This study evaluated human papillomavirus (HPV) type prevalence in 370 Scottish invasive cervical cancers (ICCs) using HPV genotyping and HPV mRNA detection.HPV 16 and/or 18 was detected in 72% of cancers overall and in 82% of HPV-positive cancers. HPV 45 and 16 were the most frequently transcribed types.A significant reduction in ICC in Scotland should be achieved through the HPV immunisation programme. Human papillomavirus immunisation is being introduced in many countries across the world. In Scotland, a national school-based immunisation programme began in September 2008 for girls aged 12–17 years using HPV bivalent vaccine. The vaccine protects against infection with high-risk HPV 16 and 18 that are thought to contribute to around 70% of cervical cancers . HoweverWe undertook a sufficiently powered, systematic interrogation of cervical cancer cases from all regions of Scotland to determine HPV type-specific prevalence. This study was necessary to gauge the potential impact of the immunisation programme, which so far has achieved high uptake (93.3% for first dose and 89.5% for second dose) for girls in second year of secondary school .The study was also undertaken to define a baseline prevalence against which to monitor changes over time in HPV types associated with cervical cancers. Acquisition of related demographic, clinical and HPV type-specific data allowed us to assess the relationship between HPV 16/18 status, disease stage (FIGO), disease grade and the expression profile of HPV types within cancers with multiple infections.A representative population-based sample of Scottish invasive cervical cancers (ICC) was achieved by selecting cases regionally from Edinburgh, Dundee, Aberdeen, Glasgow and Lanarkshire. Each site was asked to generate a sequential list of ICC cases, diagnosed from 2004 working backwards. 2004 was chosen as the most recent year for which data in the Scottish Cancer Registry were complete. The sample included both microinvasive and frank cancers, but not recurrent cancers. Review of cases was performed at each site and the relevant formalin-fixed paraffin-embedded block from the primary invasive tumour was sent to the Scottish HPV Reference Laboratory for sectioning and HPV testing.Demographic and clinical information was sent to the Scottish Cancer Registry where laboratory and registry data were linked, with a view to attaching demographic variables (including the Scottish Index of Multiple Deprivation), prognostic variables (FIGO stage and ICD-O grade of differentiation) and a unique linkage number . Where necessary, data cleansing was performed.μm paraffin sections cut with a clean microtome knife were digested overnight with proteinase K. Crude lysate (10 μl) was subjected to PCR and amplicons were hybridised to genotyping arrays capable of detecting high-risk HPV types and several low-risk types. A result of HPV ‘X’ was generated if the sample was HPV positive but could not be attributed to a type within the detection range of the assay.Typing of HPV was performed using HPV INNO-LiPA  according to the manufacturer's instructions. This is a very sensitive DNA-based assay that amplifies a 65-nucleotide fragment of the HPV L1 gene. Briefly, 10 Any cancer that tested positive for >1 type, tested positive for low-risk type(s) only or generated a result of HPV X alone was subjected to mRNA E6/E7 expression analysis, using HPV PreTect RNA Proofer  according to the manufacturer's instructions. The method involves RNA extraction before isothermal NASBA RNA amplification of full-length E6/E7. Type-specific detection was performed using molecular beacons corresponding to HPV types 16, 18, 31, 33 and 45.χ2-tests.Our hypothesis was that HPV 16 and 18 distributions in cervical cancers in Scotland was no different from collated, worldwide estimates of around 70%. To detect a prevalence of HPV type 16/18 of 70% to within 10%, we needed a sample size of 323 using a two-sided 95% confidence interval (CI). To assess how representative our study population was, it was compared with the cancer registry data on 1752 cervical cancers diagnosed in Scotland from 2000–05 . Socioecvs other or no HR-HPV and FIGO stages I–IV was assessed using a χ2-test. A similar analysis was performed to assess the relationship between HPV 16 and/or 18 positivity and ICD-O grade of differentiation (grades I–IV).Where data were available, the relationship between HPV 16 and/or 18 For mRNA expression analysis, frequency of type-specific transcripts was assessed and descriptive analysis was performed.χ2-test there was no evidence of a difference between socioeconomic status (P=0.273) or age (P=0.846) between the study population and the cancer registry data based on 1752 cases, indicating that our study population was representative. Of the 370 cases evaluated, 309 (83.5%) were squamous cell carcinoma, 54 (14.6%) were adenocarcinoma and 7 (1.9%) were ‘other’ types.Of the 375 cervical cancers collated, 370 were included after data checking in the cancer registry. Omissions included two cases from women with permanent residence outside Scotland at time of diagnosis, two related to recurrent disease and one to a case of high-grade CIN. Using a Comprehensive type-specific data on all DNA-positive cases are presented in P=0.008. The three most prevalent types after 16 and 18 were 45, 33 and 31. A result of HPV X was generated quite frequently .Multiple infections were detected in 36 (10%) cases (95% CI 7–13%). Interestingly, by combining cases in Scotland's ‘central belt’ , we found evidence of a difference in the proportion of cases with multiple infections compared with non-central belt (Aberdeen and Dundee combined) with 7.8% more in the central belt with a 95% CI of (2–13.5), vs non-HPV status according to FIGO stage, disease grade and age. There was no evidence from the available data of a relationship between HPV 16 and/or18 presence and either stage (P=0.561) or grade (P=0.553).Of the 36 cervical cases in which multiple infections were detected, 32 proved adequate for expression analysis. In 25 out of 32 cases only one transcript was detected, in 6 out of 32 two transcripts were detected and in one case no transcript was detected. It was notable that in five cases (shaded), an RNA transcript was detected that was not reflected in the equivalent HPV DNA component. In the seven cases in which a low-risk type was detected , no transcripts were detected in five cases, and two cases (both HPV 6) were invalid for expression analysis. In the eleven cases in which HPV X was detected, one tested positive for HPV 18, no transcripts were detected in three cases, six cases were invalid for expression analysis and one case had insufficient material for re-testing. The two most commonly expressed transcripts were HPV 45 (number of appearances=12) and HPV 16 (number of appearances=11).The HPV vaccine is being offered through a national programme in several parts of the world including Scotland. However, the vaccine is not a panacea for the eradication of HPV-associated cervical disease and is licensed only to protect against HPV types 16 and 18. Nevertheless, the findings of this study are very positive with 82% of a systematic collection of HPV DNA-positive ICC from across Scotland, being positive for HPV 16 and/or 18. A meta-analysis by It is notable that in our series 13 cancers (2.9%) occurred in women aged 18–24 years, with 11 out of 13 being infected with HPV 16 and/or 18. This is representative of collated Scottish Registry data from 2000 to 2005 in which 52 (2.96%) of 1752 cases were diagnosed in women <24 years. Routine cytology screening of women occurs in Scotland from age 20. Current discussion centres round the effectiveness of such a policy (It was interesting that in four cancers HPV 6 was the only HPV type detected. An explanation for this could be that there was a co-infecting high-risk type that was below the level of detection of the assay. However, in a recent review, which focussed on the classification of weakly carcinogenic HPV types, vs absence of HPV 16, again no association with stage of disease was found. Although this suggests that HPV type may have no substantive impact on prognosis, it is our intention to investigate this more directly by assessing survival according to HPV type in the study cohort as soon as sufficient follow-up time has accrued.We did not find an association between HPV 16 and/or 18 positivity and FIGO stage or disease grade. When the analysis was restricted to HPV 16 Preliminary data relating to transcript detection would indicate preferential expression of HPV 16 and interestingly HPV 45, the latter being expressed even when present in co-infections with HPV 16 and/or HPV 18. However, numbers are small and this phenomenon requires further investigation.In conclusion, this evaluation would suggest that the bivalent prophylactic HPV vaccine will eventually have a significant impact on the reduction of ICC in Scotland. In addition, the accumulated data will act as a baseline against which future changes in type-specific prevalence in cancers can be referred as HPV immunisation embeds.


