HIV-positive women are at increased risk of human papillomavirus (HPV) infection, and, especially, multiple infections compared to HIV-negative women. Whether certain HPV types have a tendency to cluster in multiple infections beyond or below what would be expected by shared risk factors  is unclear. We, therefore, investigated clustering patterns of 44 HPV types in HIV-positive women from Kenya.HPV status was assessed on cervical scrapings from 498 women using GP5+/6+ PCR and reverse line blot. Logistic regression was used to model type-specific HPV positivity, adjusted for age, specific HPV type prevalence, CD4, combination antiretroviral therapy, and, in the Full Model, individual-level random effects that represent unobservable risk factors common to all HPV types. We found a modest excess of women with co-infections with 2 HPV types  in the Full Model but no significant associations of individual types. No significant deviations of observed/expected counts were observed for any 2-way combination of HPV types at either the chosen level of significance, p = 0.00005, or at p = 0.01. Findings were substantially similar when women with CIN2/3 were excluded and when they were stratified by use of anti-retroviral therapy or CD4 count.HPV co-infections occurred at random in the cervix of HIV-positive women as previously found in HIV-negative women. The removal of HPV types through vaccination should not result, therefore, in an increase or decrease in the prevalence of HPV types not targeted by vaccination in immunosuppressed women. HIV-positive women are at increased risk of infections with human papillomavirus (HPV) compared to HIV-negative women ,2. AlthoPrevious studies among HIV-negative women -7 and meTo date, the clustering patterns of multiple HPV infections in HIV-positive women have been evaluated in only one study based in the United States . ChaturvThe study in Kenya has been described in previous publications -12. BrieStatistical methods used in the present report have been described in previous publications -8,14,15.Three models were used. The Basic model included age and specific HPV type prevalence only as covariates. The Adjusted model added CD4 count (as a continuous variable), use of combination antiretroviral therapy (cART)  and an interaction term between CD4 counts and cART use. This model adjusted for the fact that low CD4 counts were found to be associated with higher HPV positivity and that this association was weaker among women with a prolonged cART use . The FulA Bayesian approach with Markov Chain Monte Carlo simulation was used; estimates were reported as posterior means and 95% credible intervals (95% CI). Discrepancies between the data (observed counts of co-infections per subject) and the model (expected counts of co-infections per subject) were assessed by posterior predictive two-sided p-values and measured by an observed-to-expected (O/E) ratio for each HPV co-infection. Since all possible two-way interactions between the 44 HPV types were included, this generated 946 (44 × 43/2) statistical comparisons. To minimize errors due to multiple comparisons, the Bonferroni correction was used to set the p-values thresholds in order to assess statistical significance. With 946 multiple comparisons, the Bonferroni corrected p-value threshold for type-type associations was 0.05/946 = 0.00005.Overall HPV positivity in the 498 HIV-positive women was 67.5% (N = 336). Multiple infections were found in 56.8% N = 191) of HPV-positive women .Our study is the first to evaluate clustering patterns of specific HPV types among HIV-positive women in Africa. The assessment of the associations between all the different HPV genotypes requires studies with a high statistical power. The present study was relatively small compared to previous studies in the general population ,5,6,8. SA strength of our analysis was the inclusion into the logistic regression model of random effects at the individual level that allowed us to account for the unobservable risk factors shared by all HPV types. Non type-specific clustering of HPV types has often been observed in previous studies of HIV-negative women prior to the inclusion of random effects. Non type-specific clustering has generally been explained by a correlation with high-risk sexual behavior as all HPV types are sexually transmitted ,5,6,8. IIn conclusion, our present findings confirm in HIV-positive women previous results observed in HIV-negative women, providing further evidence on the lack of clustering between carcinogenic HPV types in either immunocompetent or immunosuppressed women.cART: combination antiretroviral therapy; CI: Credible Interval; HPV: Human papillomavirus; O/E: Observed-to-expected.The authors declare that they have no competing interests.MHC, HDV, NRM and SF conceived and designed the study. SV performed the statistical analyses. MHC, NRM, and SRS contributed to data collection. DAMH performed the HPV testing. All authors contributed to the interpretation of the findings, and SV, HDV and MHC wrote the manuscript. All authors approved the final manuscript.


We used a PANArray HPV Genotyping Chip for high-risk and low-risk mucosal types. Genotyping data was confirmed using a conventional direct DNA sequencing method. Two cases of extragenital Bowen's disease were positive for types 16 and 33 of mucosal HPV, respectively. None of the squamous cell carcinoma cases were positive. Neither patients with digital Bowen's disease (n = 5) nor those with squamous cell carcinoma (n = 3) showed any mucosal high-risk HPV. Mucosal high-risk HPV DNA was confirmed in 5 (55.6%) of the 9 patients with bowenoid papulosis. HPV 16 was most prevalent (n = 3), while the DNA of HPVs 35 and 67 was detected in one sample for each of the two types. Our study demonstrated that two (6.7%) of the patients with 30 extragenital Bowen's disease were positive for types 16 and 33 of mucosal HPV, respectively. HPVs belonging to the mucosal high-risk group may participate in the development of extragenital Bowen's disease. However, we could not find any relationship between the mucosal high-risk HPV and Bowen's disease or squamous cell carcinoma in the fingers.Human papillomavirus (HPV) infection has been demonstrated in some of the nonmelanoma skin cancers as well as in precancerous lesions. Multiple infections of mucosal high-risk HPV may contribute to the onset of digital Bowen's disease through, if any, digital-genital transmission. We screened for the presence of the mucosal HPV DNA in patients with extragenital Bowen's disease ( Human papillomavirus (HPV) is increasingly recognized as an important human carcinogen. HPVs can be divided into cutaneous and mucosal groups according to their target sites. HPV infection was demonstrated in some of the nonmelanoma skin cancers including squamous cell carcinoma, verrucous carcinoma, and precancerous lesions including epidermodysplasia verruciformis (EV), Bowen's disease, and bowenoid papulosis –3. WhileAmong mucosal high-risk HPVs, HPVs 16 and 18 are known as major causal factors for cervical cancer. Bowen's disease and squamous cell carcinoma in the genital area are generally thought to be associated with mucosal high-risk HPVs, as in cervical cancer . These fRecent studies have shown that mucosal high-risk HPVs are related to the development of extragenital Bowen's disease , 3, 7–10In the present study, we screened for the presence of the DNA of mucosal HPVs in patients with extragenital Bowen's disease, squamous cell carcinoma, or bowenoid papulosis. For comparison, cases of verrucous carcinoma, actinic keratosis, and basal cell carcinoma were included. In particular, we focused on any possible detection of mucosal high-risk HPVs in digital cases of Bowen's disease and squamous cell carcinoma.Cases. We examined a total of 61 formalin-fixed and paraffin-embedded resection and biopsy specimens from patients with extragenital Bowen's disease (n = 30), squamous cell carcinoma (n = 11), bowenoid papulosis (n = 9), verrucous carcinoma (n = 1), actinic keratosis (n = 5), and basal cell carcinoma (n = 5). We also included five cases of normal skin as a control. All patients were immunocompetent. As shown in n = 5), hand and wrist (n = 7), buttock (n = 3), abdomen (n = 3), back (n = 5), chest (n = 1), thigh (n = 3), ankle (n = 1), arm (n = 1), and scalp (n = 1). The sites of squamous cell carcinoma were the finger (n = 3), thigh (n = 2), ankle (n = 1), face (n = 4), and scalp (n = 1). The study was approved by the institutional review board.μm paraffin sections were gently mixed with 800 μL of xylol and 400 μL of ethyl alcohol absolute by inverting the tube several times. The supernatant was discarded after brief centrifugation and the pellet was washed with 1 mL of ethyl alcohol absolute. The pellet was dried for 10 min at 55°C after the removal of the supernatant. The tissue pellet was vortexed with 80 μL of a tissue lysis buffer  and 20 μL of proteinase K, followed by overnight incubation at 55°C. The digested samples were loaded into the Magna Pure LC instrument (Roche diagnostics GmbH). All DNA samples were stored at −70°C before use.DNA extraction was performed as described previously . GenomicThe PANArray HPV Genotyping Chip  detects 19 high-risk and 13 low-risk mucosal HPV types. HPVs 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, and 73 are high-risk HPV types, and HPVs 6, 11, 32, 34, 40, 42, 43, 44, 54, 55, 62, 81, and 83 are low-risk types. Analyses were performed according to the manufacturer's instructions  .Sequencing. Genotyping data obtained from HPV chips were confirmed using a conventional direct DNA sequencing method, as described in a previous report [s report . Type-spn = 5) or squamous cell carcinoma (n = 3) did not show any mucosal high-risk HPV. Meanwhile, mucosal high-risk HPV DNA was confirmed in 5 (55.6%) of the 9 patients with bowenoid papulosis. HPV 16 was most prevalent (n = 3), while HPVs 35 and 67 were detected in one sample each. The locations were the pubis in 3 cases and the penis in 2 cases. Cases of verrucous carcinoma, actinic keratosis, and basal cell carcinoma were all negative. Cases of normal control skin were also all negative. No positive results were found in mucosal low-risk HPVs.Two (6.7%) of the 30 extragenital Bowen's disease specimens were positive for types 16 and 33 of mucosal high-risk HPV, respectively. The locations of these positive cases were wrist and buttock, respectively. There were no clinically significant findings in mucosal high-risk HPV-positive cases compared to negative ones. The two positive patients were immunocompetent. None of the 11 squamous cell carcinoma samples were positive. Patients with digital Bowen's disease  . In contdization . In a stdization . On the We determined whether there might be characteristic clinical or histological findings among the two Bowen's disease patients with mucosal high-risk HPV. Macroscopically and histologically, however, there were no significant differences between high-risk HPV-positive and negative patients. They had no other histories of systemic disease or malignancy. Notably, the locations of the lesions were the buttock and wrist. The buttock is near the anogenital area that is the main target site of mucosal HPV. Although the detection rates of HPV in patients with Bowen's disease in the hands and feet, especially in the fingers and toes, were reported to be high , 14–16, n = 3) did not show any mucosal high-risk HPV, and all patients were immunocompetent. These results are in contrast with those of a previous report which indicated relatively high prevalences of mucosal high-risk HPVs in digital squamous cell carcinoma [in situ hybridization, or Southern blot analysis and resulted in the biased detectability of individual types [Digital squamous cell carcinoma presents a diagnostic challenge because of its relatively rare occurrence and mimicry of benign conditions. Although low-risk HPV subtypes are commonly associated with benign digital verrucae, digital squamous cell carcinoma can be associated with high-risk, oncogenic HPV subtypes. In this study, patients with digital squamous cell carcinoma  of the 30 patients with extragenital Bowen's disease were positive for types 16 and 33 of mucosal HPV, respectively. HPVs belonging to the mucosal high-risk group may participate in the development of some extragenital Bowen's diseases. However, we could not find any relationship between the mucosal high-risk HPV and Bowen's disease or squamous cell carcinoma in the fingers.


