Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%–41.5% within the assays and 8.3%–36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation.Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R In cl for use . In 2014® immunoassay (cLIA) [in situ purified glutathione S-transferase L1-based MIA (GST-L1-MIA) [in vivo protection well when the dominant antibodies in some serum samples are not the selected epitopes. VLP-MIA and GST-L1-MIA detect the total VLP-specific antibodies, which include some non-neutralizing antibodies, and might therefore overestimate the efficacy of the vaccine.In HPV vaccinology and epidemiology, serological testing methods are used to analyze the antibody levels in naturally infected or vaccinated individuals. A variety of methods have been developed for testing antibody levels, including the competitive Luminexy (cLIA) , the viry (cLIA) , and the-L1-MIA) . DependiGaussia luciferase (Gluc) [Although the level of protection afforded by neutralizing antibodies has not been determined, neutralizing antibodies are accepted as the primary mediator of a vaccine’s potency. The HPV life cycle is strictly dependent on the differentiation stage of the host cell . Native e (Gluc) ,15). Thee (Gluc) ,16, whice (Gluc) . Using tEGFP gene into the pCDNA3.1 vector , as described previously [The 293FT cell line  was maintained in growth medium . HPV L1/L2 expressing plasmids  were kindly provided by Shanghai Zerun Biotechnology . Written informed consent was obtained from all donors. Mouse sera were collected from mice intraperitoneally administered a candidate nonavalent HPV vaccine . Rabbit serum samples were collected from rabbits immunized with a monovalent vaccine kindly provided by Qiming Li , as described previously [Naturally HPV-infected serum samples were selected from a large number of samples from a blood bank . Fifty post-vaccinated human serum samples that had been collected in a phase I clinical trial of a bivalent HPV16/18 vaccine, produced in eviously . This st® , 0.1% Plasmid-Safe ATP-Dependent DNase , and 1/40 volume of 1 M ammonium sulfate. The pseudovirions were matured by incubating the cell lysates overnight at 37 °C. The lysates were then transferred to an ice bath for 5 min and the NaCl concentration was adjusted to 0.85 M. The lysates were clarified by centrifugation at 5000× g for 10 min. The three types of pseudovirions  were stored as aliquots at −80 °C until titration.HPV pseudovirions were produced in 293FT cells using a previously described method, with modification ,27. Brie4 cells/100 μL/well for 3–6 h before the pseudovirions were added. Each type of pseudovirion was prediluted 100-fold, and nine serial five-fold dilutions were prepared. After incubation for 72 h at 37 °C under 5% CO2, the percentages of fluorescence-positive cells and fluorospots were determined with FCM  and an ImmunoSpot reader . The pseudoviron titers were defined as 50% tissue culture infective dose (TCID50), calculated with the Reed-Muench method [The pseudovirion stocks were titrated as follows: 293FT cells were trypsinized and placed in the inner wells of 96-well tissue culture plates at 1.5 × 10h method .50), calculated with the Reed-Muench method [Once the pseudovirion titer had been determined, the neutralizing antibody titers of the sera were determined with a PBNA, as in previous studies ,13,15. Bh method .To select the appropriate fluorescent proteins for the multiplex PBNA, 293FT cells were transfected with different fluorescence-expressing plasmids: AmCyan1 (blue), EGFP (green), enhanced yellow fluorescent protein , RFP (red), and E2-CFP (far red). The cells were scanned with different excitation and emission wavelengths using ImmunoSpot . Only EGFP, RFP, and E2-CFP were detected with unique excitation-emission wavelength pairs. Therefore, the three reporter plasmids were cotransfected with the HPV structural genes to generate pseudovirions producing different colors . No mutuEGFP or RFP, to yield four pseudovirions: HPV16-EGFP, HPV18-EGFP, HPV16-RFP, and HPV18-RFP. The 293FT cells were then infected with the serially diluted pseudovirions and detected with FluoroSpot and FCM 72 h post-infection. The FluoroSpot counts and the percentages of positive cells correlated well, with R2 > 0.97 , and the results were compared with the FCM results. The linear correlation Rncreased . The cor for FCM .50 values for HPV16 with both reporter proteins  and red (RFP) HPV16 pseudovirions were tested against rabbit sera immunized with HPV16. The almost totally overlapping inhibition curves yielded similar ICproteins A. A simiproteins B. To invproteins C,D. The lor PBNA . In addiTo further improve the throughput of the PBNA, a third color (CFP: far red) was introduced to the assay. Nine kinds of pseudovirions were prepared: HPV16-EGFP, HPV16-RFP, HPV16-CFP, HPV18-EGFP, HPV18-RFP, HPV18-CFP, HPV58-EGFP, HPV58-RFP, and HPV58-CFP. The differently colored pseudovirions and different combinations of pseudovirion were tested against pooled serum samples from mice immunized with a nonavalent (HPV6/11/16/18/31/33/45/52/58) HPV candidate vaccine. The similar inhibition curves suggest that there were no significant differences between the single-color PBNA and triple-color PBNA in testing HPV16, HPV18, and HPV58 .50 values of 118, 97, and 55, respectively, which are similar to those reported previously [50 values of 801, 1027, and 628, which are similar to those reported previously [The single-color and triple-color PBNA were also compared using different kinds of samples, including sera from naturally infected humans, sera from immunized mice, and WHO International Standards for antibodies. For HPV16, HPV18, and HPV58, the coefficients of variation (CVs) were 18–34%, 16–51%, and 28–43%, respectively . The HPVeviously . The HPVeviously . Most im50 of 40 as the cut-off value. The sero-status assignment of the samples with the two assays is cross-tabulated in Because the antibody titers are low in individuals naturally infected with HPV, the agreement between the single- and triple-color PBNAs can be assessed most stringently with this kind of sample. Samples from 48 individuals naturally infected with HPV were used in both assays for HPV16, HPV18, and HPV58, using an ICTo determine the reproducibility of the three-color PBNA, three samples for each type were tested: two from naturally infected individuals  and one from a rabbit immunized three times with the corresponding monovalent HPV vaccines . Each serum was tested nine times in three independent runs . When thA nonavalent HPV vaccine targeting HPV6/11/16/18/31/33/45/52/58  was approved by the U.S. FDA in 2014 [Neutralizing antibodies are considered the primary protective mechanisms for most prophylactic vaccines, and PBNA is considered the gold standard method for evaluating the immune response raised against HPV vaccines ,14,30. HFluoroSpot, which is used in the ELISPOT assay, allows the analysis of single cells secreting several cytokines . Here, FAlthough the mechanisms have not been fully revealed, cross-protection has been observed with bivalent and quadrivalent vaccines ,33,34. TTo date, neutralizing methods have been used in few clinical HPV vaccine trials. Instead, antibody binding is used as a surrogate index for the immune response. The coating antigens and principles of detection differ completely between these two types of assays. No comparison data for different clinical trials are available. Placebo controls cannot be used in future clinical studies, especially for the development of second-generation vaccines. Therefore, the development of high-throughput easy-to-use protection-related PBNAs is urgently required for future clinical trials.Three triple-color PBNAs, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed to evaluate a nonavalent vaccine. This multicolor PBNA provided rapid and precise profiles of the protection-related immune response in vaccine-immunized or naturally infected cohorts. The new assay has advantage over the FCM assay for the easy-to-use procedure, which shortens the detection time from more than 3 h to 5 min for 96-well plate. Even compared to the recently developed high-throughput Gluc-based assay , triple-


Among all 85% of the women were younger than 45 years of age. Fifty-seven patients were evaluated 15 months after the base study, with initial prevalence of morbidity 49.12% (28/57) and at the end 10.53% (6/57), showing in 89.29% (25/28) negative for HR-HPV infection, 10.34% (3/28) showed persistence of infection, 17.54% (10/57) presented cytological alterations, with 80% of positivity for HPV, and a regression of 100% (10/10) of the previously identified lesions. With colposcopy, 50% (14/28) presented alterations related to HPV, of these 85.71% (12/14) showed regression of such an alteration. The cumulative incidence for HPV was 10.34% (3/29). The incidence rate was 4.23% (3/71), which is equal to 4.23 new cases of HPV infection per 100 people, per year of follow-up. In conclusion, the present work shows a high frequency of infection by high-risk HPV, with predominance of HPV18 and 16 and in general for multiple infections. Colposcopy was better predictor than the Pap smear for infection. The follow-up study revealed a low percentage of persistent infection, and a high frequency of negativity for viral infection, high regression of cytological and colposcopic lesions, a low cumulative and incidence rate similar to that reported by other Latin American countries and higher than the European countries.Cervical lesions have been associated with infection by high-risk human papilloma virus (high-risk HPV). In 409 women aged >15 years high-risk HPV lesions were identified. In a cohort of this population persistent infection was compared with cytological, colposcopic, and histological lesions. Cervical scrapes were taken and DNA was isolated. HPV was detected by PCR in the E6/E7 region. Genotyping was performed by PCR nested multiple E6/E7. HPV was detected in a 37.40% (153/409), high-risk HPV in 86% (153/178), HPV18 46.64% (83/178), HPV16 34.28% (61/178). Among these 53.93% (96/178) were multiple infections, and HPV18/16 (30/96) was the most frequent 31.25%. The cytology showed changes in 15% of positive patients. A 49.67% in women positive for HPV infection showed abnormalities in the colposcopic study, a relationship that turned out to be statistically significant (  Cervical cancer (CC) was associated with HPV infection in the 1970s, when concerns arose about the hypothesis proposed by Dr. Harold zur Hausen that this could be caused by infection with the virus identified in the condylomata acuminata . CervicaCurrently, CC represents the fourth most common type of cancer in the female population, constituting an important cause of morbidity and mortality, with an incidence of approximately 528,000 new cases and a cause of 266,000 deaths globally during the year 2012; with about 80% of these cases affecting women who reside in developing countries, where the CC totals 12% of all diagnosed neoplasms, and 7.5% of all deaths in the female population. Similarly, it has been determined that South America is included among the regions of greatest risk for the development of CC, with an age-standardised rate of 31.5/100,000 women .Studies in the course of the last ten years have shown that the prevalence of HPV infection in our country is over 25%, both in women who are asymptomatic for cervical pathology and those that have a degree of diagnosed cytological abnormality . AccordiThe progression of lesions related to cervical infections by high-risk HPV as far as CC, occurs only in a small percentage of the affected women, because of the fact that these infections are self limiting, with the average time for resolution being 6–18 months, in those cases the woman does not show any clinical symptoms and the infected area is colposcopically and cytologically normal. A possible explanation of this approach is based on the fact that some HPV genotypes are more persistent than others, an ability that is genetically coded and therefore associated with this pathogenicity .Persistent HPV infections have a high possibility of developing into cancer, if the associated genotypes are included in the oncogenic high risk group, due mainly to the fact that the persistence may induce secondary genetic changes, given the interference of the viral oncoproteins in the cell cycle checkpoints, and their ability to induce immortality in the keratinocytes –12. In tIn accordance with the concepts outlined above, it was considered important to conduct a clinical prospective epidemiological cohort study, aiming to establish prevalence of cervical infections because of HPV, demonstrate the possible relationship between high-risk HPV infection and the progression to different degrees of severity of the cytological and colposcopic lesions detected and to evaluate the persistence of the infection because of HPV and the cervical lesions by means of a cohort study, which would contribute to the epidemiology reality regarding the behaviour of the infection because of this viral agent, its impact on the health of women in our community, and the evaluation of the progression or resolution of cervical lesions associated with HPV.Type of study: This clinical epidemiological study followed the experimental design of cohort, being the prospective concurrent type.Population sample: in order to carry out the investigation, a total of 409 women were selected at random all being between the ages of 15 and 69, sexually active or who had initiated sexual activity, and had attended the gynaecology department of the Autonomous Institute University Hospital of Los Andes (IAHULA) of Mérida state, Venezuela, during the time span in which the study was conducted.Inclusion and exclusion criteria: Included in the investigation were women who had attended the gynaecology department and had undergone a semi-annual, annual, or bi-annual routine screening. Likewise, excluded from the study were all women who, for the time of the interview, and sampling, presented genital bleeding by normal menstruation, metrorrhagia or menorrhagia, and those who showed changes in the cervix because of surgical procedures that prevented proper sampling. Similarly women with diagnosed pregnancy and women who were sera-positive for HIV or undergoing immunosuppressive treatment were excluded.Bioethical standards: To all the selected participants and to the parents and/or representatives of the minors, the nature, and importance of the study was explained and were told that it would be carried out following the principles laid down by the World Health Organisation for medical research in humans [n humans ; in addiFollow-up protocol: Among them 57 of the women participating in the study underwent a monitoring protocol during 15 +/- 5 months, distributed according to the reports of HPV detection, cytological and colposcopic, to assess the occurrence and/or evolution of possible pathological endocervical alterations, and their relationship to the incidence and persistence of HPV infections, identified in this group of patients.Sampling: All participants underwent a gynaecological evaluation, which was carried out by qualified medical personnel; with the report of this study being recorded in a clinic registered designed for that purpose.in situ (CIS) [Cervical swab samples were obtained for later processing using the Papanicolaou technique. The results are expressed according to the terms of the Bethesda classification, namely: negative for intraepithelial lesions or malignancy; atypical squamous cells (ASC), including cells with alterations that are not clearly pre-cancerous; atypical squamous cells of undetermined significance (ASC-US), including all the cells that are among the cellular changes benign and intraepithelial lesion; atypical squamous cells that do not exclude a high grade lesion (ASC-H); low grade intraepithelial lesion (LEL BG), including cases of mild dysplasia/NIC I, and the cellular changes associated with HPV infections; High-grade squamous intraepithelial lesions (HSIL), cellular changes associated with moderate or severe dysplasia/CIN 2, CIN 3, and carcinoma tu (CIS) .A second cervical specimen was taken with a swab, using the DNA collection device kit ; which were used for the isolation of DNA and the identification of HPV cervical infections.Sample of cervical biopsies: The cervix biopsies of the 41 patients who had cervical lesions at the time of the gynaecological examination, were obtained by the gynaecologist; a portion of the tissue was placed in 10% buffered formalin for histology analysis, and another portion was placed in a ATL lysis solution (QIAGEN®), and were frozen at −70 °C until required for processing. The results are expressed according to the terms of the classification established by the National Cancer Institute in the USA . This assessment was carried out using staining with haematoxylin–eosin (H&E) to the tissue samples, which helps to demonstrate the presence of histopathologic lesions. The NIC is divided into grades I, II, and III. NIC I corresponds to the mild dysplasia, CIN II to the moderate dysplasia, and CIN III to the severe dysplasia and cancer in situ [ in situ .Colposcopic evaluation: All the participants in the study underwent a colposcopic exam of the cervix, vagina, and vulva. The international nomenclature established by the International Federation of Cervical Pathology and Colposcopy (IFCPC) was used, to classify the patterns observed colposcopic studies [ studies . AccordiThe colposcopic evaluation of the patients was included as a tool for the routine diagnosis of cervical atypia, in accordance with the parameters proposed for Venezuela in the Consensus Meeting on Human Papilloma Virus, 2008, published in the Caracas Medical Gazette .Isolation of DNA: The commercial kit for the QIAamp DNA Mini Kit (QIAGEN®) was used in accordance with the instructions of the manufacturing laboratory.Quantification of the extracted DNA: The concentration of the extracted DNA was determined by spectrophotometry at 260 nm, using the UV photometer T1101/1101 .Amplification of DNA fragments of HPV specifics by multiple nested-PCR, viral gene region E6/E7: This technique was applied to the detection and genotyping of HPV. The first reaction allowed the detection of specific sequences of HPV DNA, contained in a conserved region of the early genes e6/e7, using the pairs of consensus primers GP-E6-3F/ 5B/ 6B, according to the protocol previously described by Sotlar et al, 2004 [al, 2004 . As an ial, 2004  and for The multiple PCR, nested format, allowed for the identification of the high-risk oncogenic viral genotypes HVP16, 18, 31, 33, 45, 52, 56, and 58, and low-risk HPV6/11 [All of the previously described amplifications were processed in a thermal cycler model ABI 2400 (Applied Biosystems). To view the reaction products, these were subjected to electrophoresis in agarose gel to 1.20%, with 10 μL of ethidium bromide/100 mL of agar, and rear illumination with UV light. The electrophoretic run included a marker of molecular weight of 1000 pb, ladder of 100 BP , and the buffer 10 X blue juice TM loading gel buffer (Invitrogen) to verify the size of the bands obtained.Definition of persistent HPV infections: For a persistent infection positivity for the same viral genotype was taken as the same persistent infection on two specific occasions: in the first assessment and one that took place 15 months later. It is important to note that all the patients included in the follow-up were said to be sexually active; which is why the ‘sexual abstinence’ was not assessed.Collection and analysis of the data: A database in the EPI Info 2012 programme, version 7.0 was created, which was used for the descriptive statistical analysis of the results, frequency distribution of infections by HPV of high and low risk and association in qualitative scales of the different groups identified, with clinical and epidemiological variables from patients, which were summarised in proportions. Inferential statistical analysis and the definition of significant associations were conducted using the Chi-square test. The indicators rate of incidence, or incidence density, calculated as the quotient of the number of new cases of disease which occurred during the monitoring period and the sum of all the individual observation times and accumulated incidence, referred to the proportion of candidates who developed the disease during the observation period.Of 409 women studied, HPV DNA was detected in 37.40% from cervical specimens .85% of HPV infections arose in women under 45 years of age; there was a similar percentage in women 15 to 24 years of age and 25 to 34 years .Among these 85.96% of HPV found corresponded to high-risk oncogenic genotypes, from those 80.92% were genotypes HPV16 and 18, with a percentage of 34.28 for the first and 46.64 for the second .p < 0.000029 test χ2). However; when considering each genotype separately, both HPV18 and HPV16 showed greater frequency for individual infections, 58.54% (48/82) and 37.80% (31.82) respectively  correspond to mixed infections. The most frequent combinations were HPV18/16 with 31.25% (30/96) and HPV18/6y11 with 20.83% (20/96), the later with