Early detection of precancerous cells in the cervix and their clinical management is the main purpose of cervical cancer prevention and treatment programs. Cytological findings or testing for high risk (HR)-human papillomavirus (HPV) are inadequately sensitive for use in triage of women at high risk for cervical cancer. The current study is an exploratory study to identify candidate surface-enhanced laser desorption/ionization (SELDI) time of flight (TOF) mass spectrometry (MS) protein profiles in plasma that may distinguish cervical intraepithelial neoplasia (CIN 3) from CIN 1 among women infected with HR-HPV. We evaluated the SELDI-TOF-MS plasma protein profiles of HR-HPV positive 32 women with CIN 3 (cases) and 28 women with CIN1 (controls). Case-control status was kept blinded and triplicates of each sample and quality control plasma samples were randomized and after robotic sample preparations were run on WCX2 chips. After alignment of mass/charge , an iterative method was used to develop a classifier on a training data set that had 28 cases and 22 controls. The classifier developed was used to classify the subjects in a test data set that has six cases and six controls. The classifier separated the cases from controls in the test set with 100% sensitivity and 100% specificity suggesting the possibility of using plasma SELDI protein profiles to identify women who are likely to have CIN 3 lesions. Pap smear is the most commonly used diagnostic test used to identify women who are at risk of developing cervical cancer. An estimated 50 million Pap smears are performed each year in the United States  and 6% oBecause the cause of most human diseases lies in the functional dysregulation of protein interactions, advances in proteomic technologies represent exciting ways to explore the disease processes at the molecular level. The identification, quantification, classification and functional assignment of proteins are essential to the full understanding of these molecular events. Since mammalian systems are much more complex than can be deciphered by their genes alone, expression analysis directly at the protein level is thought to be necessary to unravel the critical changes that occur as part of pathogenesis of a disease .The surface-enhanced laser desorption/ionization (SELDI) time of flight (TOF) mass spectrometry (MS) is one such technique with considerable potential for the detection and quantitation of protein in a wide variety of human tissue samples  and appear to be an important diagnostic tool for a whole range of diseases. Preliminary applications of proteomic techniques are published in the area of cervical cancer using two-dimensional gel analysis  and SELDThe current study is an exploratory study to identify candidate SELDI-TOF MS protein profiles in plasma that may aid in distinguishing CIN 3 from CIN 1 among women positive for HR-HPV.The plasma samples used were from a previously conducted study of cervical neoplasia  where HPV status (as detected by the Hybrid Capture 2 assay-Digene Diagnostics) and cervical diagnoses were known . Plasma 20. The bioprocessor was disassembled and the chips were washed several times with HPLC grade H20. Each spot was allowed to air dry and 0.5 μl of sinapinic acid (energy absorbing molecule) was added twice to each spot. The chips were read on the Ciphergen Protein Chip Reader Series PBS II using the following parameters: Molecular Weight range–1000 to 15,000, High Mass–20,000, Intensity–180, and Sensitivity–7.The plasma samples were prepared in triplicate and diluted 1:10 in binding buffer  using the Biomek 2000 Robotics System (Beckman Coulter). The plasma samples (100 μl) were added to a pre-washed bioprocessor containing the WCX2, eight spot protein chips . The samples were incubated on a shaker for 2 hours at room temperature. The chips were washed twice with binding buffer and twice with HPLC grade HThe mean spectrum for an individual was obtained by averaging across the three replicate spectra for that individual. The m-z values for the CIN 3 group were slightly different from the CIN 1 group, but these differences were within the expected margin of error (0.2%) of the instrument. The average m-z values of both groups at each point were used as the estimated m-z value. The error of +/– 0.2% was used to develop a new variable index, which was the upper limit of the m-z interval within this error range. A total of 530 index variables were used to represent the 10,300 m-z values. Peaks were defined as the maximum intensity value within each set of m-z values represented by the new variable index. Each group had 530 peaks. The data sets were then randomly divided into training and test sets. Six patients from each group were randomly selected to create the test set. Therefore, the training set consisted of 28 CIN 3 and 22 CIN 1.PCA plot which places the components in terms of the proportion of variance in the data that is attributable to the particular combination was used to visually demonstrate the differences in the groups before building a classifier in order to assess the possibility of obtaining a good classifier. The PCA components are orthogonal to each other. Therefore, the first principal component accounts for the largest amount of variability and the second component accounts for the largest amount of variability unaccounted for by the first. The orthogonality of the components allows their representation as axes in a two-dimensional plot. If the variability explained by the components is the variability associated with the differences in the two groups (CIN 3 and CIN 1), and not the random noise, the principal component plot will visually demonstrate the differences between the groups.An iterative process was implemented to develop a classifier. At each iteration, a random sample of 18 CIN 1 and 22 CIN 3 was selected from the training data set. The peaks that were statistically significantly different based on a Wilcoxon Rank Sum test were then subjected to a stepwise discriminant analysis. This procedure produced a list of index values at which the intensities were most likely to separate the two groups. This list of possible discriminators was stored. The entire process was repeated 300 times in this preliminary analysis. Index values that occurred in at least 270 of the 300 (90%) lists were selected to put into the classifier. This error rate of the classifier was estimated by using the classifier to classify the groups in the test data set.The PCA was based on the entire set of 530 index values. The first two components explained 37 percent of the variability in the data. As shown in An important application of SELDI is the simultaneous analysis of multiple proteins to establish “fingerprint” profiles that discriminate disease from non-disease. This is an important approach since no single biomarker or protein by itself will improve the early detection/diagnosis of diseases including cancer or pre-cancer. Protein based approaches are utilized to study the natural history and treatment of several cancers including ovarian , breast Our preliminary study demonstrated that plasma protein profiles are significantly different between women diagnosed with CIN 1 and CIN 3. Biologically, it is plausible to see difference in protein profiles between HPV positive women who develop CIN 3 and HPV positive women who don’t develop CIN 3 because several biological pathways related to HPV (example: HPV integration which may result in development of CIN 3 lesions) could be different between HPV positive women who develop CIN 3 and who don’t. Cervical epithelial cells exposed to HPV which undergo transformation to pre-neoplastic lesions such as CIN 3 are very likely to differentially express signaling molecules responsible for host responses following HPV infection that resulted in CIN 3 and these molecules are likely to be present in the microenvironment of cervical cells and in the circulation. Identification of such molecules will have enormous clinical applications. Triage of HPV positive women by protein biomarkers could yield enormous cost savings and other important benefits include less invasive sample collection and inconvenience regarding referral.These results, however, have to be considered with caution. SELDI-TOF-MS analysis is a very sensitive method for determining differences in protein spectra of complex samples including plasma and serum. Because of this sensitivity, the method is very susceptible to the effects of various biases. Since cases of CIN 3 and of CIN 1 were selected randomly from participants from the ALTS Nutrition Ancillary Study, we know of no biases which separate the samples of CIN 3 from those of CIN 1. Similarly, because the triplicate aliquots of the samples were run randomly, there should be no bias in the analysis. Before final conclusions can be drawn, however, it is necessary to further evaluate this classification model by using an independent replication set, which will allow more precise conclusions about the accuracy of this classification. These pilot results also warrant an expanded study using SELDI-TOF-MS analysis to evaluate samples of plasma and other samples  from patients with a wide range of cervical lesions to determine if these can be classified successfully. A study is currently underway at the University of Alabama (U54 CA118948) to generate this data.


