Overall HPVpositivity detected by AmpliLute was 57.3% (30.4% as single and 27% as multiple infections) in contrast to MagNA 54.5% . Discrepant results observed in 25 cases: 11 MagNA(−)/AmpliLute(+), 10 of which had positive histology; 5 MagNA(+)/AmpliLute(−) with negative histology; 8 MagNA(+)/AmpliLute(+): in 7 of which AmpliLute detected extra HPV genotypes and 1 MagNA/AmpliLute(+) with positive histology. Both methods performed well when compared against cytological (area under curve (AUC) of AmpliLute 0.712 versus 0.672 of MagNA) and histological diagnoses (AUC of AmpliLute 0.935 versus 0.877 of MagNA), with AmpliLute showing a slightly predominance over MagNA. However, higher sensitivities, specificities, and positive/negative predictive values were obtained by AmpliLute.Nucleic acids of human papillomavirus (HPV) isolated by manual extraction method (AmpliLute) and automated MagNA pure system were compared and evaluated with cytohistological findings in 253 women. The concordance level between AmpliLute and MagNA was very good 93.3% ( It is now well established and widely accepted that virtually all cervical cancer and its immediate precancerous lesions arise from persisting cervical infection by some highly oncogenic HPV genotypes , 2. The Fifteen HPV genotypes have been to date classified as high-risk (HR) types , 3 as probably HR  and 12 as low-risk (LR)  [The majority of HPV infections are transient, but persistence of an HR HPV is a significant risk factor for the development of cervical cancer. This occurs only in a minority of infections and is an unpredicted event. It could be a genetic predisposition with an inadequate immune response and/or possible uncontrolled reaction with tumor suppressor genes , 6. Type-specific detection of HPV is increasingly important for monitoring the impact of HPV vaccine implementation and as a tool for cervical cancer screening. As a consequence, standardization of laboratory methods for HPV detection and typing is important. The commercial HPV detection kits: Hybrid Capture II , Cervista HPV HR  and Cervista 16/18 tests are approved by the FDA for use in routine screening of HPV. However, the above assays are unable to discriminate specific genotypes or to identify infections involving multiple genotypes and the Cervista assay detects only two HPV types (types 16 and 18).Various molecular assays for HPV detection and typing have been used in epidemiological studies, and they are based on two different technologies: (1) hybridization-based assays  and (2) PCR-based tests , Linear Arrays HPV test , CLART HPV2 . The advantages and disadvantages of these two basically different methodologies have been extensively discussed –19. The qualitative Linear Array HPV (LA-HPV) HPV genotyping test, developed by Roche Molecular Systems offers a reliable, sensitive, and standardized approach for HPV typing in cervical specimens. It is distributed as a research use only but it has been submitted for FDA review. This test utilizes amplification of target DNA by PCR and nucleic acid hybridization for the detection of 37 types in cervical cells collected into an LBC media. This test includes four steps: specimen preparation—DNA extraction, PCR amplification, hybridization of the amplified products with specific probes and colourimetric detection on the hybrids on strip , 20–22. The objective of this study was to evaluate and compare the automated MagNA pure DNA extraction method with the AmpliLute DNA extraction method in detecting HPV DNA form ThinPrep Pap tests using the linear array (LA) HPV genotyping and detection assays and also to correlate these results to cytological and histological diagnosis.In the present study, cervical brush specimens were obtained from women aged from 17 to 70 years who attended the gynecologic outpatient clinic of “Attikon” University Hospital, Athens, Greece, for opportunistic examination, between July 2009 and May 2010. Women considered eligible for the study if they fulfilled the following criteria: (a) they agreed to undergo colposcopy and if necessary cervical biopsy and (b) there was enough residual biological material, after cytological examination, for the two molecular assays to be completed. A total of 253 women met these criteria and were enrolled in the study. This patient population does not represent the general population of women attending public screening programs. Approval from the ethics committee was obtained before inclusion.Samples of ThinPrep Pap tests were collected by means of a Brun's-like brush. The PreservCyt vials , containing the cell samples were addressed to the Department of Cytopathology of the aforementioned hospital for preparation of thin-layer slides using the ThinPrep 2000 Automated Slide Processor  according to the manufacturer's instructions. Cytological findings were interpreted according to the Bethesda classification system and were classified as follows: (a) within Normal Limits (WNL), (b) atypical squamous cells of undetermined significance (ASC-US), (c) low-grade squamous intraepithelial lesion (LSIL), and (d) high-grade squamous intraepithelial lesion (HSIL). The cytopathologists and the biologist conducting HPV testing were all blinded to the clinical profile to ensure unbiased reporting.A cervical biopsy was performed if lesions were present upon colposcopy. All histological assessments were made blinded to the HPV DNA status of the participants. The histological evaluation revealed the following categories: negative HPV, CIN 1, CIN 2, and CIN 3. In case histology showed a CIN 2 or CIN 3, the patient was referred for appropriate treatment.μL and 1 mL were generated from each clinical specimen.After slide preparation for cytological examination, the remaining PreservCyt samples were vortexed vigorously for 15 sec to maximize homogeneity and two aliquots of 250 μL aliquot was extracted by AmpliLute liquid media kit (Roche Molecular Systems) in conjunction with a QIAvac 24 plus vacuum system, according to the manufacturer's instructions in the product insert and (ii) a 1 mL aliquot was extracted by MagNA Pure LC extraction system using the DNA-I kit (blood cells high-performance protocol) (Roche Molecular Systems). Briefly, for the manual extraction, samples and HPV positive/negative controls were processed in parallel. Clinical samples were mixed by vortexing to form a homologous state, and 250 μL were removed and lysed with proteinase K solution and buffer ATL at 56°C for 30 min. The samples underwent a second incubation at 70°C for 15 min in the presence of buffer AL containing a carrier RNA. The lysate was then transferred to vacuum columns where isolation and purification of DNA was completed via washing of different solutions to bind DNA and remove other cellular materials. Extracted DNA was eluted into 120 μL of buffer AVE. Specimens and controls were immediately stored at 2°C–8°C for up to 7 days or frozen at −20°C for up to 8 weeks.DNA was isolated using two different procedures (i) a 250 μL of sterile phosphate-buffered saline, and the procedure of automated extraction was followed according to the manufacturer's instruction, using the DNA-I kit. The method is based on magnetic-bead technology with a special buffer containing chaotropic salts and proteinase K. Nucleic acids are bound to the surface of the magnetic glass particles. Cellular debris was removed by several washing steps, and the purified nucleic acids were eluted. 100 μL in volume of extracted genomic DNA product was obtained, after the magnetic beads were separated from the solution. Specimens and controls were immediately stored at 2°C–8°C for up to 7 days or frozen at −20°C for up to 8 weeks. After nucleic acid purification all samples were analyzed by LA HPV assay for HPV genotyping.For automated extraction, the samples were prepared using a modified procedure involving the centrifugation of 1 mL aliquots of the PreservCyt samples at 13000 × g for 20 min prior to discarding of the supernatant. The resulted cell pellets were resuspended into 200 β-globin gene is included in the assay to provide a control for cell adequacy, extraction, and amplification. PCR was carried out on each of the samples and controls, using the Linear Array HPV genotyping mastermix which contains: Tris buffer, potassium chloride, AmpliTaq, gold DNA polymerase , AmpErase, (uracil-N-glycosylate) enzyme , dATP, dCTP, dUTP, dGTP, dTTP, each of upstream and downstream primers (biotinylated) and β-globin primers, sodium azide, magnesium chloride, and amaranth dye. The reaction mixture contained 50 μL of HPV mastermix and 50 μL of eluted DNA. The amplification was performed on the Applied Biosystems Gold-plated 96-well GeneAmp PCR System 9700  using the following thermal profile: 2 min at 50°C, 9 min at 95°C; 30 sec at 95°C, 1 min at 55°C, 1 min at 72°C (40 cycles) with a ramp rate set at 50% followed by 5 min at 72°C and a final hold at 72°C indefinitely. PCR amplicons were immediately denatured by the addition of 100 μL of (DN) denaturation reagent and stored at 4°C for further analysis within 7 days.The LA genotyping test use a pool of biotinylated primers designed to amplify an approximately 450 bp sequence within the polymorphic L1 region of the genome of the 37 HPV genotypes. An additional 268 bp primer pair which targets the human μL of denatured amplicon were added to the linear array strips that contain multiple copies of HPV genotype-specific probes in a defined area for all 37 genotypes and the β-globin reference lines. The HPV types detected are HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51–56, 58, 59, 61, 62, 64, 66–73, 81–84, IS39, and CP6108. The biotin-labeled amplicon was bound to the strips using a hybridization buffer in a shaking waterbath at 53°C. Once bound, the strips were washed at high stringency to remove nonbound material and streptavidin-horseradish peroxidase conjugate was then added and bound to the biotin-labeled amplicon hybridized to the oligonucleotide probes. The strips were then washed with a substrate solution containing hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine (TMB). In the presence of hydrogen peroxide, the bound streptavidin-horseradish peroxidase catalyzed the oxidation of TMB to form a blue-colored complex, which precipitated at the probe positions where hybridization had occurred (colourimetric determination). This color precipitation allowed for manual reading of the strips and genotype detection by comparison with the HPV reference guide provided. LA test does not directly detect HPV52 but combines a set of probes that detects HPV33, −35 and −58 (HPV mix). Specimens that test negative for HPV33, −35 and −58 individually but are positive for HPV mix are considered to be HPV52 positive. The specimens that test positive for HPV mix and for HPV33, −35 and/or −58 have an uncertain HPV52 status, for this analysis, these specimens were considered to be HPV52 negative, since coinfection with HPV52 cannot be ruled out by this test.The detection of the HPV genotypes was carried out using the LA HPV detection kit. Once the amplification was completed, 75 The procedure performed into two physically separated areas (pre-PCR and post-PCR) in order to avoid contamination of samples with previously amplified products. All washes and hybridization steps were undertaken in a 24-well tray with lid. The reading of the strips, produced by the two methods, was made by one well-experienced biomedical scientist. Discrepant interpretations were resolved by a second biomedical scientist and consensus review performed without knowledge of prior results. The LA-HPV test does not cross-react with a variety of viruses, bacteria, protozoa and yeast that could be present in cervical specimens.