Purpose. Human papillomavirus (HPV) as a risk factor for esophageal squamous cell carcinoma (ESCC) has previously been studied, but importance of HPV status in ESCC for prognosis is less clear. Methods. A total of 105 specimens with ESCC were tested by in situ hybridization for HPV 16/18 and immunohistochemistry for p16 expression. The 5-year overall survival (OS) and progression-free survival were calculated in relation to these markers and the Cox proportional hazards model was used to determine the hazard ratio (HR) of variables in univariate and multivariate analysis. Results. HPV was detected in 27.6% (29) of the 105 patients with ESCC, and all positive cases were HPV-16. Twenty-five (86.2%) of the 29 HPV-positive tumors were stained positive for p16. HPV infected patients had better 5-year rates of OS  and had a 63% reduction in the risk of death . Conclusions. HPV infection may be one of many factors contributing to the development of ESCC and tumor HPV status is an independent prognostic factor for survival among patients with ESCC. Esophageal cancer is the eighth most common cancer and the sixth most common cause of death from cancer, worldwide , 2. OnceThe etiology of ESCC remains unclear, and epidemiological studies suggest that tobacco smoking, heavy alcohol drinking, micronutrient deficiency , 8, and INK4A overexpression, hereafter denoted as p16, which is associated with superior clinical outcome [HPV types 16 and 18 are known to cause the majority of squamous cell carcinomas of the cervix –15 and a outcome , 22. Thu outcome –25 and p outcome , 25.Some retrospective clinical studies have consistently proved that patients with HPV-positive head and neck squamous cell carcinoma had a better prognosis than patients with HPV-negative tumors , 26–28. With the present study, we aim to determine the prevalence of HPV infection in ESCC and evaluate its clinical significance. We also sought to evaluate the effect of tumor HPV status on survival of patients with ESCC in northern China.μm sections were cut from each patient's tumor tissue. One representative section was stained with hematoxylin and eosin (H&E) to ensure the tissue derived from esophageal cancer. The other sections were prepared for detection. All slides were reviewed by a pathologist specializing in gastrointestinal pathology.A total of 279 patients with primary esophageal carcinoma treated with surgery, admitted to the Oncology Center, Qilu Hospital of Shandong University, were identified from December 2006 to January 2008. This hospital is located in Shandong province, which was a high-incidence area for ESCC in China . PatientPostoperative follow-up data were obtained from all patients. The following parameters were studied: gender, age, tumor location, postoperative pathological T and N status (pT and pN), TNM staging according to American Joint Committee on Cancer TNM staging system, differentiation grade of the tumor, adjuvant therapy (postoperative radiotherapy with or without chemotherapy), and smoking and alcohol habits. Anatomical localization of the tumor was grouped into an upper part (15–24 cm), a middle part (25–34 cm), and a lower part of the esophagus (35–46 cm). The tumor status was characterized into localized  or advanced disease (with distant metastases). Alcohol intake cutoff point was 0.025 kg/day. The cutoff value was based on the 2011 Chinese Inhabitant Dietary Guideline.All patients had a regular follow-up schedule including a complete history and physical examination every 3 months during the first 2 years after surgery and every 6 months thereafter. Routine radiological examinations and esophagoscopy were performed when necessary. Patients were followed until death or for a maximum of 5 years.All specimens were evaluated for HPV-16 and HPV-18 with using the in situ hybridization-catalyzed signal amplification method for biotinylated probes . Briefly, sections underwent conventional deparaffinization, heat-induced target retrieval was performed, and digestion using proteinase K and then HPV-16 biotinylated DNA probe  was applied. Sections were then denatured and stained with diaminobenzidine detection system. Sections were counterstained with hematoxylin. All tumors were further evaluated for HPV-18 by means of HPV-18 biotinylated DNA probe . The positive control was a cervical squamous cell carcinoma sample, whereas the negative control was obtained by omitting the HPV probe. All slides were scored as positive or negative. Brown staining confined to nuclei of infected tumor cells was defined positive. All scorings were conducted with no knowledge of p16 immunohistochemistry status.INK4A protein, Clone G175-405, Dako). A p16-positive tumor was used as a positive control; negative controls were obtained by omitting the primary antibody. P16-positive was defined as >50% of cells showing strong nuclear and cytoplasm immunolabeling. All scorings were conducted with no knowledge of clinical characteristics or outcome.P16 immunohistochemical detection was done as described previously . BrieflyP values less than 0.05 were considered statistically significant.All analyses were performed using SPSS 16 . Statistical analyses included univariate analyses of demographic and postoperative outcome data. For these analyses, the differences between the groups were tested for significance using the Mann-Whitney test for continuous variables and the chi-square test or Fisher's exact test for categorical variables and the Kruskal-Wallis test for ranked data. The Kaplan-Meier method and log-rank test were used for analysis and comparison of survival curves. The primary end point was overall survival (OS), defined as the time from date of surgery to death. Secondary end points included progression-free survival (PFS), defined as the time from date of surgery to death or the first documented relapse, which was categorized as local-regional disease  or distant metastases. Death from the primary cancer without a documented site of recurrence or death from an unknown cause was considered death from local-regional disease. PFS and its components were adopted to facilitate comparison with published meta-analyses . The CoxP = 0.66), age (P = 0.22), pT status (P = 0.18), pN status (P = 0.27), TNM stage (AJCC) (P = 0.14), differentiation grade (P = 0.21), adjuvant therapy (P = 0.41), smoking (P = 0.13), and alcohol consumption (P = 0.78) and only marginally associated with tumor location (P = 0.07).A total of 105 patients  met the protocol study criteria for analysis. The median age of the patients was 60 (range: 42–78) years at the date of surgery. Patients were divided into two groups according to the tumor HPV status. Baseline characteristics of HPV-positive and HPV-negative patients are shown in P < 0.001) (Twenty-nine (27.6%) of the 105 ESCC patients were determined to be HPV-positive by in situ hybridization, and all positive cases were HPV-16 ; none we< 0.001) .P = 0.002, log-rank test). The 5-year rates of OS were 65.9% in the HPV-positive subgroup and 43.4% in the HPV-negative one . The 5-year rates of PFS were 61.8% and 36.8%, respectively    . The 5-ynk test) .Univariate analysis was performed to evaluate factors potentially associated with OS and PFS . Gender,P = 0.003), N status , and TNM stage  were associated with degraded mortality risk after adjustment for smoking and tumor HPV status. Tumor HPV status was independently associated with mortality risk after adjustment for pT status, pN status, TNM stage, and smoking: patients with HPV-positive tumors had a 63% lower risk of death than patients with HPV-negative . After adjustment for pT status, pN status, TNM stage, and smoking, tumor HPV status was also statistically significantly associated with PFS. Patients with HPV-positive tumors had a risk of progression that was 62% lower than that of patients with HPV-negative tumors .We then performed multivariable analysis to estimate the association of tumor HPV status with survival outcomes . In thisHPV is a small double-stranded DNA virus with tropism for the squamous epithelium where it can cause hyperproliferative lesions . There aThe etiological role of HPV in ESCC is still unclear. The incidence of HPV in ESCC varies between different geographical areas . It is pP = 0.07) with tumor location. HPV infection in upper esophagus was higher than lower. Potentially possible causes were the route of HPV infection and the histological similarities between the oropharyngeal squamous epithelia and upper digestive tract. HPV is currently one of the most common sexually transmitted infections worldwide [In the present study, we also found that there were marginally significant differences between the HPV-positive and -negative ESCC  of the 29 HPV-positive ESCC cases expressed p16, while 14 (18.4%) of 76 HPV-negative subgroup. We observed strong agreement between tumor HPV status by in situ hybridization and p16 by immunohistochemistry, an established biomarker for the function of the HPV E7 oncoprotein. HPV in situ hybridization assay has sensitivity for single viral copies, and a positive result is strongly correlated with expression of the HPV E6 and E7 oncogenes which is the standard for defining a tumor as being effected with HPV , 47. A rThe prognostic value of HPV status has previously been investigated in patients with ESCC. However, the results were much controversial. Furihata et al. reported that HPV-positive patients had worse survival than HPV-negative patients with an overexpression of p53 in esophageal carcinoma patients; they concluded that HPV infection and p53 overexpression indicate poor prognosis . HippeläOn the basis of our data, tumor HPV status was an independent prognostic factor for OS and PFS among patients with ESCC. Other retrospective researches have also consistently demonstrated that patients with HPV-positive tumors have a superior prognosis than patients with HPV-negative ones , 48, 49.P = 0.06). However, patients with pack-years of smoking < 20 had a 49% reduction in their risk of progression compared with patients with pack-years of smoking ≥ 20 (P = 0.02). The link between HPV positivity in ESCC and smoking was still under investigation. Some studies have suggested synergistic effect [Smoking is associated with an increased risk of ESCC  and a poc effect , 56 whilc effect , 57. GenAdditional variables of potential prognostic importance, such as weight loss, anemia, performance status, dietary habits, and sexual behavior, were lacking in our study. Sample size limits the number of variables that could be included in our models. Factors not included in our models may be important and affect survival. Although statistically significant differences in survival were observed between HPV-positive and -negative, definitive conclusions cannot be drawn from this study due to its small sample size and retrospective nature; larger confirmatory studies are needed to provide definitive evidence.In addition, the temporal sequence of HPV infection and onset of ESCC cannot be ascertained. Therefore, a causal relationship between exposure and outcome must be a tentative one, despite the association of infection and tumor which has been observed. A prospective study would be needed to further address this issue. The role of viruses has great potential in the clinical practice, particularly when investigated in combination with other factors. This study provides a direction for future clinical research. However, given the limited sample size, the results of this study should be interpreted with caution.