Research on the use of human papillomavirus (HPV) DNA testing in the screening and management (triage) of cervical lesions began in the late 1980s with the growing evidence that certain (high-risk (HR)) HPV types were the cause of cervical cancer . Since wIn their 5-year cohort study in The Netherlands of 2810 women aged 30–60 years with normal cytology, The improvement in sensitivity and negative predictive value made possible by the addition of HR HPV testing to cytology supports the use of viral testing in order to increase the screening interval, although the findings of Also in this issue, It is important to bear in mind, however, that the detection of HPV at a single point in time (as in case–control studies) or over a few years of follow-up  whether HPV infection disappears or, in some instances, persists undetectably in the basal cells of the cervix, and (b) whether HR HPV infection without cervical abnormality should be observed or treated, and if so, when and how. These uncertainties must be resolved before HPV testing can replace cytology and new recommendations made about screening interval or early screening cessation (Several large-scale randomised controlled trials are ongoing on these issues and their final results should become available within the next 2 or 3 years . Apparen


Both parity and oral contraceptive use are associated with elevated circulating levels of sex hormones, at least transiently, and with increased risk of cervical cancer in human papillomavirus (HPV)-infected women. We directly evaluated whether elevations in the physiologic levels of these hormones predispose to the development of cervical neoplasia. We identified 67 premenopausal and 43 postmenopausal women with cervical intraepithelial neoplasia 2, 3, or cervical cancer (⩾CIN2) diagnosed during enrollment of a population-based cohort of 10 077 women. Four controls, two chosen randomly and two chosen from women testing positive for cancer-associated HPV, were matched to each case on menopausal status, age, days since last menses (pre), or years since menopause (post). Sex hormone-binding globulin, oestradiol, oestrone, oestrone-sulphate, dehydroepiandrosterone sulphate, and progesterone were measured in enrollment plasma. There was no consistent association between the sex hormones and risk of ⩾CIN2. Excluding cases with invasive disease had a minimal impact on results. Though this case–control study was based on a well-defined population, it was limited by reliance on a single measure of hormone levels taken at the time of diagnosis. Nonetheless, our results do not support the hypothesis that plasma levels of sex hormones have an important bearing on the risk of cervical neoplasia in HPV-infected women. Research of the past two decades has established that infection with oncogenic types of human papillomavirus (HPV) is the major cause of cervical cancer , and sevn=31) during enrollment of the study in the three regional hospitals or referred to three tertiary care hospitals in San Jose were invited to participate in the study.Study subjects were drawn from the enrollment phase of a population-based cohort study of cervical cancer that was launched in Guanacaste, Costa Rica, in 1993. The Guanacaste cohort has been described in detail  of the 10 769 women eligible for interview completed a brief interview that collected information on the socioeconomic, demographic, sexual, reproductive, medical, and smoking history. A total of 8582 sexually active women with an intact uterus underwent a pelvic exam, followed by a cervical scrape with a Cervex brush that was used to prepare a conventional Pap smear. Residual cells remaining on the sampling device were rinsed in PreservCyt  and used to prepare a ThinPrep slide. A second exfoliative sample was collected in specimen transport medium  for HPV testing. Then, the cervix was painted with 5% acetic acid and two cervigrams (photographic images) were obtained.In all, 93.6%  with invasive cervical cancer , 119 with histologically confirmed high-grade cervical intraepithelial neoplasia (CIN 2-3), nine with CIN 2-3 defined by reviewed and confirmed cytology, 189 with low-grade cervical intraepithelial lesions (CIN 1), 661 with equivocal lesions, and 7564 with normal final diagnoses.In all, 99.2% of the women interviewed donated a 15 ml blood sample that was collected in a heparinised tube and kept at 1–4°C in coolers in the field. The samples were transported daily to field stations, where the plasma was isolated and frozen in aliquots at −30°C. Weekly, the samples were transported on ice packs to San Jose, stored at −70°C, and sent on dry ice to the NCI repository in Maryland, where they were stored long term at −70°C.Cervical cells were tested for the presence of HPV using PCR with MY09/MY11 L1 consensus primers . All controls were frequency matched to cases by 5- or 10-year age group and menopausal status. Premenopausal controls were further matched to cases by days since the last menstrual period, and postmenopausal controls were further matched to cases by years since menopause.All 110 women diagnosed with ⩾CIN 2, who met the eligibility requirements, were chosen as cases. There were 76 cases with histologically confirmed CIN 2-3, nine cases with CIN 2–3 defined by reviewed and confirmed cytology, eight cases with invasive cervical cancer found during screening, and 17 cases with invasive cervical cancer identified in hospitals. Two sets of controls were matched to cases, each in a 2 : 1 ratio. The first set of controls, hereafter referred to as random controls, was chosen from the 6430 women with neither CIN 2–3 nor invasive cancer. The second set of controls, HPV-positive controls, was chosen from the pool of noncases and nonrandom controls, who had PCR results indicating a high-risk HPV type  for hormone analysis using in-house methods. Briefly, oestradiol, oestrone, and progesterone were measured using radioimmunoassay (RIA) following hexane : ethyl acetate extraction and column chromatography. Oestrone sulphate was measured similarly following overnight incubation with a sulphatase. Sex hormone-binding globulin was measured using a ‘sandwich’ immunoradiometric assay (IRMA), utilising a solid-phase capture antibody and a label antibody in the presence of calcium. Sex hormone-binding globulin is the major transport protein of oestradiol, and is thus inversely related to free oestradiol, the fraction of oestradiol that is not bound and thus is most biologically active. Free oestradiol was estimated by a method proposed by k-sample test . Eight premenopausal cases and 32 matched controls reported more than 40 days since their last menstrual period. Exclusion of these women, mostly lactating, had no substantial effect on the results (data not shown).Plasma levels of SHBG, oestradiol, oestrone, oestrone sulphate, DHEAS, and progesterone were not significantly related to the risk of ⩾CIN2 in this study. To our knowledge, this is the first epidemiologic study to evaluate the role of circulating hormones and ⩾CIN2, and, to the extent that associations were observed, they conflict with our initial hypothesis that elevated levels increase the risk of cervical neoplasia.−1 in cases, 81.1 pg ml−1 in controls) and oestrone  in cases than in controls. In contrast, our findings for total oestradiol, SHBG, oestrone, and free oestradiol fail to support the notion that high levels of oestrogens increase the risk of cervical cancer. Rather, the observed trends of increased risk in women with increasing levels of SHBG and decreased risk in women with increasing levels of oestradiol, free oestradiol, and oestrone imply that high levels of oestrogens may, if anything, be associated with a decreased risk of ⩾CIN 2.Since multiparity and oral contraceptive use are associated both with elevated circulating hormone levels and with an increased risk of cervical cancer, we hypothesised that high physiologic oestrogen levels would adversely affect cervical carcinogenesis . The stuThe presence of progesterone has been hypothesised to increase the risk of ⩾CIN 2 by inducing transformation of cells by HPV-16 and upregulating HPV expression in cell lines via binding and activation of glucocorticoid response elements found on the upstream regulatory region of the virus . The cases, identified through intensive screening of a large sample of the Guanacaste population and among Guanacaste residents with invasive cancer presenting at regional and tertiary care hospitals, comprised an unbiased sample of Guanacaste women who developed ⩾CIN 2 during enrollment. This surrogate end point poses a high cumulative risk of subsequent invasion, but is still intraepithelial and, thus, unlikely to affect the systemic hormone levels. Using subjects identified in a well-defined, large cohort allowed us to select controls that were representative of the underlying population from which most of the cases arose. DNA testing of study subjects allowed selection of a second control group of HPV-positive women, to assess whether hormones were related to the risk of ⩾CIN 2, given exposure to HPV. We were able to measure a panel of five hormones and SHBG using assays with good reproducibility.Several issues may have contributed to our finding of no association between sex hormones and ⩾CIN 2. Even in a large population-based study such as this, the number of cases of ⩾CIN 2 available for analysis was small, particularly after separating subjects by menopausal status. Thus, we were not well-powered to detect modest risks. Despite our focus on CIN 2 and 3, the neoplasia may have influenced plasma hormone levels. However, median hormone levels were similar among cases with invasive disease and cases with CIN 2-3, results were similar after exclusion of invasive cases, and we reiterate our belief that intraepithelial lesions are unlikely to affect the circulating hormone levels. Finally, the use of a single blood sample at one point in time to characterise the long-term hormonal environment of a woman may be problematic. Endogenous hormone levels are known to vary throughout a woman's lifetime, in response to various factors such as changes in body weight, exogenous hormone use, and during menstrual cycling. We matched controls to cases by age, years since menopause  and days since the last menstrual cycle , to account for this. Analyses including all premenopausal women and those restricted to women in the luteal phase of the cycle yielded similar results. However, we lacked information to control for other sources of variation in hormones over time, such as menstrual cycle length. Our inability to account for sources of hormone variability may have attenuated the risks and limited the ability of the study to detect some true associations (In conclusion, the data fail to support hypotheses that elevated plasma levels of endogenous oestrogens or progesterone increase the risk of cervical neoplasia. If associations between endogenous hormone levels and the risk of cervical cancer truly do exist, they are likely to be modest in size. In order to reliably document such associations, future studies of squamous cervical neoplasia would require both very large numbers of subjects and a means of more accurately characterising women's long-term hormonal milieu.