Seroconnversion (n=409), persistence (n=675), and clearance (n=541) were defined based on enrolment and follow-up serology measurements. Age-specific distributions revealed that HPV-16 seroconversion was highest among 18- to 24-year-old women, steadily declining with age; HPV-16 seropersistence was lowest in women 65+ years. In age-adjusted multivariate logistic regression models, a 10-fold risk increase for HPV-16 seroconversion was associated with HPV-16 DNA detection at enrolment and follow-up; two-fold risk of seroconversion to HPV-16 was associated with increased numbers of lifetime and recent sexual partners and smoking status. Determinants of HPV-16 seropersistence included a 1.5-fold risk increase associated with having one sexual partner during follow-up, former oral contraceptive use, and a 3-fold risk increase associated with HPV-16 DNA detection at both enrolment and follow-up. Higher HPV-16 viral load at enrolment was associated with seroconversion, and higher antibody titres at enrolment were associated with seropersistence.Determinants of human papillomavirus (HPV)-16 serological conversion and persistence were assessed in a population-based cohort of 10 049 women in Guanacaste, Costa Rica. Serologic responses to HPV-16 were measured in 7986 women by VLP-based enzyme-linked immunosorbent assay at both study enrolment (1993/94) and at 5–7 years of follow-up. Seropositive women were defined as ⩾5 standard deviations above the mean optical density obtained for studied virgins at enrolment ( Most genital human papillomavirus (HPV) infections are transient: within 2 years of incident infection, HPV DNA becomes undetectable in approximately 90% of women  was considered the reference standard of serious, newly diagnosed high-grade disease. Plasma specimens collected from women at enrolment and at the end of follow-up (concurrently at the time of diagnosis for cases) were used for measurement of serologic responses to HPV-16. Of the 10 049 women enrolled in the cohort, serology results from enrolment specimens were obtained for 9951 women. Of the 8084 women selected for follow-up   at the Johns Hopkins Medical Institutions by a VLP-based enzyme-linked immunosorbent assay (ELISA) for HPV-16. Serology measurements were conducted as previously described . To demonstrate that our results were robust, we also compared seropositive women to seronegative women, defined as less than one standard deviation from studied virgins.Consistent with our previous analysis, serology results were dichotomised as antibody positive or negative (n=1534) and seronegative (n=8417). Of the 1216 (79%) HPV-16 seropositive women with follow-up data, 675 (55.5%) remained seropositive at follow-up and 541 (44.5%) tested seronegative . Age was calculated as the woman's age at the midpoint of her follow-up.All HPV cofactors reported previously in Costa Rica .Variables independently and statistically significantly associated with seroconversion or seropersistence in our univariate analyses were included in our final multivariate models; they included number of lifetime sexual partners reported at enrolment , number of sexual partners during follow-up , smoking , OC use , and HPV-16 DNA status  as measured by PCR. Our final model also adjusted for age at the midpoint of the woman's follow-up (years), and serology batch. We excluded women who remained virgins throughout the duration of study follow-up . For these cases, the follow-up testing was carried out at the time of diagnosis before treatment. CIN3/cancers diagnosed at enrolment were censored for follow-up and did not have a second time point for which serology measurements were made; thus, they were excluded from all analyses. Statistical analyses were conducted on SAS 8.2 for Windows and STATA 7.0.Seroprevalence of HPV-16 at both cross-sectional time points  was 15%. In all, 55% (675 of 1216) of women seropositive at enrolment remained seropositive for HPV-16 at follow-up, while 6% (409 of 6770) of initially HPV-16 seronegative women were seropositive at the time of follow-up. As shown in Figure 1Univariate analyses for determinants of HPV-16 seroconversion, compared to women who remained seronegative, revealed a number of potential determinants, which were subsequently included in the final multivariate logistic regression model. After adjustment for age and serology batch, and excluding women who remained virgins for the duration of the cohort study, two or more lifetime number of sexual partners and one or more sexual partner (not necessarily new partners) during study follow-up were statistically significantly associated with an increased risk for seroconversion  was also associated with a significant higher odds ratio for HPV-16 seroconversion. Odds ratios were 3.0 (95% CI: 1.5–6.0) and 6.2 (95% CI: 3.5–11.1) for weak and strong HPV-16 PCR signal strengths, respectively. Viral load did not appear to modify the risk of seropersistence among HPV-16-positive women, with odds ratios of 2.7 (95% CI: 1.2–6.4) and 2.2 (95% CI: 1.1–4.2) for weak and strong signal strengths, respectively. Further stratification by age also did not reveal differences. Further investigation of determinants of serologic persistence also revealed that of women seropositive at enrolment (e.g. five standard deviations above the set cut-point), women with the highest mean OD measures at enrolment were most likely to remain seropositive at follow-up, as defined by the same cut-point. Specifically, 80% of women with the highest quartile of mean OD seropersisted; these women possessed a 12.7-fold increase in risk for seropersistence when compared to seropositive women in the lowest quartile. In all, 5.8- and 2.0-fold increases in risk for seropersistence were observed for women in the third and second quartiles of mean OD, respectively. In the third quartile of mean OD, 69% seropersisted, and in the second quartile 44% seropersisted.Increasing HPV-16 viral load at enrolment as measured by qualitative PCR signal strength  with the same HPV type and resultant antigenic stimulation is to be investigated in a more refined analysis of HPV DNA typing for all years of follow-up. We also found that for seropositive women, a higher OD at enrolment was associated with seropersistence at follow-up, consistent with a previous study in which HPV antibodies persisted when initial levels were high or when there was continued exposure  to define HPV-16 seropositive women. Because VLP ELISAs are still prone to misclassification including false positives, we chose to define HPV-16 seropositive women stringently, thus probably misclassifying some HPV-16 seropositive women (seroconverters or seropersisters) as seronegative and biasing our results towards the null. However, we attempted to measure the accuracy of our definition of seropersistence by a small subset of approximately 1500 women, and measurements were conducted 1 year after enrolment. While the majority of women defined as seropersistent (based on enrolment and 5–7 years of follow-up) were also seropositive at 1 year after enrolment, approximately 10% were not. This probably reflects misclassification in our definition of seropersistence and might also have further biased our results towards the null.To our knowledge, this is the largest population-based seroprevalence study of HPV-16 with follow-up serology data. Our subjects were representative of the adult female population of Guanacaste, Costa Rica and our results provide a detailed description of serologic status in this and population behavioural factors likely to influence long-term serologic status. The known waning of HPV antibody detection with age is confirmed, attributed to both decline in seroconversion and seropersistence; these age-specific data may be relevant to future plans for HPV vaccination. Because antibody titres after natural infection are lower than those following vaccination, these immunologic responses should be distinguishable. Future analysis assessing longitudinal HPV DNA status in relation to serologic status will be important for clarifying the role that transient and persistent HPV infections play in initiating and sustaining a humoral immune response.


The p16 (MTS1) tumour-suppressor gene is a cyclin-dependent kinase (cdk) inhibitor that decelerates the cell cycle by inactivating the cdks that phosphorylate the retinoblastoma tumour-suppressor gene (Rb) protein (pRb). In cervical cancers, pRb is inactivated by the HPV E7 oncoprotein or by mutations. The hypothesis of earlier reports was that the disruption of the p16/cdk-cyclin/Rb cascade is essential for malignant cervical transformation/carcinogenesis. We previously established in vitro model systems of cervical cancer representing four steps of oncogenic progression initiated by the two most common oncogenic HPVs in ectocervical and endocervical epithelial cells. This report used these systems to investigate the role of p16 in cervical cancers. A dramatic enhancement of the p16 RNA level was observed after immortalization by HPV 16 or 18. Furthermore, the p16 protein was newly observed following immortalization. However, no further changes were found for RNA or protein levels after serum selection or malignant transformation. For three cervical carcinoma cell lines, similar high levels of p16 expression were seen. Point mutations or homozygous deletions of p16 were not observed in the in vitro systems or in clinical specimens. These results suggest that the inactivation of the p16/cdk-cyclin/Rb cascade does not occur during malignant transformation but occurs during the immortalization by HPV in HPV-harbouring premalignant lesions, the in situ equivalent of immortalized cells. Also suggested is that p16 has no role in the specific malignant transformation step from immortal premalignant lesions during the carcinogenesis of HPV-initiated cervical cancers.


Oral malignancy is a major global health problem. Besides the main risk factors of tobacco, smoking and alcohol, infection by human papillomavirus (HPV) and genetic alterations are likely to play an important role in these lesions. The purpose of this study was to compare the efficacy of HC-II assay and PCR for the detection of specific HPV type (HPV 16 E6) in OSMF and OSCC cases as well as find out the prevalence of the high risk HPV (HR-HPV) in these lesions.Four hundred and thirty patients of the potentially malignant and malignant oral lesions were taken from the Department of Otorhinolaryngology, Moti Lal Nehru Medical College, Allahabad, India from Sept 2007-March 2010. Of which 208 cases were oral submucous fibrosis (OSMF) and 222 cases were oral squamous cell carcinoma (OSCC). The HC-II assay and PCR were used for the detection of HR-HPV DNA.The overall prevalence of HR-HPV 16 E6 DNA positivity was nearly 26% by PCR and 27.4% by the HC-II assay in case of potentially malignant disorder of the oral lesions such as OSMF. However, in case of malignant oral lesions such as OSCC, 32.4% HPV 16 E6 positive by PCR and 31.4% by the HC-II assay. In case of OSMF, the two test gave concordant result for 42 positive samples and 154 negative samples, with an overall level of agreement of 85.4% . The sensitivity and specificity of the test were 73.7% and 92.05% (p < 0.00). In case of OSCC, the two test gave concordant result for 61 positive samples and 152 negative samples, with an overall level of agreement of 88.3%  and the sensitivity and specificity of the test were 87.14% and 92.76% (p < 0.00).This study concluded that slight difference was found between the positivity rate of HR-HPV infection detected by the HC-II and PCR assay in OSMF and OSCC cases and the HC II assay seemed to have better sensitivity in case of OSCC. Oral malignancy is a major global health problem and it constitutes the sixth most common malignancy. More than 90% of these malignancies representing a squamous cell carcinoma (SCC), which are often preceded by pre-existing oral lesions termed as potentially malignant disorders of the oral mucosa such as oral sub mucous fibrosis (OSMF) . OSMF ocMehrotra et al reported that potentially malignant and malignant disorders of the oral mucosa were widespread in the patients visiting in the Medical College and SRN hospital, Allahabad and suggested that OSMF constituted the highest number of the patients in the potentially malignant group while in case of malignant group, OSCC was most prevalent in this region . The hab6 D. Molecular evidences also provide support to the role of high risk HPV, particularly HPV-16, in the pathogenesis of OSCC of the head and neck [Human papillomavirus is about 55 nm in diameter. It has a single circular double stranded DNA molecule and belongs to the family papillomaviridae. Its genome is made up of 7,200-8,000 base pairs with a molecular weight of 5.2 × 10HPV detection in potentially malignant and malignant oral squamous cell carcinoma showed many discrepancies. Several studies reported the presence of HPV-DNA within these lesions with variable frequency. HPV16 and 18 genotypes were the most frequently found viruses in these