In China, high-risk human papillomavirus (HR-HPV) prevalence is unexpectedly high in older women, but the possible reasons have not been well studied yet. This study investigated the age trends of HR-HPV infection in a prospective study.careHPV and careHPV16/18/45 were used to describe the HR-HPV prevalence, incidence and clearance. Risk factors associated with HR-HPV infections were explored using a logistic regression model.A total of 7397 women aged 25-65 years without cervical precancer or cancer were evaluated during 2010-2011 with a stratified sample of 2791 women re-evaluated after one year. Test results for (p for trends = 0.159). Risk factors that associated with HR-HPV infection differed between younger and older women.The overall HR-HPV prevalence was 13.1% at baseline, with a peak of 19.3% in women aged 55-59 years. The prevalence of HR-HPV (p for trends < 0.001), HPV16/18/45 (p for trends = 0.002), and HR-HPV other than HPV16/18/45 (p for trends = 0.002) generally increased with increasing age. Number of infections that cleared was generally greater than number of incident infections within age groups. One-year clearance rate decreased with increasing age (p for trends < 0.001), however, incidence rate was unrelated to age The greater HR-HPV prevalence in older versus younger women in rural China may be explained by a cohort effect, higher than expected incidence, and/or poorer clearance at older age. Previous studies of age-specific high-risk human papillomavirus (HR-HPV) prevalence have demonstrated substantial variability across geographical regions -3. TypicDue to the lack of well-organized, nation-wide HPV-based cervical cancer screening programmes, the age distribution of HPV prevalence in China has not been well demonstrated, and results from population-based studies also differed from each other -12. MoreIn particular, persistent infection with HR-HPV is the necessary cause of cervical cancer and its precancerous lesions such as cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) -16. One-careHPV , a DNA test for a pool of 13 carcinogenic HPV genotypes and one possible carcinogenic HPV genotype, which had been evaluated in multiple countries [careHPV platform (“careHPV16/18/45”). We also investigated risk factors for prevalence, incidence, and one-year clearance of HR-HPV.We therefore wanted to further characterize the age-specific patterns of HR-HPV infections in rural China. Using data from a multi-center study of lower-cost molecular HPV tests in rural China, we described the prevalence and short-term (one-year) dynamics of HR-HPV infection as measured by ountries ,21, and careHPV and Hybrid Capture 2 (HC2)  tested on both self- and clinician-collected specimens and visual inspection with acetic acid (VIA). Woman who were tested positive by any of the six tests or tested negative by all six tests but was selected as a part of ~10% random sample underwent a rigorous colposcopic evaluation using a biopsy protocol as previously described [A total of 7541 women aged 25- to 65-year living in rural villages in Shanxi, Henan and Jiangxi provinces were enrolled from October 2010 to August 2011 in a study called “Screening Technologies to Advance Rapid Testing for Cervical Cancer Prevention–Utility and Program Planning (START-UP)” Project. Participant recruitment processes and study procedures were described elsewhere . Brieflyescribed . The priescribed .One hundred and forty four women with histology confirmed CIN2+ at baseline were excluded with 7397 women in this analysis. Of the 2147 screen-positive women, 1859 (86.6%) returned for one-year follow-up; of the 5250 screen-negative women, 1014 were randomly selected for one-year follow-up, of whom, 932 (91.9%) returned in a year  institutional review boards (IRBs).careHPV was used to detect HPV DNA in both self- and clinician-collected specimens. careHPV is a CE Marked and Chinese Food and Drug (CFDA)-approved signal-amplification DNA assay that uses antibody capture of HPV DNA and RNA probe hybrids with chemiluminescent signal detection. Same as HC2 [careHPV-positive specimens were also tested periodically in a batch-wise fashion for HPV16, 18, and 45 by careHPV16/18/45.e as HC2 , the relcareHPV and careHPV16/18/45 results, while the prevalence of HR-HPV other than HPV16/18/45 (other HR-HPV) was inferred as careHPV-positive but careHPV16/18/45-negative.The prevalence of overall HR-HPV and HPV16/18/45 were demonstrated directly by 2 statistics. The HPV status in the successive visits were used to demonstrate incidence (-/+) and clearance (+/-). The number of incidence was adjusted for approximately 20% sampling fraction of baseline screen-negative women. McNemar’s ϰ2 test was used to compare the estimated number of incidence and clearance. The adjusted prevalence at follow-up was determined by the estimated number of incidence and clearance.The overall HR-HPV prevalence at baseline was standardized by using the world standard population that was reported by the WHO in 2001 . Age-specareHPV and HC2 results .The results were confirmed by using HC2 outcomes (data not shown). To increase the analytic sensitivity, these analyses were redone by combining the SPSS 17.0  was used to analyze the data. Statistical significance was assessed by two-tailed tests with an α level of 0.05.The median, mean, and age range of the participants were 44, 44.5, and 25-65 years, respectively; 27.5% of the participants were menopausal at baseline, and the mean age at menopause was 48.7 years. Most women were Han ethnicity (99.9%), farmers (75.5%), currently married (97.1%), and of less than 12 years’ education (98.2%). Most women reported that they did not smoke (99.6%) or drink alcohol (91.5%). Most women have self-reported only one sexual partner in their lifetime (87.3%).Overall, 969 of 7397 women  were HR-HPV positive at baseline, and the positive rates varied among study sites . The prevalence of HR-HPV age-standardized to the world’s standard population was 14.2% (95% CI: 13.4%-15.0%). The crude prevalence of HPV16/18/45 was 2.9%  and of other HR-HPV was 10.2%.Risk factors associated with the HR-HPV infection are described in Table Risk factors associated with HR-HPV infections have also been stratified by tertiles of age , with the prevalence of 11.4%, 12.4%, and 15.8%, respectively  months. The crude and sampling fraction-adjusted HR-HPV prevalence at follow-up was 20.6% and 11.1%, respectively. The net change in HR-HPV infection is demonstrated in Table  months. careHPV positive or HC2 positive showed similar results.The age trends of incidence and clearance of overall HR-HPV, HPV16/18/45 and other HR-HPV are illustrated in Figure The baseline risk factors of HR-HPV incidence and clearance are also investigated Table . Women wThis study investigated the age-specific prevalence of overall HR-HPV, HPV16/18/45 and other HR-HPV in 25 to 65-year-old women without cervical precancer and cancer in a prospective study in rural China. It focused on the interpretation for the peak observed in older women. Moreover, we explored the risk factors of HPV incidence and clearance using one-year follow-up data.careHPV at baseline in our study was 14.2%, which was comparable to other studies in mainland China [careHPV was similar to the prevalence as measured by HC2 in our study (p = 0.196), which suggested that the difference between the prevalence in this study and the recent pooled analysis [The age-standardized prevalence by world population of HR-HPV tested by nd China -10, and nd China . Howevernd China . We repond China ,10. Thesanalysis  was primPrevious studies in China had observed a second peak of HPV prevalence in older women, although the peak age varied between studies -10,13. IThe probable explanations for this HR-HPV peak in older Chinese women could be a cohort effect that leads to increased lifetime exposures, increased HPV incidence ,and/or increased viral persistence in older women  versus yBefore the one-child policy which had been strictly implemented in year 1979 , most woHowever, one-year follow-up was not sufficient to provide a strong evidence to explain the cohort effect. Long-term follow-up studies with birth cohort analysis are needed to further clarify this effect. With recent changes in sexual norms, we anticipate an upturn in the annual incidence of cervical cancer in China unless widespread secondary prevention through screening, diagnosis, and treatment of precancerous lesions is implemented.et al. [We expected to see a decreased trend in the incidence of HR-HPV infection in older women as reported in other studies ,37, but et al.  found thAnother explanation, a greater HPV persistence in older women, was strongly supported by our data  cohort effect; 2) higher than expected incidence; and/or 3) poorer clearance/greater persistence of HR-HPV at older ages. Long-term prospective studies with frequent follow-up intervals by HPV genotyping are needed to verify the conclusions from this study.PEC has received commercial HPV tests for research at a reduced or no cost from Roche, QIAGEN, Norchip, and mtm. He is a paid consultant for BD, GE Healthcare, and Cepheid, and has received a speaker’s honorarium from Roche. He is a paid consultant for Immunexpress on sepsis diagnostics. He is compensated as a member of a Merck Data and Safety Monitoring Board for HPV vaccines. JJ was the director of the study and received all the tests used in the study as a donation from the manufacturing companies (QIAGEN and Arbor Vita Corporation). All other authors have no competing interests.PEC, JJ and YLQ contributed to conception and design of the study. All authors were involved in the study implementation. LNK, FHZ, JJ, FC, JL, WC, XZ assisted in the data collection; LNK, JJ, PB, JL contributed to the data management. PEC, LNK designed the analysis; all authors were involved in data analysis and interpretation. LNK, PEC, and YLQ drafted manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/96/prepubAge group-specific, one-year persistence of any high-risk HPV, any HPV16, 18, and/or 45 (HPV16/18/45), and high-risk HPV other than HPV16/18/45. Additional file Click here for file


