Tennessee  (1). CouResidents of the Delta region have a poorer health status than do other US residents . ResidenA 2005 county-level analysis indicated that DRA counties had a median cancer mortality rate that was approximately 8.5% higher than in counties in Delta states outside the DRA region , particuThe Advisory Committee on Immunization Practices (ACIP) recommends routine vaccination of children aged 11 or 12 years with HPV vaccine to prevent HPV-associated cancers . DespiteThe objective of this analysis was to 1) better understand HPV vaccination coverage among adolescents in DRA areas and how it compares to the rest of the nation, 2) examine the association of sociodemographic and health care-related factors with HPV vaccination, and 3) examine HPV vaccination intentions and reasons for hesitancy among parents of unvaccinated adolescents.The National Immunization Survey-Teen (NIS-Teen) is a random-digit–dialed telephone survey of parents or guardians of adolescents aged 13 to 17 years. NIS-Teen also includes a survey mailed to all vaccination providers identified by the parent and those who consented to contact for vaccination history . NIS-TeeWe analyzed NIS-Teen data from 2015, 2016, and 2017, collected from households by way of landlines and cellular telephones ,19. DetaWe included data from 63,299 adolescents, aged 13 to 17 years, in 2015–2017 NIS-Teen. Inclusion criteria required that adolescents have adequate provider data  to determine whether they were up-to-date with vaccinations. The Council of American Survey Research Organizations (CASRO) landline response rates were 56.4% for 2015, 55.5% for 2016, and 51.5% for 2017. CASRO response rates for the cell phone sample were 29.8% for 2015, 29.5% for 2016, and 23.5% for 2017 , Delta areas outside the DRA , and non-Delta states . We examined a dichotomous outcome of HPV vaccine initiation (≥1 HPV vaccine dose or not vaccinated). We compared HPV vaccination initiation coverage estimates in the DRA region to coverage estimates in the other 2 geographic areas. We also estimated HPV vaccination initiation coverage for selected covariates, including demographic characteristics , health insurance, and access to care variables for adolescents . Among parents with adolescents unvaccinated for HPV, we examined intent to vaccinate their adolescents in the next year by asking parents, “How likely is it that [TEEN] will receive HPV shots in the next 12 months?” Response options included “very likely,” “somewhat likely,” “not sure or don’t know,” “not too likely,” and “not likely at all.” Parents who indicated the last 3 responses were asked, “What is the main reason [TEEN] will not receive HPV shots in the next 12 months?” This open-ended question allowed parents to indicate multiple reasons, and we identified the top 5 reasons from verbatim responses.t tests to identify significant differences (P < .05) in the proportion of categories between the DRA region and Delta areas outside the DRA authority and non-Delta states. We also conducted a multivariable logistic regression analysis to produce adjusted prevalence ratios (APRs) and 95% CIs by using a standard statement in SUDAAN procedures to produce such estimates (model-adjusted risk). We used χ2 tests to identify covariates that were associated with HPV vaccination initiation in each of the 3 geographic areas.We conducted statistical analyses by using SAS-callable SUDAAN release 11.0.1  to account for the complex sampling design of the NIS-Teen data. Point estimates and their 95% confidence intervals (CIs) were weighted to be representative of the areas from which the households were sampled. We used bivariate analyses to describe the distribution across selected sociodemographic characteristics. We used Demographic characteristics differed substantially between adolescents living inside DRA areas and adolescents living in Delta areas outside the DRA or in non-Delta states . We founUnadjusted HPV vaccination initiation coverage estimates among adolescents in the DRA region were significantly lower (54.3%) than in non-Delta states (61.4%) but similar to coverage in Delta areas outside the DRA region (56.2%) . These fP value <.05) among adolescents were having a mother who was divorced, widowed, or separated compared with a married mother, having Medicaid health insurance compared with having only private insurance, having received a provider recommendation for HPV vaccination compared with no provider recommendation, and residing in urban areas compared with rural areas  was found among adolescents whose mothers were high school graduates or had some college compared with mothers having less than a high school