lesions. Bouda et al suggested that high risk HPV E6/E7 transcripts and viral integration have also been detected in head and neck squamous cell carcinoma (HNSCC). Transcriptionally active HR-HPVs, particularly HPV-16 are found in a subset of HNSCC. HPV16-associated carcinogenesis is mediated by expression of the viral E6 and E7 oncoproteins, which cause deregulation of the cell cycle by inactivating p53 and pRb respectively . IntegraRecentaly, a second-generation assay with improved diagnostic sensitivity has been developed known as hybrid capture II test (HC-II) and approved by the US food and drug administration (FDA). The performance characteristics of HC-II assay and the PCR for detection of HPV DNA have been compared in cervical lesions and results shown variation. -19. ManyFour hundred and thirty patients of the OSMF and OSCC cases were taken from the Department of Pathology & Department of Otorhinolaryngology, Moti Lal Nehru Medical College, Allahabad, India from Sept 2007-March 2010, in a random manner after obtaining consent from the institutional ethical committee. Of which 208 cases were OSMF and 222 cases were OSCC. Detailed demographic information of each patient, including the patient's age, sex, addiction habits was obtained. Emphasis was given on their addiction habits. Usually those patients were considered who had no previous history of treatment for HPV infection. Figure Detailed clinical examination of each patient was done to assess the site, size and type of lesions. For confirmation of the clinical diagnosis, histopathological examination was carried out. Patients with OSMF were included in this study and lesions with an abnormal epithelial surface like erythroplakia, leukoplakia and submucosal lesions including hemangiomas, mucoceles, papilloma, aphthous ulcers, melanoplakia and fibromas were excluded due to less number. A punch biopsy was performed as per standard protocol and the tissue was processed by paraffin embedding 2-3 micrometer thick sections were cut and stained by haematoxylin and eosin (H and E). Biopsies, which were inadequate on histopathology, were excluded from the study.Histopathological examination was done and results were recorded according to the traditional grading of OSMF by Pindborg and Sirsat . All speSamples were collected from the suspicious lesions of the oral cavity by a soft brush provided in the kit. Gentle rolling strokes were made over the affected area as per the manufacturer's instructions and samples were collected in the sample collection tubes, stored at 4°C till the HC-II test was performed and also biopsies of the same site were taken in 1 × phosphate buffer saline (PBS) solution for the isolation of HPV-DNA.® (manufacturer), a FDA approved test for clinical diagnostics was utilized. Detection of HPV DNA was carried out in the presence of a probe which consist the 13 high risk HPV (HR-HPV) types . This technique is a nucleic acid hybridization assay with signal amplification that utilizes micro-plate chemiluminiscent detection.Second-generation commercial HR-HPV test, the HC II assay *Digene®). The intensity of the light was proportional to the amount of target DNA in the sample.First, double stranded DNA was denatured by using a strong alkaline denaturation solution and converted into single stranded DNA (ssDNA). This ssDNA was then hybridized in-solution to a cocktail of specific 13 HR- HPV RNA probes. The resultant DNA-RNA hybrids are captured onto the surface of a microwell plate coated with specific antibodies for DNA-RNA hybrids. The immobilized hybrids are then reacted with alkaline phosphatase conjugated antibody and detected by cleavage of the chemiluminescent substrate. The emitted light was measured as relative light unit (RLU) in a luminometer  of the sample/mean of RLU of three positive controls (PC) were taken as an estimate of approximate viral load which relates to the index of the intensity of HPV infection. This ratio of any specimen represents empirically a relative measure of the viral load in it. Cut off value (RLU of specimen/mean RLU of PC) was 1.0 pg/ml of sample.Cut-off ratio of 0 to 0.99 is negative for HR HPV; Cut-off ratio greater than 1.0 positive for HR HPV. The cut off ratio at 1.0 correspond to viral DNA load of 5,000 copies/ml of 1pg/ml at a threshold of finding a clinical disease or prognosis of an OSMF and OSCC lesions.High molecular weight genomic DNA was extracted from oral biopsies of patients by using the Qiagen QIAamp DNA tissue Kit (Qiagen Inc. USA) The extracted genomic DNA was quantified and checked for purity spectrophotometrically  Ethidium bromide (EtBr) stained 0.8% agrose gel electrophoresis was used to confirm presence of DNA in samples.PCR amplification was performed using the consensus degenerate primers (L1 gene) [MY09: 5'-GCM CAG GGW CAT AAY AAT GG-3', MY11:5'-CGT CCM ARR GGA WAC TGA TC-3' where M = A/C; W = A/T; Y = C/T; R = A/G) with an amplicon size 450 bp, as described earlier . Further2, 100 μM of each dNTPs  (Fermentas Inc.USA), 10 pmoles of oligonucleotide primers and 0.5U Taq DNA polymerase . The thermal cycler  used, where an initial denaturation at 94°C for 5 min followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min, which was extended for 5 min at the final cycle.PCR was performed in a 25 μL reaction mixture containing 50-100 ng DNA, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgClhttp://www.quantitativeskills.com/sisa/index.htm.Level of agreement was calculated by both absolute agreement and Cohen's kappa statistic (k), a measure of the agreement between two methods in excess of that due to chance . ProportFour hundred thirty cases (average age 58.0 ± 12.6 years) were incorporated in this study, of which 208 patients suffered from potentially malignant disorder of the oral cavity such as OSMF  and 222 OSCC patients . On the basis of demographic distribution of the patients with 208 cases of the OSMF, of which 105 (50.48%) and 103 (49.52%) cases were below and above the age of 45 years respectively while in 222 OSCC cases, 89 (40.1%) and 133 (59.9%) cases were below and above the age of 45 years respectively. In case of OSMF, 156 (75%), 163 (78.36%) and 165 (79.32%) subjects were addicted with tobacco chewing, cigarette smoking and alcohol drinking respectively while 52 (25%), 45 (21.63%) and 43 (20.67%) subjects were never taken any types of addiction. In case of OSCC, 176 (79.27%), 184 (82.88%) and 172 (77.47%) subjects were addicted with tobacco chewing, cigarette smoking and alcohol drinking respectively while 46 (20.72%), 38 (17.11%) and 50 (22.52%) subjects were never taken any types of addiction [Figure Total 208 cases of OSMF, 57 27.4%) were HR-HPV DNA positivite and 151 (72.59%) cases were negative while in 222 cases of OSCC, 70 (31.53%) were positive and 152 (68.47%) subjects were negative by HC-II assay. These sample were again tested by the type specific HPV 16 E6 with the help of PCR. The positivity rate of HPV 16 E6 DNA was 54 (25.96%) in OSMF and 72 (32.43%) in OSCC cases while 154 (74.03%) and 150 (67.56%) cases were negative respectively [Figure % were HRTable On the basis of addiction habits, in OSMF cases, 42 were positive by the HR-HC-II assay as well as type specific PCR. Of which, 38/42 (90.5%) tobacco chewers, 35/42 (83.3%) cigarette smokers, 40/42 (95.2%) alcohol drinkers and 04/42 (9.5%) non chewers, 07/42 (9.5%) non smokers and 02/42 (4.7%) non drinkers were HPV positive. While 139 were negative by both test. Of which, 96/139 (69.1%) tobacco chewers, 111/139 (79.8%) cigarette smokers, 105/139 (75.5%) alcohol drinkers and 43 (30.9%) non chewers, 28/139 (20.1%) non smokers and 34/139 (24.5%) non drinkers were negative.In case of OSCC, 61 were positive by the HR-HC-II assay as well as type specific PCR. Of which, 52/61 (85.2%) tobacco chewers, 51/61 (83.6%) cigarette smokers, 40/61 (65.6%) alcohol drinkers and 09/61 (14.7%) non chewers, 10/61 (16.4%) non smokers, 21 (34.4%) non drinkers were positive. While 141 cases were negative by the both test. Of which, 109/141 (77.3%) tobacco chewers, 121/141 (85.8%) cigarette smokers, 120/141 (85.1%) alcohol drinkers and 32/141 (22.7%) non chewers, 20/141 (14.2%) non smokers, 21/141 (14.9%) non drinkers were negative.On the basis of location of the lesions in potentially malignant OSMF of the oral cavity, out of 42 positive cases, 18 (42.8%) oral cavity, 12 (28.6%) tongue, 09 (21.4%) lip, 03 (70.1%) hard & soft palate were positive while out of 139 negative cases, 68 (48.9%) oral cavity, 13 (9.4%) tongue, 28 (20.1%) lip, 30(21.6%) hard & soft palate were HPV negative by the both the test HR-HC-II assay as well as type specific PCR. In case of OSCC, out of 61 positive cases, 32 (52.5%) oral cavity, 12 (19.7%) tongue, 08 (13.1%) lip, 09 (14.7%) hard & soft palate were positive while out of 141 negative cases, 72 (51.1%) oral cavity, 21 (14.9%) tongue, 20 (14.2%) lip, 29 (19.8%) hard & soft palate were HPV negative by the both the test HR-HC-II assay as well as type specific PCR [Table Clinico-pathological diagnosis of the potentially malignant disorders (OSMF) of oral cavity, out of 42 positive cases, 5 (11.9%) grade I, 09 (21.42%) grade II, 11 (26.12%) grade III , 17 (40.47%) grade IV were HPV positive while out of 139 negative cases, 18 (12.94%) grade I, 29 (18.7%) grade II, 37 (26.61%) grade III , 55 (39.56%) grade IV were HPV negative by the both the test [Table In case of OSMF, the two tests gave concordant results for 42 HPV positive samples and 154 HPV negative samples, with an overall level of agreement of 85.4% (Cohen's kappa = 66.83). Among the samples with discrepant results, 15 were positive by the HC-II assay but negative by PCR, whereas 12 samples were PCR positive but negative by HC-II assay. The sensitivity and specificity of the test were 73.7% and 92.05% (p < 0.00) respectively [Table In case of OSCC, the two tests gave concordant results for 61 HPV positive samples and 152 HPV negative samples, with an overall level of agreement of 88.3% . Among the samples with discrepant results, 11 were positive by the HC-II assay but negative by PCR, whereas 9 samples were PCR positive but negative by HC-II assay. The sensitivity and specificity of the test were 87.14% and 92.76% (p < 0.00) respectively [Table In this study we compare the data obtained by two techniques, (HC-II & PCR assay) detecting HR HPV DNA in potentially malignant (OSMF) and malignant (OSCC) disorders of the oral cavity. Use of the same samples for both DNA tests was done to avoid bias. We examined the punch biopsies sample histopathologically and to find out the final diagnosis of the oral lesions. This approach acceptable to find out the positivity rate between HPV detection and oral lesions. We did not detect any type of low risk humanpapilloma virus in these lesions. The HC-II assay used for the detection of the presence of 13 types high risk HPV types  and 18 low risk  in cervical lesions ,24. As wThis study included 430 cases (average age 58.0 ± 12.6 years), of which 208 patients suffered from potentially malignant lesions such as OSMF  and 222 OSCC patients . The overall occurrence rate of HPV 16 E6 DNA positivity was nearly 26% by PCR and 27.4% by the HC-II test in case of OSMF. While in case of OSCC the HPV DNA positivity were 32.4% by the PCR and 31.5% by the HC-II test. Therefore, the study concluded that slight difference was found between the positivity rate of HR HPV infection of the both detection methods in OSMF as well as in OSCC cases. However, Kujan et al from United Kingdom, reported that all oral samples were negative for HR-HPV using the HC-II technique [In this study we reported that in case of potentially malignancy oral lesions such as OSMF, the two tests (HC-II & PCR assay) concordant results with an overall level of agreement of 85.4% . The sensitivity and specificity of the test were 73.7% and 92.5% (p < 0.00) respectively. While in case of OSCC both the tests gave concordant result with an overall level of agreement of 88.3% . The sensitivity and specificity of the test were 87.14% and 92.76% (p < 0.00) respectively. Therefore, we concluded that HC-II assay seemed to have better sensitivity in case of OSCC. Similar result was reported by the Kalama et al in case of cervical lesions . To the This study concluded that slight difference was found between the positivity rate of HR-HPV infection detected by the HC-II and PCR assay in OSMF and OSCC cases and the HC II assay seemed to have better sensitivity in case of OSCC.(HPV): Human papillomavirus; (PCR): Polymerase chain reaction; (OSMF): Oral submucous fibrosis; (HNSCC): head and neck squamous cell carcinoma; (HC-II): Hybrid Capture; (OSCC): Oral squamous cell carcinoma.The authors declare that they have no competing interests.AKC and SP carried out the experimental work, analysis and drafted the manuscript. Mamta Singh conceived of the study, participated in its design and coordination as well as helped to draft the manuscript. MS, ACB and RM participated in coordination of the study. All authors read and approved the final manuscript.