Human Papillomavirus (HPV) high-risk (HR) types are the causal factor for cervical cancer and premalignant dysplasia. Data on frequency of HPV types provide a basis to design and evaluate HPV prevention programs. Taking into account the heterogeneity of HPV types across and within populations this study aims to access the HPV frequency in Brazilian women.Betapapillomavirus and a likely new type, previously reported, from 132 women positive for the virus analysed by Hybrid Capture II assay. These women were infected by a single or multiple HPV types and 142 HPV strains were identified. HR types were found in 75% of women and HPV types 16, 18, 45, 58, and 66 had the highest frequency. Significant differences in frequency of HR HPV types were found for presence of cervical lesions, and for different HPV species and women age.We identified 24 different types of HPV, including a Compared with previous studies in Brazil, our data indicated differences in frequency and HPV type diversity, a significant association of other HR-types but HPV16 and 18 and cervical lesions, and a trend for distinct distribution of HPV types by age. Cervical cancer accounts for the third highest mortality amongst cancers in women worldwide, with a higher incidence and frequency in underdeveloped and developing countries . The etiThe high frequency of HPV16 and HPV18 in cervical cancer and pre-cancerous lesions lead to development of vaccines against L1 viral capsid proteins of these types ,10. HoweTaking in account that the characterization of HPV types will be valuable to implement immunization polices and to monitor the presence of different HPV types, the present study aim to accesses the diversity of HPV types in women from communities of low socioeconomic status of the Metropolitan region of the city of Rio de Janeiro city, Brazil.We studied women from Duque de Caxias and Nova Iguaçu, two municipalities with low socioeconomic status in the state of Rio de Janeiro, Brazil, assisted by the governmental Family Health Program. They had been visited by trained health care professionals and invited to participate in studies for evaluating the efficacy of different methods for detecting cervical lesions , and theThe conventional cytology results was classified according to the recommendations of Brazilian Ministry of Health and Brazilian Society of Cytology , which iSamples were submitted to pH neutralization step with addition of HCl 1N. DNA isolation was carried out with QIAamp DNA Mini and Blood Kit  following the manufacturer's instructions, modified at the elution step that was performed with 30 μL of AE buffer.HPV DNA amplification was performed by nested-PCR with MY09/11  and GP05http://blast.ncbi.nlm.nih.gov/Blast.cgi and by phylogenetic analysis within the MEGA 4.0 software [Alphapapillomavirus. Sequences from Betapapillomavirus and Deltapapillomavirus were used as outgroups. The strength of each node was evaluated by bootstrap test with 1,000 replicates. HPV types were epidemiologically and phylogenetically classified following Muñoz et al [Identification of HPV types was carried out with the Blast software software  applyingoz et al  and de Voz et al , respect2 test. Mann-Whitney and Kruskal-Wallis tests were used to analyze differences between age at diagnoses and HPV type for all women.Association between HPV types and cytology results for women with single infection was performed with the χA total of 297 women positive for HCII assay had samples available for DNA isolation and 132 of these had HPV DNA successfully amplified. Despite this, there were no significant differences in respect to the cytological results  and age between women that had HPV DNA successfully amplified and those that not had. The mean age of the 132 women were 39.5 years, ranging from 25 to 59 years of age.HQ834551 - HQ834692). Infections by multiple HPV types or by different strains were found among the 39 women that could not be typed by direct sequencing due to overlap of sequence-peaks and were submitted to molecular cloning and clone sequencing. HPV typing carried out with Blast and confirmed by phylogenetic analysis showed the presence of 24 different HPV types, including HPV17, a Betapapillomavirus often identified in cutaneous lesions [A total of 123 women had the HPV type identified totalizing 142 HPV sequences corresponding to women infected with single, multiple HPV types or by different strains of the same type (GenBank accession numbers  lesions . One hunThe frequency of HPV HR-types among HPV+ women was 75% (99/132 women), with a highest frequency for HPV16 , followed by HPV18 , HPV45 , HPV58 , HPV66 , HPV31  and HPV33 . Considering only the 84 HPV+ women with normal cytology, we found frequencies of 28.6% (24/84) for HPV16 and 19.0% (16/84) for HPV18.2 test, p = 0.0411). Our data also showed that infection by alpha-7  and alpha-9 species  presented a significant distinct distribution by age at diagnosis respective to women positive for other HPV types  . However, we identified 12 HPV types  not included in that set of probes. These findings reinforced previous evidence of cross-hybridizations among probes used in HCII test [Betapapillomavirus HPV17 was also identified, a type frequently associated to cutaneous lesions, indicating that probes contained in the HCII test were capable of cross-hybridizing with viruses not belonging to Alphapapillomavirus.All cervical samples included in the present study were HPV+ by the HCII assay, which include probes for detection of 18 CII test -45. InteIn Brazil, most studies on HPV frequency used as inclusion criteria the suspicion of HPV infection, presence of cervical lesions or cancer. In our study, these criteria were not used, a reason why we compared our findings with studies with similar inclusion criteria ,19-24. TIn a study performed in Metropolitan region of Rio de Janeiro city at the Southeast region of Brazil, Oliveira et al  analyzinIn two studies performed at the South region of Brazil, the first by Krambeck et al  in the sConcerning the 84 HPV+ women with normal cytology, we found a higher frequency of HPV16  and HPV18  than in a meta-analysis, restricted to women with normal cytology, carried out for South America  with 15%e.g.: ranging from 17.2% to 78.7% for HPV16, and from 0% to 14.4% for HPV18, among HPV+ women), evidences the need to further investigations to improve information of geographical distribution of HPV types in Brazil using standardized methodologies to HPV detection and typing.Our results indicated a trend for a higher proportion of lesions in women infected by HR-types other than HPV16 and/or HPV18, indicating that other HR-HPVs must also be considered for further implement appropriate immunization and monitoring policies. Moreover, the considerable difference in frequency of HPV types amongst previous studies (AGUS: Atypical glandular cells of undetermined significance; ASCUS: Atypical squamous cells of undetermined significance; HCII: Hybrid Capture II assay; HPV: Human Papillomavirus; HPV HR: Human Papillomavirus of High-Risk for cancer; HPV LR: Human Papillomavirus of Low-Risk for cancer; HSIL: High-grade squamous intraephitelial lesion; LSIL: Low-grade squamous intraephitelial lesion; PCR: Polymerase Chain Reaction; RFLP: Restriction Fragment Length PolymorphismThe authors declare that they have no competing interests.MOS and CXL contributed to conception and design, acquisition, analysis and interpretation of data. CXL and MOS performed the molecular procedures, phylogenetic analyses, and drafted the manuscript. CRB revised the data and contributed with important intellectual content. MAMM and LMGZ conceived participated in study design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