statistical significance ( ectively .p < 0.02 test χ2)  showed normal cytological results, while 13.74% (21/153) were reported as LSIL; other categories such as HSIL and ASC-H were reported in very low percentages of 0.65% for each one. When comparing the LSIL cytological report with infection from HPV18 there was an association with statistical significance found ( test χ2) .p < 0.0019 test χ2). And also for dense and thin acetowhite epithelium ( p < 0.0019 test χ2). The association between infection from HPV and a positive Schiller’s test resulted statistically positive ( p < 0.02 test χ2)  showed abnormal colposcopy results and 47.71% (73/153) had normal colposcopy results. Within the abnormal results 96.05% corresponded to minor colposcopy atypia and 26.32% to major colposcopy atypia. Significant statistical association between infection from HPV and abnormal colposcopy results was observed, especially within the TZ (transformation zone) and the iodine-negative clear boundary area ( test χ2)  and 6.When following-up with a cohort of 57 women of the initial sample, for a period of 15 months, an initial prevalence of 49.12% (28/57 women) morbidity was observed, based on this condition, in the second phase of the study, there were findings of high negativisation of the infection, displayed by the absence of HPV DNA detection in the cervical samples of 89.29% (25/28) of the women initially infected with HPV; likewise, there were findings of low prevalence of HPV infection, 10.71% (3/28 women) and after studying the prevalence of specific genotypes, there was just one case found for HPV18 representing 16.67% (1/6) of the patients who were initially positive for this viral genotype. These observations allow the establishment of an accumulated incidence of HPV infection of 10.34% (3/29) in 15 months. Upon determining the level or density of viral infection, a value of 4.23% (3/71) was obtained, which is equivalent to 4.23 new cases of HPV infection per 100 people, per year of follow-up .In the initial sample, 17.54% (10/57) of the women evaluated, presented cytological alterations, and 80% (8/10) of them were positive for the detection of HPV DNA. Upon observing the results obtained after follow-up, there was a regression of 100% (10/10) of the previously diagnosed lesions in all patients, both positive and negative for HPV, and negativisation of HPV infection. Two patients presented new cervical lesions (LSIL in both) after time of follow-up; it is important to point out that these two patients presented new HPV infections, classified in the group of those which resulted negative for the viral genotypes researched in this study .Among the 50% (14/28) of the positive patients’ for HPV infection in the initial sample showed colposcopy lesions with few differences between the cases with minor and severe atypia. In 85.71% (12/14) of those cases, there were observations after the monitoring timeframe, of the disappearance of the initial colposcopy alterations that were found, and there was negativisation of the HPV infection .et al [The percent of viral infection detection was 37.40%, pointing out that 83.67% of these HPV positive reports were from women with normal cytology, this is a finding which is considered superior to that registered in the general population around the world, making up between 9–13% ; in addiet al  for LatiThe high percentage of HPV positive cervical samples found in our study could be because of the nature of these infections, which have displayed high variability, depending on multiple factors, such as demography, behavioural characteristics of the population being studied, and the method of diagnosis being applied . In the It is important to emphasise the high levels of HR HPV genotypes identified in the population studied in this investigation, both in multiple and single infections; in addition, it is important to point out that HPV18 was the most frequently detected viral genotype, followed by HPV16. The relative distribution of these genotypes in the studied women is similar to the data previously published in the city of Mérida, Venezuela, which shows a predominance of HPV18 infections over all genotypes studied . On the Within the samples positive for HPV DNA identified in this study, multiple infections with two or more genotypes were reported in 53.93% showing the importance of genotyping evaluations in patients with a positive report for viral detection. This relevance is based mainly on the fact that multiple infections caused by different HPV genotypes increase the probability of acquiring a high-risk genotype . In addiThe results obtained in the present study, based on cytological analysis of the cervical samples through the Pap smear technique, show that 15.03% of HPV positive patients showed cytological changes suggestive of this viral infection, emphasising the predominance of infections by HR HPV in patients with a diagnosis of LSIL. The previously presented data reflects a relationship between identified HPV genotypes and the abnormal cytological report. Consequently, these results support the widely known concept regarding the importance in the detection and genotyping of HPV in the routine gynaecological consultation. With this in mind, previous publications have shown an elevated risk of progression of cervical lesions, from CIN 1 to CC, in patients infected with HPV16 and/or HPV18; with an estimated timeframe of 12 months, in approximately 3.5–12% of the women infected with a HR HPV genotype, there occurred progression to cervical lesions of different grades of CIN . Similarly, two meta-analysis in publications with world prevalence of HPV infection and the distribution of viral genotypes in cervical samples of women with normal and abnormal cytological results concluded that the testing for diagnosis of HPV, simultaneous with the cytological evaluation, was associated with a significant reduction in the progression of cervical lesions to CC and in deaths because of this pathology , 36.On the other hand, it is currently well-known that in about 60% of women with normal cytology and a positive HPV DNA test, there is negativisation of the viral infection report in the following six months from the first report. However, when dealing with patients over the age of 40, the risk of progression from CIN 1 to CIN 3 in the following ten years increases significantly, compared with that of young women , 37. TheIt is important to mention the recommendations for CC screening tests issued by the American Congress of Obstetricians and Gynaecologists in 2012 which indicate that gynaecological evaluations with cervical cytology should be no later than one year; however, the time between evaluations could increase to an interval of 3–5 years with the realisation of cytology and the incorporation of screening tests for HR HPV infection . This suet al [The colposcopy exam done suggested the viral infection in a range superior to that obtained through the Pap smear technique, and in addition there were observations of a significant statistical association between the detection of HPV and the abnormal colposcopy findings, showing that the colposcopy had high predictive value in the diagnosis of HPV infection. These results coincide with those reported by Mitchell et al , who fouet al .In the present study, 57 participants were monitored for a period of 15 months, with morbidity rates of HPV infection of 49.12% in this group. At the end of the monitoring period it was observed that the prevalence of positive cases for HPV infection was low (10.71%); likewise, the infection persisted in specific viral genotypes; it was only identified in one patient positive for HPV18 (16.67%). The data obtained in the present study coincides with that reported in another study, in which it was shown that infection by oncogenic HPV genotypes has an average duration of eight months, while the duration of non-oncogenic HPV has an estimated duration of four months . Numerouet al [et al [Data complementary to that obtained in the present study regarding the prevalence of HPV infection shows that a generally high percentage of negativisation of the viral infection (89.29%) was determined in the 28 patients positive for HPV in the initial sample. In that sense, there is consistency between our results and the estimates of various studies done on women who were HPV positive, in which the resolution rate of the viral disease was high throughout the first year of infection as described in the work of Schmeink et al  and Muñol [et al . They reet al [et al [The cumulative incidence of HPV obtained in this study (10.34% in 15 months) differs from studies previously published in which this indicator is considerably higher, Winer l [et al  reportedet al [With regard to the rate or density incidence of the viral infection, there was an obtained value of 4.23 new cases of HPV per 100 people, per year of monitoring; these results coincide with those reported in another cohort study done in Colombia, which included 1610 women between the ages of 15–85, with abnormal cytology, who were monitored every six months for an average of 4.1 years, and the rate of incidence determined for HR HPV was five cases per 100 women a year . In addiet al , in 1383The evaluation of persistent HPV infections, diagnosed in this study, according to the reported colposcopy atypia, showed that the three patients with this condition did not present colposcopy atypia after follow-up. On the other hand, there was regression of the colposcopic alterations at an elevated percentage in the patients who presented negativisation of the viral infection. The previously presented results support the concept under which the severity of the colposcopic alterations could be related to the frequency of persistent cervical infections caused by HR HPV , 50, a fAn increased frequency of high-risk HPV infection was shown in the population of women under 45 evaluated in this study, with a predominance of HPV18 and 16 and in general multiple infections, a colposcopy was found to be a better predictor for HPV infection than cytology, with statistical significance between infection by this virus and abnormal colposcopy. The follow-up study of the cohort showed a low percentage of persistent infection, a high frequency of negativisation of viral infection, a high percentage in the regression of cytological and colposcopic lesions, a low cumulative frequency, and a similar incidence rate to that reported by other Latin American countries and higher than that of the European countries.We declare that no conflicts of interest exist among the authors of this original research article.