education, and those with a household IPR of 133% to less than 322% compared with those having a household IPR of 503% or more.In DRA areas, characteristics independently associated with higher rates of HPV vaccination initiation coverage  program in the United States , which pAlthough provider recommendation for vaccination was associated with HPV vaccination initiation in all 3 geographic areas, children inside the DRA region were less likely to have received a provider recommendation for HPV vaccination than children outside the DRA region. Previous research has indicated that both the source and manner of recommendation influence parental receptiveness to HPV vaccination ,23; physAlthough a higher proportion of adolescents with 2 or 3 providers lived in the DRA region, compared with the other 2 geographic areas, vaccination coverage was not associated with the number of providers among adolescents in the DRA region. Those who have several health care providers might have their medical histories dispersed to multiple providers, and follow-up might be difficult or unlikely. Record scattering, shown to affect vaccination coverage for young children, might also contribute to lower HPV vaccination coverage . EncouraThirty-six percent of adolescents in the DRA region received all of their vaccinations at private facilities. Receipt of all vaccinations from private facilities was more common in the other 2 geographic areas. Additional research is needed to determine if this finding results from fewer private facilities operating in the DRA or a reduced likelihood among private facilities in the DRA region to stock and administer HPV vaccines.This study has several strengths. First, NIS-Teen includes provider-reported vaccination data, which are more reliable than parental recall or vaccination shot cards. Second, multiple years of data were combined to increase sample size and study power to allow detailed analysis of this underserved geographic area. Third, although NIS-Teen was previously limited to households with landline telephones, this data set included cell phone sampling frames as well, which was instrumental in increasing how the data represented the target population.Our study also had limitations. First, incomplete provider vaccination records and lack of data on community- or county-level factors that might influence HPV vaccination could have limited the scope of this study. Second, after weighting adjustments to mitigate bias from incomplete data in the sample frame and nonresponses, some bias may remain . Third, Although factors related to HPV vaccine initiation are similar in the 3 areas studied, overall vaccination levels are lower in the Delta (both inside and outside the DRA region). Lower vaccination levels are likely correlated with the unique sociodemographic and health care characteristics of the areas, which are likely also responsible for disparities in HPV vaccination initiation across the 3 geographic areas. In the DRA region, assisting providers in effectively recommending HPV vaccination could be a primary strategy to increase coverage, as recommendations were closely associated with HPV vaccine initiation. Existing resources to help communicate HPV vaccine recommendations might need to be evaluated to ensure cultural appropriateness. To help identify strategies to increase HPV vaccination, additional research is needed to understand the barriers to vaccination in the Delta region, especially differences between uninsured and privately insured adolescents.


Human papillomaviruses are responsible for around 5% of all cancers, and to date there are no anti-viral therapeutics available for treating these cancers. In this report we demonstrate that in HPV positive cells the transcriptional repressor DREAM complex is disrupted by E7 proteins, with a resulting increase in expression of DREAM target genes. Expression of a mutant DREAM component, LIN52 S20C, competes with E7 and partially rescues DREAM complex formation. This restoration attenuates the growth of HPV positive cells, including HPV positive cervical cancer cell lines. We propose that restoration of the DREAM complex in HPV positive cancers is a novel therapeutic approach that could be adapted to aid in the treatment of these cancers.High-risk (HR) human papillomaviruses are known causative agents in 5% of human cancers including cervical, ano-genital and head and neck carcinomas. In part, HR-HPV causes cancer by targeting host-cell tumor suppressors including retinoblastoma protein (pRb) and RB-like proteins p107 and p130. HR-HPV E7 uses a LxCxE motif to bind RB proteins, impairing their ability to control cell-cycle dependent transcription. E7 