κ) statistics. A κ value of 0 indicates no agreement better than chance, and κ value of 1 indicates perfect agreement, κ values from 0 to 0.20, 0.21 to 0.40, 0.41 to 0.60, 0.61 to 0.80 and 0.81 to 1.00 indicate poor, fair, moderate, good, and very good strengths of agreement, respectively. All P values <.001 are considered statistically significant. Receiver operating characteristics (ROC) curve analysis was applied to calculate and compare AmpliLute method and MagNA pure method with cytological findings. In addition ROC analysis was applied to calculate and compare AmpliLute method and MagNA pure method with histological findings. All the statistical analyses were obtained with the statistical package for the social sciences (SPSS) computer software.Pairwise comparison of AmpliLute method and MagNA pure method was performed by using kappa   . HPV gen < .001) . Overall, 94/253 (37%) women were diagnosed with normal cytology, 50/253 (19.8%) were exhibited ASCUS, 91/253 (36%) were diagnosed with LSIL, 5/253 (2%) with LSIL but having some positions with HSIL, and 7/253 (3%) with HSIL. In 6 cases (2.4%) the cytological diagnosis was difficult due to inadequacy of the clinical samples , for ASCUS was 46%, for LSIL 77%, for LSIL/HSIL 100% and for HSIL 100%. HPV positivity detected by the MagNA pure LC method for the aforementioned categories was: 33%, 44%, 78%, 80%, and 100%, respectively, . Analytically, for the cytological category WNL, the composition of multiple infections observed by AmpliLute method was at 6.4% with HR, 4.2% with HR-LR, and 1% with LR, for ASCUS at 4% with HR, 12% with HR-LR, and 2% with LR, for LSIL at 9% with HR, 24% with HR-LR, and 9% with LR, for LSIL/HSIL at 40% with HR and 20% with HR-LR and for HSIL at 40% with HR, 20% with HR-LR. Multiple infections detected by the MagNA Pure LC method for the aforementioned categories was WNL at 4.2% with HR, 4.2% with HR-LR, and 2% with LR, for ASCUS at 4% with HR, 8% with HR-LR and 2% with LR, for LSIL at 4% with HR, 23% with HR-LR and 9% with LR, for LSIL/HSIL at 20% with HR and 20% with HR-LR and for HSIL at 14% with HR, 43% with HR-LR. The HPV type prevalence according to the two extraction methods is given below with HPV16 being the most frequent type detected, in both types of infections and by the two methods, followed by HPV31, HPV53, HPV6, HPV33, HPV45, HPV42, and HPV51 as detected by the AmpliLute method and by HPV31, HPV53, HPV18, HPV51, HPV18, HPV6, and HPV33 as detected by the MagNA method (data not showed).Within the studied population, single and multiple type infections were present in every cytological diagnosis. In total, single HPV infections detected by the AmpliLute was 28.5% and by MagNA Pure 30.4% whereas multiple type of infection was observed in 26.5% and 23%, respectively. In this study, multiple infections composed of up to five HPV genotypes in a plethora of combinations. The HPV distribution of single infections (divided into two categories according to the HPV genotype oncogenicity: LR and HR) as well as multiple infections (divided into three categories: LR (when only low-risk HPV types were present), HR-LR (when low- and high-risk HPV types were present) and HR (when only high-risk HPV types were present) in all cytological categories studied are given in P < .001) compared to AUC of MagNA pure method 0.672 .  The two extraction methods were compared against the cytological findings . In terms of processing evaluation, AmpliLute method obtained better results than MagNA pure method (AmpliLute: sensitivity (SE) = 73.2%, specificity (SP) = 69.15%, positive predictive value (PPV) = 79.43%, negative predictive value (NPV) = 61.32%; MagNA Pure: SE = 67.32%, SP = 67.02%, PPV = 76.87%, NPV = 55.75%). Both methods performed well when compared against the cytological diagnosis; nevertheless, the AmpliLute method demonstrated a slightest higher area under curve (AUC) 0.712  and it was higher compared to AUC of MagNA pure method 0.877 , P < .001).Results of biopsy reading and HPV genotyping by the two extraction methods are demonstrated in Tables Various research assays for HPV detection and typing have been used in epidemiological studies. The LA-HPV genotyping test provides a standardized, consistent and rapid means for HPV detection and genotyping. This test provides the capacity to identify 37 individual HPV genotypes within a given specimen and ascertain whether recurrent HPV positivity is, in fact, due to the persistence of a specific HR HPV genotype, meaning a substantially increased risk of disease progression –25. Curr In the present study, we assessed DNA extracts form PreservCyt cervical samples, generated by the automated MagNA pure extraction system and by the manual AmpliLute method (both recommended by Roche) for HPV testing using the LA-HPV genotyping and detection assays. In addition, we correlated those results with the cytological and histological findings of the enrolled participants. β-globin. In contrast, all nucleic acids generated from the AmpliLute protocol were valid for HPV DNA genotyping. This marginal difference in sample adequacy could be either due to the high AmpliLute protocol efficiency or to the variations in aliquoting the specific sample resulting in an inadequacy of cellular material for the automated procedure. Comparison of the HPV genotyping results, obtained with the AmpliLute DNA to those from the MagNA demonstrated a substantial level of agreement (93.3%), with κ value of 0.864. Both extraction methods, in terms of qualitative results performed equally well when compared against the cytological diagnosis, with AmpliLute method demonstrating a small predominance (AUC of AmpliLute: 0.712 versus AUC of MagNA: 0.672). Nevertheless, the AmpliLute method exhibited higher sensitivity, specificity, positive and negative predictive values as opposed to MagNA method. The same outcome, AmpliLute method being more efficient than the MagNA, was noticed when we compared the two methods with the histological diagnosis. AUC of AmpliLute was 0.935 in contrast with the AUC of MagNA which was 0.877. Among the 253 ThinPrep Pap tests analyzed in our study, only one extract from the MagNA pure modified DNA-I extraction protocol was found to be invalid due to the absence of low and high HPV types identified in individual samples by each method are largely in agreement 90.1% 228/253). In all studied cases, AmpliLute showed a slightly higher detection rate of HPV compared with MagNA. For the former, HPV overall positivity was calculated at 57.3% comprising 30.4% of HPV detected as single infection and 27% as multiple. And for the latter, the respective positivity was 54.5%, 32% as single and 23% as multiple type of infection. The prevalence of HPV-infected samples increased, in both methods, with the severity of cytological diagnoses: 30.8% of AmpliLute versus 33% of MagNA in the WNL, 46% versus 44% of ASCUS, 84.6% versus 77% of LSIL, 100% versus 80% of LSIL/HSIL and 100% by both of HSIL. Since, there are limited Greek epidemiological data available, studies that yielded similar findings in healthy women to our results, were the report by Papachristou et al.  who foun/253. In AmpliLute correctly identified 87.5% of the negative histological cases as HPV negative samples compared to 81.2% of MagNA. In addition, in cases with histological evaluation from HPV up to CIN2, MagNA missed 10 cases counting for 8% (10/123) of the population with these specific histological abnormalities, whereas all those cases were accurately detected as HPV positive samples by AmpliLute. In 144 cases with cytological findings of WNL and ASCUS, HPV was detected approximately in 52 cases (more than 50% of which were HR HPV types) and from which only 33 participants exhibited histological lesions of HPV up to CIN2. The remaining cases need to be followed up closely due to their elevated risk for developing a high-grade cervical lesion in the future. The small number of cases investigated in this present study limits our ability to conclude correct and representative epidemiological data on HPV prevalence in Greek women. Nevertheless, data of this report on HPV distribution add to a rich body on literature demonstrating that HPV 16 was the most frequent type detected in both types of infections followed by HR HPV 31, 53, 33, 45, 18, and 51. The observation of HPV 53 being among the three most prevalent HR HPV types detected is consistent with findings of previous Greek studies , 31. HowRegarding the discordant results observed between the two extraction methods, as showed in It is important to mention that the decision of utilizing the modified DNA protocol for the automated MagNA pure extraction system, was made based on a recent report. It compares DNA extraction efficiencies using the same extraction system with the incorporation of three different working DNA extraction kits: (i) blood cells high-performance protol (DNA-I kit), (ii) total nucleic acid (TNA) kit, and (iii) a modified DNA-I kit with the manual AmpliLute protocol for both AMPLICOR and LA HPV tests in 150 specimens . AlthougμL) as opposed to the automated MagNA DNA-I modified protocol (1000 μL) and equal amount of DNA extract inputs were used for PCR amplification (50 μL) and subsequently HPV detection, more HPV-positive cases were detected by the manual method. Someone would assume that increased HPV genotype detection would occur when a bigger amount of clinical sample is incorporated in the DNA extraction procedure, since more representative epithelial cells would be present in the sample tested, and thus increasing the possibility of HPV genotypes been detected by the assay used. The findings of our report, which are in contrast with previous reported one [At this point, it is important to emphasise that even though for the manual AmpliLute method we used one fourth of biological material (250 rted one , declare In the literature, there is also a report that utilizing the same MagNA pure automated extraction system, compares the AMPLICOR HPV test to the INNO-LiPA HPV genotyping test, using only the TNA extraction kit for DNA isolation for AMPLICOR test , making,Accurate laboratory assays for the diagnosis of HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. The first and most important step in molecular diagnosis of HPV infection is the nucleic acid isolation. An alternative approach to manual extraction procedures, which are time consuming and labor intense, is the automated processing of clinical specimens for HPV detection and genotyping which minimizes the potential sample contamination and the hands-on time. From our data, it was concluded that both DNA extraction methods demonstrated similar clinical performance, with no significant difference for any of the outcomes assessed even if for some outcomes the AmpliLute method exhibited higher sensitivity, specificity positive and negative predictive values as opposed to MagNA methods. Based on the results of this study, the automated nucleic acid isolation method should be tested versus other automated and manual techniques before it is routinely implemented. In addition, additional studies with larger populations are required to be carried out using the automated extraction system in order for its potential value to accurate HPV detection been determined.