P=0.004). Paired comparisons of high-risk human papilloma virus prevalence rates of the different trimesters with the postpartum rate showed (McNemar test) decreased rates: first trimester: 18% (P=0.02), second trimester: 13% (P=0.02) and third trimester: 23% (P<0.005). Such a phenomenon was not found in non-pregnant women. Pregnant women showed a trend for increased high-risk human papilloma virus clearance during the third trimester and postpartum compared to non-pregnant women (hazard ratios 3.3 (0.8–13.7) and 4.6 (1.6–12.8), respectively). These results suggest a lowered immune-response against human papilloma virus during the first two trimesters of pregnancy with a catch-up postpartum.We followed 353 women referred with abnormal cervical cytology in a non-intervention cohort study. In 91 pregnant women we compared high-risk human papilloma virus rates in the subsequent trimesters and postpartum in comparison to 262 non-pregnant women. High-risk human papilloma virus clearance was compared with 179 high-risk human papilloma virus positive non-pregnant women. Our main questions were: (1) do high-risk human papilloma virus rates change during pregnancy?; and (2) is there any difference between high-risk human papilloma virus clearance in pregnant and non-pregnant women? Women were monitored 3–4 monthly by cytology, colposcopy, and high-risk human papilloma virus testing. The median follow-up time was 33 months (range 3–74). Non-pregnant women showed prevalence rates of high-risk human papilloma virus of 64, 57, 53, and 50%, respectively, in four subsequent 3-months periods since the start of the study. These high-risk human papilloma virus rates were higher than in the three trimesters of pregnancy, and during the first 3 months postpartum, i.e. 50, 44, 45, and 31%, respectively. Postpartum only, this difference was statistically significant  87, 75–80. doi:10.1038/sj.bjc.6600367www.bjcancer.comCancer Research UK© 2002  Several epidemiological and biological studies have established the important role of infection with high-risk types of human papilloma virus (HPV) for development of cervical cancer and its precursor lesions. In women with or without abnormal cervical smears a positive high-risk HPV test result indicates an increased risk for development of high grade cervical lesions  do high-risk HPV rates change during pregnancy?; and (2) is there any difference between HPV clearance in pregnant and non-pregnant women?From June 1990 to December 1992, 353 women referred to the colposcopy clinic of the University Hospital Vrije Universiteit, Amsterdam, were followed in a non-intervention study. The characteristics of this cohort have been described previously . BrieflyNinety-one women were pregnant during follow-up. The first recorded full term pregnancy in each woman was used to computer-high-risk HPV prevalence rates and analyse high-risk HPV clearance. For the analysis of high-risk HPV clearance we excluded four pregnancies ending in abortion and four women who acquired HPV during follow-up prior to their pregnancy. One hundred and seventy-nine non-pregnant women with a positive high-risk HPV test at baseline , defined as a colposcopic impression of CIN 3 covering three or more cervical quadrants or a cervical smear suspect for microinvasive carcinoma, or at the end of the study in December 1996. At the last visit, colposcopically directed biopsy samples were taken for histological verification of suspected lesions (=end histology). In women with normal colposcopy, random samples were taken. If necessary, women were treated according to standard protocols.Women were monitored every 3 to 4 months by cytology, colposcopy and HPV testing . CliniciThe study protocol was approved by the ethics review board of the hospital, and all women voluntarily signed informed consent before enrolment.Testing for HPV was done by EIA PCR, which used HPV general-primer-mediated PCR with the general primers GP 5+/6+ to detect a broad spectrum of mucosotropic HPV types .Women were considered to be clear of infection when no high-risk HPV type from the previous visit was detected at the next visit .We compared pregnant and non-pregnant women in 3 months periods. In non-pregnant women these periods were designated from the start of the study. Age distribution at baseline and at time of pregnancy was compared by the Mann–Whitney test. The Fisher-exact test was used to calculate differences in high-risk HPV rates and abnormal cervical smears in pregnant and non-pregnant women. We also analysed the prevalence of low-risk HPV types and multiple HPV infections at different trimesters but the numbers were too small to draw any conclusions (data not shown).P<0.05 was considered significant. We did not correct for multiple testing.Not all women were seen in each trimester of pregnancy. Therefore comparisons for cytology and HPV samples between first, second, and third trimester, or postpartum were made by the McNemar test, only for those women with specimens available at both times. Being interested in the effect of pregnancy, only the first postpartum visit within 6 months after delivery was included in the calculation (range 5–25 weeks after pregnancy). The clearance of high-risk HPV in pregnant and non-pregnant women and between pregnancy groups (pregnant at the start of the study versus conceived during the study) were studied by Kaplan–Meier curves during a period of 12 months. For contrasts we used the log-rank test. The time-point of HPV clearance was taken at the mid-point between positive and negative test results for high-risk HPV . We inclP=0.23). At baseline 68% (179 out of 262) of non-pregnant women had a positive high-risk HPV test result compared to 63% (57 out of 91) of pregnant women (P=0.32). At baseline no significant difference in number of abnormal cervical smears was observed in high-risk HPV positive pregnant and non-pregnant women; 133 out of 179 (74%) non-pregnant women still had an abnormal cervical smear compared to 15 out of 25 (60%) pregnant women (P=0.13).The median age in the pregnant and non-pregnant group of women was 30 years (range 20–44 years) and 32 years (range 19–55 years), respectively (P>0.1). In non-pregnant women no significant difference in abnormal cervical smears among the different time periods was found (data not shown).Among pregnant women with cervical smears obtained in the first trimester of pregnancy 39% (21 out of 54 available smears) had abnormal cervical cytology (mild dyskaryosis or worse). Abnormal cytology in the second, and third trimester and postpartum was present in 42% (25 out of 60), 33% (13 out of 39), and 34% (27 out of 79 available smears), respectively. Comparison of paired cervical smear samples among the different trimesters with the postpartum visit did not show any significant difference  were 64, 57, 53 and 50%, respectively (Table 1Among pregnant women whose HPV samples were obtained in the first trimester of pregnancy 50% had positive high-risk HPV DNA samples . The higP=0.02). Comparing second trimester results with postpartum results (paired data available from 53 women) showed a significant decline from 45% (24 out of 53) to 32% (17 out of 53) high-risk HPV positive tests (P=0.02). A significant difference in high-risk HPV rates was also found between the third trimester and postpartum (paired data available from 40 women) from 48% (19 out of 40) to 25% (10 out of 40) (P=0.004).Comparison of paired samples of high-risk HPV among the three trimester groups in the pregnant women group did not show any statistical difference (data not shown). In 45 women with paired samples in the first trimester and postpartum a significant decline in high-risk HPV positive tests of 51% (23 out of 45) to 33% (15 out of 45) was found (Twenty-five high-risk HPV positive women (66%) were pregnant at baseline ; group BAmong 38 pregnant women with a high-risk HPV positive test at the first visit during pregnancy the cumulative 12-months incidence of high-risk HPV clearance was 42% . One hundred and seventy-nine non-pregnant women with a positive high-risk HPV test at baseline had a cumulative 12-months incidence of high-risk HPV clearance of 31% . Both clearance curves according to Kaplan–Meier analysis cross at 9 months thus excluding long term effects on prevalence rates. The crude trimester dependent hazard ratio's, and the trimester dependent hazard ratio adjusted for pregnancy groups for HR–HPV clearance (Table 2Our results show that during pregnancy in a group of women initially referred for colposcopy with an abnormal cervical smear the prevalence of high-risk HPV is higher than in the postpartum period. This effect is also demonstrated by the trimester dependent HPV clearance rates; during the postpartum period women were more at risk to clear high-risk HPV than non-pregnant women.The high-risk HPV prevalence rates in pregnant women varied between 45 and 50% during the trimesters, and 31% in the postpartum period. Similar figures were found by others. Using a variety of HPV detection techniques, several other studies also have found a significantly higher prevalence of high-risk HPV during pregnancy in comparison with the postpartum period (From recent studies we know that HPV clearance is associated with and precedes on average 3 months regression of cervical lesions (In conclusion, although at long term the course of a high-risk HPV infection is not affected we found evidence that pregnancy influences high-risk HPV clearance. Trends for a decreased high-risk HPV clearance in the first two trimesters in pregnant women in contrast with non-pregnant women are compensated by increased clearance demonstrable postpartum but already starting in the third trimester.


