The objective of our study was to assess the performance of different triage strategies for high‐risk human papillomavirus (hrHPV)‐positive results utilizing either extended genotyping or a p16/Ki‐67 dual‐stained cytology (DS) approach, with or without partial genotyping. A subset of women with hrHPV infections participating in the Addressing the Need for Advanced HPV Diagnostics (ATHENA) study were analyzed to determine the number of cervical intraepithelial neoplasia grade 3 or worse (≥CIN3) cases detected, and the absolute risk for ≥CIN3 of each genotype. A clinical utility table was constructed to compare the impact of different triage strategies. In all, 2,339 women with single‐genotype hrHPV infections were identified. Among these were 171 ≥CIN3 cases. The U.S. Food and Drug Administration (FDA)‐approved algorithm  for primary HPV screening detected 132/171 (77.2%) ≥CIN3 cases and required 964 colposcopies (colposcopies per ≥CIN3 ratio: 7.3). An approach that uses DS instead of cytology in the FDA‐approved algorithm detected 147/171 (86.0%) ≥CIN3 cases, requiring 1,012 colposcopies (ratio: 6.9). Utilizing DS for triage of all hrHPV‐positive women identified 126/171 (73.7%) ≥CIN3 cases, requiring 640 colposcopies (ratio: 5.1). A strategy that detected HPV16/18/31/33/35+ captured 130/171 (76.0%) ≥CIN3 cases, requiring 1,025 colposcopies (ratio: 7.9). Inclusion of additional genotypes resulted in greater disease detection at the expense of higher colposcopy ratios. Substituting cytology with a DS triage approach improved disease detection and the colposcopy detection rate. Further reduction of colposcopy rates can be achieved by using DS without partial genotyping. Extended genotyping strategies can identify a comparable number of cases but requires an increased number of colposcopies. What's new?Among HPV‐positive women, the goal is to maximize the number of CIN3 or greater cases detected while minimizing colposcopies. These authors compared various combinations of genotyping and dual‐stained cytology (DS) as triage strategies. From a pool of 2,339 hrHPV+ women, 171 cases with ≥CIN3 were identified. Only one algorithm they tested boosted sensitivity over the current approach without reducing specificity. This method used DS to triage women who tested positive for 12‐other HPV, and referring HPV16/18+ patients directly to colposcopy. This approach caught 147 of the 171 cases, while reducing the number of colposcopies per case from 7.4 to 6.9. AISin situadenocarcinoma ASCUSatypical squamous cells of undetermined significanceATHENAAddressing the Need for Advanced HPV DiagnosticsCIconfidence intervalCINcervical intraepithelial neoplasiaCIRcumulative incidence rateCPRcentral pathology reviewDSdual stain FDAU.S. Food and Drug AdministrationHPVhuman papillomavirushrHPVhigh‐risk human papillomavirusKPNCKaiser Permanente Northern CaliforniaNILMnegative for intraepithelial lesion or malignancyThere is an evolving worldwide consensus that the best way to screen for cervical cancer is to include high‐risk human papillomavirus (hrHPV) testing in the initial screening process. Multiple studies have documented the superior sensitivity of clinically validated hrHPV testing over Pap cytology for detecting cervical precancer and cancer.in situ (AIS), is on the order of 0.5–1%.The high degree of reassurance of a negative HPV test result is balanced by the risk of precancer or cancer in HPV‐positive women. The prevalence of HPV infections in a screening population can depend on the age distribution and sexual activity, but averages ~10% in Western countries.U.S. guidelines from 2012 apply the concept of equal management for equal risk to drive an algorithmic referral strategy for colposcopy.Triage of HPV positives can be accomplished in several ways. In almost all current algorithms, Pap cytology is applied to the HPV‐positive individuals, and any abnormal cytology result leads to colposcopy. Cytology has the advantage that it is already available in all established screening systems, and it is relatively specific for colposcopically identifiable disease, especially if the cytology is nonequivocal .However, just as cytology is less sensitive as a screening test, it may suffer in sensitivity as a triage test. Indeed, one of the criticisms of HPV primary screening with cytology triage is that the triage step may hinder the sensitivity of the algorithm. Therefore, in many settings, either repeat cytology after 6 months or partial HPV