disrupts DREAM , a transcriptional repressor complex that can include p130 or p107, but not pRb, which regulates genes required for cell cycle progression. However, it is not known whether disruption of DREAM plays a significant role in HPV-driven tumorigenesis. In the DREAM complex, LIN52 is an adaptor that binds directly to p130 via an E7-like LxSxE motif. Replacement of the LxSxE sequence in LIN52 with LxCxE (LIN52-S20C) increases p130 binding and partially restores DREAM assembly in HPV-positive keratinocytes and human cervical cancer cells, inhibiting proliferation. Our findings demonstrate that disruption of the DREAM complex by E7 is an important process promoting cellular proliferation by HR-HPV. Restoration of the DREAM complex in HR-HPV positive cells may therefore have therapeutic benefits in HR-HPV positive cancers.  Human papillomaviruses have been etiologically linked to 5% of all cancers worldwide . In partC. elegans multi-vulva class B (MuvB) genes LIN9, LIN37, LIN52, LIN53/RBBP4 and LIN54  disrupts the DREAM complex in vitro and correlates with poor clinical outcomes in breast and other cancers [The interaction of HR-HPV E7 proteins with RB family members requires an LxCxE motif in the viral protein . Other viewed in ) [11,16,iewed in . The Muviewed in ,19. In Siewed in ,13. Over cancers ,21.Only p130 and p107, but not pRb, interact with the MuvB core to form DREAM . This spTransgenic mice expressing HPV16 E7 under the keratin-specific promoter develop carcinoma tumors in oral carcinogen studies ,24. pRb Previously, we established a HPV16 life cycle model in the immortalized human foreskin keratinocyte cell line N/Tert-1 and used RNA-seq to identify transcriptome changes induced by HPV16 in these cells ,30. Genep-value = 1.00 × 10−10), as well as their upregulation in HPV+ tumors  of HPV-positive and negative head and neck cancer (HNC) TCGA samples revealed significant enrichment of the DREAM target genes in the differentially expressed gene sets  increases its affinity to p130 C (16). TTo determine whether LIN52-S20C can restore DREAM in HPV-positive cancer cells, we stably expressed WT-LIN52 or LIN52-S20C in human cervical cancer cell lines HeLa (HPV18) and SiHa (HPV16). Again, the binding of p130 to LIN52-S20C in these cells was increased compared to WT-LIN52 A,B. ThisCCNA2, CCNB1 and PLK1 were expressed at reduced levels in HeLa cells expressing LIN52-S20C compared to control cells while the expression of other genes was not repressed  driven expression of E7 provided several key insights into E7 oncogenesis. The K14-E7 mice displayed epidermal hyperplasia in multiple sites, including skin, mouth palate, esophagus, forestomach and ectocervix . DevelopGiven our previous work demonstrating the critical role of the DREAM complex  in regulating cell-cycle dependent gene expression, we investigated the mechanism of DREAM complex disruption in HR-HPV positive cells. Here we demonstrated that introduction of HR-HPV genome disrupts the DREAM complex in human keratinocytes by dissociation of MuvB core from p130, resulting in elevated expression of DREAM and MMB target genes. This finding is in agreement with previous reports of the DREAM disruption in human and mouse cell lines with overexpression of E7 protein ,42. UsinOverall, the results present evidence that disruption of the DREAM complex plays an important role in HR-HPV induced cell proliferation. Future studies will focus on modeling our results in transgenic mice, and also on identifying the LIN52 modifications for more efficient disruption of the interaction between E7 and p107/p130, resulting in complete restoration of the DREAM complex. The ability to control proliferation of the HR-HPV-positive cells without affecting “normal” cells could lead to development of virus-specific cancer treatments.Clonal cell lines containing the HPV16 genome were generated from telomerase-immortalized human keratinocytes (N/Tert-1) and grown as previously described . HeLa, St-test, or with ANOVA and Dunnett’s post hoc correction for multiple comparisons, as indicated in figure legends. To measure proliferation, 3 × 105 cells were seeded in triplicate onto 10 cm dishes, grown for 3 days, then detached, and counted using trypan blue staining. Total number of cells was recorded, and 3 × 105 cells per dish were re-plated each time on days 3 and 6. For clonogenic assays, 103 cells were seeded in triplicate wells of a 6-well plate, and colonies were grown for 10 days, stained with Crystal Violet dye and quantified using ImageJ software  [3 cells were seeded in triplicate wells in a 24-well ultra-low attachment plate  in growth medium containing 1% methylcellulose, incubated