Participants completed an interviewer-administered questionnaire survey in their preferred language . A little over half of the participants were mothers  and parents’ mean age was 43.2 years (SD = 6.4). The majority was married or cohabitating , and 39.3% (n = 22) reported having two children. Seventy-five percent (n = 42) of parents reported they had heard of the HPV vaccine. Fewer fathers were aware of the HPV vaccine  than mothers. Among parents who had heard of the HPV vaccine (n = 42), 85.7% (n = 36) reported their children had received at least one dose of the HPV vaccine. Fewer fathers reported their child had been vaccinated against HPV  than mothers. Moreover, 90% of parents (n = 18) whose children were unvaccinated reported willingness to vaccinate their adolescent children against HPV if recommended by their child’s physician. Findings indicate parents’ low to moderate awareness of the HPV vaccine, and high willingness to vaccinate their adolescent children if recommended by their child’s physician. Findings also demonstrate fathers’ lower awareness and acceptability of the HPV vaccine than mothers. Despite limitations and the need for more research, findings of this pilot study serve as a valuable first step toward building a knowledge foundation that is needed for developing future studies and interventions targeting Central American immigrant parents living in the US. Future studies can build on the findings of this exploratory study with other research designs and address its limitations by having a larger sample size and accounting for additional factors associated with Central American immigrant parents’ HPV awareness, knowledge, beliefs, attitudes, and vaccine acceptability from other communities across the US.Despite increasing interest in understanding the factors influencing awareness and acceptability of the human papillomavirus (HPV) vaccine among Latino parents, to date limited information is available specific to Central American parents living in the United States (US). Therefore, this pilot cross-sectional study was designed to explore and assess Central American immigrant parents’ awareness, acceptability, and willingness to vaccinate their children against HPV, and interest in participating in future HPV-associated cancer prevention study. Fifty-six Central American parents, majority immigrant (96.4%;  Latinos or Hispanics (hereafter referred to as Latinos) are the largest and most rapidly growing minority group in the United States (US) . The curCancer is the leading cause of death among Latinos in the US, surpassing cardiovascular disease ,6, thus Genital HPV is the most common sexually transmitted infection in the US, and an estimated 14 million persons are newly infected with HPV every year . HPV is Approximately 150 HPV types have been identified, and over 40 of which infect the genital area ,9. Genit®  and two quadrivalent HPV vaccines (HPV4), called Gardasil® and Gardasil® 9  are licensed and approved for use by the US Food and Drug Administration (FDA) for prevention of several HPV-associated cancers [HPV-associated cancers can be prevented, and research shows that the most efficient and cost-effective mechanism for combating HPV is to prevent infection through vaccination ,9,10. Cu cancers . Both th cancers . HPV typ cancers .The Advisory Committee on Immunization Practices (ACIP) and the American Academy of Pediatrics (APA) recommend routine vaccination with quadrivalent (HPV4) or bivalent (HPV2) HPV vaccine for females aged 11 or 12 years and with HPV4 for males aged 11 or 12 years ,11. FurtThe Healthy People 2020 objectives call for an increase to 80% in the percentage of female and male adolescents aged 13 through 15 years who receive 2 or 3 doses of HPV vaccine as recommended . NonetheDespite an increasing number of studies conducted to identify knowledge and awareness of HPV and the HPV vaccine among various Latino groups in the US ,25,26,27Findings from a recent systematic review conducted to identify studies exploring HPV knowledge, awareness, beliefs, attitudes, and acceptability of the HPV vaccine among Latino fathers living in the US identified only 11 eligible studies . FurtherFindings of the only study with a national sample of Latino fathers, most of whom were foreign-born (the majority were from Mexico), showed that although almost half were aware of HPV, knowledge of the HPV vaccine was low, and knowledge gaps related to transmission and risky behaviors existed . LikewisGiven the lack of studies among Central American immigrant parents living in the US, the importance of the family in the Latino culture, and the joint role parents have in health care decision-making for their children, understanding awareness and acceptability of the HPV vaccine of Central American immigrant fathers and mothers will likely make it possible to design culturally tailored interventions to meet the specific needs of this ethnic group, and to increase vaccine uptake among their children ,23. TherThe present study was conducted as part of a large ongoing, mixed-methods HPV research with multi-ethnic Latino parents . The curOur study protocol was reviewed and approved by the University of Massachusetts Boston Institutional Review Board (IRB # IRB#2017221). Written informed consent was obtained from all parents participating in this study. Parents were eligible to participate if they: (a) self-identified as Central American; (b) had at least one child aged 11–19 years; (c) lived in MA or RI; and (d) had resided in the US for at least 12 months. We recruited participants using strategies successfully employed in our previous studies with similar Latino populations, and these included: (1) posting flyers inside local Latino businesses, and community-based social and health services agencies, and (2) making announcement and posting flyers at predominantly Spanish-speaking churches ,32,33,34Data collection followed the same procedures used in our previous HPV research with Brazilian immigrant parents and included two-sequential steps . First, The brief questionnaire survey instrument included questions adapted from previous research with Latino groups in the US that assessed: (1) participants’ awareness of HPV and the HPV vaccine, (2) sources of information about HPV vaccination, (3) acceptability of the vaccine and willingness to vaccinate their adolescent children against HPV, and (4) participants’ interest in participating in a future HPV-related cancer prevention ,22,25,26Participants responded to queries concerning their awareness of HPV  and the HPV vaccine . Only those who had heard of HPV were asked if they were aware of the HPV vaccine.Participants who reported being aware of the HPV vaccine were asked to report where they heard about the vaccine and had multiple choices for responses including: (1) healthcare sources—physician, nurses, other healthcare professional, public health campaigns; (2) personal sources ; and (3) media sources ; and (4) other (specify). Participants who reported hearing about the HPV vaccine were also asked whether their children aged 11-19 years had received the HPV vaccination . In addition, parents who reported their children had not received the HPV vaccine or were unsure, were asked whether they would be willing to vaccinate against HPV if recommended by their child’s physician .Finally, participants were also asked whether they would be interested in participating in future HPV-related cancer prevention study .Participants reported their age, marital status, number of children, country of origin, years of residency in the US, primary language, educational attainment (less than high school (HS) diploma/general education degree (GED)/high school graduation/GED or higher), and annual household income . In addition, participants reported if they had regular access to a healthcare provider  and if they had health insurance .Acculturation was measured using the Short Acculturation Scale for Hispanics (SASH), a 12-item scale validated for use in Latino populations . The SASOur core data analysis approach was similar to that utilized in our prior research with Brazilian immigrant parents . Data wen = 32); and parents’ mean age was 43.2 years (SD = 6.4).The sample included 56 Central American parents, representing 56 unique families. All participants chose to complete the questionnaire survey in Spanish. As shown in n = 43), and 39.3% (n = 22) reported having two children. Nearly all parents were foreign-born , originating from four Central American countries—half were from El Salvador , 25.9% from Guatemala (n = 14), 22.2% from Honduras (n = 12), and 1.9% from Panama (n = 1). Foreign-born parents reported living in the US for an average of 13.7 years (SD = 7.2), and the vast majority  was categorized as having low acculturation levels (SASH score < 2.99) [The majority was married or cohabitating  .n = 28). The vast majority was employed , and 78.6% (n = 44) reported annual household income earnings of less than US$40,000. The majority  reported having access to healthcare through either government-sponsored health insurance   or private insurance . Furthermore, half of the study sample had completed less than high school education or obtained a high school diploma or General Education Degree (GED)  and those who reported household income less than US$40,000/year  than among parents with high acculturation levels (SASH ≥ 2.99) and parents who reported household income more than or equal to US$40,000/year, these differences were not statistically significant see . In cont> 2.99), this difference was not statistically significant  of parents reporting they had heard of the HPV vaccine . Fewer fnificant . In contnificant .Parents who were aware of the HPV vaccine  were asked where they heard about the HPV vaccine and could choose multiple sources of information . As showNon-healthcare information sources included personal social network members such as family  and friends , and media sources such as the internet , media campaigns on the TV and the radio , and billboards . As shown in Furthermore, parents who reported being aware of the HPV vaccine  were asked if their child had received the HPV vaccination, and of these, 85.7% (n = 36) reported their children had received at least one dose of the HPV vaccine. Fewer fathers than mothers reported their children had received the HPV vaccine  . Moreover, parents who