genotyping has been used to improve identification of patients most at risk for ≥CIN3.In current U.S. practice, partial genotyping means testing for HPV genotypes 16 and 18, which together account for 60–70% of cervical cancers.Extended genotyping generally refers to an approach that uses individual genotype‐specific information beyond HPV genotypes 16 and 18, that is, it offers insight into the amalgamated 12‐other hrHPV genotypes reported as a pooled hrHPV‐positive result by various U.S. Food and Drug Administration (FDA)‐approved HPV tests. Recent epidemiologic data have shown that the risk of CIN3 for some of those 12‐other hrHPV genotypes, such as HPV31 or 33, is on par with HPV18.Besides cytology and partial genotyping, a promising triage test shown to offer improvement over cytology is p16/Ki‐67 dual‐stain (DS) testing on a liquid‐based cervical cytology specimen.Current strategies for triage of hrHPV‐positive screening results include cytology, some form of genotyping, DS testing or combinations thereof. There is also growing interest in the potential utility of testing for alterations of both host and viral methylation patterns for triage.https://ClinicalTrials.gov (NCT00709891).The present study expands upon previous analyses from ATHENA, a large U.S.‐based, multicenter cervical cancer screening study in more than 47,000 women. The study demographics, design and genotype‐specific results have been described in detail previously.ATHENA has been well described in other publicationsSubsequently, 8,067 archived liquid‐based cytology specimens stored at 2–8°C for up to 5 years from women ≥25 years were utilized for p16/Ki‐67 DS testing using the CINtec® PLUS Cytology test . The CINtec PLUS Cytology test was performed as previously described.Of the evaluable patients included in the ATHENA substudy population, a further subset of women with single HPV‐positive infections by the LINEAR ARRAY® HPV Genotyping Test, who had valid test results for the cobas® HPV Test, p16/Ki‐67 DS testing, liquid‐based cytology and a valid central pathology review (CPR) result formed the basis of the present analysis , were calculated for each single genotype infection at baseline  by using the ratio of sensitivities and 1‐specificities of a potential strategy relative to the existing FDA‐approved algorithm.Sensitivity and specificity, along with 95% CIs, were plotted for each screening strategy. To compare the impact of various triage strategies on baseline disease detection, ratios of colposcopies performed per ≥CIN3 detection were calculated. For example, “14 hrHPV+ and DS+” describes a strategy whereby any case that is positive for any of the 14 hrHPV genotypes, and is also positive using DS testing, is sent for colposcopy.This approach allowed an assessment of the trade‐offs between performing an additional triage test at the lab  compared with performing additional colposcopies in the clinic .Of the 7,727 evaluable primary screening women ≥25 years old in the ATHENA substudy with a valid CPR diagnosis, 314 had nonevaluable DS test results, 4,196 tested HPV negative and 878 had infections by more than one genotype. These were excluded from this analysis. A total of 2,339 women with valid results for Pap cytology, DS testing, HPV testing and genotype testing with single HPV genotype infections were available for analysis. A total of 171 CPR‐confirmed ≥CIN3 were identified . Although the colposcopy referral rate is increased, the ratio of colposcopies performed per ≥CIN3 detected is decreased to 6.9 (compared to 7.3) because of increased disease detection. The interplay between the importance of HPV16/18 genotyping and DS testing can be seen in the strategy of utilizing DS testing on all hrHPV‐positive women. There is a large increase in efficiency relative to the FDA‐approved algorithm, measured by both a reduction in the number of colposcopies to 640 as well as the ratio of colposcopies performed per ≥CIN3 detected to 5.1. This is accompanied by a nonsignificant loss in sensitivity.vs. 147 cases) of the combined HPV16/18 and DS testing approach, albeit at a cost of 22% more colposcopies. Including seven of the hrHPV genotypes (HPV16/18/31/33/45/52/58) incorporated in the nonavalent HPV vaccine in a triage strategy detected three additional ≥CIN3 cases, but also increased the ratio for the number of colposcopies per ≥CIN3 detected from 8.4 to 8.9.The present study helps to inform the question of how many and which HPV genotypes would be