We describe type-specific progression, regression and persistence of incident human papillomavirus (HPV)-6-11-16 and -18 infections, along with type distribution in cervical intra-epithelial neoplasia (CIN) lesions.The study population consisted of 16–23 year-old women undergoing Pap testing and cervical swab polymerase chain reaction testing for HPV DNA at approximate 6 month intervals for up to 4 years in the placebo arm of a clinical trial of an HPV 16-vaccine. HPV types in incident infections were correlated with types in lesion biopsy specimens.56.7% of CIN-1 and nearly one-third of CIN-2/3 lesions following incident HPV-6-11-16 or -18 infections did not correlate with the incident infection HPV type. Cumulative 36-month progression rates to CIN-2/3 testing positive for the relevant HPV type were highest for HPV-16 infections (16.5%), followed by HPV-18 (8.2%). Overall, 26.0% of CIN-1, 50.0% of CIN-2 and 70.6% of CIN-3 biopsies tested positive for HPV-6-11-16-18 infections.Women with a given HPV type may often be co-infected or subsequently infected with other types which may lead to subsequent cervical lesions. This issue has been addressed in this study reporting data for the natural history of HPV-6-11-16 and -18 infections and is a relevant consideration in designing future studies to evaluate the incidence/risk of CIN following other type-specific HPV infections. Cervical cancer is the second most common cancer in women worldwide and is cProphylactic vaccines targeting HPV 6/11/16/18 and HPV The present study describes the type-specific progression and regression of incident HPV 6, 11, 16 and 18 infections, along with the observed distribution of these HPV types in CIN grade 1–3 lesions. A unique feature of the present study with respect to prior analyses of HPV infection natural history -15 is thThe current evaluation focuses on women enrolled in the placebo arm of a randomized double-blind clinical trial of an HPV-16 vaccine (Merck Research Laboratories). The study population and trial design have been described elsewhere -22. Brie32P-labeled β-globin or HPV 6/11/16/18 gene-specific oligonucleotide, and visualized by autoradiography. Appropriate negative and positive controls were run with each assay and any specimen testing positive for at least 2 of the 3 genes was considered positive. Specimens testing positive for only 1 gene were considered positive if, on retesting, they were positive for 2 or 3 genes or the same single gene. Laboratory validation studies rigorously evaluated assay sensitivity against known copy number type- and gene-specific plasmids. The assay was shown to have a greater than 95% probability of detecting at least 13 copies per sample, with 95% upper confidence bounds for sample false negativity and false positivity of 0.7% and 0.8% respectively. All PCR assays were performed at Merck Research Laboratories .Women underwent type-specific endo/ecto-cervical swab HPV polymerase chain reaction (PCR) testing for HPV 6, 11, 16 and 18 infections at approximate 6 month intervals through 48 months of follow-up. The HPV testing methods utilized have been described in detail elsewhere ,22. BrieAt visits where swab samples were obtained, cervical samples were also collected for liquid-based cytology  testing. An algorithm was used to guide further evaluation for cytologic findings. A single Pap test result of high-grade squamous intraepithelial lesion (HSIL), or repeated Pap tests showing low-grade lesions (LSIL) or atypical squamous cells of undetermined significance (ASCUS), prompted colposcopy. Investigators were allowed to manage single ASC-US and LSIL results based on local standards of care. All women attending the month 48 trial visit were referred for colposcopy, with biopsy performed if a CIN lesion was suspected. Cervical biopsy specimens were processed and assigned histologic diagnoses for purposes of medical management by central laboratory pathologists, and were typed by PCR for HPV 6, 11, 16 and 18 -22. A muIncident HPV 6, 11, 16 and 18 infections were identified on cervical swab or biopsy specimens among woman negative for the relevant HPV type on their first 2 study swabs and any cervical biopsy specimens obtained on or prior to the second swab. Routine cervical swab HPV testing occurred at discrete intervals within the trial (approximately every 6 months). It is likely that initial positive HPV test results observed on a particular date reflected HPV infections that originated at some indeterminate time point between the last negative and initial positive test results. Consistent with our and others' earlier work,,25,26 weIncident HPV infections were examined until either the detection of a CIN-1-3 lesion for which the cervical biopsy specimen tested PCR positive for the relevant HPV type ("progression"), or a negative cervical swab HPV test result(s) for that type ("regression"). Because women were required to have two consecutive negative swabs for a given HPV type prior to the start of an incident infection, if two consecutive negative swabs for that type were subsequently observed, the infection was assumed to have cleared at the midpoint in time between the final positive test and first negative swab result. However, there were also women with positive swab sample(s) for a given type followed by only a single negative swab prior to the trial concluding or their withdrawal. Among women with incident HPV-6 infections, 19 (18.4%) fell into this category, compared to 2 (15.4%), 16 (11.3%) and 9 (14.5%) with HPV-11, 16 and 18.Since these women lacked two consecutive negative swabs, some previous analyses have evaluated them as censored at the date of the negative swab. HoweverFrom among all placebo-arm women diagnosed with CIN during the course of the trial, the prevalence of HPV 6, 11, 16 and/or 18 infections in CIN-1-3 lesions was estimated using PCR from the first biopsy diagnosis of CIN observed in the trial for each woman. Only the first CIN biopsy specimen was chosen for each woman to avoid selecting data for the same lesion multiple times in cases in which more than one biopsy specimen was taken during the course of follow-up. If multiple grades of CIN were observed for a woman among biopsies on a given date, the most severe histologic diagnosis was selected. HPV status for types other than 6, 11, 16 and 18 was unavailable.Results were compared to data from previous US studies of the prevalence of type-specific HPV infections in biopsy-confirmed CIN lesions. Studies included performed PCR typing for multiple HPV types on cervical lesion tissue specimens. Analyses based on endo/ecto-cervical swab or lavage were excluded based on the increased potential for detecting HPV types unrelated to the cervical lesion of interest. To avoid double-counting in estimating the fraction of lesions attributable to individual or groups of HPV types, in instances where multiple HPV types were observed in a single lesion, the following procedure was undertaken. Each lesion testing positive for multiple HPV types was fractionally attributed to individual HPV types based on the relative proportion of lesions observed in each study to test positive for each single-type infection only, for that grade of lesion. For instance, if within a given study, a CIN-2/3 lesion tested positive for HPV types 6 and 16 and the number of single type-infected CIN-2/3 lesions for HPV-6 and -16 were 1 and 19 respectively, then (1/20) and (19/20) of the lesion was attributed to HPV-6 and -16.The cumulative proportion of HPV infections persisting without evidence of CIN, progressing to CIN 1, 2 and 3 and regressing, at 12, 24 and 36 months post-infection were estimated using Kaplan-Meier (K-M) methods. Each ouAmong 1,203 women in the placebo arm of the trial who underwent cervical swab HPV PCR testing at enrollment, the prevalence of individual HPV types was as follows: HPV-6 (3.2%), HPV-11 (0.5%), HPV-16 (6.8%), HPV-18 (2.6%). The combined baseline prevalence of HPV 6, 11, 16 and 18 infections was 12.1%.Characteristics of women with incident HPV-6 n = 103), 11 (n = 13), 16 (n = 142) and 18 (n = 62) infections are presented in Table 03, 11 (nth–75th percentile) of times to detection of type-concordant CIN-2/3 lesions covering the 10th to 70th percentiles of times to detection of type-discordant CIN-2/3.Following the date of their incident HPV 6, 11, 16, and 18 infections, women averaged an additional 21.2, 16.1, 22.1 and 21.4 months of follow-up respectively. The number of CIN lesions testing positive or negative for the HPV type observed in the incident infection, during the subsequent course of follow-up, is reported in Table The cumulative proportion of females with incident HPV 6, 11, 16 and 18 infections progressing to CIN-1-3 testing positive for the relevant HPV type on biopsy, persisting with type-specific infection in the absence of detected CIN, or regressing to negative for HPV infection for the relevant type on a cervical swab, at 12, 24 and 36 months of follow-up is presented in Table Among all placebo arm women diagnosed with CIN (n = 267), regardless of HPV status, there were 442 CIN-1-3 cervical biopsy specimens. From the first CIN biopsy specimens obtained for these 267 women, 22 (8.2%) lacked HPV typing. This left an eligible sample of 245 initial CIN biopsies, of which 192 were CIN-1, 36 CIN-2 and 17 CIN-3. Overall, 26.0% of CIN-1, 50.0% of CIN-2 and 70.6% of CIN-3 biopsies tested PCR-positive for HPV 6, 11, 16 or 18  in prior US studies .Over the past two decades there has been substantial research demonstrating the causal link between HPV infection and cervical neoplasia, and theThis study was supported by Merck Research Laboratories, where the authors are currently employed. Authors potentially hold stock and/or stock options in the company. Merck & Co., Inc. has developed a quadrivalent HPV vaccine.All authors participated in the design of the study and drafting of the manuscript, and have read and approved the final version. RI conceived of the study and performed the data and statistical analyses.


We conducted a study aiming to describe Human Papillomavirus (HPV) type distribution in invasive cervical carcinoma in Uganda.191 archival cervical carcinoma samples diagnosed in the Department of Pathology, Makerere University in Kampala between 1968 and 1992 were analysed using a sensitive PCR-Reverse Hybridization Line Probe Assay.Out of the 186 cases of confirmed invasive cervical cancer in the study paraffin blocks, 114 were positive for HPV DNA. Specific HPV genotypes were identifiable in 109 cases: HPV 16, 18, 31, 35, 39, 44, 45, 51, 52 and 70. These occurred as single infections in 105 cases (96.3%) and as multiple infections in 4 cases (3.7%). HPV 16 or 18 accounted for 80% (84/105) of cases with single infection.The results of this study confirm the role of HPV 16 and 18 in cervical cancer pathogenesis in the Ugandan population. The results suggest that the currently available HPV vaccines against HPV 16 and 18 could possibly prevent the majority of invasive cervical cancers in Uganda. Clinical and epidemiological studies have clearly established that infections by certain human papillomaviruses (HPVs) types are causally linked to cervical cancer development -5. Among191 samples from patients with histological diagnosis of invasive cervical carcinoma from 1968 to 1992 were obtained from the archives of the Department of Pathology, Makerere University in Kampala, Uganda. These samples accounted for less than 5% of cervical carcinomas diagnosed during the study period. Nine cases (4.7%) were from the period of 1968 to 1969, 66 (34.6%) cases from 1970–1979, 74 (38.7%) from 1980–1989 and 42 (22.0%) were from the period 1990–1992. Until the early nineties, the Department of Pathology received most specimens for histopathology services from all hospitals and health facilities in the country.Samples were received in formalin, which may not always have been buffered. Tissue specimens were usually processed within 24 hours of their arrival to the hospital, but for a fraction of samples transported from remote parts of the country, longer times may have been required. Samples were selected randomly from the register of Pathology Department starting in 1968; we oversampled of adenocarcinomas (1:1) because of the special interest to compare genotype distribution in the two major histologies of cervical cancer.When at the testing laboratory, the paraffin embedded tissue blocks were re-embedded in fresh paraffin wax and four sections (sandwich method) were cut for testing under strict conditions to avoid potential contamination. The first and the last sections were stained with Haematoxylin and Eosin (H&E) to confirm diagnosis and to ascertain the suitability of the tissue to continue for HPV testing. The sections in-between were collected in a screw-top Eppendorf tube for HPV testing. A tissue-free paraffin block was cut after each study block to avoid any HPV carry-over from block to block. A new blade was used for each block and the microtome was cleaned with a vacuum cleaner, Histoclear II and 70% alcohol. To further control for possible sources of contamination, paraffin blocks containing non HPV related lesions  were included blindly in the process at a ratio of 5%, and blank paraffin sections were simultaneously tested.450) were read on a microtitre plate reader. HPV DNA positive samples were subsequently analysed by HPV SPF10-LIPA25  [Proteinase K (Sigma) digestion for 16 hours at 56°C temperature was used to obtain a tissue lysate containing DNA from the paraffin inner 5 μm sections. SPF10 PCR was performed using 10 μl of a 1:10 dilution with water of the tissue digest in a final reaction volume of 50 μl . The amperlands) , a reverEthical approval for the reported study was obtained from Ethical Committee at Makerere University, Kampala, Uganda.Of the 191 paraffin blocks corresponding each one to a cervical cancer cases, 186 harboured invasive cervical carcinoma cells and, according to protocol, were considered suitable for HPV detection. Five were excluded due to extended necroses seen on the histological slide. 