reported they had not heard of the HPV vaccine (n = 14); or that their children had not received the HPV vaccine (n = 1); or who were not sure if their child had received the HPV vaccine (n = 5), were asked whether they would be willing to have their children vaccinated if recommended by their child’s physician. Ninety percent (n = 18) of these parents reported they would be willing to vaccinate their age-eligible children against HPV if recommended by their child’s physician, whereas 10% (n = 2) reported they were not sure . AlthougFinally, when asked about their interest in participating in future HPV-related cancer prevention study, the majority of parents reported being interested  . AlthougTo our knowledge this pilot cross-sectional study is the first study conducted in the US assessing Central American immigrant parents’ awareness, acceptability, and willingness to vaccinate their adolescent children against HPV. In addition, this study adds to a dearth of research assessing Latino fathers’ awareness of HPV and the HPV vaccine ,25,26,28Study findings found that 76.8% of parents had heard of HPV and 75% had heard of the HPV vaccine. These findings indicate that although awareness of the HPV vaccine in this group of Central American immigrant parents is lower than the target level set by the Healthy People 2020 (80%) , it is hFurthermore, the present study found that awareness of the HPV vaccine was significantly lower among parents with lower educational attainment (less than high school or diploma) than those with higher educational attainment (high school/diploma or college). Study findings also showed that awareness of the HPV vaccine was statistically significant lower among parents who reported household income of less than US$40,000/year than those who reported household annual income of more than or equal to US$40,000/year. Our findings are consistent with previous studies conducted among Latino parents ,43,44,45The majority of parents (83.3%) participating in this study reported having heard of the HPV vaccine from their child’s physician. This important finding adds to research evidence that indicates the central role physician’s play in increasing parents’ awareness of the HPV vaccine, and ultimately in influencing the uptake of the HPV vaccine for their adolescent children ,53,54. Nn = 42), the majority  reported their children had received the HPV vaccine. Although more research is needed, this finding suggests that increasing awareness of the HPV vaccine would likely increase Central American parents’ acceptability of the vaccine for their age-eligible adolescent children. Despite that, findings also showed that fewer fathers reported their children had received the HPV vaccine  than mothers. This finding concurs with that of a limited number of studies conducted with other racial/ethnic parents [A notable finding of this study is that among parents who were aware of the HPV vaccine  participating in this study whose children were unvaccinated or who were unsure whether their children had received the HPV vaccine reported willingness to vaccinate if recommended by their child’s physician. In spite of this, study findings also determined that fewer fathers  reported willingness to vaccinate if recommended by their child’s physician than mothers. Although this difference was not statistically significant, this might be influenced by the small sample size, suggesting the need for future studies with a larger sample size to further examine this difference in parental willingness to vaccinate their children against HPV. Nonetheless, our study’s findings coupled with evidence from prior research documenting lower acceptability of the HPV vaccine among fathers than mothers [Notably, the majority of parents 90%, n =  particip mothers ,25,26 unn = 49) participating in this study reported high interest in participating in future HPV-related cancer prevention study. Although fathers reported lower interest in participating in future HPV-related cancer prevention study than mothers , this difference was not statistically significant. This finding is important and suggests the need for and the viability of future HPV-related cancer prevention studies given parents’ receptivity to information about the HPV vaccine, the lack of research among Central American immigrant parents, and in particularly, the dearth of research among Latino immigrant fathers living in the US.Finally, the majority of parents (87.5%, We acknowledge that this pilot cross-sectional study has several limitations. First, findings are based on a small, convenience sample of Central American immigrant parents living in a few cities in MA and RI, which limits study generalizability beyond the sample population . In addiDespite limitations, this pilot study contributes to building an understanding of Central American immigrant parents’ awareness, acceptability, and willingness to vaccinate their children against HPV if recommended by a child’s physician. Findings are particularly important given the dearth of information specific to Central American immigrant parents, and in particular, the dearth of research on Latino fathers. This information is important and needed to develop culturally relevant educational and prevention strategies for Central American immigrant parents aimed at improving HPV vaccination rates among their age-eligible children, and, thereby decreasing their HPV-associated cancer risk and cancer health disparities.


The role of human papillomavirus (HPV) in oropharyngeal cancer (OPSCC) as a cause agent has been reported in much of the literature. As a surrogate marker, p16 immunohistochemical staining is used as the standard for classifying OPSCC, and the prognosis of p16+ OPSCC has been reported to be better than p16− OPSCC. However, it was necessary to study what is the next biomarker that could predict the prognosis after classification by p16. We assumed that SOX2 may be a potential biomarker. For each p16+ and p16− OPSCC, SOX2 was used to analyze whether the degree of expression level differed in survival and recurrence rates. The results showed that both immunohistochemical staining and mRNA expression level of SOX2 significantly affected the survival and recurrence rates of p16+ OPSCC patients in two different datasets which were constructed in differently ways. Our study presented the clinical applicability of SOX2 as a biomarker./SOX2High (HPV+/SOX2High in TCGA) showed the best prognosis among the four groups classified by SOX2 and p16 for 5-year overall survival (OS) and recurrence , but SOX2 did not make a significant difference in the prognosis of the p16− group. In the TMA cohort, SOX2High was significantly correlated with response to radiotherapy and lower pathologic T classification in the p16+ group (p = 0.001). In TCGA, correlations between SOX2 and tumor stage classification or radiotherapy were not observed; however, HPV+/SOX2High had a significantly low tumor mutation burden among the four groups . In summary, SOX2 was proven to be a potential marker to predict overall survival and recurrence in p16+ OPSCC. However, the role of SOX2 has not yet been confirmed in p16− OPSCC patients.For oropharyngeal squamous cell carcinoma (OPSCC), there are not enough additional robust biomarkers for subgrouping after the distinct classification using p16. As SOX2 is an emerging biomarker for cancer treatment, its clinical implication in OPSCC was evaluated using a consecutive tissue microarray (TMA) cohort consisting of 111 patients who underwent surgery as an initial treatment from May 2002 to December 2016 and 79 patients in The Cancer Genome Atlas (TCGA) dataset. In both datasets, p16+ Head and neck cancers, which occur at the oral cavity, oropharynx, nasopharynx, hypopharynx and unspecified pharynx, are the seventh most frequent cancer and the ninth leading cause of cancer-related death worldwide . The majSOX2, a pluripotency-associated transcription factor, is an emerging biomarker for the prediction of prognosis and therapeutic targets for cancer treatment because it plays an important role in the development and maintenance of the stem cell state and is associated with cancer progression . HoweverINK4a, which is a surrogate marker for HPV status  IHC score of pT3 and -4 vs. pT1 and -2:30  for HPV+ and −0.59 [IQR: −1.31–0.38] for HPV−, significantly different . Regarding TNM classification, 0.03 [IQR: −0.61–0.38] for T3 and -4 and 0.38 [IQR: −0.36–0.72] for T1 and -2, which was a similar trend observed in the TMA cohort but not significant in the TCGA dataset (p = 0.141), 0.01 [−0.77–0.77] for patients without nodal metastasis (N−) and 0.15 [−0.35–0.63] for patients with node metastasis (N+), which was not significant (p = 0.664).The median value of standardized SOX2High and 24 cases were SOX2Low.A cut-off value of −0.3020 was chosen using time-dependent ROC analysis to minimize false positives and false negatives, which was obtained from the overall survival data of the fourth year, which recorded the highest AUC (0.925). Accordingly, 55 cases were SOX2Low increased the risk of 5-year OS univariately (HR (95%CI): 8.98 (3.08–26.2), p < 0.001), even in multivariate analysis (HR (95%CI): 4.87 (1.44–16.5), vs. SOX2Highp = 0.011); rather, the significance of HPV− status was weakened in multivariate analysis (HR (95%CI): 3.24 (0.96–10.9), vs. HPV+, p = 0.059) when compared to univariate analysis (HR (95%CI): 7.11 (2.45–20.6), vs. HPV+, p < 0.001) : 2.82 (1.12–7.09), p = 0.028). Accordingly, 5-year OS and recurrence were significantly different between the SOX2High and SOX2Low groups (< 0.001) . For thew groups .SOX2Low increased the risk of 5-year survival in HPV+ patients (HR (95% CI): 2.34 (1.66–65.6), p = 0.012 for HPV+ and 2.52 (0.68–9.35), p = 0.168 for HPV−), and a similar result was even seen when SOX2 was set as a continuous value of standardized mRNA expression level , but there was no significant difference among HPV+/SOX2Low, HPV−/SOX2High and HPV−/SOX2Low. For 5-year recurrence, HPV+/SOX2High had the best prognosis, but only significant when compared to HPV−/SOX2High and HPV−/SOX2Low .The overall survival and recurrence seemed to be significantly different between groups according to HPV status and pression . In the SOX2High was good regardless of whether a history of RT was present  was the lowest among the four groups at a significant level; TMB of