Background. Cervical cancer caused by human papilloma virus (HPV) though preventable has claimed the lives of many women worldwide. This study was embarked upon to evaluate the general knowledge and perceptions of Nigerian women on HPV, cervical cancer, and HPV vaccine. Methods. Structured questionnaires were administered to a cross section of 737 women randomly selected from the general population in two southwestern States of Nigeria. Statistical analysis was done using SPSS computer software version 16. A P value >0.05 was considered statistically significant. Results. One hundred and seventy-six (23.9%) of the respondents had knowledge of HPV; 474 (64.3%) are aware of cervical cancer but only 136 (18.5%) know that HPV causes cervical cancer. 200 (27.1%) are aware that there is an HPV vaccine while 300 (40.7%) had knowledge of Pap smear test. Two hundred and sixty (35.3%) of the respondents know that early detection of HPV can prevent cervical cancer and in spite of this, only 110 (14.9%) have taken the Pap smear test before while 151 (20.5%) are not willing to go for the test at all. Conclusions. There is therefore the need to create proper awareness on the HPV and its possible consequence of cervical carcinoma. HPV infection, a sexually transmitted infection, occurs throughout the world and has been implicated as the principal cause of cervical cancer. More than 100 HPV types have been identified and most types infect the cutaneous epithelium where they cause common skin warts. About 40 types infect the mucosal epithelium; these are categorized according to their epidemiologic association with cervical cancer [Worldwide, cervical cancer is one of the most common cancers among women. It is estimated that almost half a million new cases and 274,883 deaths occurred in 2008, and about 86% of these cases occur in developing countries [Few years ago, some new vaccines were discovered against the HPV infection and cervical cancer which has transformed the prospects of reducing HPV incidence on a global scale . The vacThe vaccine against HPV types 6, 11, 16, and 18 has been licensed but is currently not available in Nigeria due to importation, distribution, and other regulatory requirements, as well as price negotiations issues. However, a vaccine which targets both HPV strains 16 and 18 and has proven to be 92% effective with more than four-year potency is both licensed and available in Nigeria . The vacDespite the prevalence and burden of cervical cancer worldwide with almost 80% occurring in developing countries such as Nigeria, only about 52% of Nigerian women were aware of this deadly disease . Less thThe effectiveness of a vaccine delivery program depends largely upon the awareness of the vaccine and the attitude in terms of acceptability of the vaccine . This stThis was a questionnaire-based cross-sectional survey carried out in Oyo and Osun States both in southwestern Nigeria. The study population included randomly selected women aged 18 years and above as at the time of the administration of questionnaire. Face-to-face structured interviews were conducted after the purpose of the study had been explained to the participants and their informed consent sought and gained. Women who did not give their consent were excluded from this study. The survey was conducted from September 2013 to March 2014. A standardized questionnaire assessing knowledge of HPV, cervical cancer, Pap smear test, vaccine acceptability, and willingness to participate in HPV vaccination as well as demographic characteristics related to HPV and cervical cancer was administered to a total of 737 women.Seven hundred and thirty-seven questionnaires were administered in face-to-face interviews. The mean age of respondents was 29.3 years (range: 18–54). Three hundred and forty-seven (47%) were single while 390 (52%) were married. Two hundred and fifty-one (34%) of the respondents were students, 181 (24%) civil servants, 129 (17%) into a trade or private business, 37 (5%) artisans, 57 (8%) housewives, and 82 (11%) others that may include the unemployed .Sixty-one (8.3%) had primary education, 203 (27.5%) had secondary education, and 447 (60.7%) had tertiary education while 26 (3.5%) had no education at all. Three hundred and sixty-five (49.5%) were Christians while the rest 372 (50.5%) were Muslims. Six hundred and eighteen (83.9%) of the participants were of the Yoruba tribe, 65 (8.8%) Igbo, 42 (5.7%) Hausa, and the remaining 12 (1.6%) others that may include tribes such as Edo, Tiv among others.P = 0.039), marital status (P = 0.0), occupation (P = 0.0), level of education (P = 0.002), and religion (P = 0.0) have significant effects on their willingness to partake in research studies.This study shows that sociodemographic characteristics of individuals such as age  of the respondents have no sexual partners, 421 (57.1%) had between 1-2 partners, and 103 (14.0%) have more than 3, while the rest 149 (20.2%) did not disclose.One hundred and seventy-six (23.9%) of the respondents had knowledge of HPV, 474 (64.3%) are aware of cervical cancer, 200 (27.1%) are aware of the HPV vaccine, 300 (40.7%) had knowledge of Pap smear test, 136 (18.5%) are aware that HPV causes cervical cancer, and 260 (35.3%) know that early detection prevents cervical cancer and in spite of this, only 110 (14.9%) have taken the Pap smear test before .The sources of information in the respondents include health care workers 178 (24.2%), television and radio 320 (43.4%), Internet 86 (11.7%), newspaper 28 (3.8%), and others 53 (7.2%) which may include school, friends.Four hundred and seventy (63.8%) of the respondents were willing to take the test in future, of which the rest 116 (15.7%) were not at all willing to take the test and 151 (20.5%) were not sure. Of the respondents, 569 (77.2%) were willing to take the HPV vaccine and 76 (10.3%) were unwilling, while the rest 92 (12.5%) are not sure if they would take the vaccine; this is because most are not sure the vaccine will be able to protect them against cervical cancer.This study revealed that the level of awareness of the HPV vaccine is low (27.1%) among the general population of women in southwest Nigeria; however after they were enlightened, 77.2% were willing to take the vaccine. This contradicts 62.7% awareness level reported in another study . The knoOnly 14.9% respondents have taken the Pap smear test before; this shows that the prevalence rates of cervical cancer in Nigeria may be underreported, since most women in this study do not know their status, even though a good majority of them (>90%) have at least a primary education. Most of the knowledge in respondents came from television and radio (43.4%) and the least from newspapers (3.8%); this study also shows that information delivery through the health care system is poor (24.2%). This report contradicts that of another study which reports that knowledge of HPV and cervical cancer in respondents came from the health care systems . HoweverFrom this study, it is evident that sociodemographic characteristics of individuals such as age, marital status, occupation, level of education, and religion have significant effects on their willingness to partake in research studies such as this, and this has implication on the relevance of results from similar research studies.Knowing the fact that cervical cancer if unattended may lead to more serious complications or even death, there is thus an urgent need for educating Nigerian women of child-bearing age and postmenopausal women who are not likely to get regular Pap smear tests, as well as a need to advocate for an increase in the availability and affordability of immunization facilities across the country.The level of awareness is low, so prior to the introduction of HPV vaccine in the national immunization scheme, there should be more awareness created; this should involve not only health workers but the media as well as other stakeholders at the grassroots. This would increase the acceptability of the vaccine and hence the success of immunization program .Information is power, so there is need to reorientate Nigerian women on the need to take the Pap smear test in order to know their status as this is very important for a successful nationwide immunization program. If more women take the Pap smear test and know their status, then we might have a true estimate of the prevalence levels of cervical cancer and this will help stakeholders in decision making. It is necessary however to reemphasize the efficacy of the available HPV vaccines as this would increase the willingness to take the vaccines.