There are no previous longitudinal studies on genotype-specific natural history of human papillomavirus (HPV) infections in oral mucosa of women.In the Finnish Family HPV Study, 329 pregnant women were enrolled and followed up. HPV-genotyping of oral scrapings was performed with nested PCR and Multimetrix® test . Incidence and clearance times and rates for each HPV-genotype identified in oral mucosa were determined. Predictors for incident and cleared HPV infections for species 7/9 genotypes were analyzed using Poisson regression model.Altogether, 115 baseline HPV-negative women acquired incident oral HPV infection, and 79 women cleared their infection. HPV16 and multiple HPVs most frequently caused incident infections (65% and 12%) in 13.3 and 17.1 months respectively, followed by HPV58, HPV18 and HPV6 (close to 5% each) in 11–24 months. HPV58, HPV18 and HPV66 were the most common to clear. HPV6 and HPV11 had the shortest clearance times, 4.6 months and 2.5 months, and the highest clearance rates, 225.5/1000 wmr and 400/1000 wmr, respectively. The protective factors for incident oral HPV-species 7/9 infections were 1) new pregnancy during follow-up and 2) having the same sexual partner during FU. Increased clearance was related with older age and a history of atopic reactions, whereas previous sexually transmitted disease and new pregnancy were associated with decreased clearance.HPV16 was the most frequent genotype to cause an incident oral HPV-infection. Low risk HPV genotypes cleared from oral mucosa more quickly than high risk HPV genotypes. Pregnancy affected the outcome of oral HPV infection. Human papillomavirus (HPV) infections are causally linked with head and neck cancers (HNSCC), particularly oropharyngeal cancers Oral HPV infections have been associated with the number of sexual partners, oral sex, deep kisses and hand warts The longitudinal Finnish Family HPV Study (FFHPVS) was designed to elucidate the dynamics of oral and genital HPV-infections within families The FFHPVS is a longitudinal cohort study conducted at the University of Turku and Turku University Hospital, Finland previously described in detail Oral scrapings were taken from the buccal mucosa of both cheeks and from the upper and lower vestibular area using a small brush . The oral sampling was performed mainly by a research nurse. At visit 6 oral sampling was performed by a gynecologist and at visit 7 by two dentists . All who collected the oral samples were trained by the principal investigator of the study who is a dentist specialized in oral pathology. The brush was immersed in 80% ethanol, frozen and stored at -70°C HPV-DNA was extracted from the oral scrapings with the high salt method as described previously HPV genotyping was performed with a Multimetrix kit® . Multimetrix kit® detects 24 LR- and HR-HPV-genotypes as follows: LR-HPV6, 11, 42, 43, 44, and 70; and HR-HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82. The previous nested PCR products to detect any high-risk HPVs were biotinylated by re-amplification with GP05+/bioGP06+-primers. The labeled hybrids were analyzed with a Luminex LX-100 analyzer . The assay was performed in half of the volume given in the protocol in all steps except the final one. A median fluorescence intensity (MFI) of at least 100 beads was computed for each bead set in the sample. The cut-off value for each run and HPV-type was 1.5× background MFI (negative control)+5MFI If any sample was positive for HPV16, then the protocol was repeated from the original sample using nested PCR and a bead-based HPV16 genotyping assay An incident HPV-infection was recorded when a woman who was HPV-negative at baseline acquired an incident HPV infection during the FU. The incident event was recorded only once and the type detected at that special occasion was fixed as the “incident type”, completely irrespective what the subsequent events might have been. Among all women, clearance was defined as an event (at any FU visit) when a previously HPV-positive test turned out to be negative and remained HPV-negative until the end of the FU. Fluctuation was a pattern of HPV outcome in which consecutive samples were intermittently HPV+ and HPV- with different HPV genotypes, without any two consecutive samples being positive for the same or different viral genotype. Women with fluctuation were excluded in the statistical analysis.®  and STATA  software. Frequency tables were analyzed using the χ2-test with the likelihood ratio or Fisher’s exact test for categorical variables. Differences in the means of continuous variables were analyzed using non-parametric  tests for two and multiple independent samples, respectively.All statistical analyses were performed using SPSSTimes (months) to incident and clearance events as well as genotype-specific incidence (IR) and clearance rates (CR) were calculated as recently described, expressed as events/1000 women months at risk (wmr) p-value <0.05.We analyzed covariates of incident infections and virus clearance for species 7 and species 9 genotypes only, using population-averaged (PA) Poisson regression models for panel data, clustered by mother-ID, FU visit as the time variable, independent within-group correlation structure as the covariance pattern, and robust variance estimator (vce) to account for the within-subject correlation Totally 171 mothers tested HPV-positive at some time point of the seven visits during the six-year FU. The mean FU time of the 115 women with incident infections (HPV negative at baseline) was 61.7±24.1 (SD) months . Outcomes of the study are presented in The highest IR was ascribed to HPV33, followed by HPV11, HPV56 and HPV70. The crude IRs of HPV16 and HPV18 were lower than for the other HR-HPV types. Due to this wide variation among individual types in different species, the crude IRs between the HPV species showed much less variation .The predictors of genotype-specific incident infections for species 7/9 are shown in The mean FU time for all infections was 59.1 months. Totally, 46.2% (n = 79) of the 171 mothers who tested HPV-positive at some visit during the FU, cleared their infection . The meaFive variables were significant predictors of species 7/9 clearance: 1) older age (more common with increasing age), 2) history of atopic reactions (increased clearance), 3) history of STDs (decreased clearance), 4) frequency of weekly intercourse, and 5) new pregnancy (protective) during FU . In the To our knowledge, there are no previous longitudinal studies on oral HPV-infections at the genotype level which hinder the comparison of our natural history data with that of others. As the six-year natural history of the cervical HPV-infections in these women is known Altogether, 115/308 mothers developed an incident oral HPV-infection during FU  is relatively small and not everyone had all samples available as well as some women were dropped out during the long six-year FU. This is why HPV infections with certain genotypes, e.g. HPV11, might be rare. This cohort is also unique as all women at baseline were pregnant. Hormonal changes during pregnancy might affect HPV infection and its outcome. FFHPVS included also the fathers and the infant of the index pregnancy. Thus, our woman-cohort represented mothers mostly with stable relationships. This has influence on their life style and sexual behavior which also reflect to the incidence and clearance of HPV infection. Also, the FU time of the study represents only a certain period of life of these women as all other studies as well. Thus the women’s HPV status before entering into the study is unknown as is the HPV status after the last six-year control visit. Furthermore, different body-sites  may have an impact on HPV status at the other sites, and further analysis of the FFHPVS data is warranted for this in the future”.In healthy, asymptomatic oral mucosa, the two most common non-invasive sampling methods for HPV testing are rinsing or brushing the mucosa. The rinsing of oral mucosa provides a sample of the exfoliated epithelial cells of which only part can be infected with HPV. Also the origin of the HPV infection cannot be determined as the detached cells can originate from any site of oral cavity and oropharynx. In the present study we are specifically interested in oral HPV infection. At least 100,000 cells are required for a representative sample and this may be difficult to accomplish with rinsing/gargle samples The overall clearance of oral HPV infection was nearly the same as found in their cervix (46% versus 52%). However, genotype specific differences in clearance were found; oral LR-HPV genotypes were less likely to clear than those in the cervix Pregnancy plays a special role in this cohort and a substantial proportion became pregnant again during FU. We showed previously that the prevalence of any oral HR-HPV infection was lowest during pregnancy (at baseline-visit) and increased after delivery Oral sex and open-mouth kissing have been implicated in increasing the risk of oral HPV infections Finally, women with a history of previous STDs were less likely to clear their oral HPV infections. The two feasible explanations could be that 1) these women are more promiscuous than the others or 2) there are other co-infections interfering with HPV infection. However, in this analysis, we did not explore this issue in any further detail.To conclude, this is the first study to characterize the incidence and clearance outcomes of oral HPV infections at the genotype level in a longitudinal setting. The predictors for these outcomes are somewhat similar to cervical HPV infections, being related not only to sexual behavior but also to pregnancy. Further comparative studies are needed to disclose all of the similarities and differences in the natural history of oral and genital HPV infections.