HPV+/SOX2Low . HPV−/SOX2High  and HPV−/SOX2Low . However, there was no significant difference between SOX2High and SOX2Low in HPV− groups (p = 0.279).TMB was calculated in groups classified by HPV+/− and High/Low . The TMBp = 0.001). Three markers, PD-1, PD-L1 and CTLA-4, related to immune therapy were not significantly different according to SOX2 expression in either HPV+ or HPV− oropharyngeal cancers   .Over the past decades, the overall incidence of HNSCC has been decreasing in the United States, Europe and Australia, while the incidence of OPSCC has been increasing . InfectiSOX2 has been reported to be related to HPV infection. HPV infection drives switches in SOX2 expression in the transformation zone in the uterine cervix , and SOXSOX2 mRNA expression level. This implies that SOX2, either by IHC scoring or measuring mRNA expression level, may be a potential prognostic marker for p16+ OPSCC. These results are in contrast to the known role of SOX2 because SOX2 is known to play an important role in maintaining the stemness of pluripotent stem cells, and many studies have reported that SOX2 is involved in cancer stem cell regulation [Even in the genomic era, IHC is still the most important tool because the method is quick, widely available, technically less challenging and cost-effective ,36. Heregulation . A migragulation . Neverthgulation ,18,19. Fgulation ,39. On tgulation ; howevergulation ,18, and gulation . These vgulation .In this study, one of the mechanisms underlying the poor prognosis of lower SOX2 IHC scores was that lower SOX2 scores were associated with advanced T classification (pT3 and -4), which was similar to other studies on oral cavity cancer ,18. Fu eThe second possible mechanism for the poor prognosis of lower SOX2 levels may be recurrence. When looking closely at the pattern of recurrence, the analysis revealed that SOX2 had a significant effect on locoregional recurrence independent of p16 expression level but not distant metastasis. In OPSCC, the results are still controversial, but there are several studies on the recurrence pattern according to the positivity of HPV ,44,45. HLow and p16−/SOX2High in the TMA cohort and between HPV+/SOX2Low and HPV−/SOX2High in the TCGA−HNSC dataset. These data further imply that the combination of these markers might help with more precise molecular typing for HNSCC, either in p16+ or p16−. Similar results have not yet been previously questioned or reported. Since the study population was not sufficient, further analysis to draw conclusions cannot be done, but this should be confirmed in the future.Since evidence for p16 has been established, p16+ oropharyngeal cancer is generally regarded to have a better prognosis than p16−. In this study, the trend that p16+ had a better prognosis regardless of SOX2 score and mRNA expression level was confirmed, but there was no significant difference in 5-year OS and recurrence between p16+/SOX2High was better in 5-year OS and recurrence. In the subgroup analysis by p16+/− with PORT+/−, however, these differences were not significant. Although not significant, it is noteworthy that the difference in prognosis between SOX2High and SOX2Low was more prominent in PORT+/p16− than PORT+/p16+. Because RT-related outcome was known to be worse in p16− oropharyngeal cancer than p16+ [Cancer stem cell features are generally associated with higher radioresistance . SOX2 ishan p16+ , SOX2 neSOX2High, which showed the best prognosis, had a significantly lower TMB among groups . However, these groups with higher TMB and worse prognosis might be the groups for which I-O treatment would work. Moreover, HPV+/SOX2High might not be a good target for I-O treatment in terms of low TMB. In this case, other predictors might be combined to properly predict the response. Future trials are warranted.Recently, immunotherapy has been introduced in the treatment of head and neck cancers. As the oropharynx is an immune-privileged site, OPSCC can be a candidate for immunotherapy . As TMB There are several limitations in the study. The TMA cohort consists of patients who underwent surgery as an initial treatment and the results cannot be applied to all OPSCC patients. In addition, due to the relatively small number of populations, a larger-scaled prospective study is necessary to ensure the results of the study. Finally, given that we did not detect HPV infection directly through ISH, there may be a limit to interpreting the results because p16 does not guarantee the presence of HPV infection. However, the significance of this study may remain, given that HPV positivity of OPSCC is determined by p16 in the latest cancer staging manual .SOX2 mRNA expression was also proven to be a biomarker to predict prognosis. However, the role of SOX2 has not yet been confirmed in p16- (HPV−) patients. T classification and the RT response are being considered as mechanisms for SOX2 to have different outcomes in p16+ OPSCC, however, further study is needed to prove.In p16+ OPSCC, the SOX2 IHC score can be used as a marker to predict overall survival and recurrence. From analysis of the TCGA-HNSC dataset,


Chlamydia trachomatis, Trichomonas vaginalis, and Neisseria gonorrhoeae) in women in the state of Maranhão.  To verify the association between HPV infection and the presence of coinfections (Chlamydia trachomatis), TVA5/TVA6 , and HO1/HO3 (Neisseria gonorrhoeae). DNA-HPV positive samples were subjected to automated sequencing for genotyping.  HPV-DNA detection was performed by the nested PCR, using the primers PGMY09/11 and GP + 5/GP + 6. For the identification of sexually transmitted agents, conventional PCR was performed using the following primers: KL1/KL2 . Women without cervical lesions were predominant (327/92.6%); however, the largest number of lesions was reported in women who had HPV/coinfections (18/8.8%). Multiple regression analysis showed that both HPV only and the concomitant presence of HPV/STI were able to indicate the occurrence of epithelial lesions .  Among the 353 women evaluated, 204 (57.8%) had HPV-DNA, of which 140 (68.6%) exhibited HPV/STIs, while 64 (31.4%) had the only HPV. T. vaginalis can contribute to HPV infection, and HPV/IST association may influence the development of cervical intraepithelial lesions that are precursors of cervical cancer. The findings suggest that the presence of  Human papillomavirus (HPV) is one of the most prevalent sexually transmitted infections (STIs) worldwide, both in men and women, infecting about 80% of the world's sexually active population [pulation . More thpulation .Although most cases of HPV infection are asymptomatic and transient, some viral types, such as HPV 16 and 18, are associated with an increased risk of developing anogenital cancer in men and women, including cervical cancer and penile cancer .However, high-risk HPV infection is not a sufficient cause for the development of the carcinogenic process . Other sChlamydia trachomatis, Trichomonas vaginalis, and Neisseria gonorrhoeae, may contribute to the damages caused by HPV, as well as to persistent infection and cancer progression [The relationship between HPV infection and other microorganisms plays an important role in the progression of cervical lesions to cancer . Interacgression –9.The main factor associated with the presence of coinfections is the chronic inflammation caused by these microorganisms, which can act as a promoter of carcinogenesis by inducing the occurrence of significant cellular and molecular changes. The proliferation, recruitment of inflammatory cells, and increased production of reactive oxygen species (ROS) during the inflammatory process can lead to damage and inhibition of DNA repair . These cChlamydia trachomatis, Trichomonas vaginalis, and Neisseria gonorrhoeae) in women attending the public health system (SUS) in the state of Maranhão, Brazil.Accurate estimates of the prevalence of HPV and coinfections are limited, especially in the general population. However, epidemiological data are essential for the development of effective strategies for the prevention and early diagnosis of HPV and other STI, as well as for the prevention of cervical cancer. Thus, we sought to evaluate the association between HPV infection and the presence of coinfections . All participants provided written informed consent.Conventional cytological smears were obtained with Ayre's spatula  and endocervical brush , extended in a glass slide, fixed with ethanol, and stained using the Pap technique. Cytological examinations of Pap smear were reported using the 2001 Bethesda Reporting System.Samples were collected and placed in the HC2 DNA collection buffer , and frozen at −20°C until processed. DNA/HPV extraction was performed by using QIAamp DNA Mini and Blood Kit  according to manufacturer instructions. Total DNA was isolated, eluted in 100 mL AE buffer, and stored at −20°C. Extracted DNA was quantified using the NanoVue system .β-globin amplification.The presence of DNA/HPV was detected using nested polymerase chain reaction (nested PCR) with the primer sets PGMY09/11 (first-round PCR) and GP + 5/GP + 6 (second-round PCR) using the Platinum™ Taq DNA Polymerase system  [μL of the final volume, containing 10 pmol of each primer, 10X reaction buffer containing 1 mM MgCl2, 10 mM dNTP, 1 U of Taq Polymerase Platinum (Invitrogen), and 100 ng of genomic DNA [The first-round mix was formed by 25 omic DNA . The ampμL of the final volume, containing 10 pmol of each primer, 10X reaction buffer, 1 mM of MgCl2, 10 Mm of dNTP, 2U of Taq Polymerase Platinum (Invitrogen), Milli-Q water, and 100 ng of genomic DNA [The second-round (GP + 5/GP + 6) consisted of an initial denaturation at 95°C for 4 minutes followed by 45 cycles of denaturation at 95°C for 45 seconds, annealing at 40°C for 1 minute, and extension at 72°C for 1 minute. The reaction mix was formed by 25 omic DNA .Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis was detected using polymerase chain reaction (PCR) using the primers suggested by (2013): TVA5/6, HO1/HO3, and KL1/KL2, respectively and the SuperMix Brazil system .The presence of μL, with 41 μL of SuperMix, 10°pmol of each primer, and 100 ng of genomic DNA. The amplification cycles varied according to the primer used. For amplification of T. vaginalis, the reaction occurred with an initial denaturation at 94°C for 5 minutes, 45 cycles of 94°C for 30 seconds, annealing at 47°C for 1 minute and extension at 72°C for 45 seconds, and a final extension cycle at 72°C for 10 minutes. For amplification of C. trachomatis, the reaction occurred with an initial denaturation at 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, annealing at 55°C for 1 minute and extension at 72°C for 45 seconds, and a final extension cycle at 72°C for 10 minutes. For amplification of N. gonorrhoeae, the reaction occurred with an initial denaturation at 94°C for 5 minutes, 40 cycles of 94°C for 30 seconds, annealing at 55°C for 1 minute and extension at 72°C for 45 seconds, and a final extension cycle at 72°C for 10 minutes.The reaction consisted of a final volume of 50 http://blast.ncbi.nlm.nih.gov/).PCR products amplified were purified using the Genomic DNA Purification Kit , labeled using the Big Dye Terminator v3.1 Cycle Sequencing Kit , and analyzed using an ABI Prism 3130XL Genetic Analyzer (Applied Biosystems). The sequences were edited and analyzed using 4Peaks Software . HPV genotypes were identified using the BLASTn  or Fisher's exact tests to assess the association between HPV/STI infection and sociodemographic and clinical factors. To assess the risk factors associated with HPV and their significance, the odds ratio test was used. To verify correlations and predictive variables for the appearance of cervical lesions, binary logistic regression and odds ratio were performed. A 95% confidence interval was adopted for all analyzes performed, and the data were considered statistically significant when p values ≤ 0.05. All tests were performed using IBM SPSS® Statistics version 24.0.The data considered categorical were presented in proportion, using the XIn total, 353 women  who attended public primary healthcare were included in the study. The population profile  was womep < 0.001), indicating that women with less than 1 minimum wage had higher risk of presenting HPV/coinfections. No statistically significant differences were found between the characteristics related to sexual life habits in the groups, such as condom use during sexual intercourse, the number of partners, age at menarche, and first sexual intercourse.Sociodemographic data and socioeconomic aspects, such as age, skin color, income, and educational level, were similar among women who had only HPV, women with HPV/STI coinfections, and women who were HPV negative . DespiteT. vaginalis infection was positively associated with HPV (p=0.003)  . Thus, pThe number of sexual partners and pregnancy were protective factors regarding the presence of HPV infection. These data mean that women who had fewer sexual partners and pregnancies had 0.033 and 0.301 fewer risks of presenting HPV, respectively, compared to women who reported having a higher number of partners and a higher number of pregnancies.Regarding cervical alterations, 327 (92.6%) women had no cervical lesions identified in the Pap test, both among those with HPV only and among those who had HPV and other associated STIs. Despite the reduced number of cervical lesions among the study participants, only 26 (7.4%), the largest number of alterations was reported in women who had HPV and coinfections (18/8.8%). The identified changes are atypical squamous cells of undetermined significance , low-grade squamous intraepithelial lesion , and atypical squamous cells that cannot exclude high-grade intraepithelial lesion .N. gonorrhea, T. vaginalis, C. trachomatis, and HPV/STI concomitantly is contributing factors for the development of cervical lesions (2 (5 degrees of freedom) = 61.63; p < 0.001, R2 = 0.260); however, only the concomitant presence of HPV/STI was a factor that significantly contributed to cervical lesion development . The associated presence of HPV and STIs had a positive correlation with the presence of cellular changes, which means that as the number of women infected by HPV/STIs increases, the number of cervical lesions also increases. In addition, women with HPV/STIs coinfection represent 12.63 times more likely to develop cervical alterations.Binary logistic regression analysis verified that the isolated presence of HPV,  lesions . The modIn this study, data from sexually active women were used to assess the presence of HPV and other sexually transmitted infections (STIs), as well as the association between them for the progression of cervical lesions. The study participants were women seeking for cervical cancer screening in basic health units located in regions of difficult access and social vulnerability.T. vaginalis showed a positive association with the presence of HPV, showing that this parasite increased the risk of women presenting HPV infection. Mercer and Johnson (2018) suggested that the nitrosamines produced by T. vaginalis promote cervical inflammation, thus causing wounds that allow HPV to penetrate the basal layer of the cervical epithelium. Other studies also indicate the association between T. vaginalis and the increased risk of cervical intraepithelial lesions [HPV infection was highly prevalent in the study population, and among these, HPV associated with another STI was more prevalent than HPV infection alone.  lesions –14; howeT. vaginalis, C. trachomatis, and N. gonorrhoeae, may have promoted more aggressive changes in the cervical microenvironment, thus making it difficult for the immune system to eliminate these pathogens.Regarding HPV classification, high oncogenic risk types were more prevalent in women who had coinfections (HPV/STIs), while low oncogenic risk types were generally found alone. These data suggest that, due to its different characteristics of aggressiveness, the viral type may have contributed to the presence of other STIs in the cervical epithelium, although these data were not statistically significant. In this context, women with concomitant infection by high-risk HPV and other STIs, such as Studies aiming to assess the prevalence of STIs in the general population are mainly focused on the identification of HIV and syphilis –17. DespC. trachomatis, N. gonorrhoeae, and T. pallidum in women between 16 and 24 years of age. They found that 47% of women had HPV and two STIs, 36% exhibited HPV and 1 STI, and 17% had only HPV; however, no statistically significant association was found between the infections [A study performed in South Africa evaluated the presence of HPV and five other STIs, namely, herpes simplex type 2 (HSV-2), fections .C. trachomatis, Gardnerella vaginalis, Mycoplasma genitalium, and Ureaplasma spp.) in women seeking routine gynecological care in Italy. They identified that HPV was associated with 1 STI (33.8%), and the highest prevalence rate was attributed to Ureaplasma spp. [Similarly, Magaña-Contreras et al.  assessedsma spp. .N. gonorrhoeae and HIV. Despite being a study carried out in a population of Brazilian women, the results differ from those reported in our study [A multicenter study performed in Brazil by Kops et al.  stated tur study . A possiur study , which oDespite presenting similar values regarding HPV prevalence and HPV/STI association, these studies present important differences. The main differences are the sample number, the types of STI evaluated, and the molecular techniques used, which may have different specificity in the detection of pathogens. Thus, to fully understand the influence of multiple STI for the persistence of HPV and the progression of cervical lesions, it is necessary to optimize techniques that allow the simultaneous detection of a greater number of STIs.C. trachomatis and T. vaginalis are among the main infections associated with HPV virus. Similarly, studies report that C. trachomatis contributes to the acquisition of high-risk HPV viruses; however, the present study did not observe an association between these two pathogens [Multiple STIs have the potential to influence HPV infection, although literature data report that athogens , 20–22. Menopause also demonstrated a positive association with the presence of HPV, as well as a cause/effect relationship. This means that women in menopause were 1.72 times more likely to have a viral infection, compared to women who still have menstrual cycles. Hormonal changes caused by menopause can lead to the reactivation of latent infections or, in cases of women with active sex life, contribute to the acquisition of a new infection . A high The number of sexual partners and parity represented protective factors against HPV infection, which means that women with fewer partners and fewer pregnancies had a reduced risk of contracting HPV. The high number of sexual partners and the early onset of sexual activity are established risk factors for increasing the risk of acquiring multiple sexually transmitted infections during life . SimilarC. trachomatis, N. gonorrhoeae, and T. vaginalis) increases, so does the number of women with cervical lesions. These data corroborate with previous studies, showing that the presence of multiple infections favors the persistence of HPV in the cervical epithelium, thus influencing the progression of lesions [Multiple regression analysis verified that HPV infection only and the concomitant infection of HPV and other STIs were associated with cervical epithelial lesions identified in the Pap smear among the studied participants. This means that as the number of women infected with HPV and STIs . This limitation could be solved with longitudinal studies, which could evaluate the ability of women to eliminate the HPV virus as well as other STIs in a given period of time, and the subsequent status regarding cervical alterations.C. trachomatis, Ureaplasma urealyticum, Trichomonas vaginalis, and Neisseria gonorrhoeae are among the main STIs identified [The presence of coinfections associated with HPV has been reported in other studies, due to its association with the progression of cervical intraepithelial lesions. However, the microorganisms detected vary in each study. In women with HPV, the presence of coinfections by entified , 24, 28.T. vaginalis, and Gardnerella vaginalis were associated with the occurrence of cervical lesions; however, the study did not evaluate the influence of the concomitant presence of HPV and other infections for the development of cervical lesions. A similar study performed by Lima et al. [C. trachomatis and CIN, but the concomitant infection by these two pathogens was not associated with cervical lesions.A study performed in Brazil  sought