In Germany, immunization against human papillomaviruses (HPV) is free of charge for all females aged 12 to 17 years. Since HPV infection rates rise soon after first intercourse, immunization against HPV should be completed before sexual debut. Knowledge of country-specific data on age at first intercourse and related risk factors is important to optimize prevention of HPV and other sexually transmitted infections. Therefore, the primary aim of this study was to describe sexual behavior in young women in Germany. Secondary aims were to identify factors that are (i) associated with younger age at first intercourse and (ii) with HPV vaccine uptake.Between 2010 and 2012, we conducted a cross-sectional study among randomly selected women aged 20 to 25 years in Germany. We used a structured, self-administered questionnaire to collect sociodemographic data, information on sexual habits such as age at first intercourse, and information on HPV vaccine uptake. We used univariate and multivariate logistic regression analyses to identify factors associated with younger age at first intercourse and with HPV vaccine uptake.A total of 823 women (response rate: 14.2%) participated, 785 (95.4%) of which reported having had intercourse already. 70% of these women experienced first intercourse before the age of 18 years. However, less than 5% were younger than 14 years at sexual debut. Younger age at first intercourse was independently associated with a higher number of sexual partners, smoking, and past pregnancies. HPV vaccine uptake was associated with higher education, whereas smoking and a migrant background reduced the chance of being vaccinated.In Germany, only a small proportion of women experienced first intercourse before the age of 14 years. Younger age at first intercourse was associated with behavior that might increase the risk of HPV infections or other sexually transmitted infections. Therefore, to optimize the HPV vaccination strategy, HPV vaccination series should be completed before the age of 14 years in Germany. Human papillomaviruses (HPV) are the most common sexually transmitted viruses , with a Several risk factors for HPV transmission have been identified so far, with high-risk sexual behavior, such as younger age at first sexual intercourse , 7 and hSince national data on sexual behavior are important to optimize prevention strategies against sexually transmitted infections, the primary aim of this study was to describe sexual behavior in young women in Germany. Secondary aims were to identify factors that are associated (i) with younger age at first intercourse and (ii) with HPV vaccine uptake.This study was nested in a study designed to determine HPV prevalence in Germany via home-based self-sampling . BrieflyWith a structured, self-administered questionnaire in the German language we obtained information on sociodemographic factors, data on sexual behavior (such as age at first intercourse and number of lifetime and recent (i.e. in the previous 12 months) sexual partners), medical and smoking history, contraceptive use, past pregnancies, past delivery of a child, as well as HPV-vaccine uptake (time of vaccination and number of doses). The questionnaire has been developed by the Robert Koch Institute and was previously used in another study . DetailsWe calculated proportions on young age at first intercourse, contraceptive use and past pregnancies with corresponding 95% confidence intervals (95% CI). We evaluated the lifetime number of sexual partners by estimating the median, range and interquartile bounds (IQB). For comparison of proportions of categorical variables, we used chi-squared test and Fisher’s exact test, and Student’s t-test for numerical variables. Differences between three age groups  were assessed by the Kruskal-Wallis-test for categorical variables and by analysis of variance (ANOVA) for continuous variables.The age cut-off for â€˜young’ age at first intercourse was defined as the lower quartile (age ≤ 14) and a â€˜high’ number of lifetime sexual partners was defined as the upper quartile (≥ 7 partners) in the respective variable distribution. For assessing factors associated with HPV vaccine uptake, women with at least one HPV vaccine dose were defined as “vaccinated”.In order to identify factors associated with (i) young age at first intercourse and (ii) HPV vaccine uptake, we conducted univariate analyses for all binary or categorical exposure variables and calculated odds ratios (OR) and 95% confidence intervals (95% CI). We then used multivariate logistic regression analysis and included variables with a p-value <0.1 in univariate analysis. In addition, we included important potential confounding factors on the basis of the current literature. Two-sided hypothesis tests were performed and a p-value of less than 0.05 was considered as statistically significant. Missing data were treated as such and not imputed. We used the statistical software package STATA, version 11 .We used data of the German Federal Statistical Office (as of 31 December 2011 ) to compOverall, 823 out of 5805 women with a valid postal address 14.2%) completed the questionnaire and were included in the analysis  had experienced sexual debut at the time of the study. More than half of the women were 16 years or older at sexual debut, whereas only 4.6%  were younger than 14 years Table . 73.5%  of sexually active women reported that they experienced first intercourse before or at age of 14 years  women reported usage of any type of contraception, with the contraceptive pill and condoms being the most common ones  with information on HPV vaccination status received at least one dose of an HPV vaccine and 206  received the full course. Statistically, vaccinated women were significantly younger than unvaccinated women  vs. 23.0 years, (SD = 1.4), p-value <0.01). Fifty-six  out of 186 women received the first dose of vaccination before their first intercourse. Nineteen  out of 538 unvaccinated women reported that they were still planning to receive the HPV vaccine. All of these women, except one, already had had their sexual debut.In multivariate analysis HPV vaccine uptake was independently associated with younger age and a higher educational status. Vice versa, active smoking and migrant background were associated with a lower chance of being vaccinated Table .Table 4In this study, more than 70% of women experienced their first intercourse before age of 17, but less than 5% before age of 14. Factors strongly associated with younger age at first intercourse were a high number (≥ 7) of sexual partners during lifetime, current smoking and a past pregnancy. HPV vaccine uptake was higher in women with higher educational status, whereas uptake was lower in smokers and in women with a migrant background.National data about sexual behavior in young adults is scarce in Germany. To optimize prevention strategies against sexually transmitted infections, these data is urgently needed. Our results on age at first intercourse are comparable with data obtained in other studies: Crochard et al. reported results from seven European countries where the majority of young women experienced sexual debut between 15 and 18 years (1st to 3rd quartile); less than 5% of girls in this study (4.6% in our study) were younger than 14 years . In a laThe use of contraceptives was very common in our study population, with the contraceptive pill and condoms being the most popular ones. 80% of participants reported that they would use condoms regularly during one-night stands. These findings might be a positive effect of an ongoing public campaign about condom use and prevention of sexually transmitted infections in Germany . HoweverThe fact that younger age at first intercourse is associated with risky  behavior is corroborated by other studies that have shown that early intercourse is associated with higher numbers of sexual partners, bisexual experience, smoking and early pregnancy later in life –25. In aIn line with current literature, HPV vaccine uptake was associated with higher education –31. InteThe findings of our survey have important implications for the target population for HPV vaccination in Germany: since rates of HPV rise soon after first intercourse and since HPV vaccination is less effective in infected individuals , 4, HPV Some limitations of our study need to be taken into account. First, the response rate was low (14%) with the potential of introducing participation bias. Since this study was implemented in a population based survey to evaluate HPV prevalence using a self-sampling method, the response rate was comparable to a similar study . HoweverAlthough hampered by its low response rate, our study indicate that in Germany the majority of young women experienced first sexual intercourse before the age of 18 years, but only a small proportion before the age of 14 years. Usage of the contraceptive pill and condoms was common in our study population. However, condom use may imply lesser protection against HPV than against other sexually transmitted infections, therefore HPV vaccination needs to be promoted as well. Since younger age at first intercourse was associated with behavior that might increase the risk of HPV infections, HPV vaccination series should be completed before the age of 14 years in Germany.


The diagnostic performance of the widely-used Cervista HPV HR test was compared to the Hybrid Capture 2 (HC2) test in a Dutch population-based cervical cancer screening program. In 900 scrapings of women with normal cytomorphology, specificity was 90% (95%CI: 87.84–91.87) for the Cervista HPV HR test and 96% (95%CI: 94.76–97.37) for the HC2 test with 93% agreement between both tests . The sensitivity for CIN2+ using 65 scrapings of women with histological-confirmed CIN2+ was 91% (95%CI: 80.97–96.51) for the Cervista HPV HR test and 92% (95%CI: 82.94–97.43) for the HC2 test with 95% agreement between both tests . Fifty-seven of 60 HC2 negative/Cervista positive cases tested HPV-negative with PCR-based HPV assays; of these cases 56% were defined as Cervista triple-positive with FOZ values in all 3 mixes higher than the second cut-off of 1.93 (as set by manufacturer). By setting this cut-off at 5.0, specificity improved significantly without affecting sensitivity. External validation of this new cut-off at 5.0 in triple-positive scrapings of women selected from the SHENCCASTII database revealed that 22/24 histological normal cases now tested HPV-negative in the Cervista HPV HR test, while CIN2+ lesions remained HPV-positive. The intra-laboratory reproducibility of the Cervista HPV HR test (n = 510) showed a concordance of 92% and 93% for cut-off 1.93 and 5.0  and inter-laboratory agreement of the Cervista HPV HR test was 90% and 93% for cut-off 1.93 and 5.0 . In conclusion, the specificity of the Cervista HPV HR test could be improved significantly by increasing the second cut-off from 1.93 to 5.0, without affecting the sensitivity of the test in a population-based screening setting. Population-based screening programs have led to a significant reduction of the incidence and mortality from cervical cancer Cervical carcinogenesis is strongly associated with high-risk human papillomavirus (hrHPV). Persistent infection with hrHPV can result in CIN lesions and neoplastic progression. Testing for hrHPV in cervical scrapings shows high sensitivity (94–97%) to detect CIN2+ lesions. However specificity, especially in younger women, is around 6% lower than with cytology The first 2 and mostly used FDA approved HPV tests are the Hybrid Capture 2 (HC2) and the Cervista HPV HR assay Several studies analyzed the sensitivity and specificity for either the Cervista HPV HR test or the HC2 test To compare the specificity of the Cervista HPV HR and HC2 test, 900 cytomorphological normal cervical scrapings (NILM) collected in PreservCyt of women between the ages of 30–60 years were randomly selected from the routine Dutch population-based screening program. Since women without cytomorphological abnormalities are not referred to the hospital for colposcopy, histology is not available for this group. To compare the specificity we only included women who also had a normal cervical scraping at the previous population-based screening 5 years prior and are therefore with the smallest chance of having an undetected CIN2+ lesion. Women with a history of (pre)malignant cervical lesions, abnormal cervical smears or any surgery in the area of the cervix as well as HIV-seropositive or pregnant women were