146 were diagnosed as squamous carcinomas, 35 were adenocarcinomas, 3 adenosquamous and two were undifferentiated carcinomas. Out of these, 114 were HPV positive and in 72 HPV could not be detected. All the negative samples tested negative for the presence of β-globin gene indicating a poor quality tissue. Table Specific HPV types were identifiable in 109 cases, while in the remaining five cases no specific HPV types could be identified (Type X). Ten specific HPV types: 16, 18, 31, 35, 39, 44, 45, 51, 52 and 70 were identified. These occurred as single infections in 105 cases, double infections in three cases and triple infections in one case. Analysis of the HPV genotypes distribution among the HPV positive cases showed that HPV 16 and 18 were the most frequent types, followed in descending order by HPV 45, 31, 35, 51, 39 and 52. Table In single infections, type 16 occurred in 51 of the cases, type 18 in 33 cases, type 45 in 11 cases, type 31 in four cases, type 35 and 51 in two cases, types 39 and 52 each in one case. The three double infections were found to be combinations of types 16/45, 16/51 and 45/70 while the triple infections included the HPV types16/44/52. When the analysis was restricted to the histological types of cervical carcinoma, HPV 16 was the most frequent in squamous cell carcinomas (47.8% of cases) while in adenocarcinomas, HPV 18 was the most prevalent type (50%). Of the squamous cell carcinomas, 44 of the cases presented with HPV type 16, 21 of the cases with type 18, 10 with type 45, four with type 31, two with type 35, three single infections with types 39, 51, and 52, three double infections and one triple infection. In the adenocarcinomas, 10 were found to have HPV type 18, 7 type 16, one type 45, and one type 51. Concurrent CIN lesions were seen in 18 cases with single HPV infections and one case with multiple HPV types. No differences in HPV detection were observed between the cases with and without pre-neoplastic lesions  due to HPV 45. Almost all infections were high risk HPV types, with exception of two low risk HPV (44 and 70) which were seen as part of multiple infections. There were only 5 samples classified as HPV X (HPV/DNA positive and genotype negative); we hope to be able to genotype these samples in the future. HPV 16 and 18 were the 2 most common types identified throughout the study period indicating viral genotype stability in an era in which HIV started to emerge.The prevalence of HPV DNA in cervical cancer varies in many studies, with some investigators reporting almost 100% ,4. This In our study, all the HPV/DNA negative samples were tested for β-globin gene. All were found to be β-globin gene negative, although the β-globin gene primer used was slightly longer than the very short primer used in the DEIA reaction. Thus, the low detection rate is very likely due to poor quality of the DNA in some archival tissue samples. It has been demonstrated that suboptimal processing of specimens in the histological process such as the use of unbuffered formalin as a fixative, which was common before 1970, may yield DNA that is unsuitable for HPV detection . In a suHPV 16 and 18 are the major aetiological risk factors for cervical carcinoma. Our overall prevalence of the two major HPV types (16 and 18) was over 70%, which is similar to the IARC multicentric case-control study by Castellsague et al. ,10. The The causes of the regional differences in HPV subtype prevalence need clarification, e.g. the results from Mauritius , where oOne of the interesting findings of our study was the five cases in which the samples were positive for HPV, but specific HPV genotypes could not be identified. This raises the possibility that there may be some unidentified HPV types or that some additional HPV types could have a wider range of probes. In a study in Mozambique , HPV speMultiple infections were seen in only four HPV positive cases 3.5%) in this study. This is similar to some previous studies where the prevalence varied from 2.5% in Spain and Columbia % in this, 4.3% inWhether multiple HPV infections are involved in cervical cancer pathogenesis is rather unclear. Herrero et al. found that the risk of cervical cancer associated with HPV 16 alone is similar or greater than the risk associated with multiple infections (HPV 16 plus other HPV) . The resOur study had a number of strengths. The method we used is sensitive and has high efficacy in comparison with other methods using other general HPV primer sets . In addiOne limitation of our study was the relatively small number of cases tested. Since the specimens came from different parts of the country, the duration of fixation were not uniform and could have contributed to some cases being negative for HPV. In addition, the use of non buffered formalin could also have deteriorated the DNA, resulting in false negative results.In our study we did not have information on HIV serology. HIV infection is a risk factor for pre cervical cancer lesions ,34 and aThe results of our study show that the HPV type distribution can be determined using existing archived biological materials, such as paraffin blocks, allowing evaluating changes over time, and reaffirm the role of HPV in cervical cancer pathogenesis in the Ugandan population. HPV 16 and 18 account for about 75% of cases while about 20% are due to other HPV genotypes, and the remaining due to multiple HPV types. The results suggest that more HPV types are yet be identified and recommend that future HPV vaccines be tailored according to local HPV type distribution.CIN: Cervical Intraepithelial Neoplasia; DDL: Delft Diagnostic Laboratories; DEIA: DNA Enzyme Immunoassay; DNA: Deoxyribonucleic acid; H&E: Haematoxilin and Eosin; HIV: Human Immunodeficiency Virus; HPV: Human papillomavirus; ICO: Institut Catala d'Oncologia; LiPA: Line Probe Assay; OD: Optical density; PCR: Polymerase Chain Reaction; RIS HPV TT: Retrospective International Study on HPV Distribution among Cases of Invasive Cervical Cancer; SOP: Standard of procedures; SPF 10-PCR: Short-Fragment PCR; VLPs: Virus-like particles.All authors declare no competing interests in regard to any financial or non-financial relationship.The founding institutions had no influence on study design, data collection, analysis, interpretation, writing report and decision to submit the paper for publication.MO, EW and SdS were responsible for the concept drafting, full proposal development and getting approval from the ethics committees. MO was responsible for the specimens selection and data file cleaning. JK and WQ were responsible for the HPV DNA detection and quality control analysis. MO, EW, SdS wrote the manuscript and EW and MO were responsible for the preparation of the manuscript for submission. XFB planned the international collaborative project on HPV in invasive cervical cancer, from which Uganda is one collaborative centre.The pre-publication history for this paper can be accessed here:


Testing for the DNA of high-risk types of papilloma virus (HPV) is more sensitive than cytology in detecting pre-cancerous lesions. One of the main advantages will be the possibility of applying prolonged screening intervals. However adequate screening protocols  need to be applied in order to avoid over-referral to colposcopy and over-treatment and to maintain sustainable costs. Further follow-up of running trials and research on molecular markers will better define these parameters. The new situation will require organised screening programmes with rigorous protocols and monitoring. This will be even more needed when women vaccinated for HPV 16 and 18 will be screened. Research on how to best screen vaccinated women is a priority. This paper proposes an overview of the plausible impact of new technologies in cervical cancer screening in the near future and in the vaccinated cohorts. Both studies based on double-testing of the same women ,2 and laHPV testing detects almost all high-grade CINs identified by cytology ,2. As a In women at least 35 years of age, in the "New Technology in Cervical Cancer" (NTCC) trial, HPV testing was found to be 63% more sensitive than cytology when all HPV-positive women are referred to colposcopy . At thisThus, a number of strategies to increase specificity have been or are being evaluated. The most studied strategy – that we will refer to as "cytological triage" – involves directly referring to colposcopy only HPV-positive women who also show cytological abnormalities, while the remaining HPV-positives are retested at shorter interval and referred to colposcopy only if they retest as HPV positive . This apThe persistence of lesions detected by HPV testing versus cytology is also a relevant factor. Remarkably, the two aforementioned studies ,7 showedHowever, cytological triage requires that some women are re-tested at short intervals, which is a disadvantage because compliance rarely is complete. Other strategies, based on molecular markers, are under study. These include viral load, genotyping, testing for the RNA of the viral oncogenes E6 and E7 and testing for the over-expression of the p16-INK4A protein . RecentlAmong younger women the situation is slightly more complicated. At this age infection is very frequent and HPV test specificity is lower. Nevertheless, among women 25–34 years of age, in the NTCC trial, HPV testing with cytological triage resulted in a relative sensitivity vs. cytology of 1.58 with only a moderate loss in PPV (relative PPV 0.78) [In conclusion, available results strongly support the adoption of HPV testing for cervical screening. However, before general routine implementation, the follow-up results of running randomised trials, which will be available soon, should be considered. They will also provide evidence on some aspects that need to be better defined: age at first testing, the best screening interval, the best management of HPV positive women. In the meantime, starting large pilot (demonstration) projects seems reasonable. This will allow us to estimate the impact of the application of new technologies in practice, evaluate costs (to date the major barrier to using HPV as the primary test is the price of the kit ) and setIn any case, the following issues must be taken into account:- Applying longer screening intervals represents a major advantage of HPV testing. This is also needed in order to maintain sustainable costs.- Adequate protocols need to be applied. In their absence there is a risk not only of over-referral to colposcopy but also of overtreatment , and therefore for potential harm .- Validated tests with appropriate sensitivity and more importantly, specificity must be adoptedAll of these factors indicate the need for organised screening programmes that actively invite women at the appropriate intervals, adopt well-defined management protocols, register all results (the application of protocols requires accurate knowledge of women's screening experience) and monitor performances.Two vaccines – one against the oncogenic HPV types 16 and 18 and one that also includes the non-oncogenic types 6 and 11 – proved to be highly effective in preventing HPV16/18-linked CIN2+ . In someOverall, this scenario represents an additional reason for shifting to HPV testing but will require even more conservative protocols, to achieve high PPV for colposcopy referral, both in order to avoid false positive histological diagnoses  and to HPV: Human Papilloma Virus; NTCC: New Technology in Cervical Cancer, multicentre randomised trial; CIN: Cervical Intraepitelial Neoplasia; CIN2+: Cervical Intraepitelial Neoplasia grade 2 or more severe; PPV: Positive Predictive Value.GR received minor payment by GenProbe for participating in internal scientific advisory boards. The Agency for Public Health received a grant by Sanofi Pasteur MSD for a study on the HPV-related burden of disease in Italy. PGR received travel reimbursement to present the results of the study at two international conferences and at an internal workshop.GR drafted the section on HPV testing for primary screening and PGR the section on the new scenario in the vaccine era. Both authors discussed the entire text and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Objective: This study was designed to compare the sensitivity, specificity, efficiency, positive andnegative predictive values, and ease of use for 2 commercially available hybridization kits fordetecting human papillomavirus (HPV) DNA: Oncor Southern blot (SB)  and Digene ViraType dot blot (DB) .            Methods: A total of 179 specimens  were assessed for acceptabilitybased on the presence of epithelial cells and tested for HPV by DB and SB. The results wereevaluated based on Papanicolaou-stained cervical specimens and selected risk factors.            Results: One hundred six (97.2%) of 109 results were concordant, i.e., 93 negative (85.3%) and 13positive (11.9%). Using SB as the gold standard, we found the sensitivity, specificity, efficiency, andpositive and negative predictive values for the ViraType DB to be 100%, 96.9%, 97.3%, 81.3%, and100%, respectively. Comparing the Papanicolaou smear to SB and DB, we found the sensitivity,specificity, efficiency, and positive and negative predictive values to be 33.3% (SB) vs. 44.4% (DB),89.5% vs. 87.6%, 87.3% vs. 84.2%, 11.8% vs. 23.5%, and 97.0% vs. 94.9%, respectively. The onlysignificant risk factor for predicting an HPV infection was the number of sexual partners.            Conclusions: Although SB has been considered the standard model, DB is an acceptable methodfor detecting and identifying HPV infections.