Vulvar squamous cell carcinoma (SCC) originates the following two pathways, related to differentiated (d) vulvar intraepithelial neoplasia (VIN) or to human papillomavirus (HPV)-related usual (u) VIN. Multicentric HPV infections  are common. We hypothesise that patients with a uVIN-related vulvar SCC more often have cervical high-grade squamous intraepithelial lesions (HSILs) compared with women with dVIN-related vulvar SCC.n=164) or uVIN related (n=37). Data with regard to the smear history and cervical histology were retrieved from PALGA, the nationwide Netherlands database of histo- and cytopathology. For HSIL cervical smears of which histology was taken, HPV DNA analysis on both the vulvar and cervical specimens was performed.All vulvar SCCs (201) were classified to be dVIN- . A total of 10 of the 13 HSILs were histologically assessed and identical HPV types were found in the vulva and cervix.At least one smear was available in 145 (72%) of the 201 patients. Patients with a uVIN-related vulvar SCC more often had an HSIL compared with patients with a dVIN-related SCC (35 These data emphasise the necessity to differentiate between dVIN- and uVIN-related vulvar tumours and to examine the entire lower female ano-genital tract once an uVIN-related lesion is found. A better method of subdividing the two types of vulvar SCC is on the basis of the histology of the adjacent VIN lesion (Vulvar squamous cell carcinoma (SCC) accounts for 3–5% of all gynaecological malignancies and originates following two separate pathways. One type of vulvar SCC, accounting for ∼80%, occurs mainly in older patients and is mostly of the keratinising type, associated with lichen sclerosus (LS) and/or differentiated vulvar intraepithelial neoplasia (dVIN) as premalignant lesions. The other type, accounting for ∼20%, is human papillomavirus (HPV) related, occurs in younger patients, and has usual VIN (uVIN) as premalignancy malignancies compared with women with dVIN/LS-related vulvar SCC. Moreover, we investigate which hrHPV types play a role in either uVIN-related vulvar SCCs and cervical (pre)malignancies.Between 1988 and 2007, 209 consecutive patients were treated for their primary invasive or recurrent SCC of the vulva at the Radboud University Nijmegen Medical Centre, The Netherlands. Data were stored in a database. All histopathological slides were revised to classify the adjacent lesion as uVIN, LS and/or dVIN, to categorise the vulvar cancers into uVIN- or dVIN related  with the EZ1 robot  according to the standard procedures and used for PCR analysis. A negative water control was included with each batch of 10 samples. Broad-spectrum HPV DNA amplification was performed using a short PCR fragment assay . This assay amplifies a 65-bp fragment of the L1 open reading frame, and allows detection of a broad range of high-risk (hr), low-risk (lr) and possible hrHPV genotypes  or less, and at least an HSIL. Fisher's exact test was used to compare hrHPV presence in uVIN and dVIN lesions adjacent to vulvar SCCs and to compare the different groups of vulvar SCC patients with respect to cervical abnormalities. P<0.001).The data of a total number of 201 patients were analysed. The median age of the patients at diagnosis was 71 years (range 30–92). The median age at diagnosis of the dVIN/LS-related pathway group  and the uVIN-related pathway group  differed significantly  tumours that were considered to follow the uVIN-related pathway, and 164 (82%) that were considered to follow the dVIN/LS-related pathway. Of the 164 dVIN/LS-related tumours, 54 (33%) tissues showed solitary dVIN, 22 (13%) tissues showed solitary LS and 88 (54%) tissues showed dVIN in combination with LS, as surrounding lesions. Of 168 tumours (32 uVIN and 136 dVIN/LS), both histology of the adjacent VIN lesion and HPV presence and typing could be performed. Of the uVIN-related tumours, 27 of the 32 (84.4%) were found hrHPV positive and 13 out of 136 (9.6%) of the dVIN/LS-related tumours were found hrHPV positive . For then=13) had a smear indicating an HSIL. Patients with uVIN-related tumours had significantly more cervical smears indicating an HSIL, compared with patients with LS/dVIN-related tumours, 11 of 31 (35%) and 2 of 114 (2%) (P<0.001), respectively , without any clinical indication of cervical disease; 87% of these women were older than 55 years of age from the start of the national screening program for cervical cancer in 1988, as a result of which they exceeded the age limit to be invited. Of the 145 patients from whom at least one smear was available, the most severe lesion was taken: of these patients, 9.0% ; in four cases the CIN lesion was diagnosed simultaneously with the vulvar SCC ; and in one case the CIN lesion was diagnosed 6 years after the vulvar SCC (case 5).β-globin after DNA extraction. In all tissues of the other eight patients, the same HPV types were found in both the vulvar and cervical specimens . For uVIN-related SCCs, this is a more than 10-fold incidence compared with the overall incidence of a HSIL in The Netherlands . In 19% (n=7) of the total 37 patients with a uVIN-related vulvar SCC, a histologically proven cervical HSIL or SCC was found during a follow up of 20 years. Moreover, all of these uVIN-related vulvar SCCs and cervical lesions in the same patient showed the identical hrHPV type. These identical hrHPV types were seen in vulvar SCCs and cervical lesions even after an interval of more than 10 years. This may be one long-standing infection or a new infection with the exact same type of virus. Particularly as less common types were found as well, a long-standing infection is most likely. Furthermore, if a patient's immune system is not able to clear a hrHPV infection, that patient is thought to be more prone to develop multicentric (pre)malignancies. In most cases, the cervical lesion preceded the vulvar SCC, although vulvar SCCs preceding cervical lesions were seen as well. The difficulty is whether the date of diagnosis accurately reflects the origin of the lesions. It may well be possible that these lesions had initially been asymptomatic and present for a longer period before diagnosis. Our data suggest that the identical long-standing hrHPV infection is causing these multicentric (pre)malignancies. This emphasises the concept that a patient with a hrHPV-related ano-genital (pre)malignancy is at a higher risk to develop another hrHPV-related (pre)malignancy. We previously reported that 41% of uVIN patients had a past, simultaneous or future HPV-induced cervical, vaginal or anal lesion (Our study on a large group of patients with vulvar SCCs (201 cases), with regard to cervical (pre)malignancies, showed that 9% of all patients with available cytology of the cervix had a smear suggestive for a HSIL. Divided between the two pathways, 35% of the patients with uVIN-related vulvar SCCs had a smear suggestive for a HSIL, compared with only 2% of patients whose vulvar SCC was dVIN related (P<0.001). If we compare the median age for cervical SCC, which is caused by hrHPV in nearly 100% of the cases, with the median age for uVIN/hrHPV-related vulvar SCC, we find a comparable age, 51 years and 54 years, respectively  is slightly higher than the percentage of tumours with adjacent uVIN (18.4%). These findings confirm that not all hrHPV infections finally lead to vulvar SCC. As hrHPV often resolves spontaneously, most likely these five percent additional infections were transient infections, yet to be cleared.Of the 201 patients, in 56 cases (28%) no cervical smear information was available: this may be because of the limitation in coverage of the PALGA database  or because of the fact that these women never had a cervical smear taken. Worth mentioning is that 87% of these women exceeded the age limit to be invited for the national screening program for cervical cancer. Still, at diagnosis, in 105 of the 201 (52%) cases no cervical smear was taken around the diagnosis of the vulvar SCC. Approximately one-fifth of the vulvar SCCs is uVIN related, and simultaneous cervical and vulvar infections have previously been described malignancies with identical hrHPV types in both cervical and vulvar (pre)malignancies. This points to the multicentric development of (pre)malignancies and emphasises the necessity to differentiate between dVIN- and uVIN-related vulvar tumours. The examination of the entire lower female ano-genital tract in case of a hrHPV-related vulvar lesion should therefore be obligatory.


The human papillomavirus has emerged over the past decade as the leading candidate to be the sexually transmitted aetiological factor in cervical cancer. Although it appears that papillomavirus types 16 and 18 are associated with a higher risk of advanced cervical neoplasia, most of the evidence comes from studies which do not satisfy basic epidemiological requirements, and are therefore difficult to interpret. The most significant problems are the small sample size, potentially biased selection of study subjects, the difficulties in cytologically distinguishing precancerous lesions from papilloma infection of the cervix, the unknown specificity and sensitivity of the various hybridisation methods for determining papillomavirus infection status, and the statistical analyses and presentation of results. On the basis of the existing studies, one is forced to conclude that, while experimental data suggest an oncogenic potential for HPV, the epidemiological evidence implicating it as a cause of cervical neoplasia is still rather limited.


