Infection of specific high risk Human papillomaviruses (HPVs) is known to cause cervical cancer and two prophylactic vaccines have been developed against two major high risk HPV types 16 and 18 for prevention of cervical cancer. Because of societal, religious and ethical issues associated with the vaccination of adolescent girls in India together with lack of awareness about HPV and HPV vaccines, no successful HPV immunization program has been employed in India.To determine knowledge, awareness and attitude of college students on HPV, HPV vaccine and cervical cancer.A questionnaire-based survey was conducted in a total of 1580 undergraduate students between the age group 16–26 years comprising 684 girls and 876 boys.Out of a total of 1580 students, girls had more knowledge about cervical cancer , HPV  and HPV vaccines  when compared to those in boys. However, knowledge about the types of HPV and vaccines was poor. Interestingly, students from biology-major had more knowledge and awareness about cervical cancer  and HPV  when compared to non-biology students. Girls from both biology and non-biology group had higher awareness compared to boys. Analysis of odds ratio (ORs) along with 95% CI showed older girls with 1.2 to 3 fold (p<0.05) higher knowledge than boys. All students agreed that girls should get vaccinated against HPV (p<0.001).It is suggested that there is a need for educational intervention and awareness campaigns to augment HPV immunization program for control of cervical cancer in India. Cervical cancer is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer death in women  but in IThe principal causative agent for the development of cervical cancer is the infection of specific high risk types of human papillomaviruses (HPVs). Though more than 140 types of HPV have been identified only about 40 types are sexually transmitted. Of these, two high risk HPV types 16 and HPV 18 are responsible for more than 80% of cervical cancer in India . In addiThe success and benefit of control and prevention of cervical cancer largely depend to a great extent on the level of awareness and knowledge about different aspects of the disease and the vaccine. It is therefore important to target immunizable young adult college-going girls and boys, as both are part of the infection chain and at risk for HPV infection as they are living a more independent lifestyle but have a choice to undergo vaccination with the consent from parents and are within the age group of successful vaccination outcome. Hence, assessment of their knowledge, awareness and attitude towards the causes of cervical cancer, HPV infection and vaccination available for the disease including adequate participation in immunization programs can lead to successful reduction in disease burden and control of cervical cancer in India. Along with screening, HPV detection and prevention strategies, creating knowledge and awareness among youth can help in further reduction in cervical cancer burden. Thus, the study aimed at assessing the extent of knowledge, awareness and attitude towards HPV, HPV vaccine and cervical cancer among college students in a premier private university where students come from different parts of the country but mostly from a higher echelon of the society. The results demonstrate that the awareness and knowledge of HPV, HPV vaccine and cervical cancer is low and depend mainly on age, sex and education-background of the college students.The study was carried out from January to March 2015 at Amity University Uttar Pradesh (AUUP), NOIDA campus which is one of the largest private universities of India. It is one of the biggest campuses and is located in the National Capital Region (NCR). It has students from all over India and abroad, the majority of whom belong to affluent and educated families with access to print and visual media. The students were from both urban and rural areas but majority belong to urban areas. The study was approved by the Institutional Ethics Committee, Amity University, NOIDA, India. An informed, written consent was obtained from each participant and recorded for the study. The informed consent form was also approved by the Institutional Ethics Committee, Amity University, NOIDA, India.A pre-tested questionnaire  was usedConsidering 25 percent HPV awareness with 3 percent allowable error and 95 percent CI, a sample of 800 was required. This was doubled i.e. 1600 to make it sufficient for sub-groups comparison. The pre-tested questionnaire was developed first to gather information on age, sex, religion, biology or non-biology background, place of residence and family income of the students while the second part formed a set of ten questions involving knowledge of cervical cancer, HPV and awareness about HPV vaccine and opinion about vaccination for girls and boys. The questionnaire was administered to the students and the data from questionnaire was processed anonymously by assigning random codes.Students were categorised into groups based on three factors; sex , age (younger students ≤ 19years vs. older students > 19years) and educational background (biology vs. non-biology)in order to examine which of these factors are strongly associated with the knowledge, awareness and attitude towards cervical cancer, HPV and HPV vaccination. Biology background students were pursuing undergraduate education in Biotechnology and non-biology background students were from various streams of Engineering.Chi-square and Fishers’ exact tests were used to evaluate the association among various factors related to awareness cervical cancer and HPV. Statistical analysis was carried out using Statistical Package for Social Sciences program . Multivariate analysis was applied and odds ratios (ORs) along with 95% CI were also calculated.Data collected through questionnaire were tabulated and analysed using various statistical methods. Out of a total of 1580 students interviewed, the number of girls was 684 and 876 boys who were again categorised based on their age into younger (≤ 19years) and older (> 19years) and background in biology or physical sciences. Of the total students, girls had more knowledge about cervical cancer , causes of cervical cancer  and HPV being the major cause of cervical cancer  and also about genital warts  compared to boys . Among gThe students were also categorised into two groups based on their educational subject specialization such as biology and non-biology groups . BiologyOverall, older students with background in biology showed more knowledge and awareness about cervical cancer as being the most common cancer in women and HPV as its primary causative agent compared to the younger non-biology or biology students. Multiple logistic regression analysis with respect to age, sex and educational-background resulted in adjusted odds ratio with 95% CI that showed older girls with biology-background had 1.2 to 3-fold higher knowledge about cervical cancer (p<0.05) compared to younger boys with non-biology-background . Older sCancer of the uterine cervix is a major cancer in India in women but it is a curable cancer, if detected early. The two major high risk oncogenic HPV genotypes, HPV16 and HPV 18 most commonly associated with the development of cervical cancer, are highly prevalent in India –7. IntegThe level of awareness among boys was found to be poor as compared to girls who were more aware about cervical cancer and HPV .A study In the present study, both boys and girls showed positive attitude towards girls getting vaccinated against HPV after knowing that it can provide protection against HPV that causes cervical cancer . HPV immThe study suggests that the overall awareness and knowledge about cervical cancer, HPV and HPV vaccination are poor among college students and are strongly associated with the age and educational background of the students in biology. There is an urgent need for educational intervention—both formal and informal, not only for girls and boys from both biology and non-biology background but also for parents to change their attitude towards HPV vaccination. To achieve this, both print and audio-visual media may play a pivotal role. It is suggested that there is a need for incorporation of HPV vaccination in the National Immunization Program which can be integrated with the screening (VIA/ Pap/ HPV DNA) and national cancer control program, and eliminate the limitations of vaccine cost and poor awareness for successful control of HPV- induced cervical and other cancers.S1 File(XLSX)Click here for additional data file.