A recent intriguing carcinogenetic hypothesis for lung cancer foresees its viral aetiology. The human papilloma virus (HPV) is the main virus actually recognised in the pathogenesis of lung cancer. The aim of this study was to investigate, for the first time to our knowledge, the presence of HPV in the exhaled breath condensate (EBC) of lung cancer patients.We enrolled 89 patients affected by lung cancer and 68 controls. HPV infections were investigated in their EBC, paired bronchial brushing and neoplastic lung tissue through genotyping.We were able to detect HPV in the EBC, bronchial brushing and neoplastic lung tissue. We described the presence of an HPV infection in 16.4% of the subjects affected by non-small cell lung cancer, but in none of the controls. HPV 16 and 31 turned out to be the most widespread genotypes. The HPV positivity in airways as well as in the smoking habit was seen to independently increase the individual's susceptibility to developing lung cancer.When summing up, we demonstrated the possibility to identify an HPV infection in the EBC of lung cancer patients; further, we supported the notion that the EBC is a suitable tool to study airway colonisation. That being said, although further studies are needed to confirm our results, we retain the study of HPV in EBC to be very interesting in terms of future programmes involving lung-cancer screening. A viral aetiology of lung tumours is a tantalising prospect since the virus can be regarded as a main oncogenic event or as an important co-factor . After aAt present, more than 100 HPV serotypes have been identified . The oncHuman papilloma virus infection and metaplasia in lung tissue may increase an individual's susceptibility to tumourigenesis , 2002.The presence of HPV in the airways of lung cancer patients has already been investigated through research of the virus in bronchial aspirates .Several soluble and genetic markers have already been measured in the EBC of lung cancer patients and investigated as early neoplastic markers, although none of them have been validated sufficiently for clinical application. In regards to the possibility to analyse viral or bacterial colonisation in EBC, studies on pseudomonas, burkholderia and the influenza virus were found to be unsuccessful  that consented to the study were enrolled at the Unit of Thoracic Surgery, Casa di Cura La Madonnina, Bari, as well as at the Department of Respiratory Disease, Foggia University . WrittenAll patients were enrolled in the study before pathological diagnosis. All patients also underwent standard staging procedures consisting in a physical examination, serum chemistry analysis, brain, chest and abdomen CT scans and radionuclide bone scan.All the patients underwent thoracotomy except for six of them, who were deemed surgically inoperable for reasons related to staging and/or inadequate cardiorespiratory functioning.In these cases the definitive diagnosis of malignancy derived from a positive cytohistology of the samples obtained broncoscopically. Following the histological analysis carried out on specimens, 68 subjects turned out to be negative and were considered as controls. All 68 patients were operated on and the definitive diagnosis is shown in All subjects underwent EBC and bronchial brushing collection (the latter was carried out during bronchoscopy). Their paraffin-embedded neoplastic lung tissue, collected during lung surgery, was stored for successive analysis.We acquired information on their smoking habit at the time of diagnosis. In all, 67 of the lung cancer patients were current smokers (43.5±11.6 packs per year), whereas 17 were ex smokers (42.9±18.6 packs per year) and 5 were non-smokers. On the other hand, in the controls group fifty-one were current smokers (40.6±10.3 packs per year), eight were ex smokers (39.9±16.5 packs per year) and nine were non-smokers. A detailed history relating to their family history of lung cancer or any other cancer was collected in a pre-tested pro-form. All the women enrolled underwent a PAP smear test and were evaluated as cytological using the Bethesda system for PAP smear interpretation along with colposcopic evaluation. We considered negative samples where koilocytosis was absent.Bronchial brushing specimens were collected in ice-cold phosphate-buffered saline solution during the fibreoptic bronchoscopy and were stored at −70 °C for subsequent analysis.DNA extraction was performed using qiasymphony , following the manufacturer's instructions.The above required 1 ml of EBC in one setting from each patient at the time of diagnosis.The EBC was collected by using a condenser, which allowed for the non-invasive collection of the non-gaseous components of the expiratory air . The condensate was collected in ice at −20 °C, transferred to 1.5 ml polypropylene tubes and immediately stored at −70 °C for subsequent analysis.DNA extraction was performed using qiasymphony (Qiagen) following the manufacturer's instructions.μm and used for each patient for DNA extraction purposes.Formalin-fixed and paraffin-embedded neoplastic lung tissue were sectioned at a thickness of 10 At the beginning, one slide of lung tissue was treated with ematossilin eosin for the selection of the neoplastic lung area. Next, from the area selected neoplastic material was asported and put into two separate 1.5-ml polypropylene tubes. After that samples were deparaffinised in xylene and then rehydrated through serial dilutions of alcohol. After evaporation, the DNA was extracted and purified with a DNA Qiamp mini-kit (Qiagen) that foresaw the first step of enzymatic digestion with an ATL buffer and proteinase K overnight at 56 °C. For the second step, we added lysis buffer AL and carried out incubation at 70 °C for 10 min, whereas the further step involved purification by the addition of absolute ethanol and passage through ion-exchange column chromatography followed by washing and finally the use of elution buffer AE with DNA in a volume proportional to the starting material.The HPV DNA was analysed in DNA from EBC, bronchial brushing and lung tissue.The assay for HPV genotyping was initially performed using an HPV sign .The DNA was amplified by PCR reactions with one of the primers biotinylated on Rotor-Gene TM 6000 , whereas single-stranded DNA templates were prepared using the PyroMark Vacuum Prep Workstation .The pyrosequencing analysis was performed on the PyroMarkTM Q96 ID instrument (Biotage).Pyrosequencing is a DNA-sequencing technique to determine nucleic acid sequences. This method is a sequencing-by-synthesis technique that employs a series of enzymatic reactions to accurately detect short nucleic acid sequences during DNA synthesis. In principle, when a complementary nucleotide is incorporated by DNA polymerase into an already-primed DNA template (whereby the sequencing primer is hybridised to target the DNA), an inorganic pyrophosphate (PPi) molecule is released. The released PPi is converted to ATP by ATP sulphurylase using adenosine phosphosulphate as substrate. This reaction provides energy for the conversion of luciferase to oxidase luciferin, and consequently the light produces a peak in the pyrogram (c.f. electropherogram in Sanger sequencing). Each signal peak is proportional to the number of nucleotides incorporated . Apyrase is a nucleotide-degrading enzyme, which continuously degrades the ATP and non-incorporated dNTPs in the reaction mixture. There is a certain time interval between each nucleotide dispensation to allow for complete degradation. For this reason, one nucleotide addition is performed at a time. During the sequencing-by-synthesis process, the primed-DNA strand is extended by complementary nucleotides, and the DNA-sequence signals are displayed by the signal peaks in a pyrogram on a computer monitor. Base callings are performed with integrated software with features for related SQA and sequencing analysis. HPV 16 and HPV 30 pirograms and relative melt curves are reported in The INFINITI HPV-QUAD assay was successively used to confirm the HPV results.E1 gene of the HPV genome. Multiplex PCR for five individual HPV types , five combinations of HPV types  and a β-globin internal control were performed on extracted DNA using Platinum TaqDNA Polymerase  and an amplification mix was provided with the INFINITI HPV-QUAD Assay  as previously reported  and odds ratio (OR) with relative confidence intervals (95% CI) were calculated. The t-Student's test for independent samples was used to assess the differences in continuous variables  between lung cancer patients and controls.In order to assess the association between categorical variables such as sex, smoking habit or HPV positivity and positivity to lung cancer, or between histological subtypes or TNM stage and HPV positivity, the P-value of <0.05 was considered statistically significant.An exact logistic regression model was performed in order to state the possible confusing effect among the variables, in the comparison between the NSCLC and the healthy controls. A Sensibilities, specificity, positive and negative predictive values with relative confidence intervals of 95% were calculated for cytology, as well as for cytology in association with the HPV test (in brushing or EBC). Statistical analysis was carried out using STATA 10 MP for the MAC OS X software package . The data were analysed by the Department of Medical Sciences, Section of Hygiene, University of Foggia.The number of lung cancer cases and healthy subjects enrolled in the present study was 89 and 68, respectively. Demographic and clinical data of study subjects are summarised in χ2=9.46, P=0.0021).The male sex was associated as having NSCLC  that did not allow for the evaluation of HPV positivity.P=0.002, degree of freedom=1).Eleven NSCLC patients (15.1%) turned out to be HPV positive on the EBC. Of these six were positive to HPV-16 and three to HPV-31 (genotype at high risk for cancer), one to HPV-30 and one to HPV-39. Comparing the NSCLC-positive patients with the controls who were not affected by tumours  it was shown that being HPV positive to the EBC increases the probability of being affected by NSCLC .A total of 12 NSCLC patients (16.4%) turned out to be HPV positive on the bronchial brushing and lung tissue. Of these, seven were positive to HPV-16 and three to HPV-31 (genotype at high risk for cancer), one to HPV-30 and one to HPV-39. Comparing the NSCLC-positive patients with the controls who were not affected by tumours  it was shown that being HPV positive to the bronchial brushing and lung tissue increases the probability of being affected by NSCLC  showed, except in one case, an overlap of the HPV positivity spectrum.All controls turned out to be negative to HPV both in the EBC and in the paired brushing and lung tissue.The HPV results were confirmed in all the samples of those subjects analysed with the INFINITI HPV-QUAD assay.Comparing the NSCLCs to the healthy controls  using sex as a criterion, being smokers/ex smokers, being HPV positive on the brushing and HPV positive on the EBC as variables in logistic regression models, either being smokers/ex smokers or HPV positive  will increase the risk of having an NSCLC , 3 and 4P>0.05). On the other hand, none of the SCLC patients showed HPV positivity in the EBC, brushing or lung tissue. HPV-positive patients were those with TNM stage>2 . Neither sex, age nor smoking habit were found to be associated with a more advanced stage of lung cancer or to HPV positivity. The association of cytology with the HPV test  slightly increases the sensibility of the test  slightly increases the sensibility of malignant diagnosis.The great interest in the viral aetiology of lung cancer recently produced several studies on blood-borne HPV in the other local samples of lung cancer patients , inadequate choice of primers and geographic area analysed. The complete negativity reported in controls is the proof of the specificity of HPV analysis in the bronchial brushing, lung tissue and the EBC of neoplastic patients.The findings of the involvement of HPV infection in lung cancer are often controversial. Several authors reported that HPV has no role to play in lung cancerogenesis  to moderIn consideration of the fact that HPV is the second most important cause of lung cancer after cigarette smoke, in order to better understand the co-responsibility of both of these aetiological factors we analysed the HPV infection in relation to the smoking habit and did not report any correlation. Further confirmation of our suggestion is supplied by the result of the logistic regression model applied to variables substantiating that the HPV infection is an independent factor in causing lung cancer.We reported higher percentages of HPV positivity in lung cancer smokers than in ex smokers or non-smokers. These data found a rationale in the fact that the respiratory epithelium of smokers shows micro-abrasions with squamo-columnar junctions that are considered to be the prerequisite for the spread of HPV  are more prone to integrate HPV into the tumour genome than normal cells  in the EBC, bronchial brushing and lung tissue of NSCLC patients in a future study.