The accurate genotyping of human papillomavirus (HPV) is clinically important because the oncogenic potential of HPV is dependent on specific genotypes. Here, we described the development of a bead-based multiplex HPV genotyping (MPG) method which is able to detect 20 types of HPV  and evaluated its accuracy with sequencing. A total of 890 clinical samples were studied. Among these samples, 484 were HPV positive and 406 were HPV negative by consensus primer (PGMY09/11) directed PCR. The genotyping of 484 HPV positive samples was carried out by the bead-based MPG method. The accuracy was 93.5% , 80.1%  for single and multiple infections, respectively, while a complete type mismatch was observed only in one sample. The MPG method indiscriminately detected dysplasia of several cytological grades including 71.8%  of ASCUS  and more specific for high grade lesions. For women with HSIL  and SCC diagnosis, 32 women showed a PPV  of 77.3% . Among women >40 years of age, 22 women with histological cervical cancer lesions showed a PPV of 88% . Of the highest risk HPV types including HPV-16, 18 and 31 positive women of the same age groups, 34 women with histological cervical cancer lesions showed a PPV of 77.3% . Taken together, the bead-based MPG method could successfully detect high-grade lesions and high-risk HPV types with a high degree of accuracy in clinical samples. Human papillomavirus (HPV) infection is associated with a variety of clinical conditions, ranging from asymptomatic infections to benign and malignant diseases of the genital mucosa There are currently two main molecular approaches used for HPV detection: the polymerase chain reaction (PCR) with generic primers used to amplify part of the L1 gene of the viral capsid, which is highly conserved among anogenital HPVs, and the Hybrid Capture II test , which detects 13 high risk HPVs including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The HCII allows quantification of the HPV DNA load, but lacks information on the HPV genotype. Since the oncogenic potential differs among the high risk HPV types Luminex (xMAP) suspension array technology is based on polystyrene beads that are internally dyed with various ratios of two spectrally distinct fluorophores. Different molecules such as individual oligonucleotide probes can be coupled to different bead sets with specific absorption spectra. These sets are combined to a suspension array and allow up to 100 different probes to be measured simultaneously in a single reaction (multiplexing). This technology can potentially be fully automated, dramatically decreasing the personal cost component of the assay; this assay has been used for the genotyping of 45 HPV types with PGMY09/11 PCR The purpose of the present study was to validate a bead-based MPG method developed for the identification of 20 HPVs  with sequencing. Histologically confirmed cervical intraepithelial neoplasia (CIN) grade 1 (CIN1), and CIN grades 2–3 or higher (CIN2+) were used. This study showed that the MPG method is a valuable tool for the detection of pre-cervical cancer or cervical cancer lesions (CIN2+).All women underwent cytologic testing and HPV screening. The number of sequencing positive and negative samples was 484 and 406, respectively. Among these, 484 PCR product positive samples were used for the evaluation of the bead-based MPG method as shown in The bead-based MPG method basically uses the MFI of the reporter fluorescence for HPV genotyping. That is, positivity of the respective types is calculated from the MFI over a defined cutoff value and can provide a semi-quantitative numerical output. For the calculation of the cutoff value, the biotinylated PGMY09/11 amplimers generated from 200 ng of the HPV negative human DNA were hybridized after one step PCR see . The det6 copies of HPV plasmids and 200 ng of genomic DNA from HeLa cells known to be HPV 18 positive. Positivity for the respective types was calculated from the MFI over their cutoff values. The PGMY09/11 PCR amplicons of the HeLa cells showed a high MFI value only in for the HPV 18 probe. There was a 10 fold higher MFI for GAPDH than the other PGMY09/11 PCR amplicons without GAPDH genomic DNA  and hybridized to PMGY09/11 amplicons generated from 1×10omic DNA . In addiAs shown in The accuracy of the bead-based MPG method was also estimated for multiple infections . The verThe HPV genotyping by the bead-based method and sequencing showed almost perfect agreement ; while the κ values for the detection of HPV 16 and 18 were 0.99 and 0.96, respectively. In addition, the sensitivity, specificity and accuracy of the bead-based MPG method were 90.7%, 100%, and 94.9%, respectively, for the detection of high risk HPVs when the genotype information given by sequencing was used as the standard. This suggests that the bead-based MPG method could successfully detect HPV infections and identify HPV genotypes with a high degree of accuracy.Accurate HPV genotyping has become an important prognostic indicator in the clinical screening of women due to the strong casual relationship between persistent HPV infection and high grade cervical intraepithelial neoplasia In most studies, the HPV genotyping accuracy in multiple infections has not been evaluated because the conventional direct sequencing cannot resolve the identity of genotypes in samples containing multiple infections in which viral sequences overlap This study also showed that 89.3%  of single infections were caused by high risk types; the three main high risk types were HPV-16, 18, and 53. In the contrast to other studies In this study, such relationships with multiple infections and cervical intraepithelial neoplasia grade were also observed. The PPV for HPV positive women with ASCUS is higher than the PPV for women with normal cytology, indicating that the consistency might be due to the high accuracy of the bead-based genotyping method with enough sample numbers. Among women >40 years of age, the PPV of the diagnoses HSIL and SCC for CIN2+ were 93.3% and 80.0% respectively. This suggests that women >40 years of age with HSIL and a positive HPV test can be directly referred to treatment. HPV 16 positive women have a PPV for CIN2+ of 78.1% when including all age groups, representing a higher PPV than 68.9% of the cytological HSIL diagnosis, while the PPV of the other HPV types is lower. Thus, these findings suggest that the bead-based MPG method showed a higher clinical specificity and positive predictive value, specially for CIN2+. For the fundamental understanding of multiple infections and patient follow up for CIN2+ The accurate genotyping of human papillomavirus is clinically important because the oncogenic activity of HPV is dependent on the specific genotypes. Although HPV genotyping by sequencing is the most accurate method, it is the most labor intensive and time-consuming. Therefore, the ideal HPV genotyping method should have genotyping accuracy comparable to sequencing and be automated or deployed on a high-throughput platform, features essential for an assay intended for a large volume of patients. Our findings suggest that the bead-base MPG method is a valuable strategy for an efficient HPV genotyping and risk stratification of women with a positive HPV test.All patients involved in the study had signed a declaration of consent stating that the patients specimens may be used for scientific intentions. Specimens were obtained from the patients in the Department of Obstetrics and Gynecology in concordance with procedures approved by the Institutional Review Board of The Catholic University of Korea (07BR238).A total of 890 cervical samples were tested for HPV detection with the patient's permission, in the Department of Obstetrics and Gynecology, The Catholic University of Korea. Cervical swabs were obtained with a Cerex-Brush . The brush was immediately rinsed in a vial of PreservCyt® solution . To isolate DNA, two millimeters of cell suspension was transferred to a 2-ml reaction tube and washed twice by centrifugation at 14,000 rpm for 2 min, followed by removal of the supernatant and resuspension of the pellet in 1 ml of phosphate-buffered saline. The pellet was resuspended in 200 µl of phosphate-buffered saline and used for DNA isolation with the Qiagen blood DNA minikit  according to the manufacturer's instructions.For the evaluation of specificity and cross hybridization of the bead-based MPG method, plasmid clones of the following 20 HPV types were used: 6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68 and 70.http://hpv-web.lanl.gov/)  on the basis of the DNA sequences of the viral L1 gene, which were obtained from the HPV database (nl.gov/) . All pro5′-GAGTCAACGGATTTGGTCGT-3′ for the forward primer and GAPDH R, 5′-TTGATTTTGGAGGCATCTCG-3′ for the reverse primer. Since the copy number of the GAPDH gene in the clinical sample was usually higher than that of the HPV, the GAPDH primer concentration was reduced to one fourth of the PGMY09/11 primer concentration. A biotinylated primer set was used for one step PCR reaction. The PCR components in 20 µL PCR mixture were 0.025 µM biotinylated PGMY09/11 primer, 40 µM dATP, dGTP, dTTP mixture, 10 µM biotin-11-dCTP , 75 mM Tris HCl (pH 9.0), 20 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, and 1.5 U Taq polymerase . A 5-min denaturation step at 94°C was followed by a 40-cycle denaturation step at 94°C for 1 min, an annealing step at 53°C for 1 min and an elongation step at 72°C for 1 min.Amplification of the L1 gene on 200 ng of genomic DNA isolated from cervical samples was carried out with the biotinylated PGMY09/11 primer set. PCR conditions were the same as published in the PGMY 09/11 version The type-specific oligonucleotide probes were 5′-amine modified and then were coupled to carboxylated beads  through a carbodiimide base coupling procedure. Preparation of the beads coupled with oligonucleotide followed a procedure reported previously 4 beads (2.5×103 for each HPV type and control) were added to a labeling tube containing the PCR product. The hybridization was conducted as previously reported Hybridization buffer  and 1 µL of HPV bead mix containing 6.75×10http://www.ncbi.nlm.nih.gov/BLAST).The amplified L1 gene with the PGMY09/11 primer set was purified with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions. Sequencing was performed using the BigDye Terminator v3.1 cycle sequencing kit with AmpliTaq DNA polymerase (Applied Biosystems) according to the protocols supplied by the manufacturer with type-specific primers. The sequences obtained from the sequencing were compared with the HPV sequences of known types using the basic local alignment search tool from the NCBI website Click here for additional data file.Table S2The comparison of the results of HPV genotyping with DNA sequencing.(DOC)Click here for additional data file.Table S3Type-specific oligonucleotide probes for 20 HPV genotypes.(DOC)Click here for additional data file.