Despite the human papillomavirus conferring a better prognosis in the primary treatment setting, the prognostic impact of viral status after treatment failure in oropharyngeal squamous cell carcinoma patients is poorly understood.We retrospectively identified 33 oropharyngeal squamous cell carcinoma (OPC) patients with local and/or distant disease recurrence post-treatment, and looked at metastatic patterns, time to failure and survival patterns by HPV status.Median overall survival following local failure was not significantly different by HPV status . However, following distant failures, HPV+ patients lived significantly longer than HPV- patients . HPV- patients were more likely to have locoregional failures as compared to HPV+ patients (p = 0.005), but the difference in distant failure between both groups was not significant (p = 0.09). HPV+ patients were more likely to develop metastases to sites other than the lung and bones.HPV positivity predicts a favorable prognosis with the potential for long-term survival following distant, not locoregional, failures. These results have important implications for the aggressiveness of treatment and type of surveillance imaging performed. Among patients with newly diagnosed oropharyngeal squamous cell carcinoma (OPC), those with human papillomavirus positive (HPV+) disease have a more favorable disease course and prognosis than those with HPV negative (HPV-) disease. Studies have proven that patients with HPV+ OPC have better local regional control and a longer overall survival and progression-free survival compared to HPV- OPC patients after curative therapy –5. HowevIn addition, previous research suggests that after definitive therapy, HPV positive patients may have a tendency to experience more unique patterns of soft tissue and visceral metastases –7. They We conducted an Institutional Review Board approved retrospective departmental chart review. All data was fully anonymized before access by the researchers, and the IRB or ethics committee waive the requirement for informed consent. We identified 33 patients treated between 2006–2015 with OPC, who had experienced local and/or distant failures after definitive therapy. We analyzed their demographics, clinical information, treatment type, and failure patterns. Failures were defined as either a local recurrence and/or distant metastasis of the disease after definitive therapy as determined by imaging and pathology. Persistent disease was defined as evidence of disease on imaging after finishing the prescribed course of treatment. Imaging studies used to determine distant metastasis included positron emission tomography (PET) or computerized tomography (CT).Specifically, we collected information including gender, age, HPV status, HPV serotype when available, disease stage, primary site, radiation dose, radiation volume, treatment course, smoking status, alcohol use, salvage therapy modalities used, date of failure, type of failure, date of last follow-up, sites of metastases, any subsequent episodes of treatment  and current disease status (disease free versus not). DNA polymerase chain reaction and/or p16 immunostaining were used to identify the HPV status of the entire cohort. All patients identified either underwent definitive chemoradiation therapy (chemoRT) or surgery followed by adjuvant therapy and were without evidence of disease following their respective definitive treatment. Patients with persistent disease following definitive treatment were excluded.All individuals received RT in this study and were treated with intensity-modulated radiation therapy. Definitive treatment in this cohort consisted of either definitive chemo/RT or surgical resection with adjuvant radiotherapy or chemoradiotherapy based on prognostic factors. HPV status was not taken into account when planning definitive treatment at time of diagnosis. Radiation fields and dosages, along with chemotherapy agents and surgical procedures, were similar regardless of HPV status. The treatments they received are outlined below, and are detailed in All patients in the surgical cohort received transoral robotic surgery (TORS) or traditional oropharyngeal resection including unilateral or when appropriate bilateral neck dissections. In the surgical cohort, 42% of patients received TORS. Patients undergoing surgery received radiation based on standard National Comprehensive Cancer Network guidelines for intermediate- and high-risk feature . All patCategorical variables were compared using Chi-square or Fisher’s exact tests. Continuous variables were compared using Mann-Whitney U test. Time to distant and local failures were characterized with the use of Kaplan-Meier plots and log-rank tests were used to compare the outcomes between HPV positive and negative patients. All reported P-values are two-sided, and P-values <0.05 were considered significant. All analyses were performed in SAS v9.4 statistical software .The median age of the patients was 58.8 years. The majority of patients were male (91%), and the HPV+ group was exclusively male. In patients where DNA PCR data was available HPV 16 was the most common genotype. The tonsil was the most common primary subsite for the HPV+ group, and base of tongue was the most common primary subsite for the HPV- group. The demographic and clinical characteristics of the cohort can be found in Time to failure from completion of definitive treatment occurred at a median of 8.6 months (range 1.7–26.3 months) for the HPV+ group, and a median of 4.0 months (range 1.4 to 22.0 months) for the HPV- group. This value was 5.7 months for the entire cohort. Failures were categorized as locoregional only, distant only and locoregional and distant. A breakdown of types of failure by HPV status is listed in Tables Of the 15 patients (10 HPV- and 5 HPV+) who experienced a local failure, 93% had advanced stage disease (stage III or IV). In the HPV+ group, 40% had a significant history of smoking (40+ packs/year) and 20% had a significant alcohol use history. In the HPV- group, 40% had a significant history of smoking, and 40% had a significant alcohol use history.Of note, HPV- patients have more locoregional failures compared to HPV+ patients statistically . However, the difference in distant failure between HPV groups is not statistically significant .A summary of sites of distant metastases by HPV status is available in For patients who developed distant failures, 86% received systemic therapy, and in addition, 50% received surgery. Of these surgeries, 71% were lung resections. For patients who failed locally only, 40% received salvage surgery and 27% received salvage RT. For a more complete breakdown of post-curative treatments for the patients who experienced distant and local failures by HPV status, see Among patients with locoregional failure, median survival from treatment failure did not significantly differ between the HPV positive and HPV negative groups at 17 months and 14 months, respectively p = 0.23). However, among patients who failed distantly, median survival from treatment failure was significantly longer for HPV+ than HPV- patients at 42 months and 11 months, respectively (p = 0.004). Furthermore, at the 2-year mark, survival rates for patients after distant failure were 65% for HPV+ vs. 0% for HPV- patients. Kaplan-Meier curves of survival analysis are included below in Figs 3. HoweveThis study attempts to address the prognostic value of HPV status following treatment failure. This data is derived primarily from definitive radiation-based treatment, and assumed to be true for primary surgery followed by appropriate adjuvant therapy as well ,9. In geIn our study, HPV positivity appears to influence survival following distal failure only. By contrast, prognosis following local failure does not seem to be as driven by viral positivity. The mechanism by which HPV continues to convey a better prognosis following distant failure remains unclear. A number of factors could be involved including increased sensitivity to chemotherapy and radiotherapy, generally fewer comorbidities, and a more limited tobacco and alcohol exposure history . DecreasThus, in HPV+ patients, long term survival following distant metastases was substantial in comparison to HPV- patients. Historical results in clinical trials where the majority of patients with recurrent/metastatic disease are HPV- also support this finding . In lighIn our study, local failure irrespective of HPV status portended a very poor prognosis. The feasibility of salvage surgery or radiation, acquired or intrinsic resistance to radiotherapy, and the evolution of the cancer likely drive survival following local failure rather than the viral status. However, in the era of treatment de-intensification, the poor prognosis associated with local failures even in the HPV+ patients should raise caution about the ease of salvaging them should reduced radiotherapy or CRT result in local failure. It is also important to note that a distant failure following chemo/RT does suggest that the disease is radio-resistant or chemotherapy resistant.While our data showed that HPV+ patients were more likely to have distant metastases and HPV- patients were more likely to fail locoregionally, the difference in distant failures was not significant. Regardless, in line with previous studies, we found that metastatic patterns are distinct between HPV+ and HPV- patients –6,13. MeThis study has several limitations including the relatively small cohort size and retrospective nature of the study that can lead to inherent bias. Additionally, while HPV status was not a factor in determining treatment, HPV+ patients did tend to receive more therapy when they relapsed potentially accounting for some fraction of their improved survival. While HPV subtyping was not available for all patients, it would be important going forward to ascertain how the different biology of non-HPV16 viral subtypes impact prognosis. Lastly, multivariate analysis was not performed given the limited number of deaths in our cohort, but would be worthwhile in future studies. Despite these limitations, this represents one of the largest studies evaluating failure patterns by HPV status in oropharyngeal cancer and significantly contributes to our growing understanding as to how best to treat, counsel and follow patients in the setting of treatment failure.Similar to the curative primary treatment setting, HPV positivity continues to drive prognosis following distant failure but does not appear to affect local regional failure after radiotherapy. Notably, in our cohort, HPV negative patients had a predilection to fail locoregionally. HPV+ patients can exhibit a variety of metastatic pattern, with longer periods to recurrence, but have the potential for long-term survival despite these metastases. Collectively, this has implications for imaging and the need for aggressive therapy following failure in HPV+ patients.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