Quantifying rates governing the clearance of Human Papillomavirus (HPV) and its progression to clinical disease, together with viral transmissibility and the duration of naturally-acquired immunity, is essential in estimating the impact of vaccination programmes and screening or testing regimes. However, the complex natural history of HPV makes this difficult. We infer the viral transmissibility, rate of waning natural immunity and rates of progression and clearance of infection of 13 high-risk and 2 non-oncogenic HPV types, making use of a number of rich datasets from Sweden. Estimates of viral transmissibility, clearance of initial infection and waning immunity were derived in a Bayesian framework by fitting a susceptible-infectious-recovered-susceptible (SIRS) transmission model to age- and type-specific HPV prevalence data from both a cross-sectional study and a randomised controlled trial (RCT) of primary HPV screening. The models fitted well, but over-estimated the prevalence of four high-risk types with respect to the data. Three of these types  are among the most closely related phylogenetically to the most prevalent HPV-16. The fourth (HPV-45) is the most closely related to HPV-18; the second most prevalent type. We suggest that this may be an indicator of cross-immunity. Rates of progression and clearance of clinical lesions were additionally estimated from longitudinal data gathered as part of the same RCT. Our estimates of progression and clearance rates are consistent with the findings of survival analysis studies and we extend the literature by estimating progression and clearance rates for non-16 and non-18 high-risk types. We anticipate that such type-specific estimates will be useful in the parameterisation of further models and in developing our understanding of HPV natural history. Persistent infection with high-risk human papillomavirus (HPV) has been shown to be the necessary precursor of progression to cervical cancer We have aimed to infer the viral transmissibility, rate of waning natural immunity and rates of progression and clearance of infection of 13 high-risk HPV types and for HPV-6 and -11, making use of two rich epidemiological datasets and a comprehensive survey of sexual activity from Sweden. Estimates of viral transmissibility, clearance of initial infection and waning immunity were derived in a Bayesian framework by fitting a susceptible-infectious-recovered-susceptible (SIRS) transmission model to age- and type-specific HPV prevalence data from both a cross-sectional study and a randomised controlled trial (RCT) of primary HPV screening. Rates of progression and clearance of clinical lesions were estimated from longitudinal data gathered as part of the same RCT. Where possible we compare our estimates of these parameters with those derived from similar studies.Sex in Sweden study Parameterisation and Data. These data also informed the boundaries of the age brackets.We developed a deterministic model of heterosexual partnership formation, mono-type HPV transmission and the potential progression to cervical cancer for each of 13 high-risk HPV types  and for HPV-6 and -11. We model an age- and sexual activity- structured population with two sexual activity groups for each of three age brackets  where the size of the high sexual activity group is a fixed proportion (15%) of the overall population, regardless of age. Contact rates for each of these six age/sexual activity groups were derived from the Swedish sexual activity data gathered as part of the Cervical Intraepithelial Neoplasia). Women with an initial infection may either progress to CIN1-type lesions at a rate of or naturally clear the virus at a rate of We assumed an SIRS structure for HPV infection in both men and women, allowing for an SIR structure in the limit where the rate of waning natural immunity, estimated within this analysis, tended to zero. The infected (I) compartment for females was divided into five sub-compartments, corresponding to the progression of HPV infection to disease: Initial infection, CIN1, CIN2, CIN3 and Cancer  are infectious. Also, the force of infection was calculated assuming that viral transmissibility The state variables used in the model are summarised in the Supplementary Material.andThe force of infection acting on males depends on the number of females in each of the three infectious At demographic equilibrium, Mixing in the model is determined on a proportionate random basis i.e. without accounting for either assortativity on the basis of either age or sexual activity group. In order to balance the number of partnerships between males and females we assume that females adjust their behaviour completely to the number of partnerships sought by males. This leads to the simplistic conclusion that the overall rate of partnerships formed between females of age Cervical screening was incorporated into the model according to the Swedish screening protocol. In Sweden, women are screened at 3-yearly intervals between the ages of 23 and 50 and at 5-yearly intervals between 51 and 60. Uptake is 79%  and we made the limiting assumption that this rate is independent of sexual behaviour. Treatment is provided for screened women with CIN2+ lesions and cancer, resulting in the clearance of infection; the combined rate for screening and subsequent successful treatment is π.We assumed that the rates of waning immunity and clearance of initial infection are independent of age, gender, sexual activity and personal history of HPV infection.Sex in Sweden’ Sexual behaviour in the model (e.g. partnership formation rates) was parameterised using data from a comprehensive Swedish sexual activity survey (n = 2810). Complete details of the survey have been published in ‘As described above, the rate of partner acquisition, Given a certain annual rate of partner acquisition a priori, the number We consider males and females, and each age group, separately. Defining Since we have data on the number of new partners per year, stratified by age In the parameterisation of this model, we considered two sexual activity groups, the high sexual activity group, The proposal densities were standard Normal distributions with standard deviation dependent on the sexual activity group:Since the higher sexual activity group has a smaller population, it is more difficult to fit the likelihood than for the larger low sexual activity group. For this reason we specified a prior function of exponential form: The consequence of this was to penalise higher contact rates, essentially attempting to describe the data with rates as low as possible. The resultant estimated annual rates of new partner acquisition are shown in In order to estimate the per-partnership viral transmissibility Swedescreen is a prospective randomised controlled trial of 12,527 women, aged 32–38, attending population-based invitational cervical screening in Sweden. Women were allocated in a 1∶1 ratio to either receive an HPV test as well as a cytology test (intervention) or a cytology test alone (control). HPV positive women were invited for a second HPV test at least twelve months later and women with persistent infections were invited to colposcopy. Women were then followed with comprehensive registry-based follow-up. Further details of the study have been published elsewhere The data from the Swedish voluntary Chlamydia screening programme provided information on the prevalence of each of the 13 high-risk HPV types, by age in women aged between 14 and 50 . Although attendance rates for Chlamydia screening in Sweden are high , we anticipate that the very young (<17) and older women (>35) who attend elective screening are more likely to be at higher risk. We accommodated this in the model by specifying that a higher proportion of the high than the low sexual activity groups attended Chlamydia screening.Parameterisation and Data). These rates were inferred from a corresponding vector of cumulative risks, derived from the observed 6-, 12- and 24-month proportions of viral clearance or disease progression among women with high-risk HPV infection. These rates were:Six type-specific HPV progression and clearance rates of CIN1+ lesions were estimated from longitudinal data gathered as part of a large prospective randomised controlled trial of HPV testing and screening in Sweden IN1- type lesionsμ2: the rate of progression from C(G)IN1 to C(G)IN2 - type lesionsμ3: the rate of progression from C(G)IN2 to C(G)IN3 - type lesionsμ4: the rate of progression from CIN3 or CGIN3 - type lesions to squamous cell carcinoma or adenocarcinoma respectivelyX2: the rate of clearance of C(G)IN1- type lesionsX3: the rate of clearance of C(G)IN2 and C(G)IN3– type lesionsThese parameters were informed by a corresponding vector whereAs outlined in Bogaards et al. There is an art to deciding after what time lag One limitation of the Swedescreen data was that each woman received only one HPV test as part of the Swedescreen trial – at baseline. As a consequence of this, it was necessary to assume that her history of infection and disease progression was attributable to the HPV type(s) that were identified at her baseline HPV test. Also, since it was not therefore possible to demonstrate clearance of infection, we assumed that a reversion of abnormal to normal cytology was an indicator of clearance of HPV.Within the Swedescreen study, histological tests were only conducted if the results of a cytological test were positive. To paint a fuller picture of disease progression, we decided to include both cytological and histological data in our estimation of rates from longitudinal data. To do so, we considered equivalence between the grades shown in In cases of a cancer diagnosis, we always relied on the definition from the histological test.We assumed that development of disease was attributable to a hierarchy of HPV types: if a woman was infected with HPV-16 this was considered the cause of any cytological abnormalities; if she was infected with HPV-18 but not HPV-16, HPV-18 was considered the cause. To estimate rates for the other high-risk types (OHR) independently, we considered all women who tested positive for the type in question and negative for -16 and -18. This meant that we included in the estimation of OHR type progression and clearance, women who were co-infected with a number of OHR types. This was deemed necessary since many of the rarer types were only found in cases of co-infection with several OHR types.