required to refer women to colposcopy to at least meet the performance of the current FDA‐approved algorithm. Three combinations of genotypes standout: HPV16/18/31/33/35 would capture 130 cases, albeit with an increase in the number of colposcopies and a decrease in efficiency compared to the FDA‐approved algorithm as a reference. The combination of HPV16/18/31/33/45/52 rivals the sensitivity  cytology who is HPV16 positive has been found to be at substantial baseline risk for ≥CIN3 (~15%), well above the colposcopic referral threshold of ~5% at 3 years.Given the above, adhering to the mantra of equal management for equal risk becomes increasingly important. The availability of reliable extended HPV genotyping tests has clarified the type‐specific risks associated with some genotypes whose prevalence/virulence interactions historically have been averaged or masked in the absence of genotypic information.However, extended genotyping clearly is not the only way to address the growing demand for an efficient and effective primary screening algorithm. The retrospective analysis from ATHENA demonstrated the potential for DS testing to provide remarkably superior triage compared with cytology, while still capitalizing on the power of HPV16/18 genotyping.In the present analysis, the only strategy that resulted in greater sensitivity without a loss in specificity compared with the FDA‐approved algorithm was the “HPV16/18+ or 12‐other hrHPV+ and DS+.” The data shown in Table Although DS testing is less subjective than cytology, because the interpretation relies on identifying the presence or absence of an individual cell that has both a brown cytoplasm and a red nucleus rather than on morphologic interpretation, DS testing is not a molecular assay integrated into the HPV test, and requires going back to the original liquid‐based cytology vial collected from the HPV‐positive patients, preparing a slide and performing immunocytochemistry for professional interpretation. This is in part similar to Pap cytology in terms of specimen workflow, but no doubt will differ in reagent costs compared to a Pap stain.With extended genotyping assays, the presence or absence of each genotype (or subsets of genotypes) is reported individually. Basing management on these alone can avoid the cost of an additional triage test but requires more colposcopies to find the same number of cases. Additionally, management by genotyping alone is based solely on risk without providing information regarding what is occurring within the cells. It has the potential to lead to delays in diagnosis for women who may have an infection with one of the 12‐other hrHPV genotypes associated with a lower risk but that is already on the verge of becoming a cancer. Various “costs” such as those of additional testing or risk in deferred identification  must be weighed against the algorithmic benefits of detecting more ≥CIN3, and the number of colposcopies needed per ≥CIN3 detected. This should lead to lively debate in future guideline development.Finally, the data presented add to the evolving awareness of how HPV vaccination can directly impact screening. Most of the screening algorithms today are driven by disease caused by HPV16 and HPV18. As these viruses are removed from the population, the risk associated with a positive screening test decreases, as most of the overall risk is prevalence driven. Thus, strategies incorporating DS testing, which provide genotype agnostic risk information, or strategies that provide specific individual genotypes, may potentially both become more useful, or a test such as DS that “assembles” risk of neoplasia may become preferable.A major strength of this substudy is that it is derived from ATHENA, a large clinical validation study with thorough and rigorous disease assessment with adjudicated disease endpoints. One limitation is that the present analysis was restricted to single‐genotype infections; however, sensitivity of the FDA‐approved algorithm to detect ≥CIN3 in this ATHENA study subpopulation was 75.3%, compared to 74.5% in the total ATHENA population.In summary, as the world moves toward HPV primary screening, optimal triage is needed to maximize precancer detection while minimizing colposcopy. The data presented in this retrospective analysis demonstrate how genotyping and DS testing can be used to approach this goal of optimization in a data‐driven manner.Appendix S1: Supplementary MaterialClick here for additional data file.