ta et al.  demonstrThe presence of coinfections can reduce the immune system's ability to eliminate HPV infection, in addition to favoring the progression of cervical lesions through the unregulated expression of viral oncogenes . STIs caThese pathogens can also modify the composition of the vaginal microbiota, contributing to the malfunction of the cervical microenvironment . CombineThis study provides information on HPV and other STI association in women in the Northeast of Brazil through the molecular detection of these pathogens. The Northeast region, especially the State of Maranhão, has low socioeconomic rates that contribute to the high rates of cervical cancer and sexually transmitted infections in this population.T. vaginalis may favor HPV infection, and the HPV/STI association may contribute to the development of cervical intraepithelial lesions that are precursors of cervical cancer. Importantly, our study could not evaluate the presence of persistent HPV infection among the participants, which could have contributed to a better understanding about the role of these STIs in the natural history of HPV infection as well as in the development of precursor cancer lesions. These STIs, including HPV, are an important public health problem since they are related to the development of multiple urogenital complications, such as infertility and cancer. Although the association between HPV and cervical cancer is well established, further studies regarding HPV/STIs need to be performed in order to understand the multiple and complex factors that may be involved in cervical carcinogenesis.The findings suggest that


The study sought to examine knowledge of cervical cancer and human papillomavirus (HPV) vaccines among child-bearing aged women in Hanoi, VietnamIn 2016, 807 women aged 18 to 49 years were recruited from one urban area and one rural area in 2016 and were examined through face-to-face paper-based interviews. Pearson’s chi-square test and an independent t-test were utilized to compare awareness of cervical cancer and HPV vaccination among women according residential status. Overall, 83.8% and 71.3% women had heard about cervical cancer and HPV vaccination, respectively. Mean knowledge scores for cervical cancer and HPV vaccination were 4.60±1.43 out of 7 and 1.53±1.35 out of 5, respectively. Women living in an urban area were more likely to be aware of cervical cancer and to be more knowledgeable of HPV vaccination than women in a rural area. Despite strong awareness, we found knowledge on cervical cancer and HPV vaccination to be alarmingly insufficient among Vietnamese women. Cervical cancer poses a tremendous global health challenge, especially in less developed parts of the world where cervical cancer incidence and mortality rates can reach up to 80% . Safe sex behavior, getting an HPV vaccine, and cervical cancer screening are well-recognized ways to successfully prevent this cancer and lower mortality rates . Since its launch in 2006, HPV vaccination has garnered consideration as an important life-saving intervention for millions of women throughout the world. Indeed, in the most developed parts of the world, where cervical cancer screening and HPV vaccination are readily available, cervical cancer incidence rates have dropped dramatically . In Vietnam, cervical cancer ranks as the second most common cancer among women of ages ranging from 15 to 44 years . To date, cervical cancer screening to detect cancer early is only offered in opportunistic screening programs in Vietnam, and few women elect to undergo cervical cancer screening using their own financial resources. Also, according to a report in 2016 by the United Nations, condom usage among men in Vietnam was extremely low, at less than 12% . To prevent cervical cancer in Vietnam, a pilot program for HPV vaccination was implemented in 2008; however, until now, it has not been incorporated into the national immunization program. Moreover, with limited resources dedicated to the prevention of cervical cancer, raising awareness of cervical cancer and HPV vaccination has proven to be a major challenge . The aim of this study was to examine knowledge of cervical cancer and HPV vaccination among women of child-bearing age in Hanoi, Vietnam using data collected in 2016. Understanding these data will help with designing and implementing successful vaccination programs through which to propel acceptance of HPV vaccination forward in Vietnamese society and minimizing the burden of this disease.Data sources and SettingData were obtained from a project funded by Pfizer, an American pharmaceutical company, entitled “Vaccination accessibility for child-bearing age women in low- and middle-income countries in Southeast Asia period 2016-2018,” which was conducted as a multi-country, pre- and post-evaluation, and community-based randomized control trial for improving awareness and utilization of several vaccine types  among reproductive-aged women. In the current study, data collected for pre-evaluation were utilized.Study design and sampleThe present study was conducted as an observational cross-sectional study with a convenient multistage cluster sampling method. First, two districts, representative of residence status, were randomly chosen: Dong Da (urban area) and Ba Vi . Second, two health communes were randomly selected for these two areas. Finally, lists of all women who were pregnant or had just given birth in the last 12 months at the time of the survey were received from the local health centers.The potential participants were recruited in this study if they met the following inclusion criteria: (1) women aged 18 to 49 years at the time of the survey; (2) currently living in the selected communes for at least 1 year and presence during the recruitment times; (3) pregnant or gave birth within the last 12 months ; and (4) agreed to participate in this project.Regarding sample size calculation, since no study has measured coverage of HPV vaccination among reproductive-aged women in Vietnam, we set P to equal 0.5. With 95% confidence intervals, a maximum permissible error (Δ) of 0.05 and a 10% drop out in case an individual refused to participate or was absent during the collection period, final sample sizes of 400 women in each district and 800 in total were calculated. Participants were then selected from two communal health units in Dong Da and Ba Vi from lists of 975 and 470 eligible women, respectively. A total of 807 women finally participated in this study.Study instrumentFace-to-face interviews were conducted by a professional researcher using a structured questionnaire. Guidelines from the Vietnam Ministry of Health and relevant references from the National Institute of Hygiene and Epidemiology were utilized develop this questionnaire . This questionnaire was reviewed by five experts and pre-tested by five pregnant women and five child-bearing women within a year before actual implementation. The questionnaire comprised three parts: socio-demographic information and awareness and knowledge of cervical cancer and HPV vaccination. Awareness of cervical cancer and HPV vaccination was assessed by items concerning whether the interviewee had ever heard of cervical cancer or HPV vaccination, responding either yes or no. For those who had heard about either cervical cancer or HPV vaccination, detailed questions on cervical cancer (7 questions) or HPV vaccination (5 questions) were then asked with possible responses of true, false, unknown, or no response. Sometimes the correct answer to the question was “true,” and other times it was “false.” One point was given for each correct answer, and points were summed to create a summary knowledge score. If a question had more than one correct answer, 1 point was given for at least one correct answer. Higher knowledge scores indicated having greater knowledge of cervical cancer or HPV vaccination. All summary knowledge scores were divided into two groups (good and not good) according to mean scores described in a previous study . Beyond the mean scores, we further considered the distribution of knowledge scores  to identify an appropriate cut-off point  for categorical decisions. Levels of knowledge of cervical cancer were categorized as “good” if more than five out of the seven questions were answered correctly or as “not good” if not. Levels of HPV knowledge were classified as “good” if more than two out of the five questions were answered correctly or as “not good” if not. Health status was self-reported by the participants using the scale of good, neutral, or not good.All participants provided informed consent after explaining the survey’s objectives, that participation was voluntary, the confidentiality of this survey, the procedure, and potential benefits for the individual and communities. Only those who agreed to participate in this survey and provided consent were included in the study. The surveys were administered in either a health commune or a participant’s home upon request. Each participant took about 30-40 minutes to complete the interview and received a gift  for their time spent.Ethical ReviewEthical approval from the Institutional Ethical Review Board of the Institute for Preventive Medicine and Public Health, Hanoi Medical University in Hanoi, Vietnam (No 184/HMU IRB) was obtained for the study. Verbal informed consent was obtained from all participants. Data analysisAll completed questionnaires were entered into EpiData version 3.1  independently by two people to ensure data accuracy. Descriptive analysis was conducted for socio-demographic factors and awareness and knowledge of cervical cancer and HPV vaccines. Pearson’s chi-square test and Fisher’s exact test were employed for bivariate analysis of all categorical variables, whereas an independent t-test was used to test differences in mean knowledge scores between women living in the two residential areas. All P values less than 0.05 were considered to be indicative of statistical significance. All statistical analyses were performed using STATA software, version 14 .The demographical characteristics of the respondents are presented in We assessed cervical cancer knowledge, after excluding 131 women who had never heard of cervical cancer prior to this study . We founThis is the first study to quantitatively investigate knowledge of cervical cancer and HPV vaccination among women of child-bearing