excluded. Study-specific, uniquely numbered samples with more than 12 ml residual PreservCyt solution were collected to perform Cervista HPV HR and HC2 testing.To compare the sensitivity of the Cervista HPV HR and HC2 test, we randomly selected scrapings of women referred to the University Medical Center with abnormal cytology (>BMD) during routine population-based screening. All 65 women included had histological confirmed CIN2+ lesions. Since a considerable number of CIN2+ lesions are missed by routine cytomorphological examination The Cervista HPV HR test  is a qualitative test detecting 14 hrHPV types  The HC2 test is routinely used in our (ISO15189 certified) laboratory. The HC2 test is clinically validated and FDA-approved and detects 13 hrHPV types . The HC2 test has previously been described extensively and results are interpreted as a ratio of relative light units (RLU/CO) to the positive control specimen Extra assay All 965 specimens were tested both with the Cervista HPV HR test and HC2 test. Cases with discordant results were retested for the presence of hrHPV using PCR-based HPV detection assays. The HPV-L1 consensus GP5+/6+ PCR was performed as previously described To evaluate the effect of different second threshold values for the Cervista HPV HR test we used an external patient group with histological-confirmed normal and abnormal tissue. In silico analysis of the data available from the Shenzhen Cervical Cancer Screening Trial II (SHENCCASTII) For intra- and inter-laboratory variability of the Cervista HPV HR test, 510 scrapings were selected. Seventy samples were selected from the 900 cytomorphological normal women from the population-based screening program. In the Netherlands women diagnosed with ASCUS or low-grade SIL are retested 6 months later using both cytomorphological assessment as well as hrHPV testing according the Dutch guidelines Clinicopathological data of the patients such as age, medical history, cytological and histological results were retrieved from the hospital database and the patient's pathology report, and entered into a separate, anonymous, password protected database. Protection of patient identity was guaranteed by assigning study-specific unique patient numbers ensuring that data is not traceable to individual patients. Codes were only known to one data manager. Therefore, according to the Dutch Law for Human Medical Research (WMO), no consent was necessary from the medical ethics committee for this study.Statistical analysis was performed using SPSS software, version 18 . The number of cases needed for the comparison of the specificity, sensitivity, inter- and intra laboratory variability were calculated from the power analysis described by the international guidelines for HPV DNA tests for primary cervical screening in women 30 years and older by Meijer et al In scrapings of 65 women with histological confirmed CIN2+ lesions, sensitivity of the Cervista HPV HR test was 91% (95%CI: 80.97–96.51), for the HC2 test this was 92% (95%CI: 82.94–97.43) . CompariThe specificity of the Cervista HPV HR and the HC2 test in 900 cytomorphological normal cervical scrapings was 90% (95%CI: 87.84–91.87) and 96% (95%CI: 94.76–97.37), respectively . CompariOf the total 965 cases, 66 cases showed discordant results when comparing the Cervista HPV HR and HC2 test. One HC2-negative case, showed a low gDNA outcome in the Cervista HPV HR test. Re-testing of this sample with the Cervista HPV HR test again showed a low gDNA outcome. This could be a false-negative result in the HC2 test, because of insufficient cells in the sample. Cytological examination confirmed low number of cells in the sample.Five cases were HC2 positive and Cervista negative . Using tMost discordant cases (n = 60) reported HC2 negative/Cervista positive cases. The GP5+/6+ PCR was performed to determine if hrHPV DNA was in fact present in each of these samples . Three cIn the Cervista HPV HR test, cases with a HPV FOZ ratio <1.525 are considered HPV-negative except those cases where all three mixes have a HPV FOZ >1.93, referred to as triple-positive cases We noticed that the lowest HPV FOZ mix value in the Cervista triple-positive cases varied between 1.95 and 4.60; only one case showed higher HPV FOZ mix values (6.58/6.83/6.22) . Since tTo determine the best HPV FOZ second cut-off value for discriminating between true-negative and true-positive HPV cases, we included all observed triple positive cases in this study. In addition to the 32 discordant triple-positive cases from the cytomorphological normal scrapings, in our whole cohort of 1405 samples (including samples used for intra- and inter-laboratory testing), we observed 31 additional Cervista triple-positive cases including scrapings with abnormal cytomorphology and/or HPV-positivity . In thisTo evaluate the effect of different second cut-off values, we re-calculated sensitivity and specificity of the Cervista HPV HR test on our series of 900 women with cytomorphological negative scrapings and on the 65 scrapings associated with histological proven CIN2+ lesions . IncreasHowever, improving sensitivity and specificity of the Cervista HPV HR test is not solely dependent on the HPV status of the scraping, but primarily by the presence of histological confirmed CIN2+ lesions. By law in most countries, including the Netherlands, no colposcopy is performed on women with normal cytomorphology. Consequently, in our series of 63 Cervista triple-positive cases only from 6 women histology was available. In five cases CIN2 or CIN3 lesions were detected and all showed a second cut-off above 5.0 . From thTo evaluate the effect of a different second cut-off for the Cervista HPV HR test on patients with histological diagnosis, we analyzed in silico an independent external cohort from the SHENCCASTII dataset. In the SHENCCASTII study women were referred for colposcopy if they were positive on any of the HPV tests performed. In addition, every HPV positive woman referred to colposcopy had a minimum of 5 cervical biopsies To ensure a reliable performance of the Cervista HPV HR test in clinical practice, we validated the intra-laboratory reproducibility and inter-laboratory agreement in time. The intra laboratory reproducibility (n = 510) showed a concordance of 92% and 93% with a kappa of 0.83 and 0.84 for cut-off 1.93 and 5.0 respectively (p<0.001) . The intThe aim of the present study was to compare the diagnostic performance of the Cervista HPV HR test versus the HC2 test on the same cervical scrapings from women participating in the routine Dutch population-based screening program. The sensitivity for detecting CIN2+ lesions in a cohort of women referred with an abnormal scraping was comparable between the Cervista HPV HR test (91%) and the HC2 assay (92%). The specificity in a cohort of 900 women with repeated normal cytomorphology was 96% for the HC2 test versus 90% in the Cervista HPV HR test. However, by adjusting the second threshold to 5.0 we were able to improve the specificity of the Cervista HPV HR test to 93% without affecting the sensitivity. Furthermore, reproducibility is an essential requirement of any screening test and has not yet been described before for the Cervista HPV HR test. In this study, we showed high intra-laboratory reproducibility and high inter-laboratory agreement, which even improved further by using the second threshold of 5.0.The selection of our samples was based on the international guidelines for HPV DNA testing in primary cervical cancer screening in women 30 years and older by Meijer et al One of the limitations of the current FDA-approved HC2 test is the lack of an internal control. Without an internal control a negative HPV result could be due to the fact that the sample was hypocellular, the sample contained a substance that inhibited the signal amplification reaction or was processed incorrectly. The use of an internal control in the Cervista HPV HR test protects against a false-negative results due to these problems. In this study only 1 of the 965 scrapings gave a negative HC2-result whereas the internal control of the Cervista HPV HR test indicated that the sample had too few cells for reliable HPV-testing. Other studies comparing HC2 with the Cervista HPV HR test showed that the false-negative rate of the HC2 test due to insufficient input of cells is approximately 3.2–4.1% In the cytological negative cases, 60 HC2-negative scrapings were positive by the Cervista HPV HR test. The GP5+/6+ PCR revealed only 3 HPV-positive cases suggesting a Cervista HPV HR false-positivity rate of 95% (57/60). Remarkably, of the 57 HPV-negative/Cervista-positive cases, 56% (32/57) were Cervista triple-positive defined as FOZ-ratio negative (<1.525) but considered HPV-positive because all three mixes had FOZ value higher than the second cut-off 1.93 (default setting). Comparison of the HC2-negative/Cervista triple-positive cases with the HC2-positive/Cervista-triple-positive cases revealed that changing the second cut-off to 5.0 improved the specificity significantly . While aThis new second cut-off of 5.0 for the Cervista HPV HR test was evaluated in an independent external cohort (SHENCCASTII) Improving specificity is an important issue when it comes to primary population-based HPV screening. Since the prevalence of CIN2+ lesions in a population-based screening setting is relatively low, even small changes in clinical specificity of the hrHPV test will have enormous effects on the number of unnecessary referrals to the gynecologist and associated costs.In our series of 900 cases, we observed 32 (3.6%) triple-positive cases with normal cytology (NILM). Literature shows no other studies using the Cervista HPV HR test that elaborates on Cervista triple-positive cases. In the reference-lab at the department of Pathology, Brugge Belgium that routinely uses the Cervista HPV HR test as a triage test in women with ASCUS, the prevalence of triple-positive cases (default setting at 1.93) for 2010 until 2011 was, 3.7% (73/1974 cases) (unpublished data). These data illustrate that triple-positive cases are described both in NILM and ASCUS at a rate of ∼3.6%.Re-testing the scrapings of the 32 triple-positive cases (at cut-off 1.93) revealed again triple-positivity in 56% (18/32). The inter-laboratory agreement showed that 7 of the 22 triple-positive cases detected in lab 1 were also triple-positive in lab 2. This suggests that the positivity did not occur occasionally but is associated with the sample. The triple-positive result is partly due to the presence of various different HPV types in the sample. However, since in almost all cases with threshold <5.0 no HPV could be detected with HC2 and highly sensitive PCR-based consensus HPV tests, an HPV-unrelated factor might result in the increased FOZ value in all three mixes. In our series of 900 women with NILM scrapings no association was observed with age when comparing the triple-positive group  with the total group . Although the use of vaginal anti-fungal creams or contraceptive jelly (not available from this study) did not seem to affect the positivity rate of the Cervista HPV HR test The reproducibility of the Cervista HPV HR test has not been described before. In this study we reported a high intra-laboratory reproducibility  and high inter-laboratory agreement . Using the second threshold of 5.0 the intra-laboratory reproducibility improved to 93% (kappa 0.84) and the inter-laboratory agreement to 93% (kappa 0.85).In conclusion, the performance to detect hrHPV using the Cervista HPV HR test is comparable to the HC2 test regarding the sensitivity for detecting CIN2+ lesions. Data from this study in addition to external validation using the SHENCCASTII dataset demonstrate that increasing the second cut-off from default setting (1.93) to 5.0 will significantly improve the specificity of the Cervista HPV HR test.Table S1Characterization of discordant cases using the analytical-sensitive GP5+/6+ PCR, HPV-typing with the INNO-LiPA and Cervista retesting.(DOC)Click here for additional data file.Table S2Summary of the 32 Cervista triple-positive cases using the analytical-sensitive GP5+/6+ PCR and HPV-typing with INNO-LiPA analysis.(DOC)Click here for additional data file.Table S3Summary of the 31 additional Cervista triple-positive cases using the analytical-sensitive GP5+/6+ HPV PCR and HPV-typing with INNO-LiPA analysis.(DOC)Click here for additional data file.Table S4SHENCCAST data of 28 triple positive cases with available histological results.(DOC)Click here for additional data file.


CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection  similar to the subset of four CDKN2A-specific CpG loci  with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products.Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level . From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four  Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease associated with improved survival and response to therapy, and carries distinct pathologic and molecular characteristics, including positive immunostaining for the p16 protein CDKN2A gene, which codes for both the p16 protein and its alternate reading frame (p14ARF) is frequently affected by epigenetic gene silencing and deletion in other head and neck cancers, and both have been established as known tumor suppressors in head and neck carcinogenesis HPV detected in OPSCC tumors  is frequently integrated and transcriptionally active, with the majority of HPV+ tumors expressing the viral oncoproteins E6 and E7 CDKN2A transcripts that is separate from tumor suppressor proteins p53 and pRb, and may involve direct epigenetic regulation of ARF transcription.As in other cancers, multiple genetic and epigenetic events have been implicated in head and neck carcinogenesis All patients consented to participation in this study under protocols approved by the Institutional Review Boards of the participating institutions. All primary tumor and histologically normal adjacent tissues were snap-frozen in liquid nitrogen within 30 min of surgical resection or biopsy and kept at −80°C until further processing. Adjacent mucosa was taken at a grossly unremarkable site away from the tumor identified by the surgeon  intraoperatively or by the pathologist (resection specimen) when processing the specimen for frozen section or final diagnosis as described previously β-globin gene. In addition, HPV-positive and negative control samples were included in each PCR using DNA from a cervical carcinoma cell line (SiHa) with two copies of HPV16 per cell and a HPV-negative cell line HepG2.All patient samples were tested for HPV infection using multiple testing methods. The presence of HPV DNA was assayed by MY09/11-PCR (degenerate and type-specific HPV16-L1 fragments) utilizing AmpliTaq Gold  and dot-blot hybridization on fresh frozen specimens as described previously E6 and E7 oncogenes GAPDH) as an RNA control . Assays were performed in triplicate on the StepOnePlus™ Real-Time PCR system. Only patients testing positive by all PCR methods were classified as HPV positive . RNA extractions were tested for HPV16 transcripts using oligonucleotide primers that span the 204–525 base-pair regions of the P-value of 0.03 as the cutoff for sample inclusion Genome-wide profiling of DNA methylation in oropharyngeal primary tumor samples was carried out as described previously μm thickness and were placed on the positively charged slides. Slides were placed in a 60°C oven for an hour. Sections were deparaffinized and rehydrated through a series of xylene and graded alcohols . Endogenous peroxidase was blocked in 3% H2O2 for 10 min. Antigen retrieval was performed by placing the slides in an Oyster vegetable steamer with Dako Target Retrieval Solution . The immunoperoxidase staining procedure was performed in an automatic slide stainer (Dako Autostainer Plus) using the Dako universal staining system. The primary mouse monoclonal antibody p16  was applied in a dilution of 1:50 for 30 min at room temperature. After primary antibody, the slides are washed buffered solution, a secondary antibody  was applied for 30 min. DAB Substrate kit  was used with 3,3′-diaminobenzidine as chromogen. Slides were counterstained with Surgipath Hematoxylin , dehydrated through graded alcohols, cleared in xylene, and cover-slipped with cytoseal 60 .Initial measurements of p16 protein positivity in OPSCC tumors were assayed using standard immunohistochemical techniques. All tissues were routinely fixed in 10% buffered formalin and embedded in paraffin. The paraffin sections were cut at 4-CDKN2A mRNA splice variants in oropharyngeal tumors and normal tissues were carried out using the real-time quantitative PCR measurements (qRT-PCR) with the Applied Biosystems®  TaqMan® RNA-to-Ct™ 1-Step Kit. Four primers were used to distinguish the mRNA transcripts: Hs00924091 (p14 ARF), Hs00923893 (p12), Hs02902543 (p16 gamma and INK4A), and Hs00923894 . Sample cycle threshold (Ct) values were normalized to the GAPDH control, and the samples with no mRNA detection were set as the baseline. Relative Ct values were converted to a relative expression (log2-Ct). To graph the expression ratios, the log2 was used for each tumor:normal pair. To graph the HPV+ tumor samples only, the relative expression (log2) was used. All samples were examined in triplicates and the mean value taken.Subsequently, measurements of individual CDKN2A beadchip measurements was carried out by bisulfite sequencing. Genomic DNA was bisulfite converted as described above. Two regions located within a downstream CpG island that is shared by ARF and INK4A were targeted using this approach. Primer sequences, PCR conditions, and amplicon sizes are shown in Validation of the M  for the 25,593 CpG loci on the beadchip that passed initial quality control . To identify a training sample set, we overlayed HPV data, based on both DNA and E6 mRNA PCR results, with p16 protein tumor expression by immunohistochemistry. Samples showing concordant results were selected including 13 OPSCC with strong HPV DNA and RNA positivity and p16 protein staining, and 10 HPV−, p16- OPSCC. P16 protein immunohistochemistry results were initially missing for 10 OPSCC cases included in our previous analyses previously P-value as well as the magnitude of difference in methylation changes between the two groups. From the training set, we identified 23 CpG loci with a P-value of less than 1 × 10−4, of which 22 showed at least a 1.5-fold difference in average ΔM between HPV+/p16+ and HPV−/p16− cases. A heatmap for these 22 highest ranked CpG loci was then generated using the Cluster program with median centering of CpG loci, hierarchical clustering, and Euclidian distance For each tumor:normal tissue pair, we calculated ΔCDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites.Differences in methylation and expression between HPV+ and HPV− cases were assessed using parametric and nonparametric univariate tests. Nonparametric receiver operating characteristic (ROC) plots reflecting combined test sensitivity and specificity were generated to illustrate the degree of association with HPV and p16 status in OPSCC, including an additional 22 cases not tested in the training set. Cumulative methylation scores were generated based on tumor-to-normal methylation (ΔM) levels across the 22 CpG panel and separately for the four https://tcga-data.nci.nih.gov/tcga) (Tablehttps://tcga-data.nci.nih.gov/tcga/tcgaCancerDetails.jsp?diseaseType=HNSC&diseaseName=Head and Neck squamous cell carcinoma). Methods for sequencing and data processing of RNA using the RNA-Seq protocol have been previously described for TCGA https://cghub.ucsc.edu/docs/tcga/UNC_mRNAseq_summary.pdf). Expression results were presented as normalized read counts. DNA methylation and gene expression data were not normalized or processed by our group in any way prior to analysis.We validated the performance of the methylation panel in an external cohort of 33 HPV+ and HPV− OPSCC cases from TCGA project (ga) Table. Tumor mDNMT1), the major eukaryotic DNA methyltranferase   between tumors and adjacent normal mucosa compared to HPV− oropharyngeal, oral cavity, and laryngeal cancers 001) Fig. supportiCDKN2A-specific loci located downstream of the p16(INK4A) transcription start site, two loci located within the CpG island of the GALR1 gene, and one associated with the PPP1R3D gene.By analyzing genome-wide DNA methylation between tumor and matched normal tissue samples for a subset of OPSCC patients, 22 CpG loci were identified that showed a statistically significant difference in methylation (ΔM) when comparing HPV+ to HPV− OPSCC Fig.. Among tP < 0.00005), which extended to both subsets of CpG sites within the panel that showed either hypermethylation (P < 0.00005) or hypomethylation (P < 0.00005) in HPV+ versus HPV− OPSCC cases. We also observed significant correlations between DNA methylation levels and HPV16 oncogene expression levels measured by qRT-PCR for both the E6  and E7 oncogenes .We then extended our analyses to an additional 22 OPSCC patients, including: 9 new HPV−/p16- tumors plus 8 ambiguous cases (1 HPV DNA-/p16+ tumor and 7 tumors with indeterminate or low levels of HPV DNA and RNA), and 4 HPV16 DNA/RNA++/p16+ tumors plus 1 case positive for a related high-risk type (HPV35) and p16. We observed significant differences in overall DNA methylation levels between HPV+ and HPV− OPSCC cases for the 22 CpG panel  was also significant  and statistically equivalent to the full 22 CpG signature (P = 0.084).We generated ROC plots to illustrate the degree of association with overall HPV detection in OPSCC, including an additional 22 cases not tested in the training set as well as the HPV35+ case Fig.. As reflCDKN2A gene: five in the CpG island (9:21958106-21958899) located downstream of the p16(INK4A) transcription start site , of which four were identified in our HPV panel, and another four located within the CpG island of the CDKN2A promoter region . We compared the tumor-to-normal methylation (ΔM) ratios for each of the nine individual loci to p16 protein expression on histology using ROC analyses  with AUC ranging from 0.79 to 0.87. In contrast, those located within the CDKN2A promoter region showed no association or an inverse association with AUC ranging from 0.49 to 0.19.The Illumina HumanMethylation27 BeadChip contains nine CpG sites associated with the yses Fig.. CpG sitCDKN2A (expression probe ILMN_1717714), GPSM1 (ILMN_1709307), FLJ33790/KLHL35 (ILMN_1693471), and SYCP2 (probes ILMN_2095704 and ILMN_1765770) CDKN2A, KLHL35 and SYCP2, there were significant positive correlations between observed levels of DNA methylation and expression of the corresponding genes. In particular, methylation of the four CpG loci located in the CpG island (9:21958106-21958899) downstream of the p16(INK4A) and p14(ARF) transcriptional start sites , were significantly correlated with expression of the CDKN2A gene as measured by probe ILMN_1717714  comparing beadchip probe fluorescence intensities for tumor and normal tissue mRNA samples collected on the same OPSCC patients, including CDKN2A region is highly complex, coding for mRNA transcripts corresponding to multiple splice variants, including p14(ARF), p12, p16(gamma), and p16(INK4A) , as well as five to seven additional CpG loci located within the same subregions. A total of 17 OPSCC tumor samples from the training set (used to develop the methylation panel) and 5 OPSCC tumors from the test set were sequenced. For both subregions, bisulfite sequencing revealed an increase in DNA methylation in HPV+ compared to HPV− OPSCC tumors  Fig.. We evalmors Fig.. This paCDKN2A, RNA expression of p16(INK4A), p16(gamma), p14(ARF), and p12 splice variants in OPSCC tumors and adjacent normal tissues were measured by qRT-PCR , we observed a significant increase in p14(ARF) expression  in HPV+ compared to HPV− OPSCC (P = 0.004).In order to examine which transcript variants were specifically affected by DNA methylation in -PCR Fig.. AlthougP = 0.007 and P = 0.006, respectively), but little difference in overall expression of p16(INK4A) between tumor and adjacent normal samples within the same subject for either HPV+ or HPV− OPSCC cases. In contrast, we observed a significant increase in p14(ARF) expression in HPV+ tumors compared to HPV− tumors (P = 0.001), whereas p14(ARF) expression levels in the adjacent normal tissues from HPV+ and HPV− cases were not statistically significant different (P = 0.3). Therefore, the changes in DNA methylation observed within the downstream CpG island of the CDKN2A locus are correlated with a tumor-specific increase in p14(ARF) mRNA expression. Further studies are required to detail the mechanism by which the downstream region regulates the expression of these two mRNA transcripts.Since these differences in the expression ratios could be attributed to changes in either tumor expression, normal tissue expression, or both, we repeated the analysis of gene expression looking at these tissues separately Fig.. When anCDKN2A were not present in the Illumina platform used to generate TCGA data sets, we confirmed the association between methylation of the downstream region of CDKN2A and HPV status by using an alternate CpG locus in the same CDKN2A downstream region not present in our panel but found in the TCGA data set (TableCDKN2A downstream locus (cg12840719) showed significantly higher methylation levels  in HPV+ compared to HPV− OPSCC cases from TCGA , whereas median p16(INK4) expression counts increased from 23 in HPV− cases to 906 in HPV+ cases (P < 0.001). In summary, we observed that differential DNA methylation of CpG loci from the same regions identified in our novel panel, and in particular the CDKN2A-specific downstream region, was significantly associated with HPV detection in OPSCC in two independent cohorts. Moreover, these changes correlated with an increase in expression of the CDKN2A splice variant p14(ARF).Given the limited availability of DNA methylation data related to normal adjacent mucosa from TCGA, we applied our methylation panel to primary OPSCC tumor samples alone with known HPV status . Hierarchical clustering using tumor DNA methylation levels revealed two clusters that segregated samples by HPV status Fig.. Since tDNMT1, the major mammalian DNA methyltransferase, in HPV+ tumors. Further analysis identified a subset of 22 CpG that were significantly associated with detection of HPV in OPSCC.The experiments described here assess DNA methylation and gene expression difference in OPSCC patients characterized by HPV DNA and RNA detection and p16 protein expression. Our previous work using the Illumina HumanMethylation27 beadchip established a significant increase in DNA methylation in HPV+ oropharyngeal cases as compared to HPV− cases CDKN2A locus. One of the CDKN2A products (the p16INK4A protein) is commonly used as a clinical diagnostic, and has been advocated as a surrogate marker for HPV in OPSCC CDKN2A, have been observed in head and neck cancer cell lines, as well as in cervical cancers CDKN2A locus, which is normally repressed in cycling cells by EZH2 via H3K27 methylation, and frequently undergoes DNA hypermethylation in cancer, is often overexpressed in HPV+ carcinoma CDKN2A region, within nonoropharyngeal head and neck cancer cases is ongoing.Other studies have shown that HPV+ OPSCC tumors have higher levels of gene promoter methylation compared with HPV− tumors It is known that there are both protein mediated and epigenetic mechanisms that regulate the transcription of p14(ARF). For example, the HPV16 oncoprotein E7 causes the release of E2F-1 from its repressor Rb CDKN2A gene as a potential mechanism for increased p16 expression in HPV+ OPSCC tumors. However, the mechanism by which the hypermethylated downstream CpG island region leads to altered expression of transcripts originating from the CDKN2A locus is not clear. Our assessment of the region reveals a large number of Sp1 binding sites, a pattern observed previously in downstream CpG island regions involved in transcription initiation, particularly those of some long noncoding RNAs TGF-β1 promoter by HPV16 E6 and E7 in cervical cancer CDKN2A transcripts ) in OPSCC.Overall, the results implicate hypermethylation of these downstream CpG sites in the A number of limitations should be considered when interpreting these results. While the 22 CpG loci panel best distinguished HPV infection in this cohort, we are cognizant of the fact that host genetic events such as mutations observed in p53 and p16 (possibly caused by tobacco carcinogens) may confound associations between HPV infection, panel DNA methylation, p16 expression, and clinical outcome. The specific genotyping of this population as it relates to those events will be the focus of future work by our group. While the agreement in associations seen with TCGA supports our findings, it should be noted that our study sample was small and represented a subset of cases from a larger cancer patient cohort; its probable additional methylation changes may better resolve ambiguous primary tumors for which there is discordance between HPV assays. It should also be pointed out that our study was focused specifically on host genome DNA methylation. DNA methylation of the HPV viral genome will be the focus of a future study. And finally, it is not clear whether viral proteins are directly involved in the methylation of the CpG loci downstream of the CDKN2A gene promoter, or whether this relies on other ancillary proteins.DNMT1 expression compared to HPV− cases. Further whole-genome DNA methylation analysis identified a subset of 22 CpG that were significantly associated with HPV detection in OPSCC in two independent cohorts. Within this panel, we identified a hypermethylated region downstream of the CDKN2A gene that was associated with p16 protein expression in OPSCC and is correlated with an increase in tumor-specific expression of the CDKN2A transcript variant p14(ARF).In summary, we observed that HPV-positive OPSCC has significantly increased