To explore the relationship between the viral load reflected by the Ct value of Cobas 4800 HPV test and cervical lesions, and the effectiveness of the viral load for secondary triage of HPV-positive women.The Chinese Multi-Center Screening Trial (CHIMUST) evaluated both self-collected samples and physician-collected samples from women, aged 30 to 59, who were screened for cervical cancer in 6 regions across China. Using physician collected samples, the relationship between the HPV-Ct values of different subtypes and the cervical lesions was analyzed. Then the combined use of the HPV-Ct values with the HPV subtypes was evaluated as a secondary screening algorithm for the women who were HPV positive.r = -0.429, P<0.001; 12 other HR-HPV subtypes: r = -0.099, P<0.01). The HPV18-Ct value was not correlated with cervical lesion(P>0.05). Compared with HPV16/18 and cytology (HPV16/18 positive and 12-type pool plus cytology ≥ ASC-US), the sequential secondary screening using HPV16/18 and the viral load of 12-type pool (cut-point HPV-Ct≤31) had equal sensitivities for CIN2+ and CIN3+ , with slightly lower specificities  and higher colposcopy referral rate , but required no cytology.The Ct values of HPV16 and 12 other HPV subtypes(12-type pool), tested with Cobas decreased with the progression of cervical lesion  as the secondary screening for HPV positive women. This algorithm may be useful in low resource regions. Type specific persistence of high-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer . Therefo® 4800 (HR) HPV  is the first HPV testing technology approved by the US FDA for primary cervical cancer screening. It specifically reports the presence of types 16 and 18, and then detects 12 other high-risk types as a pooled sample. Although Cobas is a qualitative testing technology for HPV, it can provide the cycle threshold  of the virus copy number; that is, the number of cycles experienced by the fluorescent signal in each tube when it reaches the set threshold. There is a linear relationship between the Ct value and the logarithmic value of the number of HPV viral copies in the sample. The larger the initial viral copies, the smaller the Ct value [The Cobas® solution . The physician-sampling was performed following self-sampling. The physician placed a vaginal speculum to expose the cervix, then obtained a cervical exfoliated cell sample at the squamocolumnar junction of the cervix with a sampling brush and then placed the brush into a 20mL PreservCyt® solution (DOC sample) for testing. All samples were tested with the PCR-based high-risk HPV assays: Cobas and SeqHPV . The physician-collected samples were also tested by Cytology using the Hologic I2 imager system (computer assisted cytology). Cytology was used as a secondary screening to triage HPV positive women.  The protocol of this trial was approved by the ethics committee of Peking University Shenzhen Hospital(IRB:PUSH2016001) and Cleveland Clinic Institutional Review Board (IRB:15–1549). A total of 10,000 non-pregnant sexually active women, ages 30 to 59 years were recruited. Study entry required no prior pelvic radiation, no hysterectomy, and no cervical cancer screening in the past 3 years. Women were recruited from 6 provinces and cities in China. All participants signed an informed consent document before enrollment. All participants provided a self-collected vaginal sample and a physician-collected endocervical sample. The sample on the self-sampling brush was transferred to the POI card  . It can be used for full-automatic sample preparation and real-time PCR amplification to detect a total of 14 HR-HPV subtypes, including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, and provides the results of HPV 16 and HPV 18, and the pooled results of the other 12 subtypes in the assay. It can also provide the cycle threshold  of the virus copy number,that can reflect the viral load. All procedures were carried out in strict accordance with the working manual of the testing technology and the guidelines for the companion kit.Patients testing HPV positive for any HPV sample (self or direct on either assay) were called back for colposcopy, and evaluated using POI biopsy protocol of directed and random biopsies plus endocervical curettage (ECC) . Both coP<0.05, the differences