Sexually transmitted infections (STIs) such as herpes simplex virus (HSV)-2 are associated with an increased risk of HIV infection. Human papillomavirus (HPV) is a common STI, but little is know about its role in HIV transmission. The objective of this study was to determine whether cervico-vaginal HPV infection increases the risk of HIV acquisition in women independent of other common STIs.This prospective cohort study followed 2040 HIV-negative Zimbabwean women  for a median of 21 months. Participants were tested quarterly for 29 HPV types (with L1 PCR primers) and HIV (antibody testing on blood samples with DNA or RNA PCR confirmation). HIV incidence was 2.7 per 100 woman-years. Baseline HPV prevalence was 24.5%, and the most prevalent HPV types were 58 (5.0%), 16 (4.7%), 70 (2.4%), and 18 (2.3%). In separate regression models adjusting for baseline variables , HIV acquisition was associated with having baseline prevalent infection with HPV 58  or HPV 70 . In separate regression models adjusting for both baseline variables and time-dependent variables , HIV acquisition was associated with concurrent infection with any non-oncogenic HPV type , any oncogenic HPV type , HPV 31  or HPV 70 . Detection of any oncogenic HPV type within the previous 6 months was an independent predictor of HIV acquisition, regardless of whether HPV status at the HIV acquisition visit was included  or excluded  from the analysis.Cervico-vaginal HPV infection was associated with an increased risk of HIV acquisition in women, and specific HPV types were implicated in this association. The observational nature of our study precludes establishment of causation between HPV infection and HIV acquisition. However, given the high prevalence of HPV infection in women, further investigation of the role of HPV in HIV transmission is warranted. Human papillomavirus (HPV) is a common sexually transmitted infection (STI) and has been implicated as a causative agent of anogenital cancers including cervical, vaginal, vulvar, and anal cancer Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalisImmune-compromised individuals such as those with HIV suffer from higher rates of HPV infection and disease, higher rates of relapse after treatment of HPV-related diseases, and accelerated development of HPV-associated cancer. Little is known, however, about the converse relationship, i.e. the effect of HPV infection on risk of HIV acquisition. Several viral and bacterial STIs, both ulcerative and non-ulcerative, have been implicated as risk factors for HIV acquisition including syphilis, herpes simplex virus (HSV)-2 This study was performed as a substudy within a randomized trial studying the effect of latex diaphragm and lubricant gel provision on HIV acquisition in the setting of counseling and provision of male condoms in Zimbabwe , a questionnaire on demographics and sexual behavior was administered, and participants were tested for serologic evidence of HIV and herpes simplex virus (HSV)-2; a blood sample was obtained for syphilis testing, and a urine specimen was obtained for PCR testing for Women testing positive for a curable STI were treated when the positive test result was reported. Cervical cytology was interpreted locally in Zimbabwe (R.M.) and reported using the Bethesda system Cervical swabs for HPV DNA were collected by clinicians under direct visualization of the cervix at the enrollment, 12-month and exit visits, as previously described At screening and quarterly, two HIV rapid tests were performed on finger-prick or venipuncture blood samples using Determine HIV-1/2  and Oraquick . Discordant results of rapid tests were confirmed on serum samples by ELISA . Timing of HIV acquisition was assessed by RNA or DNA PCR testing on Dry Blot Spot or serum samples from that visit and from prior visits until a negative PCR test was obtained. The HIV acquisition visit was defined as the visit at which the first positive HIV PCR test was detected.a priori analytic plan. In addition to evaluating potential associations with any HPV type, separate analyses for non-oncogenic and oncogenic HPV types were also performed, given that oncogenic and non-oncogenic HPV types have different biologic behaviors as reflected by rates of persistence Analyses were performed to look for associations between HIV acquisition and HPV infection following an Both self-collected and clinician-collected swabs were used for defining HPV status. If results from both collection methods were available at the same visit, a positive result from either test contributed to the definition of a positive HPV test. Baseline HPV status for particular types or groups of types was defined using HPV tests from the study enrollment visit, and used as a fixed covariate in analyses. We defined a cumulative HPV status indicator of ever having tested positive for a type or group of types at baseline or during follow-up (up to and including the first HIV positive visit for HIV converters), and considered this as a fixed covariate in analyses. We also defined a status indicator of persistent or non-persistent HPV infection and these were considered as fixed covariates in analyses. A type-specific infection was defined as persistent if any 2 consecutive tests were positive for a specific HPV type, or if any of the following conditions were met (if positive tests were not consecutive): for participants with 2 to 6 tests, ≥2 tests were positive but not >3 consecutive negative tests; for participants with 7 to 9 tests, ≥3 tests were positive but not >3 consecutive negative tests; for participants with 10 tests, ≥4 tests were positive but not >3 consecutive negative tests; for participants with 11 or 12 tests, 4 tests were positive but not >4 consecutive negative tests. A type-specific HPV infection that did not meet the above criteria was considered non-persistent. Participants with no visits positive for type-specific HPV were considered uninfected. Participants with HPV data from only one study visit were excluded from this analysis.excluding the HIV acquisition visit. A participant visit was excluded from the analysis if there were no HPV results for a particular visit (for “concurrent HPV infection”) or at any time in the preceding 6 months (for “recent HPV infection”), resulting in different numbers of women-visits contributing to different analyses. Observations for visits for women who acquired HIV were censored after the HIV acquisition visit.The following measures of HPV status were also considered as time-dependent covariates: Women were defined as having a “concurrent HPV infection” if HPV testing was positive at the HIV acquisition visit. Women were defined as having a “recent HPV infection” if they were positive for HPV at any visit within 6 months up to and including the HIV acquisition visit. We also performed a “sensitivity analysis of recent HPV infection” of women positive for HPV at any visit within 6 months but Cox proportional hazards regression models were used to evaluate associations between HPV status and HIV acquisition. Because of the discrete nature of follow-up, the Efron correction for tied failure times was employed in parameter estimation. The validity of the proportional hazards assumption was evaluated via a test of interaction between HPV status indicators and duration of follow-up. HPV status variables (described above) were included as either fixed  or time-dependent covariates (for concurrent or recent infection). Although the data were pooled from the 2 arms of a negative randomized trial, we included study arm as a variable in the analyses in order to control for a possible effect of the intervention. Separate models were fitted for different HPV types. Models controlled for potentially confounding variables, including baseline demographic characteristics and biological/clinical measures .Of 2089 HIV-negative female participants in MIRA who were offered participation in the HPV sub-study, 2040 women (97.6%) consented and 1918 (94%) of participants completed the study. The mean age of study participants at enrollment was 27 years (range 18–49 years), and the median follow-up period was 21 months (range 12–24 months). Detailed demographic characteristics of participants have been published elsewhere Characteristics of women with prevalent, incident and persistent HPV infections in this cohort have been described elsewhere We first examined whether prevalent HPV infection (defined as HPV detection at baseline) was predictive of HIV acquisition. In regression models adjusted for baseline variables associated with HIV acquisition, being positive at baseline for any HPV type or for any oncogenic HPV type was marginally associated, and being positive for HPV type 58 or 70 was significantly associated with HIV acquisition during the study .every visit) or HPV infected (positive for type-specific HPV at any visit up to and including the HIV acquisition visit). In regression models adjusted for baseline variables associated with HIV acquisition  was associated with an increased risk of HIV acquisition. For this analysis, each participant was defined as HPV uninfected  and HIV acquisition during the study; having an abnormal cytology result  was not significantly associated with an increased risk of HIV acquisition . Persistent type-specific HPV infection is a well-established risk factor for development of cervical intraepithelial neoplasia. Therefore, we explored the association between persistent HPV infections and HIV risk in study participants. We defined an HPV infection as being persistent or non-persistent based on the proportion of positive type-specific tests for each participant. In regression models adjusted for baseline variables, persistent infections had no effect on HIV risk, but non-persistent infection with either oncogenic or non-oncogenic HPV types were both significantly associated with an increased risk of HIV acquisition . In analTo assess the effect of HPV infection at the time of HIV acquisition, we analyzed the effect of concurrent HPV infection on the risk of HIV acquisition. In unadjusted analyses, HIV acquisition was significantly associated with having concurrent infection with any non-oncogenic HPV type, with any oncogenic HPV type, or with HPV type 31, 58 or 70 . Given texcluding the current visit; having an HPV infection with any non-oncogenic or oncogenic HPV types was independently associated with an increased risk of HIV acquisition  were implicated in the association. These results suggest a previously unrecognized role of HPV in HIV acquisition among women. An obvious explanation for the observed association is that it is attributable to a partner with both infections, and we cannot definitively rule out the effect of sexual exposure to both infections as the explanation for the association. However, demographic information collected at enrollment and at each quarterly visit allowed us to control for time-dependent variables such as new sexual partners, HSV-2 serostatus, new STIs, or high-risk behavior by the participant or her regular partner. In addition, the results from our sensitivity analysis suggest that HPV infection preceded HIV acquisition.Most HPV infections are cleared by innate and adaptive immune mechanisms Our finding of HPV type-specific associations  raises the possibility that biological processes might underlie the association with HIV risk. HPV 70 is a non-oncogenic HPV type in clade A7 whereas HPV 31 and 58 are oncogenic HPV types in clade A9. HPV16, another member of clade A9, is the most common HPV type in the African continent versus the detection of HPV DNA due to deposition in the genital tract during a recent sexual encounter. For this reason and due to the observational study design, these results cannot establish causation between HPV infection and HIV acquisition, but they suggest that HPV is a potentially significant risk factor for HIV acquisition in women. To date, little is known about the association between HPV and HIV transmission. In a recent study in men who have sex with men, anal infection with 2 or more HPV types significantly increased the risk of HIV infection  The strengths of our study are the large sample size, the high retention rate, and the abundance of data about HPV status and risk factors collected at frequent intervals over time. A limitation of this study is that the commonly used definition of HPV infection cannot differentiate between the presence of biologically active HPV