Visual inspection of the uterine cervix by paramedical personnel has been proposed for the early detection of cervical cancer, as an alternative to routine cytology screening in developing countries. We evaluated the performance of this procedure in detecting precursor lesions and cancer in a study involving 2843 married women in Kerala, India. Two thresholds were used to define a positive test. In the lower one, any abnormality was considered as positive. The cut-off point for the high threshold was one or more of the high-risk findings: bleeding on touch, suspicious growth/ulcer and hard, irregular, oedematous cervix. A Pap smear was performed on all subjects, and a biopsy was done for those with moderate dysplasia and above. A combination of cytology and histology findings was used as the 'gold standard'. Using the low threshold, 1279 (45%) women were positive on visual inspection, and with the higher threshold 179 (6.3%) were positive. There were six moderate dysplasias, nine severe dysplasias, ten carcinomas in situ and 13 invasive carcinomas. With the lower threshold, sensitivity and specificity to detect moderate dysplasia and above were 65.8% and 55.3% respectively; the values for severe dysplasia and above were 71.9% and 55.3% respectively and for invasive cancer were 92.3% and 55.2% respectively. With the higher threshold, the sensitivity decreased considerably  and the specificity increased to approximately 94%. At a lower threshold, the sensitivity was not satisfactory, and the test was highly non-specific; at a higher threshold sensitivity was even lower. Thus, the test characteristics of visual inspection are not very promising either as a preselection procedure for cytology or as a low-technology measure for cervical cancer screening in developing countries.


Human papillomavirus (HPV) infection causes cervical cancer and premalignant dysplasia. Type-specific HPV prevalence data provide a basis for assessing the impact of HPV vaccination programmes on cervical cytology. We report high-risk HPV (HR-HPV) type-specific prevalence data in relation to cervical cytology for 24 510 women  recruited into the ARTISTIC trial, which is being conducted within the routine NHS Cervical Screening Programme in Greater Manchester. The most common HR-HPV types were HPV16, 18, 31, 51 and 52, which accounted for 60% of all HR-HPV types detected. There was a marked decline in the prevalence of HR-HPV infection with age, but the proportion due to each HPV type did not vary greatly with age. Multiple infections were common below the age of 30 years but less so between age 30 and 64 years. Catch-up vaccination of this sexually active cohort would be expected to reduce the number of women with moderate or worse cytology by 45%, but the number with borderline or mild cytology would fall by only 7%, giving an overall reduction of 12% in the number of women with abnormal cytology and 27% in the number with any HR-HPV infection. In the absence of broader cross-protection, the large majority of low-grade and many high-grade abnormalities may still occur in sexually active vaccinated women. Human papillomavirus (HPV) infection has a central role in the aetiology of cervical cancer  is being conducted within the routine NHS Cervical Screening Programme in Greater Manchester to evaluate the effectiveness of HPV testing in primary cervical screening. The randomised comparison will determine whether combining HPV testing with liquid-based cytology offers added sensitivity over cytology alone and, if so, whether this would be cost-effective. Our first report described the baseline prevalence of HPV16, 18 and all other HR-HPV types in relation to age, cytology and histology  test according to the manufacturer's instructions, as described previously , 50 mM KCl, 4 mM MgCl2, 500 nM each of biotinylated HPV primers PGMY09 and PGMY11, 25 nM each of biotinylated human beta-globin primers PC04 and GH20, 200 μM each of nucleotides dATP, dCTP and dGTP, 600 μM of nucleotide dUTP and 7.5 U of Amplitaq Gold. Following heating at 95°C to activate the Amplitaq Gold polymerase enzyme and render the native DNA single stranded, nucleic acid was amplified by 40 cycles of 95°C/30 s to allow strand denaturation, 55°C/1 min for primer annealing and 72°C/1 min for primer extension. This cycling program was followed by an elongated primer extension period of 56 min at 72°C. Amplified product was stored at 4°C. Following denaturation, amplified product was hybridised to oligonucleotide-coated genotyping strips before colour development and interpretation using the template provided.Genotyping of all hc2-positive samples was carried out using the prototype Roche Line Blot Assay (LBA). Polymerase chain reaction (PCR) amplification and product detection for 37 anogenital types were essentially performed as described previously (n=102) of these samples was examined using GP5+/6+ PCR  were HPV-positive by hc2, but 40 (1.0%) of these either gave negative results for beta-globin gene amplification and were reported as inhibitory or were of insufficient volume for further testing. These 40 were excluded, and all the results were based on the remaining 24 470 women. Crossreactivity with low-risk or HR types not included in the hc2 probe mix was observed in 417 (11.1%) hc2-positive samples. A broad range of HPV type crossreactivity occurred. This was particularly noticeable for HPV types 53, 66 and 70, which were frequently detected. A further 772 (20.5%) hc2-positive samples did not hybridise to any of the LBA probes. These 1189 samples were classified as hc2 positive but HR-HPV negative. The remaining 2584 hc2-positive samples (68.5%) were positive by LBA for one or more of the 13 HR types included in the hc2 HR probe mix. Of those hc2-positive samples giving a low RLU/Co value between 1 and 3, 26.7% contained an hc2 HR type; 16.2% crossreacted with other types and 57.1% failed to type. The corresponding figures of those hc2-positive samples giving a high RLU/Co value of ⩾100 were 91.9, 4.8 and 3.3%. In total, 50% of hc2-positive LBA-negative samples had an RLU/Co value between 1 and 2.11. On testing a subset of 102 hc2-positive LBA-negative samples by GP5+/6+ PCR, 39.2% were found to be HPV positive. Multiple HR-HPV types were detected in 680 (18.0% of hc2-positive samples) and infection with a single HR-HPV type was detected in 1904 (50.5%) samples.Prevalence rates for each HR-HPV type are shown in The HPV prevalence rates by age group and cytology are shown in P=0.06). The corresponding proportions were 6.3 and 3.7% for HPV33 (P=0.001), 2.4 and 4.1% for HPV35 (P=0.003) and 4.5 and 6.7% for HPV45 (P=0.005). No other type showed significant variation with age. The proportion of women with a single HR-HPV type who had moderate or worse cytology  and 83 out of 225 (37%), respectively, were identified in high- and low-grade cytological abnormalities, which were HR-HPV positive but types 16/18 negative.The ARTISTIC trial cohort (see P<0.001).The five most prevalent types  together account for 60% of the 3512 HR-HPV infections detected ; HPV16 aThe failure of the LBA to confirm that 31.5% of the hc2-positive samples contain hc2 HR-HPV types is a cause of concern, especially if this assay was to be used as a frontline screening test. This is due, in part, to the demonstrated crossreaction of the hc2 test with other putative HR as well as low-risk types. However, the fact that 20.5% failed to yield any detectable HPV type is more problematic. As the whole of the PGMY-amplified product generated during LBA testing was denatured, further analysis by gel electrophoresis was not possible. Further work using another well-documented primer system (GP5+/6+ PCR) followed by gel electrophoresis suggested that 39.2% of these hc2-positive LBA-negative samples might in fact contain HPV. Confirmation of this observation would require further analysis on a larger number of samples. The 13% originally selected merely provides an insight into the true HPV status of these samples. The use of the improved, commercially available linear array assay  women aged below 30 years with low-grade (borderline or mild) cytology, and in only 111 out of 1720 (6.5%) aged 30–64 years. In the absence of broader cross-protection, the large majority of low-grade and many high-grade abnormalities would still occur in vaccinated women. This is consistent with the data emerging from clinical trials of prophylactic vaccine, which show much greater efficacy in preventing CIN2+ than for low-grade abnormalities. Potential cross-protection involving HPV31 and 45 has been demonstrated by More detailed age-specific analysis of these data will help to validate models of the possible impact of vaccination on subsequent cervical screening before long-term follow-up of current trial cohorts. The follow-up of ARTISTIC patients to the subsequent routine screening rounds will also provide estimates of type-specific risk over 3–6 years in women with normal baseline cytology.