were interpreted as statistically significant.SPSS v.22.0 software was used for all data analysis in this study with the significance level α = 0.05. Quantitative data were described by mean ± standard deviation and compared by one-way ANOVA. Pearson correlation analysis was used to analyze the correlation of quantitative data. A logistic regression model was used to analyze the relationship between covariates and the probability of CIN2+ in HR-HPV positive women; the OR was adjusted for covariates. McNemar was used to analyze the differences between the sensitivity and specificity for CIN2+ detection and CIN3+ detection in the paired sample groups, and when All relevant data are within the manuscript. Authors did not access to information that could identify individual participants during or after data collection.From August 2016 to January 2018, 10885 eligible women were screened with an average age of 43.9 years. 80.6% (1650/2046) of the participants testing positive on one or more tests returned for colposcopy. Except for 396 cases of colposcopy without returning, 90 cases of any HPV assay failed,10399 had complete data and are therefore included in this analysis. The pathological diagnosis showed: normal cases , CIN1 (177), CIN2 (88), CIN3 (49) and cervical cancer (5). The enrolled subjects were divided into different arms according to the pathological results: 1,508 cases of ≤CIN1 , 142 cases of CIN2+ , and 54 cases of CIN3+ (CIN3 and cancer).Cobas-HPV positive rate was 10.78% (1121/10399); The positive rates of HPV16, HPV18 and non-16/18 HPV were 1.96% (204/10399), 0.72% (75/10399) and 8.94% (930/10399) respectively; 136, 52 and 850 patients were only infected with HPV16, HPV18 and the other 12 types respectively.P<0.001). After pairwise comparisons, it was found the Ct values of HPV16 and 12 other HR-HPV subtypes in ≤CIN1 lesions were higher than those in CIN2 and CIN3+ lesions, but there was no statistically significant difference between CIN2 and CIN3+ lesions (P>0.05). The Ct values of HPV16 and 12 other HR-HPV subtypes gradually decreased with the increase in the grade of cervical lesions . HPV18 infection caused only 7 cases of CIN2+ lesions. There was no statistically significant difference in the HPV18-Ct value between all grades of cervical lesions (P>0.05) and there was no correlation between the HPV18-Ct value and the grade of cervical lesions . See There were significant differences in the Ct values of HPV16 and 12 other HR-HPV subtypes in cervical lesions of different grades . With the quartile of the Ct values of HPV16 and 12 other HPV-Ct subtypes as the threshold, the HPV-Ct values were divided into three levels, namely, Q1: ≤25% percentile, Q2: ≤50% percentile, Q3: ≤75% percentile. See The risk of CIN2+ in HPV16-positive women increased significantly with the decrease of HPV-Ct value  as compared with that in HPV16-negative women. The risk of CIN2+ in women who were positive for 12 other HPV subtypes also increased with the decrease of HPV-Ct value  as compared with that in women who tested negative for 12 other HPV subtypes. The risk of CIN2+ in women who were positive for 12 other HPV subtypes at all levels of Ct values was lower than that in women who were positive for HPV16. See Studies on viral load must be based on genotyping to obtain meaningful data concerning the correlation between viral load and cervical pre-cancer and cancer. The relationship between viral load and cervical lesions has been reported to be related to HPV type . In the In the Provisional Guidelines for Cervical Cancer Screening issued by the Society of Gynecologic Oncology and the American Society for Colposcopy and Cervical Pathology in 2014, sequential screening of combined HPV16/18 and cytology was recommended as a secondary triage plan for women who were HPV positive in primary screening ; the reaThe results of this study have confirmed that the Ct values of the 12-type pool in Cobas are closely related to the grade of cervical lesions. Therefore, we explored different threshold Ct values of the 12-type pool to triage women infected with 12 other types of HPV among the primary screening population who were HPV positive. Algorithm 5 of direct referral for colposcopy for HPV16/18-positive women and then referral for colposcopy for