Benign or malignant lesions caused by Human papillomavirus (HPV) produce considerable health burden worldwide. Since limited cross-protection would be offered between HPV types, heterogeneity in HPV type-specific distribution should be taken into account when predicting the effect of current prophylactic vaccines and forming the basic for the second-generation vaccines targeted to specific regions. Here, we investigated the prevalence of cervical HPV infection, as well as predictors concerned, in Zhejiang Province, southeast China.Totally 4987 cervical samples from five randomly chosen counties in Zhejiang Province were detected. The overall HPV prevalence was 13.3%. Established high-risk (HR) HPV prevalence was 10.2%. HPV-52 was the most prevalent type (3.1%), followed by HPV-16 (2.5%), -58 (2.1%), -68 (1.0%) and -81 (0.9%). HPV-16 or -18 were present in 3.1% of the detected samples, while 174 specimens (3.5%) were positive for any of HPV-6, -11, -16 and -18. The prevalence of HPV vaccine types was consistently low across age groups. Bimodal age distribution in HR-HPV, including established HR and probably HR-HPV, was observed, with a clear second peak in perimenopausal women. Multivariate unconditional logistic regression models revealed that partner's lifetime number of partners was the only common independent predictor of overall, established HR, established low-risk, single-type and multiple-type HPV infection in current study.We have observed low prevalence of HPV vaccine types and relatively high prevalence of HPV-52 and -58 in our population. Our findings support universal "catch-up" vaccination of sexual experienced young women in Zhejiang Province, as well as enhance the hypothesis that the second-generation HPV prophylactic vaccines including HPV-52 and -58 may offer higher protection for women in China and other Asian areas. Furthermore, our data support close surveillance of perimenopausal women with HR-HPV infection. Papillomaviruses (PVs) comprise of a large group of non-enveloped double-stranded DNA viruses that infect a variety of vertebrate species, including humans. There are 16 genera of PVs and five of these genera, alpha, beta, gamma, mu and nu, are human papillomaviruses (HPVs). HPV infection may exist asymptomatically or induce the formation of benign or malignant lesions of human mucosal and cutaneous epithelia. HPV has been shown to be the etiologic agent of anogenital, especially cervical, cancers and a fraction of oropharyngeal carcinomas. Other cancers causally linked to HPV include non-melanoma skin cancer and conjunctiva carcinoma . More th®, GlaxoSmithKline Biologicals, targeted at HPV-16 and -18) and the quadrivalent vaccine , have shown prominently type-restricted prophylactic efficacy for genital lesions related to targeted types in women who were naive to the respective types at enrollment [Both the two available prophylactic HPV vaccines, the bivalent vaccine  in Chinese women (V501-041-00) was conducted in Zhejiang Province, a coastal region in southeast China. To the best of our knowledge, no epidemiologic data on HPV genotypes in general female population were reported from Zhejiang Province, which highlights the need for timely population-based study in this region. Due to the feasible anatomic site for sampling and the well-established sampling method for cervix, here, we investigated the cervical HPV infection in asymptomatic general female population to be conscious of the overall, type-specific and age-specific HPV prevalence, as well as determinants of HPV infection, in Zhejiang Province before large scale vaccination programs occur.Since March 2009, a randomized, double-blind trial testing the safety and efficacy of the quadrivalent vaccine  were randomly recruited to ensure geographical and social diversity. A woman was eligible to be study objective if she: (a) was mentally and physically competent; (b) was aged between 20 and 79 years; (c) was a permanent resident of local area; (d) had no history of abnormal cytology or cervico-vaginal dysplasia; (e) had no abnormal vaginal bleeding and contact bleeding; (f) had no visual cervical lesions at simple visual inspection during gynecological examination; (g) had no history and associated symptoms of other HPV-related diseases; (h) was not a virgin; (i) was not presently pregnant; (j) had not undergone a total hysterectomy; (k) had no use of vaginal medication and no sexual intercourse in the previous three days. Eligible women were invited for a face-to-face interview and a gynecological examination in local hospitals.Of these 7500 enumerated women, 247 stayed away from home at interview time, 376 did not meet the inclusion criteria, 317 could not undergo gynecological examination because of menstruation, and 1502 refused to participate mainly because they did not have enough time or did not think they needed gynecological examination in the absence of symptoms. Totally 5058 women were enrolled into the study.All participants agreed to participate in the present study and signed informed consent forms. The study project was approved by The Human Research Ethical Committee of Women's Hospital, School of Medicine, Zhejiang University.An interviewer-administrated structured questionnaire was designed to collect information on socio-demographic characteristics, lifestyle, menstrual status, reproductive history, contraception and sexual behavior. Trained research nurses questioned participants face-to-face in the local dialect of Chinese.Samples of exfoliated cervical cells were collected with DNAPap cervical sampler (Digene) during gynecological examinations. The sampler was inserted 1-1.5 cm into the endocervical canal and rotated 3-5 full turns in counterclockwise direction. The tip containing cellular material was then placed into transport medium tube and stored at 4°C immediately. All specimens were coded without knowledge of the subjects.HPV DNA was extracted with phenol-chloroform, resuspended in 100 μL elution buffer, and stored at -30°C. The quality and integrity of sample DNA for polymerase chain reaction (PCR) was verified by amplification of a 268 base pair region of the human β-globin gene . Specime2, 200 μM deoxynucleotide triphosphate, 2 U Taq polymerase, 10 pmol of each primer, and 5 μL template DNA. The PCR protocol was: preheating at 94°C for 7 min, followed by 36 cycles of denaturation at 94°C for 45 sec, annealing at 56°C for 45 sec, and extension at 72°C for 45 sec, at last a final extension at 72°C for 10 min. Products were identified by electrophoresis in 2.0% agarose gel stained with ethidium bromide. For every PCR assay, a negative control and a positive control (HPV DNA from SiHa) were run to control for possible contamination and accuracy.PCR was performed in 50 μL reaction mixture containing 10× PCR buffer, 25 mM MgClHPV GenoArray Test kit makes use of both DNA amplification and Hybribio's proprietary Flow-through Hybridization Technology to simultaneously identify 21 HPV genotypes: HPV-6, -11, -16, -18, -31, -33, -35, -39, -42, -43, -44, -45, -51, -52, -53, -56, -58, -59, -66, -68 and -81 . The test employs a gene chip with a nylon membrane onto which type-specific oligonucleotides probes have been immobilized. The final results were detected by colorimetric change on the chip under direct visualization. Test was performed according to the manufacture's instructions.et al. [The products generated by MY09/11 L1 consensus primers were typed with RFLP as described by Hong et al. .http://www.ncbi.nlm.nih.gov/BLAST/).MY09/11 consensus PCR-amplified products were sequenced in the 3730 ABI Prism sequencer (Applied Biosystems). The obtained sequences were compared to known HPV sequence databases  in one single reaction by real-time PCR equipment. Test was performed according to the manufacture's instructions.Positive result by any assay mentioned above was regarded as HPV positive. For samples inconsistent between HPV GenoArray Test and sequencing, the genotypes were identified according to sequencing. Multiple-type infection was separated into constituent types, thus type-specific prevalence included that in both single-type and multiple-type infection. Based on epidemiologic classification associated with cervical cancer, HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59 were classified as established high-risk (HR) types, HPV-26, -53, -66, -68, -73 and -82 were classified as probably HR types, and HPV-6, -11, -40, -42, -43, -44, -54, -61, -70, -72, -81 and -89  were classified as established low-risk (LR) types .2 test and, where appropriate, Fisher's exact test were used to compare HPV prevalence across age groups. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using unconditional logistic regression to estimate independent predictors for HPV infection. Analyses were performed for overall, established HR, established LR, single-type and multiple-type HPV infection, respectively. Age group and variables associated with HPV positivity in age-adjusted model were included as candidates in multivariate model. Final model was determined by forward elimination of candidate variables based on likelihood ratio tests. In all of the logistic models, HPV-negative women were used as the reference group. All hypotheses testing were two-sided. A level of 0.05 was chosen to indicate statistical significance.Data were key-entered twice and analyzed using SPSS software for Windows (version 16.0). The χP < 0.001). Figure Of 5058 women who provided cervical cell samples, 71 were excluded because of negative β-globin. In the leaving 4987 samples, 615 (12.3%) were HPV GenoArray-positive and 591 (11.9%) were MY09/11 consensus PCR-positive. There was a 97.5% overall agreement between these two assays  were HPV positive. The corresponding prevalence, age-standardized to the world population, were 13.5% overall. Of these 665 positive samples, 508 (76.4%) were established HR-HPV and 176 (26.5%) were multiple-type HPV infections. Totally 35 different genotypes, grouped in 11 alpha species, were identified. The five most prevalent types were HPV-52 (3.1%), HPV-16 (2.5%), HPV-58 (2.1%), HPV-68 (1.0%) and HPV-81 (0.9%)  , while overall and established LR-HPV infection showed relatively flat curves   and -6  were similar to the established HR and probably HR-HPV infection, with two peaks, while the curves for alpha-7  and 3  were more flat  years. Figure ) Figure . Similar) Figure . The aget Figure . The preIn age-adjusted models, no significant associations were found between HPV positivity and smoking, income, age at menarche, age at marriage, reproductive history, condom or intrauterine device use as the main contraceptive method in the recent two years, history of malignancy, history of subtotal hysterectomy and history of Papanicolaou screening (data not shown). In multivariate models, marital status, residence, passive smoking, menstrual cycle, outcome of initial pregnancy, oral contraceptive use as the main contraceptive method in the recent two years, age at sexual initiation and lifetime number of sexual partners were eliminated from the final models. The independent predictors for overall, established HR, established LR, single-type and multiple-type HPV infection were listed in Table To date, no type-specific HPV testing is approved by the Food and Drug Administration or is used widely. HPV GenoArray-test, which has already been used for quite a while in European and Asian countries, showed high level agreement (97.5%) with MY09/11 consensus PCR system. The concordance of HPV GenoArray-test with Amplicor HPV test  or RocheCompared with similar studies in China, age-standardized HPV prevalence estimation in Zhejiang Province (13.5%) was similar to that in a central rural area of China (14.2%) , but lowet al. [et al. [et al. [The five most common HPV types in general female population worldwide were HPV-16, -18, -31, -58 and -52, while the rank varied by region . Contraset al.  found th [et al.  found th [et al. ,17-19,27 [et al. ,28,29, wFor the current available prophylactic vaccines, the prevalence of vaccine types was consistently low across age groups. Since a proportion of older women might have already been infected and cleared previous infections, the actually exposed rate to vaccine types might be higher than observed in older women. Considering that HPV prophylactic vaccines do not have clinical benefit in women who have been infected with vaccine types at the time of vaccination, we assume that younger women would draw more benefit from "catch-up" HPV vaccination programs.et al. also suggested a stronger role for viral persistence than for acquisition of new infections in women aged 45 years or older in Costa Rica [Bimodal age distribution of HR-HPV, which was not common in mainland China ,18,26, wsta Rica . Accordiet al., the age-related changes in cervix influence HPV type-specific detection at the cervical os, where cervical specimens are routinely collected [Alpha-6, -7, -9 species comprised mainly of established HR and probably HR-HPV types, while alpha-3 species comprised mainly of established LR-HPV types. However, alpha-7 species were more closely genetically related to alpha-3 than to alpha-9 species . Consistollected . What isollected ,35. WhetWe also explored the independent predictors for categories of HPV positivity. Previous studies showed that high-risk sexual behavior, including early sexual initiation, multiple sexual partners and sexual partner as an HPV carrier, was the pivotal determinant for HPV infection among women -38. HoweThis study provides a unique opportunity to gather baseline data on cervical HPV prevalence, as well as predictors concerned, in general female population in Zhejiang Province, southeast China. The data showed low prevalence of HPV vaccine types and relatively high prevalence of HPV-52 and -58. Our findings support universal "catch-up" vaccination of sexual experienced young women in this region, as well as enhance the hypothesis that the second-generation HPV prophylactic vaccines including HPV-52 and -58 may offer higher protection for women in China and other Asian areas. Furthermore, our data also support close surveillance of perimenopausal women with HR-HPV infection.PV: papillomavirus; HPV: human papillomavirus; HR: high-risk; LR: low-risk; PCR: polymerase chain reaction; RFLP: restriction fragment length polymorphism; OR: odds ratio; CI: confidence interval.The authors declare that they have no competing interests.JY carried out the study, performed analysis of data and drafted the manuscript. XDC participated in the study design and coordination. XJC carried out the MY09/11 consensus PCR and the PCR-RFLP assays. FY contributed to study design and developed the assay protocol. WGL participated in the study design, sample collection and coordination. XX conceived the study, provided consultation and revised the manuscript. All authors have read and approved the final manuscript.