for 14 days and imaged. The colony diameter and number of colonies > 30 μm per 100 visible cells were measured using ImageJ.All cell-based assays included 3 biological repeats. Quantification data were analyzed and plotted using Prism software version 8 . Statistical significance for comparisons with control was determined using Prism 8, either with Student’s 2-tailed MD, USA) . The ancMD, USA) . BrieflyTCGA RNAseq data (PanCancer Atlas) for HPV+ and HPV- HNC squamous cell carcinoma samples  were anaSYBR Green RT-qPCR gene expression analysis was done as described using primers from In conclusion, we found that oncogenic E7 interacts with p130 when expressed at physiologically relevant levels in HPV-positive cells. Furthermore, our data demonstrates the mechanisms by which E7 competes with the MuvB core component LIN52 to bind p130 via an LxCxE-like binding motif, resulting in disassembly of the DREAM complex, deregulation of cell cycle gene expression and increased cell proliferation. The E7-competitive mutant LIN52-S20C is able to partially rescue DREAM complex assembly and suppress proliferation of the cells driven by HPV16 and HPV18, supporting the role of DREAM as an important HPV oncogenic target. Reversal of E7 disruption of DREAM is a novel therapeutic target for combating HPV cancers.


Men who have sex with men (MSM) attending university are a high-risk population for human papillomavirus (HPV) infection and are a neglected population of HPV vaccination programs in China. To provide evidence for HPV vaccination policies, we conducted this study to examine the prevalence and factors associated with anal HPV infection among MSM attending university in China. A self-administered online questionnaire was conducted to collect information on social demographics and sexual behaviors. A self-collected rectal swab specimen was collected to test for 37 HPV types. A total of 426 participants were tested for HPV. The median age was 20 years. HPV prevalence was 37.5% for any type, 29.8% for nine-valent vaccine types, 24.6% for four-valent vaccine types, 11.5% for HPV-16/18, and 15.7% for HPV-6/11. Men enrolled in a technical diploma, living in Northern China, having more than two sex partners, being bottom or versatile in anal sex, and having a human immunodeficiency virus (HIV) testing history were more likely to have positive anal HPV of any type. Our study found a high prevalence of anal HPV infection among MSM attending university in China, with HPV vaccine-preventable types being the most popular types in this group. Thus, our findings highlight the urgency of promoting HPV vaccination among teenage MSM. Human papillomavirus (HPV) has been identified as one of the most common sexually transmitted infections (STIs) worldwide . Most HPAlthough there is an abundance of literature on HPV among men who have sex with men (MSM), data on HPV among very young MSM are still lacking. A systematic review that only included 698 MSM ≤ 25 years of age, all of whom were from North America and Europe, found that the rate of anal HPV-16 infection was up to 16% among MSM aged ≤ 20 years and up to 18% among those aged 21–25 years . It was MSM are a neglected population in HPV prevention programs around the world. Three prophylactic HPV vaccines, directed against certain HPV types, are currently available for the prevention of HPV-related disease: the bivalent vaccine is recommended to females, and the quadrivalent and the nine-valent vaccines are recommended to both males and females . HoweverThe methodology of the University Student HIV Test Intervention Study (the UniTest Study) has previously been published elsewhere . BrieflyParticipants were required to finish a self-administrated online questionnaire to collect information regarding sociodemographics, sexual behavioral, HIV testing history, and alcohol and recreational drug use.Participants conducted a self-collected anal swab sample for HPV testing. The polymerase chain reaction (PCR) HPV genotyping test  was used to determine the HPV-type distribution in specimens. The PCR test amplified the target HPV DNA for 37 anogenital HPV types, which included HPV-6, 11, 16, 18, 26, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59 61, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, and 84.