The true HPV prevalence in the foreskins of infants and children has been little documented, but reporting on this prevalence is of great importance given its impact on the rationale for treating asymptomatic boys. We searched multiple databases from 1960 to 2016 for observational or prospective studies that reported on HPV prevalence in foreskins. We conducted a meta-analysis using a random-effects model to pool for HPV prevalence in the foreskins of infants and children. Eight studies, with a total of 556 infants and children with phimosis, were eligible for the meta-analysis. The pooled overall prevalence of general HPV, high-risk HPV, low-risk HPV, HPV 16/18, HPV 16, and HPV 18 were 17.3 (95%CI: 0.8–46.3), 12.1 (95% CI: 0.9–31.5), 2.4 (95% CI: 0.0–11.2), 4.8 (95% CI: 0.0–16.8), 1.7 (95% CI: 0.0–5.1), and 0 (95% CI: 0–0.5), respectively. The estimated HPV prevalence in foreskins was not zero among infants and children, which implies HPV transmission other than by sexual contact. Considering that high-risk HPV is detected in asymptomatic infants and children, future studies are warranted to determine whether preventive treatments in asymptomatic infants and children could be effective in preventing persistence or transmission of high-risk HPV. Among these, cervical cancer is a great burden and a source of significant fatality. Nearly all, 99%, cervical cancer cases involve malignant genetic types of HPV2. In recent decades, studies on HPV have been confined to sexually transmitted HPV and can be categorized into one of two topics: investigating the detailed mechanism of malignant HPV in inducing or aggravating cervical and penile cancer, and the potential role of preventive treatment including circumcision and vaccination.Human papillomavirus (HPV) infection is a threat to public health, causing medical problems from warts to cancer including cervical and penile cancer4. However, there still remains great controversy on performing circumcision to prevent genital infection, which could help to prevent cervical and penile cancer. There are several systematic meta-analyses on this topic, but they present different outcomes. The two most recent meta-analyses, by Van Howe et al.5 and Zhu et al.6, indicate a limited role of circumcision in preventing genital HPV infections; the authors concluded that there is no association between circumcision and protection against HPV infection because even reduced HPV prevalence by circumcision does not reflect reduced HPV acquisition and clearance.Circumcision is a relatively simple procedure without severe complications, and it is recommended because of its benefits in preventing sexually transmitted disease including human immunodeficiency virus (HIV) and HPVThe reasons for the controversy about this issue lie in the nature of the studies conducted, including few randomized clinical trials (RCTs), selection bias, reporting bias related to sampling, and measurements. Clinicians have never focused on the fact that all these studies have had selection bias in that most have included males with a sexual contact history. The preventive role of circumcision for HPV infection could compromise or overestimate the effects of circumcision because of possible preexisting HPV or pre-malignant lesions. Moreover, there are issues of sample accuracy unless specimens are acquired by circumcision because the foreskin has great importance in carrying HPV.8.To demonstrate this issue, RCTs are necessary with samples of infants or children with extremely long follow-up; however, such studies would be both costly and difficult to execute. Before such RCTs are performed, and also to discuss the role of circumcision in preventing genital HPV infection related to cervical and penile cancer, the first step would be to investigate the true HPV prevalence among males without a sexual contact history. Although clinicians seldom focus on non-sexual routes of HPV transmission, there is evidence of other modes of transmission including vertical and horizontalThe aim of this study was to investigate the real prevalence of HPV in foreskin samples from males without previous sexual contact. This investigation could remind clinicians of the importance of circumcision in HPV transmission.9 and with previous experience with meta-analysis10.To report our prevalence data, we performed a systematic review and meta-analysis without language restrictions in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statementWe included both observational and prospective clinical studies that addressed foreskins and HPV prevalence. Our sole inclusion criterion was being an infant or child on the assumption that very young children would not have had any sexual contact. Moreover, to obtain normal samples, we excluded penile inflammation. We collected the HPV DNA from foreskin tissue samples.We conducted a cross-search of all related literature in MEDLINE through June 2016 and used an optimally sensitive Cochrane Collaboration search strategy using MeSH headings including “papillomavirus infections,” “papillomaviridae,” “foreskin,” and “circumcision.” For natural language headings, we included “papilloma virus infection,” “papilloma virus infections,” “papilloma virus,” “papilloma viruses,” “papillomavirus,” “papillomaviridae,” “wart virus,” “HPV,” “human papilloma virus,” “human papilloma viruses,” “human papillomavirus,” “foreskin,” “prepuce,” and “circumcision.” We also searched EMBASE from 1980 to June 2016 and the Cochrane Library.We included studies if they met the following criteria: (i) HPV prevalence determined in infants and children without a history of sexual contact; (ii) specimens had been obtained from foreskins; and (iii) the infants and children had asymptomatic foreskins. From each study, we recorded data on first author, publication year, study location, sample size, participant age range, and investigated HPV type. We extracted the data on HPV prevalence for general HPV (any type), high-risk HPV (HR-HPV), low-risk HPV, HPV 16, HPV 18, and HPV 16/18 (HPV 16 or HPV 18).The initial screening of the electronic databases for study selection was based on information in the title and abstract. Two independent authors (D.I. Kim and J.H. Kim) conducted the screening; we screened the studies and reviewed the complete study reports for selection. In cases of insufficient data, the authors reviewed the full text of the article for further information which was either reported directly or reported that could be converted to the required values. All authors discussed the studies before final selection. The authors carefully cross-checked the references and data for each included study to ensure that no overlapping data were present and to maintain the integrity of the meta-analysis.11; arcsine transformations contribute to stabilizing the variance of simple proportions. We calculated the pooled prevalence as the back-transformation of the weighted means of the transformed prevalences, using Dersimonian-Laird weights for the random-effects model. The reason we used a random-effects model was the expected heterogeneity in the study for locations, populations sampled, and HPV types investigated.For prevalence of type-specific HPV, because we only included studies that tested for a particular HPV type in our analyses of that type, sample size varied by HPV type. To calculate the pooled prevalence of HPV, we first transformed the prevalence from each study using Freeman-Tukey double arcsine transformationWe conducted meta-regression analysis for HPV prevalence to examine the potential moderators. We analyzed the variability in the effect sizes due to differences between the moderators  with a restricted maximum likelihood estimator of the variance of the true effects.P with the Q statistic and the I2 statistic, categorized as follows: <30% not important; 30–50% moderate; 50–75% substantial; and >75% considerable12. We used Galbraith plots to spot the outliers and conducted sensitivity analyses by examining the effects of excluding those outliers. To assess the effects of individual studies on the pooled estimates, we conducted influential analysis by omitting each study and re-estimating.We assessed heterogeneity across studies using When we were synthesizing the effect sizes of each study, we eliminated the outlier data to obtain more valid effect sizes. First, we examined the presumed effect sizes of any extreme data to determine whether they were outliers; then we judged whether to include or exclude the data during synthesis. We performed all analyses using R software version 3.1.3 .13.Two reviewers independently assessed the methodological quality of the studies and the data extraction, and discrepancies were resolved by consensus. We assessed risk of bias using the Quality in Prognosis Studies (QUIPS) toolP-values for the two statistical tests for all antibiotic types.We conducted meta-analyses for small study effects using Begg and Mazumdar’s rank correlation test and Egger’s linear regression method test for publication biases. In particular, we show separately the 21 contained detailed research duration and subject description information . After we eliminated 434 studies that contained overlapping data or that appeared in more than one database and after we screened the titles and abstracts, we identified 701 studies as eligible for intensive screening. Of these, we eliminated 53 studies relating to penile or cervical cancer; 161 on circumcision and HPV clinical studies in adults; 76 on other diseases with circumcision; 51 case reports; 126 non-clinical studies; and 201 other types of studies including commentary, reviews, and letters. A total of 33 studies fit the selection criteria, but 25 of them included cases with possible sexual contact. Finally, we used 8 studies that met all the inclusion criteria. These 8 studies comprised a total of 556 subjects for HPV prevalence . A detailed flow chart showing the selection process is shown in Fig. 16 in which the authors included normal foreskins but some subjects had foreskin problems. Outcome measurement bias was low in all studies except for one14 in which the authors did not provide a detailed description of how they measured outcomes.Table Detailed findings for HPV prevalence are described in Table 21. Although the study by Verit et al.21. had a marginal risk of being an outlier, Fig. et al.21 when we analyzed HR-HPV prevalence, we described overall HR-HPV prevalence differently in Table To control for heterogeneity and also to investigate the residual influential effects of each study, we conducted sensitivity analyses Fig. , and allTo determine the potential moderating factors for HPV prevalence, we conducted meta-regression Table  and estaThe publication bias findings for all HPV types are shown in Fig. 22. This phenomenon warrants circumcision or HPV vaccination before a boy reaches adolescence. Although this study does not show direct evidence of the preventive role of circumcision or HPV vaccination before adolescence, our estimated findings