In order to identify potential markers of prognosis in breast cancer, representative cDNA libraries were constructed using RNA isolated from primary breast tumour tissue associated with good and poor prognosis. Cross-screening of these libraries repeatedly identified cloned mRNA species associated with the immune system, in particular B-cells, in libraries derived from tumours of poor prognosis. We have used one of these a kappa IV light chain cDNA probe, in two complementary studies to investigate the relationship between immunoglobin gene expression and prognosis. The results obtained using a combination of S1 mapping, RNA blotting and in situ hybridisation demonstrate that the presence of plasma cells, as defined by infiltrating cells which express high levels of immunoglobulin kappa-chain mRNA, is associated with a poor prognosis.


HPV has been found repeatedly in esophageal carcinoma tissues. However, reported detection rates of HPV DNA in these tumors have varied markedly. Differences in detection methods, sample types, and geographic regions of sample origin have been suggested as potential causes of this discrepancy.HPV L1 DNA and HPV genotypes were evaluated in 435 esophageal carcinoma specimens collected from four geographic regions with different ethnicities including Anyang in north China, Shantou in south China, Xinjiang in west China, and the United States. The HPV L1 fragment was detected using SPF1/GP6+ primers. HPV genotyping was performed using genotype specific PCR.Two hundred and forty four of 435 samples (56.1%) tested positive for HPV L1. Significant differences in detection rate were observed neither among the three areas of China nor between China and the US. HPV6, 16, 18, 26, 45, 56, 57, and 58 were identified in L1 positive samples. HPV16 and 57 were the most common types in all regions, followed by HPV26 and HPV18.HPV infection is common in esophageal carcinoma independent of region and ethnic group of origin. Findings in this study raise the possibility that HPV is involved in esophageal carcinogenesis. Further investigation with a larger sample size over broader geographic areas may be warranted. Esophageal carcinoma is a common malignancy and its mortality rate is among the highest for cancers overall . There iIn 1982, Syrjanen found that HPV infection caused pathological lesions in esophageal cancer specimens . Since tIn this study, we evaluated tissues from 435 esophageal cancers collected from four different regions with multiple ethnicities including Han, Kazakh, American Japanese, American Caucasian, and African American for HPV DNA. We found comparable rates of HPV detection and similar HPV genotypes in the esophageal cancer samples from all of these geographic areas.A total of 435 esophageal cancer samples collected in an unselected manner were used in this study. Of these, 166 were from Anyang, a city in northern China, 103 were from Shantou, a city in southern China, 78 were from Xinjiang, an autonomous region in western China, and 88 were from the United States (US). Among the study subjects, 61.9% were Han, the majority ethnic group of China, 17.9% were Kazakh, a minority group of Chinese living in western China with a high prevalence of esophageal cancer, 9.4% were Caucasian, 9.0% were Japanese, and 1.8% were African-American. The male to female ratio was 2.4:1  DNA mixed together from several tumors, which had previously tested positive for HPV L1.Assay results were interpreted and used for this study only when controls met the following criteria: 1. all seven negative controls were negative; 2. ESCC DNA was positive; and 3. at least one set of plasmid controls was positive and the intensity of the positive signal corresponded with the input copy number. In cases where any negative control was positive, testing was repeated.DNA from 88 US samples was extracted in the NCI laboratory  using the FlexiGene kit . DNA from all other samples was prepared using the H.Q. & Q. Tissue DNA Kit . DNA was diluted to 35 ng/μl, and 3 μl (~100 ng) of DNA was used for each of the various PCR analyses.2, and 16 pmol of each primer. Enzyme activation was carried out at 95°C for 15 minutes, followed by 45 amplification cycles at 95°C for 40 seconds, 49°C for 50 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes.A set of primers, SPF1/GP6+, which amplify an L1 fragment of approximately 184 bp were used. These primer sets were reported previously to be highly sensitive . The polTo determine the HPV genotypes in samples for each region from different geographic areas, L1 positive samples were mixed in equal molar ratios for each region and cloned into a TA vector. Forty-eight colonies from each batched cloning were sequenced.Type-specific detection of HPV was performed with type-specific PCR primers Table  for genoPCR products were purified using a PCR clean-up gel extraction column  according to the manufacturer's instructions and were directly sequenced using a capillary sequencer (ABI Prism 3100).P values of less than 0.05 were considered to be statistically significant.Univariate comparisons of rates were performed using the chi-square test. Unconditional multivariate logistic regression was used to compare the rates of multiple type infections and HPV detection rates among different regions adjusting for potential confounders. All statistical analysis was done using SAS 9.1.3. P = 0.131) (Table P = 0.522)  were HPV L1 positive. Stratifying by region, HPV L1 was detected in 86 (51.8%) of the Anyang samples, 62 (60.2%) of the Shantou samples, 42 (53.8%) of the Xinjiang samples, and 54 (61.4%) of the US samples. There were no significant differences among the detection rates in the samples from the three areas of China (P = 0.003). HPV57 was the second most prevalent genotype, and was found in 46 (23.6%) of the China samples, of which 27 were from Anyang, 8 were from Shantou, 11 were from Xinjiang, and in 14 (20.9%) of the US samples. In addition, HPV26 was also common both in China and US with a prevalence of 9.2% in China and 13.4% in the US. HPV18 was found in three of the four study areas with a detection rate of 14.5% in Anyang, 8.1% in Shantou, and 7.5% in the US. HPV6, 45, 56, and 58 were much less common. Three cases (4.0%) from Anyang were HPV45 positive. Nine (20.0%) and 4 (8.9%) cases from Xinjiang were HPV6 and HPV56 positive respectively  or between China and the US (P = 0.834).Among L1 positive samples, 43 (17.6%) were co-infected with more than one HPV type. Multiple HPV genotypes were found in 30 (15.8%) cases from China, of which 9 were from Anyang, 15 were from Shantou, 6 were from Xinjiang, and in 13 cases (24.1%) from the US among the L1 positive samples. In addition, no significant differences in multiple infection rates were observed among three areas of China (P = 0.200). The detection rates in these 2 types of carcinomas therefore showed no statistically significant difference. Tumor site data was available only for squamous cell carcinomas from the Anyang and Xinjiang regions, and evaluation of the association between anatomic location and HPV prevalence was carried out in these cases. L1 was detected in 7 (33.3%) of carcinomas located in the upper third of esophagus, 59 (56.7%) in the middle third and 27 (58.7%) in the lower third. No statistically significant relationship between HPV infection and tumor site was found in this study (P = 0.115).HPV infection in esophageal adenocarcinomas and squamous cell carcinomas was compared among the samples from the US. L1 was detected in 18 (52.9%) of esophageal adenocarcinomas and 36 (66.7%) of esophageal squamous cell carcinomas . First, real time PCR revealed that the HPV load in esophageal specimens was at least two orders of magnitude lower than that in cervical cancer. In addition, HPV genotypes found in esophageal specimens were different from those found in cervical cancers. Based on these observations, we proposed in a previous study that the low HPV copy number might therefore result in a falsely low detection rate in esophageal cancer. To provide evidence for this line of reasoning, a series of HPV 16 positive cervical cancer specimens were employed to test a mimic esophageal cancer model. The copy number of HPV/cell in these cervical cancers was first determined using real time PCR. The samples were then diluted to 0.1-0.01/cell which was similar to the average copy number in esophageal cancers and tested for L1. Of 24 diluted samples, 6 were negative in the first round of PCR. An additional round of PCR was carried out, and all six samples which were negative in the first PCR analysis were positive, indicating that repeat testing may decrease the false negative rate in samples with low HPV copy numbers. On this basis, an analytic strategy for HPV detection in esophageal cancers was formulated.Briefly, L1 detection was performed twice in each esophageal sample. L1 positivity in either test was counted as positive for calculation of the detection rate. A batched TA cloning assay was used to evaluate the HPV genotypes in the sample population. Utilizing this approach, HPV57 which is not common in cervical cancers was identifiable in esophageal specimens. Genotype specific E6 or E7 PCR was performed to determine the genotypes in L1 positives.It is of interest to note that HPV57 presented as the second most common genotype and accounted for 22.9% of L1 positive esophageal cancer tissues from all four areas under investigation. HPV57 was originally identified in benign, premalignant, and malignant lesions of the nasal cavity . The sigThe associations between HPV infection and age, gender, tumor TNM stage, tobacco smoking and alcohol use were analyzed. Of these factors only alcohol use was associated with HPV positive esophageal cancers as compared with HPV negative subjects. However, there was no statistical evidence of such relationship when the analysis was limited to US samples only, but the possibility that the limited US sample size might have contributed to this result cannot be excluded. Alternatively, as samples from the US included both esophageal squamous cell carcinomas and adenocarcinomas, it is possible that these two types of cancers may arise from different carcinogenic pathways and thus confound the data. As such, further investigation with a larger sample size should be carried out to further evaluate the