® Determine HIV-1/2, Alere Medical Co., Ltd., Matsudo, Japan). Participants that tested positive for HIV were referred to local centers for disease control and prevention (CDC) for confirmation testing and subsequent services.HIV testing was conducted by trained doctors with rapid HIV testing ), dwelling location, sexual orientation, and the gender of sex partners. Sexual behaviors included age of first anal intercourse, number of sexual partners in the past 3 months, commercial sex history, compulsive sex history, engaging in group sex in the past 3 months, role of anal sex in the past 3 months, unprotected anal sex in the past 3 months, and condom use during last anal sex encounter.The HPV detection variables consisted of type-specific HPV prevalence, any HPV type, high-risk HPV types, low-risk HPV types, single infection types, multiple infection types , and vacp < 0.10 in the univariate analyses were included in the multiple logistic regression model to determine factors associated with HPV infections. The chi-square test with trends was used to demonstrate the significance of the trends in HPV prevalence with the time since first anal sex. Analyses were conducted using STATA MP 16 .Descriptive statistics included summary measures of the mean or median and interquartile range (IQR) for continuous variables  and frequency tables for categorical variables . HPV prevalence was given as a percentage and corresponding 95% confidence intervals (CIs) for binomial proportions. Adjusted odds ratios (AORs) and 95% CIs of HPV infections were calculated according to characteristics using multivariate logistic regression models. Predictors with A total of 447 MSM attending university were enrolled in the study from January to April 2019. The demographic characteristics of the participants are shown in p = 0.018) and having > 2 sex partners . Participants with a positive HPV diagnosis practiced significantly more receptive anal sex . Compared to HPV-negative participants, HPV-positive participants had a greater history of HIV testing . About one in five of all participants  had sex after drinking alcohol. Approximately one-third of all participants  reported recreational drug use.A total of 431 anal swabs were collected and tested for HPV types—Of which, five failed to test, with unsatisfactory samplings. p < 0.05).We found positive trends between positive anal HPV infections and time since first anal sex . The proWe found a high HPV prevalence among MSM attending university in China. The great majority of infected HPV types could have been prevented by HPV vaccination. To our knowledge, this is the first study to characterize the prevalence and types of anal HPV infections in this young population in China.Our study found that the prevalence of any anal HPV infection among MSM attending university was 37.5%, which was lower than the overall prevalence of 52–66% among MSM with HIV-negative status in the general population in China ,19. The According to our results, the majority of participants were infected with HPV vaccine-preventable types, indicating that promoting HPV vaccinations could potentially be an effective way to prevent HPV infections among this young population. In our study, HPV-16 and 18 were the most prevalent high-risk HPV types. Studies have found that the persistence of high-risk HPV types, specifically HPV-16/18, is an important risk factor for the development of anal cancer . FortunaOur study also found some risk factors associated with anal HPV infections. The proportion of anal HPV infections increased with time since the first anal intercourse among MSM attending university. With the increase of sexual activity, they are more likely to be exposed to HPV, which correspondingly increases the risk of HPV infection. Similar to the results in the general MSM population, playing a receptive role in anal sex and having multiple sex partners are also risk factors of anal HPV infections among MSM attending university ,26. FurtWe found that participants enrolled in technical diploma programs were more likely to be infected with HPV than undergraduates. In the Chinese education system, the admission scores and learning tasks of technical diploma programs are lower than those of undergraduates . StudentOur study had limitations. Firstly, this study was cross-sectional, thus limiting our ability to differentiate between transient HPV infections and persistent HPV infections. HPV detection at a single time cannot represent persistent HPV infections. A prospective study found that 66% and 90% of HPV infections would be cleared within 12 and 24 months, respectively, in men . ConsecuOur study found a high prevalence of anal HPV infections among MSM attending university, especially for HPV vaccine-preventable types, suggesting that this population are a potential target group for HPV vaccinations. Prospective studies are needed to understand the natural history and influencing factors of HPV infections in this young population and to evaluate the effectiveness of HPV vaccinations in young males in China.