The aim of this study is to evaluate different options for introducing liquid-based cytology (LBC) and human papillomavirus (HPV) testing into the UK cervical cancer screening programme. These include options that incorporate HPV testing either as a triage for mild and borderline smear abnormalities or as a primary screening test. Outcomes include the predicted impact on resource use, total cost, life years and cost–effectiveness. Extensive sensitivity analysis has been carried out to explore the importance of the uncertainty associated with disease natural history and the impact of screening. Under baseline assumptions, the cost–effectiveness of different options for introducing LBC appears favourable, and these results are consistent under a range of assumptions for its impact on the diagnostic effectiveness of cytology. However, if we assume a higher marginal cost of LBC in comparison to conventional methods, primary smear testing options are predicted to be more cost-effective without LBC. Combined LBC primary smear and HPV testing with a 5-year interval is similar in both cost and effectiveness to the other 3-yearly options of primary smear testing or primary HPV testing alone. However, both primary HPV testing and combined options would give rise to a far greater risk of inappropriate colposcopy throughout a woman's lifetime. Liquid-based technology has been developed for the purpose of improving the preparation of cervical smear samples, and a range of different methods are available which include AutoCytePrep, Easy Prep, Cytoscreen and Thinprep. Each involves the preparation of potentially better and more representative smear samples for cytological examination than with conventional means, with a consequent reduction in the number of inadequate specimens and an increase in sensitivity. In practice, however, the actual impact on the performance of cytology is still a matter of some debate since different studies have reported very different findings. Currently, in the UK, around 10% of all smear samples are classified as inadequate, and the recent Pilot Site evaluation of liquid-based cytology (LBC) in the UK found that the new technology reduced this figure to 2% . SimilarAs human papillomavirus (HPV) testing technology has developed, the inclusion of HPV testing within cervical screening programmes has been suggested, either as a triage for women presenting with borderline and mildly abnormal smears . This maThe purpose of this study is to evaluate different options for introducing LBC within the UK, in terms of the impact on resource use, cost, effectiveness and overall cost–effectiveness. The merits of liquid-based approaches are also compared with other changes that could be made to the screening programme keeping conventional techniques, including the introduction of HPV testing. This is the only study that compares such a range of options simultaneously. The study does not investigate one specific liquid-based method but a range of approaches defined by different characteristics of cost and impact. We have used a mathematical modelling approach based upon methods developed by the authors in previous studies  with HPV testing as a triage for mild and borderline smear results during routine screening. If the HPV test is positive for high-risk types, women are referred for colposcopy, otherwise they have repeat cytology as in (i);option (i) until the age of 30 years after which routine smear testing is replaced by primary HPV testing with cytology as a triage for women found to be positive for high-risk HPV types. If the cytological result is abnormal, women are referred for colposcopy, otherwise they have a follow-up HPV test in 6 months. If this repeat HPV test is negative, women revert to routine screening;option (i) until the age of 30 years, after which primary smear testing is combined with HPV testing. Women with moderate or severe smear abnormalities are referred for colposcopy. Women with mild or borderline smear results, or a negative smear and a positive HPV test undergo repeat combined testing at 6-month intervals. Women revert to routine screening after a combination of a clear smear test and negative HPV result.The screening options evaluated in this study include:Option (i) approximates to current recommended screening policy in the UK. The other options are based upon suggested strategies for including HPV testing as a triage . In line with current UK recommendations, future costs and life years were discounted at 3.5% per annum, along with the consequences of no discounting and of discounting costs and life years at 6% per annum .Incremental cost–effectiveness ratios are defined as the ratio of the difference between cost and outcome of moving from one option to the next in order of increasing cost. In calculating incremental cost–effectiveness ratios, all dominated and extendedly dominated options are excluded. An option is ‘dominated’ if there is another option which is both cheaper and more effective. An option is ‘extended dominated’ if it is not dominated by a single other option, but there is a combination of two options that is both cheaper and more effective (With the baseline assumptions, average lifetime costs and life expectancy from age 15 years are shown in Table 4Other outcomes are shown in The results of one-way sensitivity on the impact of LBC and discounting are shown in Table 5Figure 3The purpose of this study has been to evaluate and compare options for introducing LBC and/or HPV testing into cervical cancer screening programmes. All the screening options have been chosen to reflect those that have been previously proposed or evaluated. However, this is the only study that has evaluated all screening options in combination.Under baseline assumptions, the cost–effectiveness of different options for introducing LBC appears favourable, and these results are consistent under a range of assumptions for its impact on the diagnostic effectiveness of cytology. However, the marginal cost of LBC does affect outcomes. If we assume a higher marginal cost of LBC in comparison to conventional methods, primary smear testing options are predicted to be more cost-effective without LBC. The cost–effectiveness of primary HPV testing with LBC appears less sensitive to changes in the LBC marginal cost, but this is because in our one-way sensitivity analysis, variability in cost is only associated with the cytology laboratory costs and not HPV costs. Combined primary smear and HPV testing with a 5-year interval is similar in both cost and effectiveness to the other 3-yearly options. However, both the primary HPV testing and combined options would give rise to a far greater risk of inappropriate colposcopy throughout a woman's lifetime.Adding LBC to the current programme is predicted to match or improve effectiveness under the full range of assumptions used for sensitivity analysis, and, likewise, if it was further modified by adding HPV testing as a triage for mild or borderline smear abnormalities. Changing to the combined smear and HPV testing option and raising the screening interval to 5 years, could improve effectiveness, similarly if the programme is changed to primary HPV testing. However, under these options, the predicted impact is less robust to the assumptions we have made: their effectiveness being very sensitive to the relative diagnostic accuracy of the HPV test in relation to cytology, HPV prevalence and whether there is any consistent difficulty in detecting HPV infections in some women.Since a modelling approach has been used, there has been scope for analysing a wide range of options. Several different scenarios have been analysed to allow for the uncertainty regarding the model parameters. This uncertainty not only reflects the range of estimates reported in the scientific literature, but also the imprecise knowledge regarding the natural history and age-related prevalence of HPV infections and cervical precancers both now and in the future. Evaluating different scenarios has enabled us to investigate how robust predicted outcomes are to changes in model assumptions, to identify areas of uncertainty that have a large influence on outcome, and identify circumstances under which particular options are less favourable.The costs used in the model are based on best estimates currently available, although no account has been taken of the training and transfer costs associated with moving from the current screening programme to one including LBC and/or HPV testing and neither have transportation costs for HPV samples been included. It is recognised that the prospective resource utilisation data come from a relatively small number of practices in the UK and the cost data for LBC are based largely on assumption.Previous studies have analysed the impact of LBC on cervical screening programmes (The mathematical model is a tool for making the best use of the available information, yet the results cannot be treated as definitive. There are further factors that have not been investigated which could influence the outcomes. Women are assumed to be either always compliant or not, and no account has been taken of how compliance, and hence effectiveness, could be improved by reducing the number of tests a woman is required to attend, for example, by extending the screening interval, combining smear and HPV tests and reducing the number of inadequate smears. The performance of screening tests has also assumed to be the same for all ages, although there is evidence that cytological accuracy declines among older women (The aim of this study was to evaluate screening in the context of the UK, and care would need to be taken before translating its results to other settings. International comparisons of cost–effectiveness are hampered by differences in health systems, resource and cost structures, chosen comparators and recommended methods for evaluating technologies. Guidelines on appropriate discount rates differ between countries; in the United States for example, the current recommendations are 3% for both costs and life years (Since cost and life years saved are not the only outcomes of importance, other measures could come to the fore in the decision making process. In terms of the psychological impact on women, high false positive rates for referral to colposcopy may count against the acceptability of primary HPV testing and combined primary smear and HPV testing. This may also be an unnecessary use of expensive resources. The HART study, however, suggested that this problem could be overcome in primary HPV testing by annual monitoring of women who are HPV positive with borderline or negative cytology (


Human Papillomavirus-32 (HPV-32) has traditionally been associated with focal-epithelial-hyperplasia (FEH). It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR) assay.An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization) using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples .The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard.Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts. Human Papillomavirus (HPV) is the most common sexually transmitted viral infection in the world with greater than 100 genotypes described to date . High riHPV-32 has only been well described in the oral cavity. In a study of 67 high-risk individuals composed of more than 85% HIV-positive individuals HPV-32 was the most prevalent type detected in oral lesions 40%)[0%[4]. ItThe prevalence rate of HPV-32 infection in the oral cavity of HIV-positive and HPV-negative populations without obvious lesions has not been well studied. One study did show a prevalence rate of 1.6% in a cohort of normal, healthy individuals from South Africa  utilizinThe New Orleans and South African studies described above used PCR assays utilizing primers designed to detect genital HPV types (MY09/11 and PGMY primer sets). Although these methods are capable of detecting HPV-32, the sensitivity has not been determined but is expected to be low. With an increase in oral HPV infection and disease in HIV-positive individuals on HAART, and with HPV-32 being prevalent in the oral cavity, improved methods to detect HPV-32 are warranted. This study describes the development of a type-specific PCR-based assay to detect HPV-32 for the application in a natural history study of HPV-32 and oral wart progression.Previous work in our laboratory  has note). The assay was optimized by varying the magnesium concentration and annealing temperature (data not shown). Due to the primers need for a precise magnesium concentration for optimal sensitivity a commercial master mix could not be utilized. To limit variation in PCR reactions and pipeting errors a master mix for each PCR was produced and aliquoted appropriatelyPCR primers were designed using PrimerSelect in the LaserGene 6 DNA and Protein Analysis Software  to generate a 100–200 bp amplicon and have no cross-reactivity with other known DNA sequences. The HPV-32 specific primers  was used to determine the sensitivity of the HPV-32 specific assay. These experiments showed that the HPV-32 specific PCR was reproducibly sensitive down to 5 copies of the HPV-32 L1 gene Figure .7 billion copies) was used as the template for four separate PCR reactions and in all cases, HPV-42L1 gene was not amplified, while only 5 copies of the HPV-32 L1 gene was detected (data not shown). Next, using clinical material, six archival samples that had previously tested positive for HPV-42 by the Roche reverse line blot assay were identified and retested positive for HPV-42. These six samples were then subjected to PCR amplification using the HPV-32 L1 specific primers. Two of the six samples amplified. The assay volume was quadrupled (100 μL), reamplified, and the resultant amplicons sequenced. They were found to have the best homology (85.4% and 98.7%) with HPV-32. This implied that this clinical sample contained both HPV-32 and HPV-42. Finally, clinical samples (45 subjects) that were previously found to contain HPV types (number): 6 (n = 5), 16 (8), 18 (2), 26 (10), 33 (1), 35 (2), 39 (19), 45 (21), 51 (1), 53 (2), 55 (17), 58 (5), 59 (13), 62 (6), 66 (3), 69 (1), MM4 (12), MM7/83 (24), MM8 (13), and MM9 (7) were amplified via the HPV-32 Specific PCR and were found to be negative.Specificity of any HPV PCR assay is of significant concern because genotypes are defined by only a 10% difference in the nucleotide sequence of their E6, E7, and L1 open reading frames (ORFs). For this reason a number of experiments were performed to test the assay's specificity. First, because of the potential of cross-priming, the primer's sequences were compared to other HPV types. This analysis noted that the HPV-42 L1 gene was the most closely related to HPV-32. HPV-42 has no mismatches in the forward primer and only four mismatches in the reverse primer. Purified and quantified pMJ42L1 (10Subjects (n = 89) were enrolled in a study on oral HPV infection from the New Orleans HOP clinic . Reproducibility of the assay was assessed by retesting 14 patients (111 samples) with 57 of these samples positive for HPV-32. Overall, 94.6% of the samples were reproducible. Gingiva, tongue, sublingual, and saliva sites each had a single sample not reproduce, while the hard palate site had two samples not reproduce  from nine subjects (10%). All but one (23) of these HPV-32 positive samples were positive by the HPV-32 specific PCR Table .The new HPV-32 specific PCR system targeting the L1 gene demonstrated significantly increased sensitivity as compared to the laboratory's previous gold standard of MY09/11 amplification and dot blot hybridization. This could be due to the robustness of this new assay or due to laboratory contamination. Review of the PCR runs noted no false positive in no template controls making lab contamination less likely. A verification assay was developed which targeted the HPV-32 E6/E7 region. In a group of 45 oral samples from 5 patients, the E6E7 PCR assay confirmed 22 of the previously identified HPV-32 positive samples by the L1 assay. The E6E7 assay detected a positive sample among the 23 believed to be negative by the L1 assay. The agreement was almost perfect with a kappa of 0.96 ± 0.148. It is concluded that the increased sensitivity seen is due to the robustness of the HPV-32 L1 PCR assay implying that this assay is the new gold standard.Previous studies in the Hagensee laboratory have shown HPV-32 to be the most common HPV type found in oral warts in HIV positive individuals. The natSensitivity, specificity, and reproducibility are critical to the development of any new assay. The sensitivity of the HPV-32 assay was determined using purified HPV-32 DNA and was determined to be 5 copies of cloned DNA. This was significantly more sensitive than both the PGMY and MY09/11 based PCR amplification assays . The superior sensitivity was confirmed with clinical samples whereby 33 subjects with a total of 78 samples that were negative by current detection assays were found to be positive for HPV-32 by the HPV-32 specific PCR assay. This increased sensitivity was confirmed by a second assay which detected a different gene in HPV-32. This sensitivity increase is predictable as it has been observed in genital HPV prevalence studies. A healthy non-immunosuppressed female population has genital HPV-16 infection rates varying from 2.0–5.4% using consensus/degenerate primers  while ty7 copies of HPV-42 L1 thereby showing the stringent specificity of the PCR. Clinical samples positive for HPV-42 either did not have HPV-32 in them or were found to be co-infected with HPV-32. There was no evidence of cross-priming with the HPV-32 primers and amplification from a HPV-42 DNA template.An HPV genotype is defined as a ≥ 10% difference in E6, E7, and L1 open reading frame sequences. The specificity was tested theoretically by genomic alignments, as well as empirically and found to be excellent. Due to the higher sensitivity of the HPV-32 specific PCR, this HPV type was detected more readily in clinical samples; thereby, showing lower specificity, sensitivity, and agreement  with the current detection methods. The issue of cross-reactivity was tested utilizing the most closely related DNA sequence known (HPV-42 L1) which has only four mismatches within the primer regions. The HPV-32 specific PCR did not amplify in the presence of > 10The reproducibility of the assay was shown by the ability to repeatedly detect HPV-32 in clinical samples. All oral cavity sites sampled showed high reproducibility (> 92%) with the exception of the hard palate (84.6%). The lower rate of reproducibility of the hard palate may be due to a low level of infection which would be at the level of detection of the HPV-32-specific PCR assay. This is supported by the relatively lower rate of infection (10.1%) at the hard palate and is also supported by the faint bands seen in 5 out of the 6 discordant results implying low viral loads.Although this assay demonstrated increased sensitivity, excellent specificity and reproducibility, it is not optimal. Most modern PCR assays utilize a standardized universal master mix to which the specific primers are added. However, this approach does not allow one to vary the magnesium concentration which was critical to the optimization of the HPV-32 specific L1 assay in order to achieve the maximum sensitivity. The current assay could also be improved by the use of dUTP and addition of U-glucosidase step which would degrade any pre-existing amplicon DNA from previous amplification. This feature could further limit any additional laboratory contamination.In conclusion, the HPV-32 specific PCR assay has been shown to have increased sensitivity, and excellent specificity as compared to current assays. In addition, this assay is highly reproducible and is less labor-intensive. This makes the assay ideal to assess the natural history of HPV-32 in the oral cavity.E. coli, and extracted via a ProMega MiniPrep Kit according to their protocol. The DNA concentration was quantified using a DU 530 Life Sciences UV/Vis Spectrophotometer and confirmed by Eppendorf BioPhotometer.HPV-32 and -42 L1 genes were cloned directionally into pMJ601 , grown in + T-cell count and HIV viral load was collected from each subject as well as current HIV medications.For the clinical samples utilized, subjects were enrolled in the Medical Center of Louisiana at New Orleans (MCLNO) HIV outpatient program (HOP) Clinic in New Orleans, LA, from May 2002 to December 2003. One-hundred-seventy subjects whose oral samples were previously tested for HPV DNA were utilized. Subjects gave informed consent by a trained professional according to the LSUHSC Institutional Review Board (IRB) and the MCLNO HOP research committee accepted protocol. All subjects were given a comprehensive exam for all oral lesions by a health professional. In addition to the oral samples, basic demographic information, peripheral CD4Cells were collected from the buccal mucosa, labia, gingiva, tongue, sublingual surface, hard palate, and tonsils by vigorously rubbing the surfaces with a sterile swab . These sites were chosen for their association with HPV associated lesions and head and neck squamous cell carcinomas. The swabs were stored in Specimen Transport Medium  at 4°C. Unstimulated expectorated saliva (5 mL) was collected in a 50 mL conical tube. Five milliliters of sterile saline was gargled and expectorated into a 50 mL conical tube. Both saliva and gargle samples were stored at 4°C as well. Saliva and gargle samples were processed within 4 hours, and sample DNA extracted within 18 hours of collection.Saliva and gargle specimens were processed by homogenizing the sample through an 18 gauge needle and syringe. The specimen was then centrifuged at 1,260 × g for 10 minutes. Supernatants were removed, and the cell pellet was resuspended in 1 mL of sterile PBS and stored at 4°C until extraction.DNA from all samples was extracted using the QIAGEN Blood Extraction Kit (Germany), according to the manufacturer's protocols. Extracted DNA was stored at -20°C until HPV detection by PCR was performed.HPV genotyping was done using the Roche HPV Reverse Line Blot system according to the manufacturer's protocol . Briefly, multiplex PCR amplified a 448 bp sequence of the L1 Gene and 268 bp sequence of β-Globin (housekeeping gene) using biotinylated PGMY09/PGMY11 consensus/degenerate primers and GH20/PCO4 primers, respectively . Thermoc2, 7.5 U AmpliTaq™ Gold and 5 μL of template DNA. The thermocycler protocol was: 50°C for 2 minutes, 95°C for 9 minutes, 40 cycles of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, extension at 72°C for 5 minutes, followed by a 15°C hold. The 448 bp amplicons were detected and visualized as detailed above.Samples that were gel-positive and line blot-negative were then tested for HPV-32 via dot blot. This was performed by re-amplifying samples with the MY09/11 primers (table MY09/11 amplicons (10 μL) from positive patients were denatured for one hour in 100 μL of denaturation buffer . This mixture was applied to a nylon membrane  that was pre-moistened in 6× SSPE using a Dot Blot apparatus . The DNA was then UV crosslinked to the membrane in a UV Stratalinker 1800  using 120 mjoules and dried overnight. The nylon membrane was put in pre-hybridization buffer (0.1× SSPE-0.5% SDS) for one hour at 65°C, followed by a one hour incubation at 65°C in hybridization buffer (4× SSPE-0.5% SDS). The biotinylated HPV-32 probe (5'-Biotin-AGG TGC TGT TAC CTT AGC TTG-3') was mixed with hybridization buffer at a concentration of 0.5 pmol/mL, and the membrane was soaked in the probe solution for 30 minutes in a 53°C shaking water bath. The remaining procedure is done using the reverse line blot procedure as described above.2, 0.625 U AmpliTaq™ Gold and 5 μL of template DNA. Thermocycling protocol was performed as follows: 50°C for 2 minutes; 95°C for 9 minutes, 40 cycles of 95°C for 1 minute, 55°C for 10 seconds, and 72°C for 30 seconds, extension at 72°C for 5 minutes, followed by a 15°C hold. The 268 bp amplicons were detected and visualized as described above.The integrity of the stored DNA samples was verified via PCR amplification and the detection of the 268 bp amplicon of the β-globin gene. Final concentrations in the 25 μL reactions were 1× PCR Buffer II, 0.25 μM of each primer  and 5' GAT GCC CAA CAG CCA AAA G 3' (HPV-32 Det Rev). The final concentrations for the 25 μL HPV-32 Specific PCR reaction were 1× PCR Buffer II, 0.5 μM of each primer, 200 μM of each dNTP, 1.5 mM MgCl2, 0.625 U AmpliTaq™ Gold and 5 μL of template. Thermocycling was performed as follows: 95°C for 9 minutes; 40 cycles of 95°C for 1 minute, 58°C for 10 seconds, and 75°C for 30 seconds; extension at 75°C for 5 minutes, followed by a 15°C hold. The 382 bp amplicons were detected and visualized as described above.The primer sequences for the HPV-32 E6/E7 specific PCR were 5' TAT AAC GGA CGG CAT TTC AGA TTC 3' (HPV-32 E6E7 For) and 5' GTC ACT CCA CGC AGG CAC AC 3' (HPV-32 E6E7 Rev). The final concentrations for the 25 μL HPV-32 E6E7 Specific PCR reaction were 1× PCR Buffer II, 0.5 μM of each primer, 200 μM of each dNTP, 2.0 mM MgClContamination was controlled for all work by using barrier pipet tips. Lab benches were bleached after each extraction and prior to any PCR work. DNA extraction, PCR setup, and the addition of template was performed in separate rooms to minimize cross contamination. All plasmid work was done in a separate room as well. Possible contamination in extractions and PCRs was monitored through the inclusion of extraction controls for each extraction and no template control that went through the same mechanical manipulations as samples after every third sample on all PCRs.The authors declare that they have no competing interests.NRH: Developed and ran the HPV-32 specific PCR assay, did all statistical analysis, and prepared the manuscript.NH: Extracted the clinical samples and tested them via the PGMY and HPV-32 dot blot assay. JEC: Developed and validated the HPV-32 dot blot assay used at the comparison assay for the HPV-32 specific PCR. JL: Provided subjects and performed oral examinations of all subjects in the study. MEH: Responsible for obtaining funding for the project and guidance for its direction, and preparation of the manuscript. All authors have read and approved the final manuscript.