age in Vietnam, where cervical cancer remains a major public health concern. This study highlights limited understanding of cervical cancer and HPV among these women who are primarily responsible for caring for their families. Therefore, tailored interventions are needed to increase public awareness and knowledge of cervical cancer and its vaccination.As expected, the majority of respondents answered that they had heard about cervical cancer and HPV vaccination. More women said that they had heard about cervical cancer than the HPV vaccine. Similarly, previous studies have also reported greater awareness of cervical cancer than vaccination of HPV in other countries and ethnic groups . This can be explained by the fact that cervical cancer is currently a great concern in many countries; however, HPV vaccines are not often covered in national immunization programs. In Vietnam, a government program implemented in 2008 was designed to cover all aspects required for the application of an HPV vaccination program; however, even though the feasibility of HPV vaccination delivery was proven, a national program for HPV vaccination in Vietnam has yet to be fully implemented. This may account for the poor awareness and knowledge of HPV vaccination among Vietnamese women. Regarding knowledge of cervical cancer, scores for correct answers to a total of seven questions were recorded in the current study, with a mean of 4.60 ±1.43 questions answered correctly. This mean score is much higher than reported in studies in other countries . Recently, with greater coverage from broadcasting programs, as well as the internet, the spread of information has become relatively easier to achieve than ever. Further, women of reproductive age might have a better knowledge of cervical cancer, as they may be more concerned with reproductive health.Concerning HPV vaccination, the majority of respondents lacked knowledge thereof, with a mean of 1.53 (SD=1.35) questions answered correctly for a total of five questions. Similarly, low mean scores have often been reported elsewhere . Furthermore, in the current study, knowledge scores for HPV vaccination were markedly lower among women living in a rural area than among those residing in an urban area. Interestingly, while the women residing in an urban area showed relatively greater HPV vaccine knowledge, they had a lower understanding of cervical cancer, compared with women who lived in a rural area. There may be several possible explanations for this: The first explanation may be related to differences in education and health promotion programs in each area. In urban areas, where various prevention services are present, education and health promotion programs for cervical cancer typically focus on HPV vaccination, whereas, in areas where prevention programs are not well organized, educational and promotional materials mainly concentrate on features and risk factors for the disease. Another explanation may be that greater cervical cancer knowledge, which was noted for women living in a rural area, might be associated with higher incidence rates of cervical cancer in rural areas . Taking these findings together, we suggest that a comprehensive educational campaign on HPV vaccination that describes the number of required HPV vaccine shots, recommended ages for vaccination, and possible side effects is needed. Initial efforts that enable future education programs ought to be built on existing knowledge, and simultaneous attempts to address common misconceptions or unknown factors could be beneficial. Trusted persons, such as health workers or teachers, as well as influential individuals, may prove effective in disseminating such information. Meanwhile, education programs should seek to take advantage of communal health centers in order to foster community awareness, and distributing information via pamphlets or leaflets at local health centers in the countryside has been recommended as a means of increasing the acceptability of HPV vaccination in other countries .Some limitations to this study warrant consideration. First, our data were employed in a cross-sectional study, and thus, it is beyond the present scope to determine any causal relationships. Second, the study results do not represent the entire Vietnamese population, because we targeted only two regions in this study. Lastly, this study did not include women of younger age , which is the target age for receiving HPV vaccination. Additionally, the study population of pregnant women and women who recently gave birth do not represent the entire population of Vietnamese women. Despite these limitations, our study has several strengths. First, this is the first study to investigate the associations between socio-demographic factors and levels of knowledge on cervical cancer and the HPV vaccine among Vietnamese women. Our findings indicate a need for public health policymakers to push forward step-by-step strategies for cervical cancer prevention in the near future. Second, women of child-bearing age, who are primary caretakers of children and protectors of the health for each family, were recruited, and we assessed their awareness and levels of knowledge on cervical cancer and HPV vaccine, as their knowledge about cervical cancer and HPV vaccination can strongly affect their intentions to receive vaccination themselves and for their daughters.In conclusion, despite high levels of awareness, knowledge on cervical cancer and HPV vaccination waslacking among women of child-bearing age in both urban and rural areas of Hanoi, Vietnam. Our findings underscore a need to develop well-designed educational programs on cervical cancer and HPV vaccination in Vietnam and other countries.


INK4A overexpression. Different survival outcomes were compared among patients with HPV-driven and non-HPV-driven tumors. Overall, 42/130 (32.3%) patients with newly diagnosed OPC during the period 2000–2018 resulted HPV-driven; HPV16 was involved in 37 cases (88%), HPV33 in 3 cases (7%), HPV58 and HPV18 in 1 case each. Over time, HPV-driven cases raised from 16.7% (6/36) during 2000–2006 to 46.1% (24/52) during 2013–2018 (p < 0.001). The increase in HPV-driven OPCs was more marked in females than males (p = 0.010), and the frequency of HPV-driven cases was similar in the different age groups. In comparison to cases with non-HPV-driven tumors, a significantly (p < 0.001) better progression-free and overall survival were recorded among patients affected by HPV-driven OPC. The prevalence of HPV-driven OPC cases has been significantly increasing during the last two decades also in North-East Italy and was associated with favorable outcome. OPCs driven by non-HPV16 oncogenic types were restricted to patients older than 68-yrs.HPV-driven oropharyngeal carcinomas (OPCs) show geographical variations with increasing temporal trends in several areas. We investigated their frequency and clinical outcomes within a prospective multicenter cohort study in North-East Italy. A tumor was defined as HPV-driven by using at least two different biomarkers, usually HPV-DNA positivity and p16 In comparison to chemical-induced tumors, HPV-driven cancers represent a separate entity, characterized by a distinct molecular pathway, a better response to treatment and an improved prognosis3. In particular, patients with HPV-driven OPC show good response to chemotherapy and radiotherapy with a good control of loco-regional relapse, but not of distant metastases. Among the factors influencing the prognosis, stage and smoking habit are well recognized5, while studies on the search for biomarkers able to predict patients’ risk of relapse are ongoing6.Oropharyngeal squamous cell carcinomas (OPC) are causally associated to chemical exposure  or persistent infection by high-risk HPV (human papillomavirus) types7. In particular, within Europe the frequency was reported to range from 24.2% to 56.5% in Southern and Northern countries, respectively8. Data on HPV prevalence in OPC cases showing differences among Italian regions have also been reported, with figures ranging from 11% to 75%12; small groups of patients were included and different HPV detection methods were used.Large variations on the frequency of HPV-driven OPC in different geographic areas exist, with highest (50–60%) figures in United States, Canada and Northern Europe, and lower in Southern Europe including Italy; in this latter the frequency is around 25–30%14.In most areas the incidence of HPV-driven OPC is on the rise, a trend more pronounced in the countries with higher frequencies15, predictive biomarkers of relapse5, and possible modifications of the therapeutic regimens to reduce correlated morbidity16.The improved prognosis of HPV-driven OPC has prompted investigations on aspects such as the best strategy to correctly distinguish HPV-driven from non-HPV-driven casesThe present study reports a time-trend analysis of HPV-driven cases over roughly two decades and analyzes the clinical outcome in relation to viral and cellular parameters.A total of 130 patients with primary OPC , were included in the study; they were predominantly males, no peak age at diagnosis was observed, and the tonsil was the most frequently involved sub-site. Anatomo-clinical data are summarized in Table Overall, 42/130 (32.3%) cases resulted HPV-driven  during 2000–2006 to 46.1% (24/52) during 2013–2018  was detected.No correlations with socio-demographic and clinical characteristics emerged  in 7 cases. HPV16 DNA was detected at low viral load (2 copies/cell) in one p16-negative, non-HPV-driven case.The expression of p16 protein was evaluated in 66 cases (30 HPV-driven and 36 non-HPV-driven). Overexpression was recorded in all HPV-driven and in 5/36  non-HPV-driven tumors tested  death occurred in 70/130 (53.8%) patients; 8/42 (19.0%) of HPV-driven and 62/88 (70.4%) of non-HPV-driven cases, respectively.Different socio-demographic and clinical characteristics were evaluated and correlated with clinical outcome Table . TNM Tu; PFS and7, but the results of this study demonstrate that in this area it has significantly increased over the last two decades (raising from 17% in 2000–2006 to 46% in 2013–2018), thus reinforcing our previous observations17. Also in a previous Italian cancer registry-based study covering the period 1988–201218, we showed a rising trend of HNSCC carcinomas potentially related to HPV (i.e. arising