effect of alcohol use on HPV infection in the esophagus.We recognize that there are several limitations in this study, one of which is selection bias. As a hospital-based study, these cancer patients were all recruited from local hospitals and as a result the population from which we obtained these samples cannot be precisely defined. Patient self selection of a hospital for treatment may have also introduced selection bias. These factors may limit the sample's representativeness and capacity for inference. Another limitation is that some crucial information which would potentially allow broader conclusions was not obtained for this study . In this study we demonstrated for the first time that HPV DNA is both relatively common in esophageal cancer, and is independent of geographic region and ethnicity of tested subjects. In addition, HPV infection appears to be independent of all previously identified risk factors for esophageal cancer except alcohol use.This observed consistency of the prevalence of HPV infection in esophageal carcinoma from different geographic areas may warrant further investigation with larger numbers of subjects over additional areas. However, the findings here serve to raise the possibility that HPV plays a role in the etiology of esophageal carcinoma.The authors declare that they have no competing interests.XW and XT carried out HPV detection and typing. FL performed the statistical analysis. YZ, and MS participated in HPV detection. DC participated in the design of statistical analysis. CL, ZW, XS, QZ, DZ, ZS, FL and CH participated in sample preparation, data validation and study coordination. HC and YK conceived the study, and participated in its design and coordination and prepared 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/1471-2407/10/19/prepub


No recent data exist on human papillomavirus (HPV) infection in Beijing, People's Republic of China.We interviewed and examined a representative, randomly selected sample of 5552 sexually active women aged 25–54 years. Cervical cell samples were analysed for HPV DNA by a MY09/11-based PCR assay.Human papillomavirus prevalence was 6.7% overall and 4.8% among women without cervical abnormalities. Of the 21 subtypes identified, HPV16 was the commonest type , followed by HPV 58 (1.0%), 33 (0.8%), 43 (0.7%) and 56 (0.7%). High-risk HPV types predominated in all age groups. Human papillomavirus prevalence was highest in young to middle-aged women. Marital status, number of husband's sexual partners, age at sexual debut and nulligravidity were all associated with being HPV positive.In our survey, HPV 16, HPV 58 and HPV 33 were the most prevalent HPV types in Beijing, indicating the potential for the prophylactic HPV 16/18 vaccine in China. Human papillomavirus (HPV) has been established as a necessary cause of cervical cancer, found in 99.7% of invasive cervical carcinomas in the best-studied series (http://www.stats.gov.cn). However, despite representing an appreciable portion of the nation's population, no recent data on HPV prevalence and type-specific distribution exist for Beijing women.The People's Republic of China, with a population of about 1.3 billion, is the world's most populous country. Although Chinese women have been considered to have a relatively low risk (LR) of developing cervical cancer, there are approximately 100 000 new cases and 30 000 deaths per year in China . In BeijIn this study, we report a population-based study of the type-specific and age-specific distribution of cervical HPV infection and of its risk factors in Beijing, China.We carried out the present survey between September 2006 and December 2008 in Beijing, which is the country's political, economic and cultural centre; it consists of 18 districts, four of which are urban, four suburban and 10 outer suburban. A total of 5681 women aged 25–54 years were recruited from the population list of four urban, four suburban and four randomly chosen outer suburban districts in Beijing, China. On the basis of data from the 2005 census published by the National Bureau of Statistics of China, our representative sample of the adult female population of Beijing was stratified by age and regional distribution using a four-stage cluster sampling method. In the first stage, 12 of the 18 districts of Beijing were randomly selected. In the second stage, 4–20 townships were randomly selected from each district according to the proportion of each district's population. Then in the third stage, one neighbourhood was randomly extracted from each township. Finally, from the lists provided by local neighbourhoods, we derived a final random sample of 50 women per neighbourhood. On the basis of the age ratios of the 2005 census, we strove to enroll approximately 10 women aged 25–34 years, 14 women aged 35–44 years, 13 women aged 45–54 years and 13 migrant women aged 25–54 years from each neighbourhood.According to the inclusion criteria, women who (1) lived in Beijing for more than half a year; (2) had no history of cervical surgery; (3) were not pregnant; (4) had no history of pelvic radiation therapy; (5) did not have a gynaecological examination and/or treatment in the past 2 years; and (6) were physically and mentally able to undergo an interview and a pelvic examination were invited to participate in this study. After selecting our randomised sample of the population, we made house-to-house visits to recruit these women and invite them to visit their local Maternal and Child Health Hospitals to participate in this study. Here, the interview was administered to the women by two–four experienced gynaecologists, covering socio-demographic information and obstetric and gynaecological history, including number of sexual partners ; participants then had a gynaecological examination and a cervical cell sample taken for HPV DNA analysis.Written consent was obtained from all participants according to the regulations of the Beijing O&G hospital and 12 various district Maternal and Child Health Clinics, which approved the study.A total of 5681 women underwent a pelvic examination; a sample of exfoliated cervical cells for liquid-based cytology and HPV testing was collected later. First, a cytobrush was inserted into the endocervical canal, and rotated gently in a clockwise direction five times. A cytobrush was then inserted into the bottom of a preservation solution vial  and swirled 10 times vigorously. Finally, the brush was discarded and the vial contents were sent to Beijing O&G Hospital for cytological analysis. All smears were reported by two cyto-technicians separately, the abnormal/indeterminate ones were reviewed by a senior cytologist for final diagnosis. The results were classified according to the Bethesda System. All women with an abnormal cytological test (ASCUS or more severe) were referred to the study colposcopist and a biopsy was performed. All abnormal smears and 10% of normal smears chosen at random were sent for review by an experienced cytopathologist. Cervical biopsies were reviewed by a senior pathologist. All confirmed or highly suspicious high-grade or invasive lesions were treated at the Beijing O&G Hospital with loop excision, surgical conisation, hysterectomy or radiotherapy, according to protocols. In this study, cervical abnormalities were defined as the presence of histologically confirmed cervical intra-epithelial neoplasia (CIN) 1 or worse.β-globin PCR analysis was performed on each sample and only β-globin-positive samples were included in further analyses.DNA was extracted first from the remainder of the Thinprep sample. To confirm the presence of human DNA, The overall presence of HPV DNA positivity was determined by PCR using 5′-biotinylated MY09/11 consensus primers, as described previously (P for trend) was assessed by treating ordinal variables as continuous.Statistical analysis was performed using SPSS version 15 software. Odds ratios (ORs) for HPV positivity and corresponding 95% confidence intervals (CIs) were calculated using unconditional, logistic regression, adjusted for age groups . The statistical significance of trends for ORs  had histologically confirmed cervical abnormalities, including 210 CIN1, 37 CIN2, 18 CIN3 and 3 microinvasive carcinoma. Meanwhile, HSIL was diagnosed in 0.27% (n=15) and the proportion of HSIL that was histologically confirmed was 93.3%. The mean age of the total screened population was 39.7 years.Of the 5681 women who provided cervical cell samples, 32 had inadequate cytology results and 97 had Overall, the prevalence of HPV of any subtype was 6.7% . The increasing age at first intercourse was inversely associated with being HPV positive .vs ⩾30 years=2.09, 95% CI: 1.01–4.32; OR for 25–29 vs ⩾30 years=2.00, 95% CI: 0.97–4.12). Oral contraceptive use was not associated with HR and LR HPV infections, but condom use was somewhat protective against HPV infection. No significant association was found between HPV positivity and race, occupation, education level, age at menarche, marriage or menopause, or history of spontaneous or voluntary abortion (data not show). In addition to age, when marital status, number of sexual partners, husband's sexual partners and smoking were included in the same multiple logistic regression, the corresponding ORs did not change materially.Nulliparous women were more likely to be infected with HR HPV than women who had been pregnant earlier . However, there was no association of birth numbers with HPV positivity (data not shown). Age at first pregnancy was unrelated to HPV infection, whereas a trend of increased HPV positivity with younger age at first pregnancy was seen  have only one sexual partner in their life. As expected, marital status of women and age at first intercourse were risk factors for HPV infection. Similar to previous studies (Comparisons of the distribution of common HPV types among pathological grades, HPV 16 and HPV 18 were frequent with CIN3/CC relative to their prevalence in normal, CIN1 and CIN2 grades, reflecting their predominant roles in invasive carcinomas in Beijing. In most countries, HPV 16 is by far the most common type in cervical carcinoma, followed by HPV 18 (In conclusion, the commonest HPV types in the general female population of Beijing are HPV 16, 58 and 33, and the HPV 16/18 vaccination is expected to substantially reduce the cervical cancer burden in China.