μ3), we considered not only the women who were found to have CIN2 lesions at the baseline of the Swedescreen study, but all women who were found to have CIN2 lesions for the first time within the study; essentially the clock starts ticking as soon as her CIN2 diagnosis is made.In order to increase the effective sample size of women progressing from one state to another, we considered the first occurrence of a certain histological state for each woman to be her ‘baseline’ test at that level. For example, to estimate the rate of progression from CIN2 to CIN3  but since it was found that the resultant posterior distributions were not noticeably responsive to these parameters, the rates were fixed at their median values. The following Bayesian model framework was used:Type-specific estimates of the per-partnership transmissibility The likelihood function for the Chlamydia screening programme HPV prevalence data, The function whereX1,X2,…,nX. are a set of random variatesThus we are able to jointly consider the ‘success’ probability of being in the susceptible state, the initial infection state, the CIN1 state and so forth.Each of the 13 high-risk HPV types was modelled separately. Estimates of the three natural history parameters were derived from posterior distributions of the Markov Chain Monte Carlo (MCMC) chains  using the Metropolis-Hastings algorithm In each iteration of the algorithm, a proposal for th and 97.5th percentiles of all iterates after the burn-in period.For each type the algorithm was run for a chain of length n = 20,000. Visual inspection confirmed that the Metropolis-Hastings algorithm converged swiftly to the posterior distribution and parameter estimates were made rejecting a ‘burn-in’ period of 100 iterations. Examples of the MCMC traces are shown in Figures S1 and S2 for HPV-16 and HPV-45 respectively. We report 95% posterior intervals for the posterior distributions of all MCMC-estimated parameters. These are obtained from the 2.5Supplementary Material; further data available on request). The prevalence of HPV-16 infection in women taking part in the Swedescreen trial was higher than those of the same age (32–38) attending Chlamydia testing, HPV-18 and HPV-33 were observed at closely comparable levels in both studies but all other high-risk types were less prevalent in the former group. This disparity probably represents the higher representation of highly sexually active women in the voluntarily attending Chlamydia screening in their mid-thirties.The observed age-dependent prevalence of high-risk HPV infection was accurately replicated by the appropriate type-specific model for each type see . In geneThe median estimated rates of progression and clearance of CIN1-type lesions in four high-risk HPV types, each with a 95% adjusted bootstrap confidence interval, are given in The progression and clearance rates did not differ significantly between HPV types. However, HPV-16 appeared to progress from initial infection to CIN1- type lesions at an annual rate of 0.026 (0.007–0.045), somewhat more slowly than HPV-18 which had an annual rate of 0.058 (0–0.179) but at a similar rate to other high-risk types (OHR) which had an annual rate of 0.02 (0.004–0.04).The rate of clearance of type-specific initial HPV infection was well characterised by the posterior distribution of the MCMC chain, with a median posterior/prior ratio of 0.320 across all HPV types. The median values for the rate of clearance of initial infection did not differ significantly between types see . Our estAlthough few estimates have been made of the annual clearance rates of some of the rarer high-risk HPV types, more cohort studies have concentrated on estimates for HPV-16, -18 and the grouped other high-risk (OHR) types. In The median estimated rates of transmissibility in the thirteen high-risk HPV types, each with a 95% adjusted bootstrap confidence interval, are given in The estimated rates of waning natural immunity (or resistance to subsequent infection) are shown in Sex in Sweden’ sexual behaviour survey, longitudinal data on HPV prevalence and clinical disease progression from the randomised controlled trial of HPV testing, ‘Swedescreen’, and HPV type-specific prevalence data gathered as part of the Swedish voluntary Chlamydia screening programme. The analysis was formed of two stages. Firstly, rates were estimated for the stepwise progression of different HPV types to disease of increasing clinical severity and of clearance of different grades of late-stage infection. Secondly, these estimated rates were incorporated into a monotype SIRS-structured deterministic model of the transmission and progression of HPV. This model was then fitted to HPV prevalence data using a Markov Chain Monte Carlo (MCMC) routine, in order to estimate the transmissibility, rate of clearance of initial infection and rate of waning natural immunity of each type in turn.We have made estimates for the transmissibility, clearance and progression rates and rates of waning natural immunity for 13 high-risk types of HPV by using Swedish behavioural and epidemiological data to fit an ensemble of deterministic models of HPV transmission and progression. The datasets used included the ‘It was observed that the models over-estimated the observed prevalence of four of the high-risk types. We suggest that this is an artefact of modelling the HPV types independently. Three of these types  are, in fact, three of the most closely phylogenetically-related to HPV-16; the most prevalent type. HPV-45 is the most closely related to the second most prevalent type, HPV-18. It is feasible that this similarity leads to cross-immunity between the types, effectively reducing the number of young women susceptible to infection with types -33, -35, -45 and -58.We found variation in the rates of clearance of initial infection to be a very informative parameter in the fitting of type-specific models of HPV prevalence to data, indicating that this may be an important determinant in the differing prevalences between types. The lowest rates of clearance of initial infection were observed in HPV-16, -18 and -31, the most prevalent of the high-risk HPV types. This would again support the theory that higher prevalence is associated with persistence of infection, though we would agree with Bogaards et al. Our estimates of transmissibility did not differ significantly between types although there remained a large degree of uncertainty. Our inferred values were in line with those estimated in previous studies: the median per-partnership transmissibility of HPV-16 was estimated to be 0.718 with a 95% posterior interval of 0.286–0.999) compared with 0.8 estimated by Hughes et al. The rate of waning immunity was estimated to be very low; the 5-year cumulative proportions of clearance varied between 6%–12% across the 13 types. This would imply that few women become susceptible to re-infection with any given HPV type and that the observed decrease in the force of infection in the early twenties is more likely to be due to immunity than to decreased sexual activity. Indeed, our estimates of the rate of waning immunity would imply that the protection conferred by infection with HPV is effectively close to life-long.Swedescreen with those collected as part of other studies elsewhere. Such collation would increase the power of the analysis, ensuring more certainty in the inferred rates.We have also further contributed to the literature estimating the rates of progression and clearance of clinical lesions due to HPV, extending the field by deriving type-specific estimates of progression rates up to CIN3-grade lesions for type -31 and 33 and clearance thereof. Our results implied that the rate of progression of other high-risk types from CIN1 to higher grade lesions was significantly lower than for HPV-16 and HPV-18, indicating that their less prominent role in the cause of cervical cancer may be due not solely to lower prevalence but also to an inherently lower oncogenicity. Our estimates are similar to others in the literature although they are subject to a large degree of uncertainty, highlighting the benefit that would be derived from pooling data on disease progression from Although this analysis provides some useful new insight into HPV-related parameters, it has some limitations. Firstly, the nature of deterministic compartmental models means that it is not possible to consider diversity in individual-level behaviour. For example, we estimate here the per-partnership transmissibility of HPV infection. In order to derive from this a per-act transmissibility requires us to assume an ‘average’ partnership between a susceptible and an infected partner. Also, we assume that all women have an equivalent approach to screening and subsequent treatment. Such an assumption belies the true underlying heterogeneity; in fact, it is those women who do not attend screening who are more likely to go on to develop high-grade clinical lesions and cervical cancer. In considering the more subtle questions policy-makers face in terms of vaccination and screening programmes it is common for complex individual-based models to be used. However, the fitting of these models is similarly complex due to the long run times and inherent stochasticity of these models. The type-specific estimates we have made here for HPV transmissibility and rates of progression and clearance will facilitate the parameterisation of such individual-based models by, for example, providing prior ranges for natural history parameters. These models will then, in turn, allow a more thorough understanding of the complex natural history of HPV infection and its implications for the efficacy of vaccine and screening programmes in this new era of HPV prevention.Figure S1MCMC traces (left) and posterior distributions (right) for estimation of HPV-16 parameters. The MCMC chains  and posterior distribution density are shown for, in order, the transmissibility (β), cumulative clearance rate of initial infection (X1) and rate of waning of natural immunity X1..X3 for HPV-16. The fourth row shows the trace and distribution of the log-likelihood. The figures show a rapid convergence.(TIFF)Click here for additional data file.Figure S2MCMC traces (left) and posterior distributions (right) for estimation of HPV-45 parameters. The MCMC chains  and posterior distribution density are shown for, in order, the transmissibility (TIFF)Click here for additional data file.Supplementary Material S1(DOCX)Click here for additional data file.