Human papillomavirus (HPV) seroprevalence data have not previously been reported for different geographical regions of China. This study investigated the cross-sectional seroprevalence of antibodies to HPV 6, 11, 16, and 18 virus-like particles in Chinese women.Population-based samples of women were enrolled from 2006 to 2007 in 3 rural and 2 urban areas of China. Each consenting woman completed a questionnaire and provided a blood sample. Serum antibodies were detected using a competitive Luminex immunoassay that measures antibodies to type-specific, neutralizing epitopes on the virus-like particles.A total of 4,731 women  were included, of which 4,211 were sexually active women (median age 37) and 520 virgins (median age 18). Low risk HPV 6 was the most common serotype detected (7.3%), followed by HPV 16 (5.6%), HPV 11 (2.9%), and HPV 18 (1.9%). Overall HPV seroprevalence to any type was significantly higher among sexually active women (15.8%) than virgins (2.5%) (P = 0.005). Overall seroprevalence among sexually active women gradually increased with age. Women from rural regions had significantly lower overall seroprevalence (Odds Ratio (OR) = 0.7; 95% CI: 0.6-0.9, versus metropolitan regions, P < 0.001). With increasing number of sexual partners, women were at higher risk of seropositivity of any type . Wives were at higher risk of seropositivity for HPV 16/18/6/11 when reporting having a husband who had an extramarital sexual relationship . There was a strong association between HPV 16 seropositivity and presence of high-grade cervical lesions .HPV seroprevalence differed significantly by age, geography, and sexual behavior within China, which all should be considered when implementing an optimal prophylactic HPV vaccination program in China. The etiological role of human papillomavirus (HPV) infection in cervical precancer/cancer is supported by numerous biological and epidemiological studies -3. OncogRecent advances in technology have improved our ability to detect HPV cumulative exposure by measuring the antibody response to virus-like particles. HPV DNA is often transient in exfoliated cells or tissue and thus cannot provide a reliable indicator of past exposure . HPV DNATherefore, in this report, we measured seroprevalence of four common HPV types  in a population-based sample of women from five regions of Mainland China, stratified by histological grade of cervical intraepithelial neoplasia (CIN). The purpose of this study was to evaluate HPV seroprevalence and estimate HPV cumulative exposure among Chinese women, as well as to identify its risk factors.This cross-sectional study was performed within Mainland China between May, 2006 and April, 2007, and included population based samples of women from 5 areas of China: Shanxi , Beijing , Xinjiang , Henan  and Shanghai , as previously described . SubjectConsenting women were enrolled in an age-stratified quota sample, with aimed maximum of 125 women in each of eight 5-year age strata  per geographic area. We encountered difficulties, however, in recruiting women aged 15 to 19 years, as most women in this age group are not married and therefore not willing to undergo gynecological examinations. Due to the relatively small number of subjects in 15-19 year age group (n = 27), we combined this group with the 20-24 age group for data analyses.There were 4,372 sexually active subjects according to self-reported sexual history, who provided serum samples. Basic sociodemographic data, menstrual and reproductive history, sexual history and other behavioral information were collected during the visit when the plasma was obtained. Additionally, 649 subjects self-reported with no prior history of sexual intercourse were interviewed for demographic and behavioral information.Nine ml of blood was taken from all consenting females. Blood samples were centrifuged for 10 minutes and aliquoted into plasma, buffy coats and red blood cells. Aliquots of blood were stored at -80° C. Consenting sexually active women also provided self- or physician-collected exfoliated cervical cells for HPV DNA detection by HC2 .A threshold value of at least 1.0 pg/ml was used as the cut-off for HC2 positivity, according to the manufacturer’s instructions. Details on HPV DNA measurement are previously described . Liquid Yeast-derived VLPs were coupled to a set of four distinct fluorescent Luminex microspheres using conjugation chemistry as previously described . AntibodThe HPV 6, 11, 16 and 18 complex Luminex immunoassay (cLIA) was performed in a 96-well microtiter plate. Samples were tested at a 1:4 dilution. Serum samples and detection antibodies were added to each well, followed by the VLP-microspheres for types 6, 11, 16 and 18. The plates were sealed with foil covers and incubated overnight for 15 to 25 hours. Following incubation, the plates were washed 3 times and then analyzed on a BioPlex (Luminex) instrument. High, medium, low and negative controls used for this assay were collected from humans that were either HPV sero-negative, had low antibody titers from natural infection or had medium to high antibody titers to HPV L1 VLPs following vaccination. Details of cLIA are described elsewhere ,18.The statistical database was created using Microsoft Visual FoxPro (version 8.0). Odds ratios (OR) for HPV seropositivity and corresponding 95% confidence intervals (CI) were calculated by non-conditional logistic regression, with adjustment for age , geographic site  and HPV HC2 DNA status. The adjustment over HPV DNA status considers potential association between HPV serology and HPV DNA results. Chi-square test was conducted at significance level of 0.01. Basic statistical analyses were conducted using STATA (version 9.0) and R (version 2.4.0).Among 4,372 sexually active subjects, 4,212 women had finished questionnaire and complete HPV DNA data and cytology results. One woman had missing serological result and was therefore not included in the analysis. Thus 4,211 women  were included for final statistical analyses  restricted to sexually active women. Of 649 virgins, 520 subjects  provided blood samples and had serological results available for analyses.A total of 4,731 participating females had HPV serology results measured and validated by serology assay and completed questionnaire information with a median age of 35 (range 14-54 years). Overall, oncogenic HPV 16 seroprevalence was higher  than that of HPV 18 seroprevalence . Low risk HPV 6 was the most common serotype detected , and notably more common than HPV 11  , while most were married (94.8%), never smokers (96.9%), never drinkers (79.4%), and reported a history of contraceptive method use (89.5%). Most women (79.9%) had started their sexual life before 25 years of age, and reported a single lifetime sexual partner (77.3%).Overall and type-specific HPV 6, 11, 16 and 18 seroprevalence limited to 4,211 sexually active women are shown in Figure In terms of sociodemographic and sexual risk factors for type-specific HPV seropositivity, females over 35 years old had a higher seroprevalence of HPV 6/11/16/18 (OR = 1.3 [95% CI: 1.0-1.8] versus 15-24 years after controlling for geographic site and HPV DNA positivity) . A significant increase in seropositivity for HPV 6 or 16 was observed in women reporting having more than one lifetime sexual partner or their husband having had extramarital sexual relationships (P < 0.001). Other factors were not found to be significantly associated with seropositivity for HPV 6 or 16 among Chinese women.Table This report of population-based seroprevalence of HPV among Chinese women, to our knowledge, is the first to be conducted. Within five different geographic regions across Western to Eastern China, we found a higher HPV seroprevalence in women from metropolitan areas, Uyghur ethnic groups, and in Shanghai city. Differences in sexual behavior, as well as differences in underlying population-based seroprevalence, are likely causes of differences in HPV seropositivity across regions. Overall seroprevalence of HPV 6, 11, 16 and 18 among Chinese women increased with age, and were strongly correlated with higher grades of cervical lesions.A relatively low seroprevalence to HPV 16 and 18 was found among Chinese females, as compared to other areas worldwide . Given tThe age trend of HPV seropositivity in our study was characterized by a peak for anti-HPV seroprevalence to any type in the late 40s, and a relatively flat curve for individual HPV types across different age groups. Such age-specific differences are possibly due to the fact that HPV seropositivity reflects cumulative exposure, which is higher among older women. High-risk HPV DNA prevalence peaks in both the early twenties and early forties in Chinese women  and low-The association with HPV seropositivity and the subsequent risk of cervical neoplasia and cervical cancer remains inconsistent in natural history studies. However, several studies have shown positive associations between HPV seropositivity and cervical precancerous lesions -38. VerySeroprevalence among virgins was low though detectable (0.6%), as compared to other studies which have reported a range from 0-3% of HPV 16 ,41,42. GThe “male factor” is an important source of cumulative exposure of HPV to women. Though reports of husbands’ extramarital relationship were provided by wives via questionnaire and might be inaccurate, associations between HPV seropositivity and husbands’ sexual activity were still