for non-zero HPV prevalence in infants and children could reflect two main lessons: the possibility of non-sexual HPV transmission routes and the importance of HPV prevalence in asymptomatic infants and children. Both of these warrant future studies on the preventive role of circumcision or HPV vaccination and the long-term prevalence of HPV in those ages.The current systematic review with meta-analysis regarding the issue of circumcision and HPV features two salient points: first, we only included subjects who had not had sexual contact, and second, the investigated anatomical target was foreskins. A recent study supports our analyses by showing that HPV 6 and 11, the most common genotypes, can be transmitted earlier in life and persist for long periods including through adolescence, which is the most common period of sexual contactIn our study, by estimating non-zero HPV prevalence in asymptomatic infants and children, it is clear that there are transmission routes other than sexual contact. To date, this is the first meta-analysis to analyze quantitative and qualitative data on HPV prevalence. Despite having limited data and heterogeneity, we provided firm academic arguments for the need for preventive treatment including circumcision and vaccination in asymptomatic infants and children with no sexual contact history.23 on the transmission routes in infants and children, but prevalence alone is important to consider given the abovementioned persistence of HPV. Although HPV infection could be temporary and incidental, persistent infections are reported as being 10% for oral lesions and 2% for genital lesions. For transmission routes, there are three main categories to consider, vertical and horizontal transmission and autoinoculation. A recent meta-analysis showed that vertical transmission accounts for approximately 20% of total HPV cases22. Hence, by analogy, the assumed prevalence of persistent genital HPV infection is 0.4% for total HPV infections. Considering the high prevalence of HIV infections and cervical cancer, 0.4% is not a small number.There are still controversies23; and third, perinatal transmission by direct contact with HPV during birth through the female genital organs24. Horizontal transmission includes skin-to-skin transmission. The prevalence of skin HPV infection is relatively high for children older than 5 years, when they go to school for the first time25. Another horizontal transmission mode is nosocomial infection by medical instruments7. Although we did not demonstrate systematic results for specific transmission routes, all the included studies had strict inclusion of non-sexual routes of transmission.Vertical transmission has three components: first, transmission during fertilization; second, prenatal transmission with HPV being detectable in the amniotic fluid, fetal components, cord, and placenta22. Among these factors, use of sampled lesions is of great importance because only foreskin samples are related to circumcision. HPV prevalence in oral or anal lesions is not particularly high, and the possibility of obtaining false rates is high for samples from those areas; however, the readings for HPV prevalence in foreskins could be more accurate given the persistent characteristics of HPV detected in foreskins8. As described in Table The second salient point of this study was that we investigated the HPV prevalence in foreskins. The inconsistent results of many clinical studies and meta-analyses regarding the beneficial role of circumcision can be attributed to various reporting heterogeneities within the studies themselves. These heterogeneities could have been from using inconsistent definitions of HPV infection, differences in the samples used , the HPV DNA detection assay used, and whether or not sampled lesions were used7. Moreover, the mucosa of the foreskin does not contain squamous epithelial keratinization, covering the penis shaft and protecting it from micro-injuries26.The anatomical features of the foreskin as a positive reservoir of HPV include redundant skin layers and also moisture, which could result in skin scratches or micro-trauma and infection by other organisms. Usually, HPV itself cannot penetrate the skin epithelium, but with trauma, HPV can reach skin basal cells1. Moreover, HPV prevalence studies exist that do not include circumcision, and the value of sampling from other sites including the penis shaft and scrotum is controversial. However, there is certainty in the value of using foreskin samples. On this issue, Castellsague et al. reported that male circumcision had a protective effect for cervical cancer risk in female sexual partners without considering the penis shaft and scrotum4. HPV in the penis shaft or scrotum could be clinically irrelevant to sexual transmission22.The inconsistent results for the protective effects of circumcision also lie in the reporting bias of HPV prevalence, with many studies having reported on HPV prevalence not only in the foreskin but also in other sites including the coronal sulcus, penis shaft, and urethra27. Moreover, the current detection technique has the intrinsic limitation of assaying for HPV DNA but not discriminating for transient infections or contamination from true HPV infections22.Although this study has attributes including non-sexual origin samples for HPV from infants and children and investigating HPV prevalence in foreskins from circumcision specimens, there were still several limitations. First, we only included a small number of studies that had small sample sizes, and limited information including specific proportions of study samples could affect standard estimates, although the impact is greater for sample size. In this meta-analysis setting, insufficient sample sizes are the main reason for possible increased standard estimates. Second, our study reports on the variability of HPV prevalence. Although we adjusted for the two main issues by including infants and children who had had no sexual contact and by using foreskins as the sampled tissue, there were too few included studies to represent a worldwide pattern of HPV prevalence. In addition, most studies were from only certain countries, and although we primarily included studies that used random sampling, the wide range in the subjects’ ages is another problem. Lastly, we could not predict the long-term beneficial effects of circumcision for HPV infection, which was the fundamental goal of this study. However, only a few studies have researched the long-term effects of circumcision among randomly sampled subjects with HPV that was not transmitted sexuallyThe striking features of researching non-sexually contracted HPV infection include the ability to determine a true prevalence using foreskins from infants and children and the persistence of HPV infection in foreskins. Although we could not conclusively determine the prevalence of non-sexually contracted HPV infection, we did conduct pioneer work in this area. For the issue of persistent HPV, additional studies are required. In particular, longitudinal studies are needed to confirm the role of circumcision and also ascertain the natural history of non-sexually contracted HPV infection in infants and children.In this study, for the first time, we revealed the general HPV prevalence and HR-HPV prevalence of non-sexually contracted HPV in infants and children; we demonstrated the existence of non-sexual transmission routes including vertical and horizontal transmission. Although there is heterogeneity in gauging HR-HPV prevalence, considering the persistence of HPV in foreskins, circumcision could still be a valuable option.


Cervical cancer is the most common cancer in women living in Vientiane, Lao People’s Democratic Republic (PDR). This study examines cervical cytology using a liquid-based cytology (LBC) method and reports the presence of high-risk (HR) human papillomavirus (HPV).We collected cervical samples from 1475 asymptomatic and healthy volunteers from six hospitals in Lao PDR. A total of 1422 volunteers  were included in the final analysis. We performed HPV typing using the polymerase chain reaction technique to detect HR-HPV samples with abnormal cytology.The overall rates of abnormal cytology and HR-HPV–positive in the samples were 9.3% (132/1422) and 47.7% (63/132), respectively. The samples with abnormal cytology included 13 high-grade squamous intraepithelial lesions and one squamous cell carcinoma case. The results showed that the most common type of HPV was HPV16 (20.5%) followed by HPV58 (9.1%).Healthy women in Vientiane, the capital of Lao PDR, have high rates of abnormal cervical cytology and are likely to be HR-HPV-positive. A system for detection and prevention of cervical cancer in these women should be developed in the near future.The online version of this article (10.1186/s12885-017-3900-6) contains supplementary material, which is available to authorized users. Althougl cancer . Unfortul cancer .Lao PDR is a landlocked country with a population of 6.37 million in Southeast Asia; the capital is Vientiane. This nation has been developing at a rapid pace. Cervical cancer was estimated to be the most common cancer among women in Lao PDR in 2010 , but 201Human papillomavirus (HPV), the most common sexually transmitted infection (STI) worldwide , has beeWe previously reported on a cervical cytology screening program for healthy Lao women aged 30 years and older using a self-collecting instrument in cooperation with Laotian pathologists and Japanese pathologists in 2007-2010 , 14. In The study design is shown in Fig. Cervical specimens collected using Kato’s device  were plaAva II, Afa I, Bgl II, Acc I, and Ava I, and small DNA fragments are yielded by digestion of restriction enzymes. The DNA fragments were confirmed on agarose gels, and the HPV genotypes were identified.DNA was extracted using the phenol-chloroform protocol. The target HR-HPV DNA  was amplified by polymerase chain reaction (PCR) using TaKaRa PCR Human Papillomavirus Typing Set , according to the manufacturer’s protocol. The PCR products were confirmed using agarose gels. Restriction enzyme digestion of PCR products can also be confirmed because PCR products recognize the sites of P < 0.05.All data were analyzed using GraphPad Prism . Statistical significance was defined as We collected and examined specimens from 1475 volunteers; due to inadequate specimens, our analysis was limited to 1422 women  , 3.8%. Although it is desirable to perform LBC and HPV testing for cervical cancer screening, lack of resources makes this type of program unlikely in Lao PDR. Even when using the cheaper conventional PAP technique, there are some problems that can occur due to poor cytological technique as well as a lack of cytopathologists and cytotechnicians. These findings indicate that it is necessary to built a histopathology and cytology education system for medical staff in the future. In the meantime, we will continue PAP and HPV tests in Lao PDR using a self-collection procedure.