Sensitive and specific detection of human papillomaviruses (HPV) in cervical samples is a useful tool for the early diagnosis of epithelial neoplasia and anogenital lesions. Recent studies support the feasibility of HPV DNA testing instead of cytology (Pap smear) as a primary test in population screening for cervical cancer. This is likely to be an option in the near future in many countries, and it would increase the efficiency of screening for cervical abnormalities. We present here a microarray test for the detection and typing of 15 most important high-risk HPV types and two low risk types. The method is based on type specific multiplex PCR amplification of the L1 viral genomic region followed by ligation detection reaction where two specific ssDNA probes, one containing a fluorescent label and the other a flanking ZipCode sequence, are joined by enzymatic ligation in the presence of the correct HPV PCR product. Human beta-globin is amplified in the same reaction to control for sample quality and adequacy. The genotyping capacity of our approach was evaluated against Linear Array test using cervical samples collected in transport medium. Altogether 14 out of 15 valid samples (93%) gave concordant results between our test and Linear Array. One sample was HPV56 positive in our test and high-risk positive in Hybrid Capture 2 but remained negative in Linear Array. The preliminary results suggest that our test has accurate multiple HPV genotyping capability with the additional advantages of generic detection format, and potential for high-throughput screening. Human papillomavirus (HPV) infects mucosal and cutaneous epithelia and is one of the most common sexually transmitted infections. Currently there are over 120 known HPV genotypes HPV testing is currently largely based on either direct liquid hybridization or PCR amplification of polymorphic L1 or E1/E6/E7 viral genomic region using consensus or multiplex primers. The current standard non-PCR molecular test, the Hybrid Capture 2 assay, is based on liquid hybridization of RNA probes to target DNA followed by antibody detection. It is able to differentiate between low-risk and high-risk HPV infections but cannot determine specific HPV genotypes. The Linear Array HPV Genotyping test is capable of differentiating 37 HPV types but is laborious and has limited throughput capacity. The demand for high-throughput genotyping has motivated the application of oligonucleotide DNA microarray technology Here we report a highly sensitive and specific DNA microarray method for genotyping of 15 different hrHPVs and two low-risk HPVs (lrHPVs). The method is based on multiplex PCR amplification of the viral L1 region using an improved set of type-specific PGMY primers  and the National Supervisory Authority for Welfare and Health (2461/04/044/07). A written informed consent was signed by all patients participating in the study. All data were analyzed anonymously.Cloned plasmids of full-length HPV 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 51, 52, 56, and 58 were generously provided by Drs E.-M. de Villiers, W. Lancaster, A. Lorincz, T. Matsukura, G. Orth and S. Silverstein. Additionally, the PCR target sequences of HPV types 45, 59, 68, 73, and 82 were synthesized and cloned into the pCR2.1 vector .HPV samples sent to the Department of Virology for laboratory diagnostics of HPV infections using the Hybrid Capture 2 test  were used in the study.Extraction of DNA from patient samples on the MagNAPure LC instrument  was optimized for HC2 samples. The MagNA Pure LC Total Nucleic Acid Isolation Kit  turned out to be optimal. For each nucleic acid extraction, 50 µl of HC2 sample was used. For samples tested with Linear Array, 200 µl of HC2 sample was used.The HC2 test kit for hrHPV was purchased from Qiagen  and was used according to manufacturer's instructions. The LA test kit was purchased from Roche  and was used according to manufacturer's instructions.5′-GAAGAGCCAAGGACAGGTAC-3′  and 5′-CAACTTCATCCACGTTCACC-3′  HPV plasmids and patient samples were amplified with Qiagen Multiplex PCR kit in a 25 µl reaction volume containing 12.5 µl of 2× Master Mix, 5 pmol of each type-specific PGMY-t primer and 1 ng of plasmid DNA or 8.5 µl of extracted patient sample DNA. In each PCR reaction using HPV plasmid template, forty nanograms of extracted DNA from HPV-negative human placenta was added to mimic an environment containing chromosomal DNA. Amplifications were performed in a thermal cycler  starting with polymerase activation at 95°C for 10 min and 40 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min followed by a final extension at 72°C for 10 min. The amplification reaction mixtures were stored at 4°C. Five-µl aliquots were visualized in agarose gels, and 20 µl were purified using the PCR Purification Kit  and eluted in 30 µl distilled water. The PGMY-t HPV multiplex primer mix sequences are listed in m for each discriminating probe and common probe was set to approximately 55°C as computed by OligoCalc Nearest Neighbor algorithm The viral genome sequences obtained from NCBI database were managed using Staden Package Gap 4 v4.8b1 software The LDR  was carr5′-TCAATGCACTGAGCCCGAGA-3′) The 16 compartment microarray slides were spotted with 119 different complementary ZipCode oligonucleotides  in triplicate. Each cZipCode oligo also contained a sequence complementary to the hybridization control probe (The ligation reaction (20 µl) was diluted to obtain 40 µl of hybridization mixture containing 5X SSC and 10 µg of herring sperm DNA. After heating the mixture to 94°C for 2 min and chilling on ice, 5 pmol of the complementary hybridization control probe was added and the mixture was applied onto the microarray slide according to manufacturer's instructions. Hybridization was carried out in the dark at 55°C for two hours in a temperature controlled hybridization oven. After hybridization, the microarray was washed for 3×15 min in 0,1× SSC, 0.1% SDS and 3×5 min in distilled water. Finally, the slide was dried in a table top centrifuge. For Agilent microarrays, hybridization was performed according to the manufacturer's instructions. Briefly, hybridization mixture containing 1× GEx hybridization buffer , 1× GEx blocking reagent , 18 µl of ligation reaction and 1 µl (5 pmol) of control oligo was applied on each subarray and hybridized for 17 h in the dark at 65°C at 10 rpm rotation. The slide was washed with Gene Expression wash buffer 1  for 1 min at RT and Gene Expression wash buffer 2  for 1 min at 37°C. 10% Triton X-102 was added to both washing solutions to final concentration of 0.005%. The fluorescent signal was detected at 5 or 10 µm resolution using a GenePix Autoloader 4200AL laser scanning system with green laser for Cy3 dye  and blue laser for 6-FAM  marrayL′i = Lilog(Li/Ci), where Li is the raw probe channel signal value at feature i and Ci is the adjusted control channel signal value at feature i.The microarray data were managed using R-software The background signal distribution in HPV detection was determined by selecting the normalized signals of all non-specific probes for each template from each microarray used in the type specificity experiments and excluding signal values over 2000. The background signal distribution served as a reference against which probe signals were compared with one-sided statistical testing. The background distribution was adjusted for each individual microarray hybridization by computing the difference between the background distribution median and the median of three highest values in known negative probes . Three one-sided statistics, t-test with unequal variances (Welch's test) and two non-parametric tests (Mann-Whitney and Kolmogorov-Smirnov), were evaluated in their ability to differentiate true positive  and true negative  signals from 23 replicated microarrays. The performance of the tests was evaluated by ROC with p-values between 0 and 1 and by computing the overlap of p-value distributions. Mann-Whitney with p-value limit of 0.005 was used in final testing.In order to establish functionality and type specificity of the test, we prepared ligation reactions for each HPV type individually using 40 fmol HPV plasmid PCR products as templates for the ligation probe pool. The majority of the probes produced specific signals on their cognate templates easily distinguishable from the background of non-specific probe signals . HoweverAfter initial testing of MY09/11, GP5+/6+ In order to determine the sensitivity of the method, we performed a dilution series experiment using HPV 16 and 18 plasmids as templates for PGMY-t HPV PCR. The plasmid amount ranged from 1 pg to 0.1 fg. The amplicons were detected by HPV LDR probes on microarray. One fg was the lowest detectable amount of plasmid, after which the signals from HPV 16 and 18 probes were indistinguishable from other HPV LDR probes . SignalsIn order to classify signals into positive and negative categories, we compared three statistics in their ability to differentiate true positive and true negative signals. The signals from the background and true negatives distributed similarly close to zero, as expected, and the true positives distributed more widely partially overlapping with the background . All thrTwo sets of patient samples were tested in order to evaluate the performance of LDR. First, a set of 16 samples was tested with LDR and the widely used non-genotyping Hybrid Capture 2 test (HC2) . Sample Testing for hrHPV DNA in primary screening, cytology triage and follow-up of treated patients has gained widespread acceptance as it has been shown to increase the accuracy of screening in silico alignment to target genomic sequences. Furthermore, we found the multiplexing capacity of our PGMY-t primer set superior to other tested primer sets. This is in agreement with Gheit and coworkers, who reported that type-specific multiplex PCR primer pool performs better than GP5+/6+ primers Specific LDR probes were found for all 15 hrHPV types and two lrHPV types included in the study, showing good overall performance of our test using plasmid templates. However, design of new probes to replace non-functional ones is needed for the detection of three lrHPV types  and for those types with only one functional probe. It should be emphasized that each probe requires individual experimental validation because current tools do not allow reliable testing of probe candidate functionality on the basis of For any test in routine diagnostic use, an objective standard for target presence or absence is necessary to eliminate problems of interpretation. Typically these criteria have not been discussed in reports of diagnostic HPV microarrays but instead obtained signals are interpreted more or less visually as a sign of presence of a given type Studies comparing a HPV DNA microarray with other detection methods like sequencing In conclusion, we have demonstrated sensitive and accurate HPV genotyping by type-specific multiplex PCR followed by ligation reaction and microarray detection, together with objective criteria for the presence or absence of HPV types. Preliminary evaluation demonstrates the feasibility of our test for clinical samples. In future, HPV genotyping is likely to be launched to clinical use for the management and follow-up of patients with cervical epithelial abnormalities requiring accurate and economical high-throughput testing.File S1Sequences of PCR primers, LDR probes and ZipCodes.(XLS)Click here for additional data file.File S2Patient sample p403-2z triplicate. The HPV probe pool signals in three hybridizations of sample p403-2z and associated p-values are shown.(PDF)Click here for additional data file.File S3Sequencing results of two different patient samples with HPV type-specific primers. The used primers are the same as in PGMY-t multiplex pool. The files are AB1 trace files.(TAR)Click here for additional data file.File S4Comparison of PGMY-t and original PGMY multiplex primer systems at 100 pg and 10 pg template concentrations. This figure complements data presented in (PDF)Click here for additional data file.File S5Results of dilution series experiment using HPV 16 and 18 plasmids. Boxplots showing the signals of HPV 16 and 18 HPV LDR probes and other HPV LDR probes.(PDF)Click here for additional data file.File S6Linear Array results of 16 patient samples. The figure shows scanned pictures of each LA genotyping strip with reference guide for 16 patient samples.(PDF)Click here for additional data file.File S7LDR results of 16 patient samples. The figure shows boxplots of each HPV LDR probe for 16 patient samples that were also analyzed with LA and HC2 (see  HC2 see .(PDF)Click here for additional data file.