observed when husbands were reported to have extramarital sexual relationships. Induced abortion might be a marker of a greater number of pregnancies or number of lifetime sexual partners, thus explaining its association with higher anti-HPV 16 levels, results of which are consistent with a Mongolian study . EducatiThe multiplexed Luminex immunoassay can simultaneously quantitate neutralizing antibodies to multiple HPV types in a large sample size, which is suitable for our study design . Cross-rThe strength of our study is that it was a multi-center study with a large sample size. The study covered five sites across China, including both rural and urban areas. We also included women with a wide age range from 15 to 54, stratified into five-year age groups. The number of virgins we recruited was also relatively large, providing source data for future planning of a vaccination program. Also, both the DNA and serology test used in this study are well validated and standardized. Among study limitations, data obtained from the five geographic sites might not be representative of the entire nation of China, particularly given the variation of HPV seropositivity across the sites or age groups. Also, women were recruited through posters or advertisement, but not by random sampling, indicating possible selection bias in our study. This bias is, to some extent, offset by the relatively large sample size, wide age range of participating women, as well as the inclusion of multiple geographical sites. Self-reported sexual behavior and their husbands’ extramarital sexual behavior by female subjects may also have introduced some bias.Seroprevalence of HPV 6/11/16/18 among sexually active women or virgins in China was not systematically investigated prior to this study. HPV seroprevalence provided a picture of cumulative exposure to HPV over a certain period, rather than cross-sectional transient exposure that could be measured by HPV DNA detection. Our findings can be used for design and implementation of cervical cancer control and prevention programs via future prophylactic vaccination programs, in regards to regional and age distribution of immunological responses to these four HPV types. HPV seroprevalence is relatively low and varies by region in China. There is constant exposure to HPV infection, particularly HPV 6 or 16, among women at age of 20 or above, and the chance of exposure increases with age. For optimal public health benefits in China, vaccination programs should target young women prior to their becoming sexually active and the potential exposure to HPVs. Virgins in our study with median age of 18 showed significantly lower HPV seroprevalence, compared with sexually active women. We therefore suggested girls in the age range of 14 to 18, primarily junior or senior high school students, to be targeted for vaccination programs. Approximately half of women with a pathology grading of CIN3 or above were seropositive for HPV 6/11/16/18 in our study. We presumed prevention of about 50% of CIN3/cancer can be accounted for if prophylactic vaccination of these four types can be successfully implemented. These data on population-based HPV seroprevalence in representative samples of women throughout China can be used as baseline data to assess future changes in seroprevalence over time. These data will be particularly important in the context of future evaluation of HPV prophylactic vaccines as they become more accessible and utilized in China.HPV: Human papillomavirus; VLPs: Virus-like particles; LSIL: Low-grade squamous intraepithelial lesion; ASCUS: Atypical squamous cells of undetermined significance; PE: Sphycoerythrin; mAbs: Monoclonal antibodies; cLIA: Complex Luminex immunoassay; OR: Odds ratios; CI: Confidence intervals; HSIL: High-grade squamous intraepithelial lesion; CIN: Cervical intraepithelial neoplasia.Merck & Company Inc. funded this study. This work was supported in part by the Fogarty International Clinical Research Scholars and Fellows Program at Vanderbilt University (R24 TW007988) and the American Relief and Recovery Act, as Jia Ji was supported by above Program as Fogarty International Clinical Research Scholar when involved in this work. Mark T. Esser was a Merck employee and owned stock in Merck when this study was conducted. Jennifer S. Smith has received research grants, honoraria, or consultancy fees from GSK or Merck within the last five years. All other authors declare that they have no competing interests. A Merck representative read the article before submission for publication but had no role in study design, analysis of data, or the decision to submit the manuscript for publication.JJ did data analysis and interpretation, drafted and revised the manuscript. HS did statistical analysis and revised the manuscript. JS conceived of the study, participated in its design, helped to interpret data, and revised the manuscript. HW participated in subject enrollment, collected demographic data and participated in drafting the manuscript. ME carried out antibody measurement. SH managed database. RP participated in conception and design of the study. WC managed sample collection and storage. JB conceived of and designed the study, participated in data collection, and revised the manuscript. YQ conceived of and designed and coordinated the study, and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/137/prepub


Head & Neck Cancer (HNC) is one of the most common malignancies worldwide and among oral neoplasias about 90-92% are squamous cell carcinomas (OSCC). Alcohol and tobacco consumption have been recognized as the main risk factors for development of OSCC. However, 10 to 20% of patients suffering from OSCC have no history of use of these substances. Clinico-pathological evidence suggests that we are dealing with virally-induced cancers, and that HPV should not be a relevant candidate. A systematic search of HPV in OSCC has no real relevance in current clinical practice even although it is still relevant in organized research protocols. Further studies are ongoing, with the aim of identifying other infectious agents, including viruses, in OSCC. The search for an infectious cause of human cancers remains a matter of great interest in medicine. The discovery of a viral agent in a given tumor may have crucial implications not only for diagnosis and better understanding of the tumor’s physiopathology, but also on its prevention and/or treatment. This has been well illustrated in the link between cervical carcinoma and some high-risk types of human papillomavirus (HPV), and for liver cancer and hepatitis B/C infection. However, it is probable that other cancers have infectious origins that remain to be identified .Head & Neck Cancer (HNC) is one of the most common malignancies worldwide, and is a high priority for public health authorities. Among oral neoplasias, about 90-92% are squamous cell carcinomas (OSCC), developing from the mucosa, with the remaining 8-10% being predominantly made up of lymphomas, sarcomas, and salivary gland tumors. For many years, alcohol and tobacco consumption have been recognized as the main risk factors for the development of OSCC. However, 10 to 20% of patients suffering from OSCC have no history of use of these substances. In this comment, we ask if, based on current available data, it is clinically relevant to carry out systematic HPV searches in cases of OSCC. The debate will be opened on the need to search for other infectious agents in OSCC.i.e., the tonsils, the base of the tongue, and the oropharyngeal walls). Strictly speaking, the base of the tongue and the palatine tonsils are part of the anterior and lateral wall of the oropharynx [Different viruses have been looked for in HNCs. Most of the studies, probably because of a certain analogy and similarity to the situation found in cervical carcinoma, have focused mainly on papillomaviruses, especially the high risk ones. Human papillomavirus has been strongly associated with oropharyngeal squamous cell carcinoma (OPSCC). However, the prevalence of HPV infection in OSCC is quite heterogeneous, varying from 17% to 85% ,3. Anatoopharynx , and belopharynx . The impBesides alcohol and tobacco consumption, considered as the historical and still major etiological factors for OSCC, it is fair to hypothesize that infectious agents (and mainly viruses) could be associated and/or causally linked with a subset of OSCC. This hypothesis stems from the fact that OSCC in non-smoker non-drinkers is more frequently reported in an older population than alcohol-tobacco-related cancer . This isOther clinico-pathological evidence suggests that we are indeed dealing with virally-induced cancers. Some histological studies have shown that HPV-related OPSCCs are non-keratinizing tumors, whereas non-HPV-related OPSCCs are keratinizing . This coIn conclusion, recent epidemiological data tend to indicate that the low diagnosis of HPV in OSCC is linked with an actual low prevalence, independent of technical and anatomical biases. Given our current knowledge, a systematic search for HPV in OSCC is therefore a waste of health service resources, and is only relevant in the research environment, such as in organized research protocols. However, further studies continue to search for other infectious agents, including viruses, in OSCC.The authors declared that they have no competing interest.All authors participated in the preparation and writing of this comment.


Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV.These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG. In this study, it is documented for the first time in intact lungs that the membrane-permeable analog of DAG, OAG, induces a vasoconstriction in WT mice which is mediated via TRPC6. This can be concluded from the observation that the DAG-induced vasoconstriction was absent in TRPC6-/- mice, although it has been previously demonstrated that TRPC6-/- mice do not lack non-hypoxia-induced vasoconstrictor responses i was used as a readout for HPV) indicates that the oxygen sensing process underlying HPV resides in the PASMC. This is well in line with numerous previous findings [The specific role of DAG in mediating acute HPV shown in our isolated lung experiments (where the endothelium is present) and in our previous study  in isolafindings -50 and tfindings ,15,17,51findings ,52-54 anTo summarize, DAG was identified as an important mediator in the signaling pathway underlying acute HPV. Moreover, these data indicate that DAG activates TRPC6 as an essential step in the mechanism of acute HPV.The authors declare that they have no competing interests.-/- mice and developed the hypothesis together with NW and BF. Their contribution was critical for intellectual content and designed portions of the experiments. NW designed and supervised the study, interpreted the data and drafted the manuscript together with BF and AD. All authors read and approved the final manuscript.BF participated in the design, analysis and interpretation of experiments, performed statistical analysis, and designed and drafted the manuscript together with NW and AD. MR performed most of the experiments and has made substantial contributions to analysis and interpretation and statistical analysis of the data. HAG, RTS, WS, and FG participated in the design of the experiments and in the interpretation of the results. TG and AD supplied and/or generated the TRPC6


Vulvar cancer in older women is seldom associated with human papillomavirus infection.We present the case of an 80-year-old Greek Caucasian woman with an undetermined obstetric and gynecologic history. The patient underwent radical vulvectomy and bilateral inguinal lymphadenectomy for a vulvar carcinoma. A human papillomavirus infection was suggested on the basis of histological and cytological examinations followed by human papillomavirus DNA typing, which revealed the presence of human papillomavirus-66.Even though human papillomavirus-16 and human papillomavirus-18 are most frequently implicated in the pathogenesis of vulvar carcinoma, human papillomavirus-66 can also be regarded as a causative factor. Suspicious lesions should be biopsied, and in the presence of carcinoma, vulvectomy with bilateral lymphadenectomy, if necessary, must be performed. Furthermore, polymerase chain reaction assay analysis with clinical arrays in cytological samples is an accurate test for the detection of a wide range of human papillomavirus genotypes and can be used to verify the infection and specify the human papillomavirus type implicated. Verruca vulgaris [Vulvar carcinoma in older women is seldom associated with any type of human papillomavirus (HPV) infection, representing less than 15% of reported cases -5. HPV-6vulgaris ,8. On thvulgaris .In this report, we describe the case of an 80-year-old woman of Greek Caucasian origin, gravida 2 para 2, with an undetermined obstetric and gynecologic history. After her second delivery, no data referring to the clinical history of the patient was available because the patient deferred any preventive or diagnostic medical examination until she presented to our hospital. Her last menstrual period had occurred 28 years ago. Her medical history included hypertension and angina pectoris.The patient was examined in the gynecological oncology outpatient department and was found to have chronic pruritus vulvae and a recent onset palpable inflammatory lesion on the left labium majus. The lesion bled occasionally. During her gynecological examination, a 4 cm × 5 cm warty lesion with ulcerous and hemorrhagic areas Figure  was founInitially, multiple biopsies from the center of the lesion and from the lateral margins were obtained, the histological examination of which revealed the presence of squamous cell carcinoma. At the periphery of the lesion, the squamous stratified epithelium exhibited abnormalities consistent with vulvar intra-epithelial neoplasia  grade I/II). Numerous lesional cells showed koilocytic atypia, which is representative of HPV-related infection. However, initial hybrid capture 2 testing for HPV was negative in all the samples tested, which were obtained from either the center or the periphery of the lesion. A pre-operative computed tomographic scan of the abdomen and inguinal areas showed bilaterally enlarged inguinal lymph nodes with central fusion.The patient underwent radical vulvectomy extending centrally to the level of the perineal membrane, as well as bilateral inguinal lymphadenectomy. Post-operatively, the woman had no major complications and was discharged 14 days after the procedure.A histopathological examination of the excised specimen verified the presence of squamous cell carcinoma grade II/III with superficial ulcerations Figures  and 3. CIn the tissue specimen obtained pre-operatively for the performance of liquid-based Cytology , we used a polymerase chain reaction (PCR) assay with CLINICAL ARRAYS Human Papillomavirus Kit  and a Hybrid Capture 2 HPV DNA test . The latter test is an accurate qualitative and quantitative method used to detect five low-risk types of HPV  and 13 high-risk types of HPV . It can also determine the viral load. This technology is based on hybrid analysis to identify indentifying a signal enhancement in a microplate using chemiluminescence. Samples containing the target DNA are hybridized with a specific detector HPV RNA. Hybrid-produced RNA-DNA binds to the surface of a microplate coated with specific antibodies for RNA-DNA hybrids. The immobilized hybrids react with alkaline phosphatase (ALK)-conjugated antibodies and are detected with a chemiluminescence substrate. While the binding ALK cleaves the substrate, it attracts light, which is measured by chemiluminescence in relative light units .For the purpose of this study, we also used the CLINICAL ARRAYS Human Papillomavirus Kit , which is a commercially available HPV genotyping microarray test Figure . The kitIn our case, none of the above-mentioned HPV types was detected with the use of hybrid capture 2 testing. The DNA of HPV-66 was the only one detected using the CLINICAL ARRAYS Human Papillomavirus Kit.On the basis of these results, a standard PCR assay was also performed. Four histological specimens from four different levels of the lesion, as well as from a lymph node with metastasis, were examined. Extraction of total DNA from formalin fixed, paraffin-embedded sections was performed using the QIAamp DNA Mini Kit  according to the manufacturer's instructions. The quality and integrity of extracted DNA were assessed by PCR amplification of an amplicon of the interferon (IFN)-γ gene and by agarose gel (0.6%) electrophoresis, respectively. A nested, multiplex, highly sensitive PCR method  was used for HPV detection and genotyping. In this assay, consensus primers for first-run amplification of a broad spectrum of mucosal HPV genotypes, including all high-risk HPV genotypes, were combined with nested PCR amplifications of type-specific primers. Despite robust detection of the IFN-γ gene product amplification, no positive signal for HPV presence was found in any of the examined samples, even after substantially increasing the PCR cell cycles. Considering that there were clear indications of HPV infection in the microscopic examination of the specimens, the PCR assay was unable to detect the DNA of the virus, most probably because of low viral load in the specimens examined, which could have been caused and amplified by inadequate superficial lesional tissue. Furthermore, tissue fixation techniques could have a negative impact on the preservation of the viral genome.The presence, coexistence and possible cause of HPV infection in women's anogenital squamous neoplasia have been studied extensively over the past decade. It must be emphasized that the presence of condylomatous lesions does not exclude the possibility of a coexisting invasive malignancy. However, the correlation appears to be stronger as far as intra-epithelial lesions (VIN grade III) are concerned.HPV-66 is an α-papillomavirus considered to belong among the potentially high-risk mucosal HPV types. Nevertheless, it can also be encountered in benign lesions. Even though this HPV type is reported to be associated with cervical squamous carcinomas, little is known concerning vulvar squamous cell carcinomas. On the other hand, vulvar carcinomas are mainly correlated with HPV-16 and 18 genotypes. In our case report, we describe the case of a woman with a HPV-66-related vulvar carcinoma. This diagnosis was made on the basis of the use of PCR with the CLINICAL ARRAYS Human Papillomavirus Kit.In conclusion, in patients with HPV-66 infection the possibility of a coexisting invasive malignancy, albeit rare, should be considered even in the presence of benign lesions. Caution should be taken, especially in older women with cancer, as the majority of these cancers are HPV-negative. In cases that raise clinical suspicions of HPV, examination of tissue using PCR with the CLINICAL ARRAYS Human Papillomavirus Kit should be considered in patients with histological features of HPV infection and negative Hybrid Capture 2 assay testing, as it detects a wider range of HPV genotypes than other types of testing, including HPV-66. A standard PCR assay of formalin-fixed samples seemed to be less effective, as it appeared to be affected by sampling, tissue fixation and/or viral load. Patients should be followed up meticulously at short time intervals.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.IK conceptualized the case report, collected and analyzed all data and wrote the major part of the manuscript. GT corrected the initial manuscript and wrote parts of the manuscript. DE was the major gynecologist (in cooperation with IK and GT) who cared for and conducted patient follow-up. DG and AN performed the histological examinations and corrected the pathological parts of the manuscript. Also, DG wrote parts of the manuscript and gave final approval of the manuscript. PF performed the HPV typing (Hybrid Capture 2 assay and CLINICAL ARRAYS Human Papillomavirus Kit testing) and corrected the cytological parts of the manuscript. VK reviewed the literature and wrote some parts of the Introduction. All authors read and approved the final manuscript.


Although great scientific emphasis has been placed upon HPVas the primary cause of cervical cancers and its involvement in carcinogenic progression of other cancers, less attention has been focused on the secondary factors that are associated with progression from subclinical HPV infection to invasive carcinoma. Among the secondary factors that limit virus production and carcinogenic progression is CpG methylation of the HPV genome. Several studies now confirm that CpG site-specific methylation of HPV DNA, mediated in part by folate availability, is sufficient to suppress neoplastic progression. In contrast, demethylation or hypomethylation of HPV-DNA sequences is required for transformation, revealing the importance of preferential DNA methylation at CpG sites in the HPV long control region (LCR) between L1 and E6 HPV genes, in addition to the tumor suppressor sites in p53 exons 248 and 273. Because HPV has the potential to initiate oncogenesis, and also to modulate oral cancer growth and folate plays a central role in mediating the availability of methyl groups for CpG-specific DNA methylation (modulating both p53 and HPV mRNA expression) – an investigation of these inter-connected and inter-related mechanisms in oral cancers must be undertaken. In addition, ethanol and acetaldehyde may also play critical roles in determining folate availability, and are primary risk factors for the development of oral cancers, which makes the evaluation of these interconnected metabolic pathways critically important.in vitro assays, including proliferation, viability and mRNA analysis using RT-PCR, distinct effects of ethanol and acetaldehyde administration were observed in the oral cancer cell lines, CAL27, SCC15 and SCC25. In addition, the growth modulating effects of HPV infection and FA supplementation were also examined.Using a comprehensive series of integrated p53 mRNA transcriptional down-regulation. None of these cell lines were found to harbor the common C677T mutation in methylenetetrahydrofolate reductase (MTHFR), which can reduce FA availability and may increase oral cancer risk.Both high-risk HPV strains 16 and 18 induced robust growth-stimulating effects in CAL27 cells, while strain-specific responses were observed in SCC25 and SCC15 cells. FA administration [0-400 µg/mL] significantly increased the growth rate of all cell lines evaluated. In addition, FA administration induced broad, general increases in cell viability among all cell lines that were associated with Administration of ethanol or acetaldehyde [10 – 100 µM] significantly inhibited oral cancer cell growth in all cell lines tested. Moreover, the growth inhibiting properties of ethanol and acetaldehyde also mediated and reduced the HPV- and FA-induced growth of CAL27, SCC15 and SCC25 cells. These changes were associated with a transcriptional up-regulation of alcohol dehydrogenase (ADH) and other enzymes involved in alcohol metabolism.in vitro and may be associated with up-regulation of metabolic pathways involved with alcohol metabolism.Although tobacco and alcohol use are the main risk factors for developing oral cancer, HPV infection and FA availability may modulate their growth and progression. This study provides preliminary evidence that alcohol and acetaldehyde may mediate and down-regulate the growth enhancing effects of either HPV or FA