Cervical cancer rates in Latin America are higher than those in developed countries, likely because of the lower prevalence of screening. Specifically, less than 40% of women in Guatemala are regularly screened and even fewer women are screened in indigenous communities. Current screening strategies—Pap smears and visual inspection with acetic acid—might not be the most effective methods for controlling cancer in these settings. We thus investigated the potential of self-collection of cervical samples with testing for human papillomavirus (HPV) to help prevent cervical cancer in an indigenous community in Guatemala.A community representative random sample of 202 indigenous women age 18 to 60 years residing in Santiago Atitlan, Guatemala, were surveyed to assess knowledge of and risk factors for HPV and cervical cancer. Women were then invited to self-collect a cervical sample using HerSwab collection kits to assess the prevalence of HPV and the acceptability of self-sampling.Of 202 women who completed the survey, 178 (89%) provided a self-sample. In all, 79% of these women found the test comfortable, 91% found the test easy to use, and 100% reported they were willing to perform the test periodically as a screening method. Thirty-one samples (17%) were positive for at least one of 13 high-risk HPV types, and eight (4.5%) were positive for HPV 16/18.HPV testing by using self-collected samples was well accepted, suggesting that it is a plausible modality for cervical cancer screening in indigenous communities. Further studies are needed to assess rates of follow-up after a positive test and to determine whether these findings extend to other indigenous and nonindigenous communities in Guatemala and Latin America. Pap smears, the most common form of screening, allow physicians to detect and manage pre-cancerous lesions before they develop into CC.3 Nonetheless, CC is the third most common cancer worldwide and a leading cause of death among women in low- and middle-income countries (LMICs).4 Unfortunately, Pap smears are infrequently used in LMICs because they are expensive and require physicians, pathologists, and cytotechnicians to perform the procedure and interpret the results.5 Even in LMICs with screening programs, rates of participation tend to be low6 because Pap smears must be collected and analyzed at hospitals or other high-resource health facilities that women may not have access to.Because of the success of screening programs that use the Pap smear, CC rates are low in most high-income countries.7 The logistics of sample collection by health care providers, which then must be sent to laboratories, tested, and returned, can also be challenging in these settings. There are also cultural barriers that preclude the use of screening methods associated with sexually transmitted diseases (STDs).In addition, if women have abnormal results, they must return for follow-up assessment and/or treatment, which creates greater time and financial burdens.9 In addition, VIAs give the option to treat women with cervical lesions immediately. Thus VIA is often called a “see/screen-and-treat” or “one-visit” approach.7 Previous studies have shown that VIA screening helps reduce CC incidence and mortality in low-resource settings.8 However, VIA shares some of the same barriers associated with Pap smears, so despite these efforts, CC incidence and mortality remain high in many LMICs, presumably because of persistent low rates of screening with either approach.Hence, many LMICs have adopted CC screening programs that use visual inspection with acetic acid (VIA). VIA involves placing acetic acid on the cervix and looking for a change in color to detect lesions. This procedure is less costly and invasive than Pap smears and can be performed by trained laypersons in low-resource health facilities.11 There are 13 types of high-risk HPV associated with development of CC.12 Of these, types 16 and 18 account for approximately 70% of all cases.13 Cervical HPV tests have high sensitivity (approximately 90%) and specificity (> 80%).15 Women who test positive for high-risk HPV should follow up with a Pap smear and/or VIA or treatment, depending on each country’s setting and resources,16 but a negative test means the risk of developing CC in the next few years is minimal, lower than the risk after a negative Pap smear.17 Furthermore, when Pap smears are performed only on women who have tested positive for HPV, the relatively low sensitivity of screening by using Pap smears is significantly improved.18 Thus, primary screening for high-risk HPV before referral for Pap smear or VIA has been proposed as an alternative CC screening method. Unfortunately, HPV testing is expensive and requires infrastructure not readily available in many LMICs. Nonetheless, research is underway to develop low-cost HPV tests that can be used with minor infrastructure requirements.23Human papillomavirus (HPV) infections are responsible for more than 90% of CC cases.30 Some studies have provided women with self-collection kits, but at medical facilities before collection by a physician rather than at the woman’s home. In these studies, self-collection has been shown to have sensitivity similar to that of physician-collected samples,28 and self-collection has been found to be highly acceptable in many settings.31 This suggests that self-collection could be helpful to increase CC screening rates in LMICs, once cost- and infrastructure-efficient HPV tests have been developed. However, few studies have provided participants with the opportunity to try these in community settings outside of medical facilities; thus, it is not clear whether they would be an accepted form of primary CC screening.Self-collection HPV kits have been developed to allow women to collect their own cervicovaginal samples at home and send these to a testing facility through the mail or by other means. Studies in several countries have compared the accuracy of HPV self-collection samples with samples obtained by a physician and have assessed the acceptability of self-collection in different populations.11 largely because less than 40% of Guatemalan women  have ever been screened for CC.35 There have been self-collection studies conducted in Latin America, a region in which CC morbidity and mortality are particularly high,39 although few have tested the acceptability of HPV self-collection in community rather than clinical settings. Moreover, HPV self-collection has not been studied in indigenous populations in Latin America, who tend to have less access to health facilities and higher levels of stigma associated with physician-administered vaginal and STD tests.40 Thus, it is important to assess the acceptability of HPV self-collection kits and tests and determine the potential of HPV testing as a screening modality in these settings.37 We thus conducted a cross-sectional study in an indigenous population in Lake Atitlan, Guatemala, to assess knowledge of HPV and CC, provide women with the opportunity to collect a self-sample in their home and report their feelings and experiences, and assess HPV prevalence in indigenous populations.Guatemala has one of the highest levels of CC morbidity and mortality in the region. Age-standardized annual incidence and mortality rates are 22.3 and 12.5 per 100,000 women, respectively,We conducted a cross-sectional study in Santiago Atitlan, an indigenous community of 45,000 residents in Guatemala. Data were collected by using electronic surveys and self-collection kits.cN to each neighborhood according to its relative population size  and then randomly selecting a sample of cN blocks. One house was randomly selected per block, in which one woman was interviewed.This community is almost exclusively Tz’utujil, a Mayan indigenous group. We sampled 212 women age 18 to 60 years from nine neighborhoods that encompass 85% of the population of Santiago Atitlan. Population data were obtained from the local municipality. We followed a stratified sampling approach by first allocating samples of size If more than one woman in a house was eligible, the woman who had the next upcoming birthday was selected. Only women ages 25 to 54 years were eligible to provide a self-collected sample for HPV testing because women outside these ages are not eligible to receive screening using Pap smears or VIA according to Guatemalan CC screening guidelines. Menstruating and pregnant women were also excluded from self-collection. We chose to interview women outside the screening range because, although the focus of the study was on acceptability, we were interested in learning about the health practices and risk factors for all adult women.41 Four trained community health workers (CHWs) fluent in Tz’utujil and Spanish conducted the surveys.The survey component was designed by using the Qualtrics application. It included 143 questions about demographics, preventive health care practices, and HPV and CC knowledge and risk factors. The survey also assessed the acceptability of and feelings toward HPV self-collection. Questions were developed by using the STEPwise Approach to Surveillance (STEPS) survey, The University of North Carolina’s Family Health Study Survey, and the University of Michigan’s Michigan HPV and Oropharyngeal Cancer study.The survey was written in English and then translated to Spanish by native speakers from the study team. The survey was piloted in Guatemala City and in households in Santiago Atitlan. After each pilot, surveyor notes were reviewed and appropriate revisions were made. At the end of each day, surveys were uploaded to the server, ensuring that the participant’s data could no longer be accessed, except by members of the study team.We used Eve Medical HerSwab self-collection HPV kits. Each kit came with an instructions card written in Spanish with step-by-step infographs explaining the collection process. The CHWs were trained on the procedure and on how to explain the instructions to the participants in their native language.Upon interview completion, each eligible participant was asked about her interest in collecting a sample for HPV testing. If the participant agreed, the CHWs explained the instructions, and the participant collected a sample in a private room in the household. The collection kit comprised a plastic handle and brush. The woman inserted the brush into her vagina and then turned a crank on the handle to extend the brush. The woman then removed the brush and cranked it back by using the handle. She then returned the kit to the CHWs. Afterward, each participant completed a five-question survey assessing the level of ease and comfort associated with the collection and her willingness to self-collect periodically as a form of CC screening. Finally, CHWs encouraged participants to attend free VIA screening clinics at their local public hospital.42 HPV-28 kit, which tests for 13 high-risk HPV types according to the International Agency for Research on Cancer classification,12 as well as 15 low-risk types (Data Supplement).Samples were sent to an independent, nonprofit laboratory in Guatemala City  