L1, E6, E7 and long control region (LCR) of HPV 52, which have been identified in women from Northeast China by HPV GenoArray test, were analyzed. Twenty-five mutations were identified in the regions examined. Of the mutations found in the L1 gene, three novel nonsynonymous mutations of C5640T, A5641T and G5642A were located within the region that encodes the binding domain of neutralizing antibodies against HPV 52. Although four variations were identified in HPV 52 E6 and E7 genes, no significant association was found between the mutations and the cytological lesion of the patients. Eight mutations, including a novel CTT7681–7683 deletion, found in the LCR of HPV 52 encompassed the known transcription binding sites, which may possibly affect the transcription of the oncogenic genes of E6 and E7. The most prevalent HPV 52 variant in women from northeastern China belongs to clade L1-LN-A. The genetic variations of HPV 52, including three novel nonsynonymous mutations of C5640T, A5641T and G5642A in the L1 gene and a novel CTT7681–7683 deletion in the LCR, were first documented in strains from women in Northeast China. The statistical result showed no associations between the variants and the severities of the infected women. These findings provide new data regarding gene variations of HPV 52.Human papillomavirus (HPV) 52 is an oncogenic HPV type prevalent in Asia. The aim of the study was to analyze HPV 52 genetic variations in women from Northeast China. To explore the intratypic variants of HPV 52, the genomic regions of  Oncogenic human papillomavirus (HPV) is a major cause of cervical cancer. HPV 16 and HPV 18 are highly prevalent in all regions of the world , followeLCR) may affect virus replication rates and transcriptional activity of E6 promoters [LCR, E6, E7, and L1 genomic regions of HPV 52 was analyzed. Studies on HPV 52 variants in strains from cervical disease patients may improve the understanding of the molecular mechanisms underlying disease progression and transformation.Amino acid changes may affect the transforming activity of the E6 and E7 oncoproteins; those in the L1 protein may affect the efficacy of viral infection or alter viral antigenicity ,13. Mutaromoters ,15. In tL1, E6, E7 and LCR genes were performed on all the single HPV 52 positive samples where sufficient materials were available. From the 72 samples, 60 sequences of L1 gene, 66 sequences of LCR, E6 and E7 were obtained, respectively. L1, LCR, E6 and E7 genes, respectively, compared with the HPV 52 prototype. The histologic diagnoses of the women whose infected strains were sequenced successfully for the L1 gene were as follows: 11 normal cases (18.3%), 30 cervicitis (50.0%), 6 Ascus (10.0%), 3 CIN 1 (5%), 8 CIN 2 to 3 (13.3%) and 2 invasive cervical cancer (ICC) (3.33%) , 34 Cervicitis (51.5%), 7 ASCUS (10.6%), 3 CIN 1 (4.55%), 8 CIN 2 to 3 (12.2%) and 2 ICC (3.03%)  were solely infected by HPV 52. Samples with multiple HPV positives were excluded to avoid confounding the results. Analyses of the /815 were (3.33%) ; those f (3.03%) .p = 0.305), 1.741  and 1.278 , respectively  in HPV 52 LCR variants, compared with two variation sites (0.45%) over 447 nt in E6 variants, two variation sites (0.67%) over 300 nt in E7 variants and thirteen variation sites (0.82%) over 1590 nt in L1 variants. Deletions were found only in LCR. Based on the variations of E6, E7 genes and LCR, the strains were clarified into four groups (p = 0.512) and 0.714 , respectively.Based on their variation rates among the four analyzed genes, the proportion of polymorphic nucleotides was significantly greater in the r groups . The staL1 gene revealed 13 different nucleotide mutations. Except for C6917A, all the other mutations in HPV 52 L1 gene were not reported previously. Among them, three (23.1%) novel mutations of C5640T, A5641T and G5642A, which led to the Q26L amino acid change, were identified in nine strains  in the E6 gene (A379G (K93R) mutation was located in the strand H1 and the third predicted zinc finger of E6 protein. Analysis of the complete E7 open reading frame showed two synonymous substitutions of C751T and A801G.The complete  E6 gene . The A37LCR sequences had eight nucleotide mutations .Compared to the reference sequence, utations . Among tLCR encompassed, and thus potentially affected, the proposed binding sites for transcription factors. In particular, the nucleotide substitutions of G7622A and T7624G/C were located in the TATA binding site, as well as C/EBP and SRY binding sites. The CTT7681–7683 deletion was located in the NF-E2 and AP-1 binding sites; the nucleotide substitution of G7861A was located in the Oct-1 binding site. The variations of A7657C, T7659C, G7712C and A7865G, which were predicted at the binding sites for AP-1, HSF, MAT alp, Skn-1 and HFS, introduced additional putative binding sites for cellular proteins, such as Cap, Cdxa, Skn-1, and Oct-1 or HSF. Putative binding sites for cellular proteins SRY, and others, were also affected by less frequently encountered variations (data not shown). However, the binding sites for the HPV E2 protein were conserved in all the strains.All of the eight point mutations identified in Globally, the most prevalent HPV type is HPV-16, detected in approximately 40% of high-grade cervical lesions , and folHowever, the prevalence of other high-risk HPV types varies among different countries and even throughout regions of the same country . In receLCR, L1, E6 and E7 genes of HPV 52 was analyzed. Compared to the published data [LCR and the coding regions of HPV type 52.Researches on genetic variability of HPV variants may increase the understanding of the molecular mechanisms underlying disease progression and transformation. Indeed, a number of studies suggest that variants of the same HPV type are biologically distinct and may confer differential pathogenic risks . In thished data –26, seveIn a recent study, an increase prevalence of CIN3 was reported to associate with HPV 52 lineage C . ResultsL1 gene were not described elsewhere [C5640T, A5641T and G5642A, which lead to amino acid change of Q26L, were detected in nine strains from patients with HSIL. The Q26L mutation was located in the strand β-B1 of the L1 protein, which may suggest that the variants were established to escape neutralization. This mutation may influence the folding of the L1 protein, with possible consequences on the immunogenicity of neutralizing epitopes, thus favoring persistence of the infection by viral evasion from neutralizing antibody responses [Except for C6917A, all the other mutations in the HPV 52 lsewhere . The novesponses ,29.E6 and E7 genes are believed to be the main oncoproteins. In this study, the E6 and E7 gene sequences were relatively conserved, for only four mutations were identified in most of the studied HPV 52 strains, and no significant trends accordant with severity of cervical neoplasia were observed. Among the four mutations, only A379G (K93R) was nonsynonymous. The mutation G350A detected in the E6 gene has been reported as G350T in the study of Ding et al.[A379G (K93R) variation was located in the strand H1 and the third predicted zinc finger of E6 protein and occurred in almost all of the HPV 52 positive samples.HPV ng et al.. In accong et al., the uniLCR revealed one covariation pattern. Notably, the novel CTT7681–7683 deletion is firstly reported being detected in all the studied strains in Northern China. The biological meaning and geographical areas of such finding remains to be established. In addition, some of the mutations identified in LCR encompassed the proposed binding sites for YY1 and SRY transcription factors. It is suspected that these mutations may affect the transcription of the downstream E6 gene, though it remains to be determined by in vitro studies. Among HPV 52 isolates, the LCR T7624G/C substitution was detected in all of the samples tested. Interestingly, this mutation was suggested to be associated with the persistence of HPV 52 infection by Aho et al. [Analysis of HPV 52 o et al. .The genetic variations of HPV 52 were first documented in this report in Northeast China. Additionally, the frequency distributions of HPV 52 variants in Northeast China were different from those reported in European and American populations.Cervical swabs of the studied populations were collected from patients who referred to the Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University for routine gynecological detections. The median age of the studied populations was 38 years  at the time, the cervical scrapings were obtained. After giving informed consents, a total of 815 patients without a history of hysterectomy received an examination with the papanicolaou (Pap) smear, which collected cervical cells using ViraPap kits  at study entry. Pap smears were graded according to the Bethesda system, which categorizes cytology grades into normal, atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesion (LSIL), high grade cervical squamous intraepithelial lesion (HSIL) and cancer. Patients with LSIL or worse lesions were referred for a biopsy and treatment. Histological data were reviewed by expert pathologists to confirm the disease outcomes. Subjects gave a signed informed consent. The study protocol was approved by institutional ethical and research review boards of the participating institutions in the northeast of China.Viral types were detected using the HPV GenoArray test kit  according to the manufacturer’s instructions as described in the previous study . In thisLCR fragment and complete E6, E7 and L1 ORF, respectively. PCR reactions were done in a 50 μL reaction volume containing 1× PCR buffer, 200 μM of each dNTP, 2 mM MgCl2, 20 pmol of each primer and 1 unit of Taq DNA polymerase . PCR amplicons were separated on 2% agarose gels and visualized by ethidium bromide staining under UV transillumination. In the case of no observed band on the gel, 2 μL PCR products obtained with outer primer pairs were used as a template for amplification with inner primer pairs. The primer sets and PCR amplification profiles are shown in the Samples positive with HPV 52 were further amplified with primer sets specifically designed for the regions containing the partial PCR amplicons were purified and applied to enzymatic extension reactions for DNA sequencing using the ABI PRISM Big-Dye Terminator Cycle Sequencing Ready Reaction Kit . Both strands of the recovered DNA were sequenced with the same forward and reverse primers as those used for PCR amplification. The sequencing reactions were run on an ABI 3730 XL DNA Analyzer (Applied Biosystems). The obtained HPV 52 sequences were aligned with the reference sequence (Genbank Accession No.NC_001592) on NCBI . The mulLCR regions were searched by the TFSEARCH software, including sites for AP-1, E2, GRE, NF-1, Oct-1, TATA, YY1, C/EBP, Sp1, SRY, AML-1a and c-Myc/c-Max. Cut-off values and coincidence levels between consensus binding sites and the LCR sequence of HPV 52 type were adjusted in order to minimize both the number of negative and positive faults [Potential binding sites for cellular and viral transcriptional factors within the HPV 52 e faults .p < 0.05 was considered to be statistically significant.Statistical analysis was performed using the SPSS software (version 17.0) . The magE6, E7, L1 genes and LCR variants were found in our strains, no variant was found to have a significant association with the severity of cervical lesions of infected women. Because the number of women with CIN and more severe lesions was limited in this study, whether certain variants could increase the severity of cervical neoplasia still needs to be confirmed by future studies with a larger number of cases.In summary, the distribution of HPV 52 variants was investigated in a cohort of Chinese women. Some new variations were found among the studied strains. Although high ratios of HPV 52