in the oropharynx). Altogether these observations indicate that a real increase of HPV-driven OPC cases is occurring in Italy. Moreover, our data highlight other two interesting aspects. First, the increase in HPV-driven cancers is more marked in females than males; second, HPV-driven OPC cases are no longer a disease restricted to the younger ages.The incidence of HPV-driven OPC in North-Eastern Italy is lower than in Northern Europe and North America13. According to Combes et al.19, the male/female prevalence ratio we observed (0.49) is consistent with an excess of tobacco and alcohol related OPC in men compared to women in our geographic area. In Italy, both smoking and alcohol consumption are more prevalent among males than females: 25.5% versus 17% are current smokers, and 78.4% versus 46.1% are drinkers, respectively; moreover, alcohol drinkers are most frequent among current smokers (OR = 2.17), and alcohol consumption is more frequent in Northern than in Southern regions21. In relation to the temporal variations of HPV-driven OPC cases, an estimated higher prevalence in women than in men was reported and shown to be statistically significant in all European regions (except Northern Europe)2. In Italy, the age-adjusted incidence trends of SCC arising from HPV-related sites registered  during the 1988–2012 period18 were stable in males and increased in women.Differences by gender of HPV-driven OPC prevalence and time trends, with differential geographical distributions, have been already reported, and related to environmental risk factors and behavioural habits22, we recorded HPV-driven cases with similar frequency in the different age groups. Indeed, while HPV-driven OPC was initially described as a new entity affecting mainly men aged 40 to 59 years23, in recent years it is becoming more common in older patients, and this change has been related to birth cohort effects24. The increase in the proportion of HPV-driven OPC cases among elderly patients bears the clinical challenge of treating patients with comorbidities for whom the survival advantage of HPV pathogenesis might be attenuated, and portend the need to include patients over 70 into the clinical trials assessing therapeutic and post-treatment surveillance protocols.In line with recent reports15. The presence of a single marker, i.e. HPV DNA positivity or p16 overexpression, does not always reflect a pathogenetic role of HPV. The detection of HPV-DNA and mRNA sequences or of HPV-DNA at high viral load has been shown to reliably identify transcriptionally active infections25. Moreover, since the HPV causality bears a prognostic significance, it would be important to define and standardize the laboratory protocol for the correct identification of HPV-driven OPC, also because many HPV-DNA detection assays, highly different in technical and analytical characteristics, are commercially available. Indeed, the clinical outcome reported for patients with discordant results for HPV-DNA and p16 is most often similar to non-HPV-driven cases27. The eighth edition of the AJCC/UICC  TNM staging system demonstrated a better prognostic capacity than the seventh edition for HPV-associated OPC, but confirms the concerns previously expressed28 on using p16 overexpression as a stand-alone test to define an OPC as HPV-driven. Indeed, in line with data of other studies29, in our study population the five patients with HPV-DNA-negative/p16-positive OPC showed an unfavorable clinical outcome, with local and regional, but not distant, disease progression , and non-16 types were found in older patients; similarly to what observed for cervical carcinoma, these observations confirm HPV33 as a highly oncogenic type31, and the incidence at younger age of HPV16-induced tumors32. Among the high-risk types, HPV16 is the most carcinogenic; in the cervix HPV16 infections are characterized by a much higher degree of developing high-grade disease within a shorter period of time than other high-risk types32. Whereas a poorer OS was observed in Norwegian patients with OPC associated with non-HPV16 types33, no differences in relation to HPV type emerged among our cases, but we analyzed a lower number of cases.HPV genotyping identified type 16 in 88% of cases, and types 33, 58 and 18 in the remaining 12%, a distribution similar to that recently reported in Eastern DenmarkStrengths of our study are the use of two biomarkers for HPV-driven determination and the long period of patients’ enrollment and follow-up. A limitation is the relatively small number of patients included in our cohort.In conclusion, we have observed an age-independent increasing prevalence of HPV-driven OPC over the last two decades also in North-East Italy. Patients with HPV-driven tumors defined by HPV-DNA positivity and additional markers  showed an improved prognosis. Conversely, p16 positive/HPV-DNA negative patients had a worse outcome, underlying the importance to use additional markers to define the etiological role of HPV in these malignancies. Similar to cervical cancer, infections by non-HPV16 oncogenic types were restricted to older OPC cases. Systematic review and meta-analysis are required to investigate the prognostic impact of non-HPV16 oncogenic types in patients with OPC.Aims of the study are to evaluate the temporal trend of HPV-driven OPCs in North-East Italy over a 18-year period, and to compare the clinical outcome of HPV-driven and chemically induced cases, also in relation to the HPV type. The OPC cases included in the present analysis represent a subset of a multicenter ongoing prospective cohort study on HPV involvement and prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC) patients initiated in the year 2000. Patients were treated at 3 Ear, Nose, and Throat (ENT) Units located in North-East Italy .th Edition and staged according to the AJCC 7th Edition classification with reclassification also according to the 8th Edition, that introduces a separate staging algorithm for HPV-driven OPC34.All the available records of patients with OPC managed with curative intent from September 2000 to August 2018 were reviewed. Treatment planning was discussed by a multidisciplinary tumor board in order to decide the appropriate strategy, and management decisions were not guided by knowledge of HPV status. All patients underwent a regular follow-up until death or 31 July 2019, except one who at this date was still under treatment. Clinical, epidemiological, socio-demographic and behavioral data were also retrieved , tobacco smoking, alcohol drinking). Histological diagnoses were determined by the local pathologists. All tumors were classified according to the WHO  436. We confirm that all experiments were performed in accordance with relevant guidelines and regulations.The whole study was approved by the ethic committee for clinical experimentation (CEP) of Treviso (Ethic votes: 345/AULSS9 and 421/AULSS9). All patients signed an informed consent. Data on 63 patients have been included in previously published reportsFresh frozen (FF) and/or formalin-fixed paraffin-embedded (FFPE) specimens of the neoplastic lesions  were collected.et al.17 or by the QIAamp DNA kit (Qiagen), according to the manufacturer’s instructions.DNA was extracted by the phenol-chloroform (PC9) method [as previously described by Baboci 17. Quantitative real-time PCR (qPCR) was performed on HPV16-positive samples by using primers and probe specific for HPV16-E6, according to Peitsaro et al.37; two standard curves were obtained by amplification of 10-fold serial dilutions of full-length HPV16 genome  and the beta-globin gene . The E6 copy number and the amount of beta-globin were calculated; the viral load was expressed as HPV16 copies/cell.Search and typing of HPV DNA sequences were performed by PCR using MY09/MY11 primers and restriction fragment length polymorphism (RFLP) analysis of the amplified products, as previously describedINK4a protein (indicated as p16) was performed on FFPE sections by immunostaining using the primary antibody CINtec for V-kit , as previously described11, or the BD Pharmingen IHC Detection kit, according to the manufacturer’s instructions. The expression results were scored as positive by using a 70% cut-point and considering nuclear and cytoplasmic stain distribution.The expression of p16INK4A overexpression; alternatively, positivity for HPV-DNA and a different biomarker  were used, as previously described17.A tumor was defined as HPV-driven by using at least two different biomarkers, usually HPV-DNA positivity and p16th ed.) and alcohol drinking. A similar multivariate model was generated in order to investigate survival outcomes  in HPV-driven tumors vs. non-HPV-driven tumors, adjusting for gender, age and year of diagnosis to obtain crude estimates, and also for T status, N status and alcohol drinking for adjusted estimates.To facilitate statistical analysis due to the relatively small number of patients, clinical and socio-demographic data were divided into categories. HPV-driven and non-HPV-driven cancers were compared for each variable. Cochran-Mantel-Haenszel and Fisher exact tests were used, as appropriate. Similarly, median value of HPV16 copies for HPV16 positive cancers were compared for each variable, using Kruskall-Wallis test. For each patient, person-time at risk was calculated from diagnosis to event of interest or last available follow-up, whichever came first. Follow-up was truncated at 5 years. Event was defined as cancer recurrence at the UADT (Upper Aero Digestive Tract) site for mucosal control; neck lymph node failure for regional control; local cancer recurrence or regional failure for loco-regional control; distant metastasis for distant control; second primary tumor (SPT) for SPT survival; mucosal, regional or distant recurrence or death for progression-free survival (PFS); death for overall survival (OS). Survival probabilities were estimated according to Kaplan-Meier method and differences according to strata were tested through log-rank test. Progression-free survival and overall survival in the different categories identified by the clinical and socio-demographic data were evaluated by a multivariate model using Cox proportional hazards regression, adjusted for gender, age, year of diagnosis, TNM stage  of Treviso (Ethic votes: 345/AULSS9 and 421/AULSS9). All patients signed an informed consent.Supplementary Information.