for testing and were tested by using the Anyplex IITo ensure the privacy and confidentiality of the participant’s information, given the sensitivity of the survey questions and the HPV test, no contact information was collected in this pilot study; thus, participants could not be contacted by the study team with their results. Instead, participants were told to call for their results 10 days after collection by using an identification number. Announcements were made daily on the local radio for 1 month after the end of recruitment that reminded women to call for their results. Participants were informed only if they tested positive for one of the 13 high-risk types.Post–self-collection survey questions were analyzed to determine the acceptability of HPV self-collection as a form of CC screening. Two additional outcomes were analyzed: positive HPV results and previous Pap smear or VIA results. Crude comparisons between these and relevant covariates were run by using log-binomial regression, and then models were run adjusting for other covariates. Statistical analyses were conducted by using SAS software Version 9.4 .The University of Michigan Institutional Review Board approved study protocols (HUM00096559). All participants gave oral, informed consent before participation. The consent was documented by signature from one of the CHWs.Of 481 women who were asked to participate through door-to-door recruitment, 212 women enrolled (44% acceptance rate), with 202 (95%) completing the survey. Ten women chose to withdraw, and their data were destroyed. Participants’ mean age was 34.5 years, and more than 80% had primary education at most . One hunOf these 178 women, 79% found the kit comfortable to use, and 91% found it easy to use. Upon collection, 100% reported that they were willing to use the test periodically as a form of CC screening, and more than 80% said they preferred to screen themselves at home rather than with a physician in a doctor’s office  and 3. BThirty-seven (21%) of 178 women tested positive for one of 28 types of HPV, and 31 (17%) tested positive for a high-risk type . HPV 16 The number of lifetime sexual partners was significantly higher in women who tested positive for HPV. Characteristics comparing women by HPV test results can be found in P = .03; adjusted PR, 1.42; 95% CI, 0.68 to 2.97; P = .34; regression tables are provided in the Data Supplement).After adjustment, the association became statistically nonsignificant, but it did show a prevalence ratio (PR) greater than 1 .After adjustment, the association between use of health services and having had a previous Pap smear or VIA remained greater than 1 but was no longer significant  might be a stronger determinant of HPV risk in this community.We assessed the acceptability of HPV self-collection as an alternative to screening by using Pap smears or VIA in an indigenous community in Latin America. We found that self-collection kits had high acceptability and were preferred to physician screenings; a majority of women found the test kit comfortable and easy to use. We found a 17.4% prevalence of high-risk HPV, which is consistent with previous studies reporting a 16.1% prevalence for Latin America.This study was intended to serve as a first step to determine the potential of HPV screening in indigenous populations and also to provide baseline data for future longitudinal studies assessing the efficacy of HPV testing versus other screening modalities. Perhaps the most relevant finding is the high acceptability of self-collection and the willingness of the participants to engage in the study. In fact, 95% of participants completed the survey, 93% were interested in collecting self-samples, and more than 90% called to receive their results, numbers higher than expected. The study was very well received in the community, with strong support from local and health authorities, suggesting the potential to eventually implement HPV screening programs in this and other similar settings.Strengths of the study include the multiclustered community design, which allowed us to obtain a representative sample of the population, provided an opportunity for participants to try self-collection in their homes rather than at a clinic, and allowed local CHWs to perform recruitment and interviews. Because of the latter, interviews were conducted in the participants’ native language, potentially making them more comfortable answering sensitive questions. In addition, data were collected electronically, which eliminated the risk of errors from manual data entry. However, there are also important limitations. Given the cross-sectional design, participants might have misreported their history of screening and other risk factors, especially if there had been community educational programs or interventions that suggested that women should be screened for CC. Women may not have accurately remembered whether they had previously had a Pap smear or VIA  or may not even be aware of whether these procedures had been performed on them. Another limitation is that we were unable to assess whether HPV-positive women followed up on their results. This is the topic of our current work in multiple communities in Guatemala with a new study population that will be followed up after 6 months and 1 year.46 Although these studies did not specifically discuss HPV and CC, the exposure to health interventions could be reflected in the women’s knowledge of health issues and their willingness to try self-collection. In the future, it will be important to assess the acceptability of these tests in other indigenous communities with less exposure to studies and interventions.In addition, this community has been exposed to prior health interventions and studies from multiple institutions.29 However, to the best of our knowledge, this is the first study to assess self-collection in indigenous populations in Latin America. This is also one of the first studies to provide an opportunity for participants to collect a sample in a community setting rather than simply sharing their feelings toward self-collection or collecting at a clinic.The study results are consistent with those of previous studies conducted in Asia and Africa on the acceptability of self-screening for HPV.47 It is also important to continue developing new affordable and easy-to-use tests that could readily be implemented in low-income settings.22This work assessed the acceptability of HPV self-screening in one community in Guatemala. Guatemala is a country with 23 languages and even more distinct communities, so our findings cannot be generalized to the whole population. It will be important to attempt to replicate the study in other parts of Guatemala and Latin America. Although it does seem that HPV self-collection screening could be a useful alternative to Pap smear or VIAs in these settings, this information alone does not allow us to make any determinations about whether this method of screening will reduce CC rates in developing countries. Women who tested positive for HPV should follow up with a doctor to receive Pap smears or VIAs or treatment. Hence, a logical next step would be to conduct longitudinal studies that compare rates of follow-up care among women who have tested positive with rates for those who have not been screened for HPV, as well as head-to-head comparisons between HPV-based versus Pap smear and VIA screening programs.48 Following Pan-American Health Organization and WHO guidelines, the ministry has compiled a list of screening programs, some including HPV testing, that could be adopted. It will be the responsibility of each province (department) to determine which program best fits their needs and resources. We hope that our study, along with future evidence,49 will help local and regional authorities identify the best CC screening alternative for their own settings.The Ministry of Health in Guatemala is in the process of refining the National Cervical Cancer Prevention and Control program.


Acquisition of genital human papillomavirus (HPV) infection is common among the young, sexually active population. Genital HPV infections do not always lead to seroconversion. We aimed to assess the association between cervico-vaginal high risk (hr) HPV DNA and type-specific antibodies in an ethnically diverse cohort of young women.Women of Dutch, South-Asian Surinamese, African Surinamese, Ghanaian, Moroccan and Turkish origin participating in a large-scale multi-ethnic population-based cohort (the HELIUS study) provided vaginal self-samples and blood samples, and completed a questionnaire regarding demographics, lifestyle and sexual health. Vaginal swabs were tested for HPV using the highly sensitive SPF10-PCR DEIA/LiPA25 system (version1). Serum samples were tested for type-specific L1 antibodies against 7 hrHPV types  with multiplex serology. We assessed the association between vaginal HPV DNA and type-specific seropositivity with logistic and linear regression, using generalized estimating equations (GEE). We determined whether this association varies by ethnicity by adding an interaction term.p = 0.47 and p = 0.57, respectively).We selected 532 women who completed the questionnaire, provided a vaginal swab and a blood sample. Their median age was 27 years (interquartile range 24–31 years). Prevalence of DNA of any of the 7 hrHPV was 22 %; HPV-52 was most common. Prevalence of antibodies against one or more hrHPV types was 24 %; HPV-16 seropositivity was most common. In multivariable logistic regression analysis using GEE, adjusting for other determinants, vaginal HPV DNA detection was associated with type-specific HPV seropositivity . In multivariable linear regression analysis using GEE, the geometric mean of type-specific antibody reactivity was 1.15 (95 % CI 1.04-1.27) times higher in women positive for HPV DNA compared to HPV DNA-negative women. There was little evidence that ethnicity modified the association between HPV DNA, and type-specific seropositivity, or with antibody reactivities (In this multi-ethnic group of young women in Amsterdam, cervico-vaginal hrHPV DNA detection was an independent determinant of type-specific HPV seropositivity. Due to increased globalization, European populations are becoming more ethnically diverse, and health risks and outcomes differ between ethnic groups . AcquisiMost infections with oncogenic HPV are cleared spontaneously and pass without malignant sequelae. Estimates of proportions of women who seroconvert after infection range from 55–85 %, depending on study design and methods employed –6. The mWe aimed to assess the association between prevalent cervico-vaginal HPV DNA of seven high risk HPV (hrHPV) types and type-specific antibodies in serum, and to evaluate whether this association varies by ethnicity.The HEalthy LIfe in an Urban Setting (HELIUS) study is a multi-ethnic cohort study conducted in Amsterdam which has been described in detail elsewhere . Brieflyn = 959). We randomly selected a maximum of 7 women per life year per ethnic group, resulting in a selection of 610 women. Participants who were not tested for HPV antibodies (n = 60), who reported HPV vaccination (n = 10) and participants with an invalid HPV DNA test result (n = 8) were excluded, resulting in a study sample of 532 women. The study protocol was approved by The Academic Medical Center Ethics Review Board , and all participants provided written informed consent (reference number METC 10/100# 17.10.1729).The selection process for this study has been described in detail elsewhere . Briefly, we selected all women aged 18–34 years from the HELIUS participants who were enrolled until December 2013, who completed the questionnaire on demographics, health behavioral characteristics, and sexual behavior, provided a self-collected vaginal swab, and of whom a blood sample was available  were stored at 4 °C for a maximum of 5–6 days at the research location and sent to the Public Health Service of Amsterdam where they were stored at −20 °C until further analysis. Swabs were eluted in 600 μL PBS and DNA was extracted from 200 μL of each specimen using the isopropanol method  with an Serum samples were stored at −80 °C at the Academic Medical Center of Amsterdam until sent for analysis to the German Cancer Research Center, Heidelberg, Germany. Antibodies to the L1 proteins of HPV-16, 18, 31, 33, 45, 52 and 58 were analyzed by multiplex serology assay based on glutathione S-transferase (GST) fusion protein capture on fluorescent beads  . AntibodDifferences in baseline characteristics between ethnic groups were assessed using the Pearson’s chi-squared or the Kruskal-Wallis test as appropriate. The statistical analysis was restricted to HPV types 16, 18, 31, 33, 45, 52 and 58.First, we assessed type-specific HPV seropositivity on a dichotomous scale among both HPV DNA positive women and HPV DNA negative women per individual HPV type by performing standard univariable logistic analyses. As a woman can be infected with multiple HPV types simultaneously we also analyzed data on all 7 HPV types with univariable and multivariable logistic regression models using generalized estimating equations (GEE). We used a binomial distribution and an exchangeable correlation structure with robust variance.We then investigated the association between cervico-vaginal HPV DNA and type-specific antibody reactivities using linear regression. For this purpose, we transformed antibody reactivities to their natural logarithm. We first undertook standard univariable and multivariable linear regression for individual HPV types. Next, we proceeded with univariable and multivariable linear regression using GEE. Because the mean and standard deviation of antibody reactivities of the 7 hrHPV types differed, we standardized the natural logarithm of antibody reactivity levels by calculating Z-scores. We used a Gaussian distribution and an exchangeable correlation structure, with robust variance. Because we included a log-transformed outcome variable in the linear regression analysis, we back-transformed the regression coefficients in order to ease interpretation. As a result, the coefficients can be interpreted as a multiplicative factor of the geometric mean; in other words the geometric mean of an antibody reactivity in women who are positive for cervico-vaginal HPV DNA, is equal to that factor times the geometric mean of women who are HPV DNA negative.All multivariable models were adjusted for an a priori defined set of variables, i.e. age, ethnicity, marital status, smoking in pack years, and number of lifetime male sexual partners. We included age as a continuous variable using restricted cubic splines with 4 knots. All variables other than age were included as categorical variables. No backward selection was done.Only one sexual risk variable could be included in the model because of multicollinearity between these variables. Also, only smoking in pack years and not smoking status was included in the models because of multicollinearity. For all other variables multicollinearity did not appear to be a concern.As a last step we investigated whether the association between HPV seropositivity and HPV DNA varied by ethnicity. To do this we tested for interaction by adding an interaction term to the multivariable model. Stata Statistical Software release 13.1  was used for statistical analyses.A total of 532 women were included in the analyses Table . The aveVaginal HPV DNA of at least one of the 7 hrHPV types was detected in 115 22 %) women of whom some had multiple infections with different HPV types; DNA of HPV-52 was detected most often , but this was only significant for HPV-33, 52 and 58  .We examined whether the effect of HPV DNA on seropositivity differed by ethnicity by adding an interaction term to the models. Neither in logistic, nor in linear regression analyses using GEE did we detect significant effect modification prevalences may not be as straightforward as sometimes is depicted, i.e. even though there is a significant positive correlation between HPV DNA and HPV seropositive participants, being HPV DNA positive does not imply that a person inevitably will be or will become HPV seropositive, while being HPV seronegative does not necessarily mean an individual has never been exposed. Concordance between HPV DNA and seropositivity therefore has little clinical application as a diagnostic tool. However it may be valuable in describing the natural history of HPV infection.Because seropositivity to HPV is a measure of lifetime exposure to HPV we expected that age would be associated with HPV seropositivity. In our sample we found a significant positive association with age 19 and seropositivity relative to the reference age of 24 years, whereas the odds for seropositivity seem to not vary much with 29 and 34 years. A similar trend has been reported by Scherpenisse et al. (2012); seropositivity percentages rise sharply in the early twenties, then stabilize and do not vary much until 59 years of age [Both significant and non-significant associations between cervical HPV DNA and type-specific seropositivity have previously been reported –15. We fSome previous studies differ in methodology to ours in several respects, in that they were conducted amongst women at higher risk for HPV infections  , 13, 14.The fact that we could only include women who provided a cervico-vaginal swab might have introduced some selection bias into our study. Alberts et al. (2015) have shown that women of Turkish and Moroccan origin from the same study population who declined to provide a sample exhibited less sexual risk behavior . However, no significant differences were observed between women who did and did not provide a self-swab for the other ethnicities suggesting limited bias for these groups. The ethnic diversity of our sample, and the concurrent analysis of 7 hrHPV types are strengths of this study. Because seropositivity is a measure of lifetime exposure to HPV and not necessarily the result of a current infection, an association between cervico-vaginal HPV DNA and seropositivity can be difficult to interpret.In conclusion, we observed a positive association between cervico-vaginal DNA detection and type-specific seropositivity. Moreover, we demonstrated higher type-specific antibody reactivities in women who are HPV DNA positive compared to HPV DNA negative women in a multi-ethnic urban population of young women. These associations were independent of demographic and behavioral factors. There was little evidence that ethnicity constituted a significant effect modifier in our population which suggests that effect modifiers other than genetic variation are at play that result in differing HPV infection outcomes in ethnic minorities.


Cancer is a complex disease that can be caused by a multitude of factors. HNSCC is complicated because some of these cancers are clearly associated with HPV, while others have no viral involvement. Determining the pathways that are commonly altered in both types of HNSCC, as well as those that are unique to viral and nonviral tumors, is important for a basic understanding of how these cancers arise and progress and critical to the development of targeted therapies. In this work, we show that all HPV-associated tumors have increased expression of E2F target genes, indicating that the tumor suppressor function of Rb is blocked. Importantly, Rb is also inhibited in a subset of nonviral tumors, suggesting that mutations present in these cancers mimic the action of the HPV E6 and E7 oncogenes. +) and HPV-negative (HPV−) HNSCC tumors. We created a computational approach that effectively categorizes gene expression as unchanged, downregulated, or upregulated by comparing the gene’s mRNA levels in the tumor to the corresponding mRNA levels across normal tissue samples. Our findings suggest that there are three major HNSCC subtypes, defined by differences in the presence of HPV and in E2F-regulated gene expression. Most HPV+ HNSCC tumors show upregulation of E2F-regulated genes, which is consistent with inactivation of Rb by the virus-encoded E7 protein. In contrast, many HPV− HNSCCs show little or no change in the Rb-E2F pathway. However, we also identified a set of tumors that show alterations in the Rb-E2F pathway in the absence of HPV. Thus, one class of HPV− HNSCCs arise without significant alterations of the Rb-E2F pathway, while a second class of tumors appear to deregulate this pathway independently of the presence of HPV.Human papillomavirus (HPV) is present in a subset of head and neck squamous cell carcinomas (HNSCCs). The cell cycle regulatory Rb-E2F pathway is a major target of HPV and is perturbed by these viruses in cell culture and animal models, as well as in human tumors. In this study, we examined differences in the Rb-E2F pathway displayed by HPV-positive (HPVIMPORTANCE Cancer is a complex disease that can be caused by a multitude of factors. HNSCC is complicated because some of these cancers are clearly associated with HPV, while others have no viral involvement. Determining the pathways that are commonly altered in both types of HNSCC, as well as those that are unique to viral and nonviral tumors, is important for a basic understanding of how these cancers arise and progress and critical to the development of targeted therapies. In this work, we show that all HPV-associated tumors have increased expression of E2F target genes, indicating that the tumor suppressor function of Rb is blocked. Importantly, Rb is also inhibited in a subset of nonviral tumors, suggesting that mutations present in these cancers mimic the action of the HPV E6 and E7 oncogenes. These studies concluded that the presence of HPV represents a distinct subgroup of the cancer with its own unique genetic signatures, specifically through its modification of the cell cycle regulatory pathway.