Although all 11- or 12-year-olds in the US were recommended to receive a 3-dose series of the human papillomavirus (HPV) vaccine within a 12-month period prior to 2016, rates of completion of the HPV vaccine series remained suboptimal. The effects of the Affordable Care Act (ACA), including private insurance coverage with no cost-sharing and health insurance expansions, on HPV vaccine completion are largely unknown. The aim of this study was to examine the associations between the ACA’s 2010 provisions and 2014 insurance expansions with HPV vaccine completion by sex and health insurance type.Using 2009–2015 public and private health insurance claims from Maine, New Hampshire, and Massachusetts, we identified 9-to-26-year-olds who had at least one HPV vaccine dose. We conducted a logistic regression model to examine the associations between the ACA policy changes with HPV vaccine completion (defined as receiving a 3-dose series within 12 months from the date of initiation) as well as interactions by sex and health insurance type.Over the study period, among females and males who initiated the HPV vaccine, 27.6 and 28.0%, respectively, completed the series within 12 months. Among females, the 2010 ACA provision was associated with a 4.3 percentage point increases in HPV vaccine completion for the privately-insured  and a 5.7 percentage point increase for Medicaid enrollees . The 2014 health insurance expansions were associated with a 9.4 percentage point increase in vaccine completion for females with private insurance  and a 8.5 percentage point increase for Medicaid enrollees . Among males, the 2014 ACA reforms were associated with a 5.1 percentage point increase in HPV vaccine completion for the privately-insured  and a 3.4 percentage point increase for Medicaid enrollees . In a sensitivity analysis, findings were similar with HPV vaccine completion within 18 months.Despite low HPV vaccine completion overall, both sets of ACA provisions were associated with increases in completion among females and males. Our results suggest that expanding Medicaid across the remaining states could increase HPV vaccine completion among publicly-insured youth and prevent HPV-related cancers.The online version contains supplementary material available at 10.1186/s12889-021-10328-4. In the US, an estimated 34,800 cancers were attributable to the human papillomavirus (HPV) annually during 2012–2016, with cervical and oropharyngeal cancers being the most common . RevisedThe Patient Protection and Affordable Care Act (ACA) rolled out a series of provisions that removed barriers to accessing the HPV vaccine. In September 2010, the ACA required non-grandfathered private plans to cover the HPV vaccine with no patient cost-sharing , which rEvaluations of the ACA have only examined the effects of the 2010 provisions on HPV vaccination among females. Using self-reported data from national surveys, both studies found that the ACA increased HPV vaccine initiation and completion , 16. HowWe used All Payer Claims Databases (APCD) from Massachusetts , Maine  and New Hampshire , which collect health insurance claims from insurance companies operating in a given state. APCDs are mandated by law in these states, which ensures that most of the privately- and publicly-  insured populations are included. While the standardized data submission requirements allowed us to pool data from the three states, the length of the study sample differed: MA provided data from January 2011 through December 2015, and ME and NH provided data from January 2009 through December 2015. For NH there were no public claims available from April 2013 through November 2013; personal communication with Rose Hess on April 17, 2018. Data are accessible to researchers upon application and costs vary by state.Our analytic sample included children and young adults aged 9 to 26 years who had at least one HPV vaccine dose during the study period based on claims associated with Current Procedural Terminology codes 90649 (Gardasil), 90650 (Cervarix) and 90651 (Gardasil 9). We restricted the study period to September 2009 through June 2014 (from September 2011 in MA). By excluding 8 months from the beginning and 18 months from the end of the study period, we could ensure the full time period allocated for series completion at either end of data collection. Due to low HPV vaccine initiation prior to the ACIP recommendation for males, we excluded males before October 2011 , 18.Among the 385,998 youth who received at least one dose of HPV vaccine over the study period, we excluded 2701 individuals who received more than 3 doses because we were unable to observationally distinguish data submission errors from intentional repetitions. This resulted in a total sample size of 383,297 9–26-year-olds, with 194,407 females and 188,890 males. The Boston College Institutional Review Board reviewed this study and considered it exempt.2, 5 we constructed an individual-specific completion measure by aggregating all HPV claims received for a given person. Our primary outcome variable was a binary indicator of HPV vaccine completion, defined as receiving a 3-dose series within 12 months from the date of initiation  is the cumulative distribution function of the logistic distribution, YFSi denotes year of first shot, ME and NH refer to states (MA omitted), AGE1518i and AGE1926i refer to age categories (ages 9–14 omitted), Mi refers to gender male and PVTi refers to private insurance (Medicaid omitted). ACA2010i and ACA2014i are indicators of the ACA policy changes in 2010 and 2014, respectively.Next, we conducted an individual-level regression analysis to examine the associations between the insurance-related ACA policy changes with HPV vaccine completion within 12 months. Since our analytic sample consisted of multiple HPV claims for most individuals, we collapsed our dataset to a single observation per person that contained information of the characteristics of the entire series of HPV claims . We estimated logistic regression models where we modeled the probability of series completion as a function of the ACA policy breaks and demographic characteristics. For the dependent variable, we used a binary indicator which takes the value one if our completion definition is satisfied, zero otherwise. In our first, simplest specification, we ran stratified models by sex to identify any heterogeneous policy effects across males and females. Then, we constructed a combined model to directly compare our estimates across these groups. In the final specification we included three-way interaction terms for each policy break by sex and insurance type . The model also controlled for the participant’s age group, state and year fixed effects. The final estimates were obtained from the following logistic regression specification:We then conducted two sensitivity analyses in which we repeated this series of analyses by first, extending the definition of our vaccine completion measure and second, reducing the dose schedule. The former outcome was defined as completion of the 3-dose series within 18 months from the date of initiation . In respi = 1, i.e. the HPV series was completed, while holding other covariates constant. We calculated differential responses by sex and insurance type as our stratified analysis revealed significant heterogeneity among these groups. Finally, we calculated predictive margins and used an adjusted Wald test to assess the statistical significance of differences by sex and insurance type. We conducted analyses using Stata statistical software version 15.1 .We report average marginal effects which compute the partial effects of each explanatory variable, i.e. implementation of each ACA policy, on the probability that the observed dependent variable, YOverall, among females and males who initiated the HPV vaccine over the study period, 27.6% 53,591/194,407) and 28.0% , respectively, completed the series within 12 months. Among those who completed the HPV vaccine series, the mean difference in time between the first and last HPV doses was 6.2 months (standard deviation 3.1 months). The prevalence of HPV completion was higher among females and males who initiated the series between ages 9 and 14 compared with those who initiated at older ages and privately-insured youth were more likely to complete the series than Medicaid enrollees .Among females, the introduction of the ACA in 2010 was associated with a 4.3 percentage point increase in HPV vaccine completion for the privately-insured  and a 5.7 percentage point increase for Medicaid enrollees  (Table p = 0.05), the 2014 insurance reform had a larger effect among both privately- and publicly-insured females than males .Among males, the 2014 ACA reforms were associated with a 5.1 percentage point increase in HPV vaccine completion for the privately-insured  and a 3.4 percentage point increase for Medicaid enrollees  (Table In the first sensitivity analysis with the extended HPV vaccine completion window to 18 months, 32.5% of females and 34.3% of males completed the 3-dose series. The pattern of associations between participant characteristics and HPV completion within 18 months were consistent with completion by 12 months . SimilarIn the second sensitivity analysis with the 2-dose schedule, 46.9% of females and 46.5% of males completed the series within 12 months. The pattern of associations between participant characteristics and HPV completion within 12 months were consistent . SimilarWe have shown that despite low HPV vaccine completion overall, the 2010 ACA provision was associated with a modest increase in vaccine completion among females and the 2014 ACA-related health insurance reforms were associated with further increases in completion among females and males. While both privately- and publicly-insured youth benefited from the ACA, the 2014 insurance reforms had a larger effect among females than males. Furthermore, extending the HPV vaccine completion window from 12 to 18 months was associated with an increase in completion from approximately 28% to 33–34% in females and males. Despite minimal sex differences in HPV vaccine completion in these three New England states, publicly-insured youth continued to have lower completion than their privately-insured counterparts. Our findings suggest that Medicaid expansion in the 14 remaining states that have not yet expanded Medicaid [State and national estimates of HPV vaccine initiation and completion are based on self-report of the number of HPV doses received without specifying a time frame for receipt , 15, 16.This study also contributes to the literature in its inclusion of males. Prior evaluations of the 2010 ACA provision on HPV completion have been limited to females. Lipton and Decker found that the ACA increased HPV vaccine completion by 5.8 percentage points among women aged 19–25 years compared to 18- or 26-year-olds . CorrierThis is one of the first studies to evaluate the 2014 ACA-related health insurance reforms on HPV vaccine completion. We found that the reforms were associated with an increase in vaccine completion among both privately- and publicly-insured females and males. For the privately-insured, state and federal Marketplaces offered publicly-subsidized private health insurance plans as well as the protections for those with pre-existing conditions. For the publicly-insured, Medicaid expansion due to the ACA provided preventive services with no cost sharing for those who qualified. Our findings demonstrate that increasing health insurance coverage, access to preventive services and reducing costs through the ACA directly benefited youth by increasing HPV vaccine completion rates.In addition to the lack of insurance coverage and cost –12, haviThere are a number of limitations to note. APCDs only record insured enrollees, so uninsured youth, recognized to have the lowest uptake of HPV vaccine initiation and completion , were noAmong cancers attributable to HPV in the US, 92% are associated with HPV types targeted by the 9-valent HPV vaccine . Using AAdditional file 1 Table S1. Average completion of the HPV vaccine series stratified by state, sex, and insurance type across study years. Table S2. Descriptive statistics for HPV vaccine completion defined as completion of the 3-dose series within 18 months. Table S3. Descriptive statistics for HPV vaccine completion defined as completion of the 2-dose series within 12 months.