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is associated with exposure to various risk factors, such as tobacco or human papillomavirus (HPV) infection . In rececyclin-dependent kinase (CDK) complexes, which inactivate Rb through phosphorylation, and growth-inhibiting signals promote the expression of cyclin kinase inhibitors (CKIs), which interact with cyclin/CDK complexes and prevent them from phosphorylating Rb, thus inhibiting ERG expression  pathways is based on cell culture and animal models. The TCGA data provide an opportunity to explore the role of these pathways in the context of human tumors. Thus far, most research on the TCGA data has focused on genomewide analyses. In this study, we focus specifically on the Rb-E2F/p53 pathways to identify HNSCC subtypes.+ and HPV− tumor samples with boxplots . Interestingly, while E2F1 to -3 are all known as activators of cell cycle progression  in these pathways . This an10.1128/mSphere.00580-17.2FIG S1 + tumors as a unique group based on RNA expression levels in the Rb-E2F/p53 pathway. Gene expression in each tumor was categorized , and the 25 genes in the Rb-E2F/p53 pathway  were clustered based on the categorized RNA levels using the pheatmap R package. Each gene in every tumor is defined as upregulated (red), unchanged (yellow), or downregulated (blue). Separately, consensus clustering was applied to the Rb-E2F/p53 categorized gene expression data using the ConsensusClusterPlus R package. The hierarchical clustering algorithm was used with Euclidian distance, and the parameters were set to 80% item resampling, 100% gene resampling, a maximum evaluation of 10 clusters, and 500 subsamples. The results identified 6 groupings of the tumors to be the most significant clustering level of the data. These tumor groupings were added to the heatmap as an annotation and show that the HPV+ tumors are classified as a significantly unique cluster as verified by the consensus clustering algorithm. Download FIG S1, PDF file, 0.1 MB.Consensus clustering identifies HPVCopyright © 2018 Johnson et al.2018Johnson et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The combination of increased levels of E2F1 and E2F2 with HPV-mediated degradation of Rb should lead to increased expression of an array of genes whose expression is dependent on E2F. Therefore, we analyzed the RNA levels of ERGs in HNSCC tumors. We used a specific list of 325 ERGs  ERGs upregulated in each tumor, with three low outliers  ERGs upregulated in each tumor. The two tumor groups were significantly different from each other . The range in ERG upregulation suggests that three groups of tumors can be defined based on ERG upregulation: HPV+, high-ERG tumors (more than 175 ERGs upregulated), HPV−, high-ERG tumors (more than 175 ERGs upregulated), and HPV−, low-ERG tumors (less than 175 ERGs upregulated).In HPVoutliers . The hig−, high-ERG tumors and HPV+ tumors seem to be affecting the Rb-E2F pathway in similar ways, we wanted to learn which pathways the HPV−, low-ERG group may be uniquely affecting. We searched for genes regulated exclusively in the HPV−, low-ERG subset of tumors. We used an expression cutoff of 1 to remove genes that had negligible RNA levels in both normal tissue and tumors . We then performed analysis of variance (ANOVA) on the categorized RNA levels of the remaining genes that had measurable expression levels, searching for significant differences in RNA levels between the three ERG tumor classes: HPV−, low ERG; HPV−, high ERG; and HPV+, high ERG. ANOVA identified 2,513 genes with significantly different mRNA levels (P < 1e−10) between the three groups. The Tukey test was applied to the ANOVA results to calculate the differences between pairs of groups for each gene. Out of these 2,513 genes, 586 genes  but similar RNA levels in HPV+ and HPV−, high-ERG tumors (P > 0.01).Since HPV+ and HPV−, high-ERG tumors clustered together and apart from the HPV−, low-ERG tumors. For the HPV+ tumors, the different HPV species that we detected did not cluster together (data not shown). In the HPV−, low-ERG tumors, a group of 45 genes with unique upregulation was identified (Table S4 in P = 9.0e−6) and keratin (P = 4.3e−2), cytoskeleton (P = 5.4e−2), and protein complex (P = 1.3e−1) assembly. Genes uniquely upregulated in the HPV−, low-ERG tumors were also analyzed with Ingenuity Pathway Analysis software , which revealed two major networks: dermatological diseases/inflammatory response and cell death/survival/development  than the HPV−, low-ERG group. Previously identified HPV+ HNSCC clinical associations were also verified, such as a high presence in the base of the tongue and the tonsil, a better vital status, and a higher occurrence in males  (+ tumors at the RNA level (P = 2.8e−18)  .Next, we examined the levels of the phosphorylated pRb and p16 proteins in HNSCC tumors using normalized reverse-phase protein array (RPPA) data from TCGA. Due to HPV E7’s ability to cause degradation of Rb, we expected to see low Rb levels in HPV 8.0e−8) . We were2.8e−18)  translat 5.9e−7) . Previouase KDM6 –19. We ase KDM6 –. No sign10.1128/mSphere.00580-17.3FIG S2 y axis, and patient sample data were clustered on the x axis, using the pheatmap R package. Each gene in every tumor is defined as upregulated (red), unchanged (yellow), or downregulated (blue). HPV presence is added as an annotation. Download FIG S2, PDF file, 0.1 MB.Clustering of HOX/KDM6 genes shows insignificant association with HPV presence and CDKN2A upregulation. HOX genes were analyzed as they are known to be regulated by the KDM6 demethylase. (A) HOX gene expression and (B) CDKN2A, KDM6A, and KDM6B gene expression was categorized as described in Materials and Methods. The categorized gene expression data were clustered on the Copyright © 2018 Johnson et al.2018Johnson et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + tumors indicates that Rb-mediated transcriptional repression is impaired in these samples. Furthermore, the HPV E6 protein present in these tumors should block p53 function. This is consistent with the gene expression profiles described above.E7 inactivates Rb function by binding it and stimulating its degradation. Accordingly, the presence of the HPV E7 protein and upregulated ERGs in HPVRB1, CDKN2A, or TP53 in HPV+ HNSCC tumors. To test this, mutation data from TCGA were analyzed for each tumor. If a gene had one or more nonsynonymous mutations, it was scored as mutated. Otherwise, it was considered wild type. All genes in the Rb-E2F pathway were analyzed (Table S9 in P < 0.05). No mutations were detected in CDKN2A in the presence of HPV (0/68), but CDKN2A mutations were found frequently in HPV− HNSCC tumors   had a TP53 mutation, whereas the majority of HPV− tumors (359/440) had a TP53 mutation (P = 1.2e−40)  . While t1.2e−40) .RB1 appears to be mutated in almost 10% of the HPV+ tumors (6/68) and in a few of the HPV− tumors  . Finally, we explored the idea that E7 may not be highly expressed in tumor samples harboring RB1 mutations. We compared E7 RNA levels between HPV+, RB1 mutant samples and HPV+, RB1 wild-type samples. The results show similar levels in both types of HPV+ tumors: the RB1 mutant, HPV+ tumors have an E7 mean value of 244.2, while the RB1 wild-type, HPV+ tumors have an E7 mean value of 240.7   . The hig regions . Thus, i = 0.98)  and C. S+ tumors .+ tumors were identified by aligning DNA and/or RNA-seq reads to sequences in HPV databases. Consistent with previous reports, we found that 14.2% of HNSCC tumors contained HPV  and CIP1/WAF1 (p27 and p57) families, were elevated in HPVependent  and 2A.RB1 was mutated in about 10% of HPV+ tumors and in 2.7% of HPV− tumors. In these cases, the nature of the mutations suggests that they would inactivate RB1 function. Examination of the HPV E7 proteins in these cases indicated that they were wild type with an intact LXCXE motif, indicating they should be fully capable of inactivating pRb. One interesting possibility is that the mutations of RB1 occurred before HPV infection and integration. In such cases, the RB family members p130 and p107 might compensate for the loss of pRb. The subsequent appearance of E7 would then add an additional selective advantage by eliminating p130 and p107. Alternatively, the E7-mediated inactivation of pRb in these tumors may be inefficient for some unknown reason, thus providing a selective pressure for mutation. While early studies showed no evidence of mutations in these pathways, interestingly, pRb mutations were found in HPV+ cervical carcinomas in a recent study . However, this website is no longer functional; thus, we are keeping a local copy of the data for posterity. Protein data  were downloaded through Firebrowse (http://firebrowse.org/?cohort=HNSC) at the following URL: http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/HNSC/20160128/gdac.broadinstitute.org_HNSC.Merge_protein_exp__mda_rppa_core__mdanderson_org__Level_3__protein_normalization__data.Level_3.2016012800.0.0.tar.gz.Transcriptome  and mutation data (version level 2.1.4.0) for 499 tumor and 43 normal tissue samples from HNSCC patients were downloaded from the TCGA data portal  of the 43 normal samples were calculated. Median was chosen instead of mean because outlier values could skew the mean significantly. Then, the tumor expression of each gene in 499 HNSCC patients was compared to the median of its expression in normal tissue and categorized accordingly: upregulated if the tumor expression value for that gene is greater than the gene’s normal median plus 1 MAD, downregulated if the tumor expression value for that gene is less than the gene’s normal median minus 1 MAD, and normal if the tumor expression value of the gene falls anywhere within 1 MAD of the gene’s normal median. We found this categorization method to be more effective than the TCGA expression values in predicting HNSCC subtypes see .+ tumors together better than other clustering distance methods. The categorized data (described above) were clustered using specific genes of interest. Data for genes with similar patterns of expression across tumor samples were clustered on the y axis, and data for patient tumor samples with similar patterns of expression across the genes of interest were clustered on the x axis. HPV presence was detected in TCGA BAM files as described previously (Gene expression patterns were clustered using the R pheatmap package with the default clustering distances. The default Euclidian distance clustered HPVeviously . A virusz score compared against the expression in other HNSCC tumors. The Rb protein measured was phosphorylated (S807/S811) Rb. A gene was considered mutated if one or more nonsynonymous mutations were found in that gene. No mutation was defined by zero nonsilent mutations or only silent mutations.Protein expression in tumors was measured as a P < 0.01). The Fisher exact test was also used for the mutation contingency analysis.To verify the association of clusters seen in the heatmap representation, correlations were done using the chi-square test or the Fisher test in R. Specifically, this tested for significant association of the genes’ mRNA levels  with the presence of HPV or mutations  was used to identify pathways and diseases that are associated with a group of genes.To gain a better understanding of the biological function and significance of groups of genes, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) was utilized . Genes w