those who were positive for 12-type pool , had low sensitivities for CIN2+ and CIN3+, and a missed diagnosis rate of 33% for CIN2+ and nearly 20% for CIN3+, with 10 missed CIN3 cases. Due to the low natural reversal rate of CIN3, missing 33% is not suitable for triaging HPV-positive women. Algorithms 6 and 7 of direct referral for colposcopy for HPV16/18-positive subjects and referral for colposcopy for those from the 12-type pool with HPV-Ct values ≤31/35, had similar sensitivities and slightly lower specificities for CIN2+ and CIN3+ as compared with the guideline (Algorithm 4). Algorithm 6 had a reduced colposcopy referral rate of 6.59% as compared with primary screening (10.78%) while Algorithm 7 had a colposcopy referral rate which is reduced by 2.2% as compared with primary screening and increased as compared with Algorithm 6. In addition, Algorithm 6 had a similar sensitivity for CIN3+ as compared with Algorithm 7. As a result, the Algorithm 6 was considered to be better than #7, although #6 had slightly lower specificities for CIN2+ and CIN3+ as compared with the program recommended by guideline (#4), but it had similar sensitivities. The critical benefit is that the secondary screening can be completed with a single test, without adding cytology. It may reduce the number of return visits and the cost of a second examination depending how the primary sample was managed, and is clearly advantaged in areas lacking cytological resources. Therefore, the sequential screening with combined HPV16/18 and 12 other HPV types with the Ct value ≤31 can be considered as an alternative to cytology-based secondary screening.This study is the first to use HPV-Ct value to reflect the viral load as a secondary triage for HPV positive women The results may be biased due to the difference in the count of cervical exfoliated cells in the clinical sample. Therefore, in the large sample population screening, we obtained cervical exfoliated cell samples by a standardized sampling procedure of removing cervical secretions and rotating brush 3 times. Although we recognize this method cannot guarantee obtaining the same number of cells.In conclusion, the secondary screening algorithm of combining HPV16/18 subtype and the viral load of 12 other HPV subtypes (cut-point HPV-Ct≤31) for women who are HPV positive in primary screening, yields the same sensitivity as the currently used sequential secondary screening strategy of HPV16/18 and cytology, and no cytology examination is required. The secondary screening can be completed with a single test. As a result, it is especially suitable for areas lacking cytological resources.S1 Checklist(DOCX)Click here for additional data file.S1 File(RAR)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.


Using HPV testing to triage ASC-US still has some problems of unnecessary colposcopy in many cases. A previous study reported that methylation of CCNA1, a tumor suppressor gene, can differentiate between low and high grade lesions. This study was designed to evaluate the diagnostic values and application of CCNA1 methylation in the patients with ASC-US group. Cross sectional analytic study was conducted in the patients with ASC-US cytology. HPV DNA testing and CCNA1 promoter methylation testing were performed. The patients were sent for colposcopic examination and biopsy. Biopsy results were considered as gold standard. Diagnostic test of HPV test and CCNA1 methylation test were calculated for sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), likelihood ratio for test positive and negative and 95% confidence interval. One hundred and seventy patients were enrolled. Mean age was 39.7 years old. HR-HPV was positive in 70% of the patients. HPV type 16, type 18 and non-16,18 were 12.4%, 4.7% and 42.4%, respectively. CIN2+ were found in 12.4% (21 cases). CCNA1 promoter methylation was positive in 5 cases. CCNA1 had high specificity 99.3%, NPV 89.2% and PPV 80% in detection of CIN2+ but sensitivity was 19%. Likelihood ratio for positive test was 28.4 and likelihood ratio for negative test was 0.8. HPV test had sensitivity of 90.5% and NPV of 95.9% but low specificity and PPV as 31.5% and 15.7%, respectively. CCNA1 promoter methylation testing had very high specificity, likelihood ratio for the positive test and PPV . Therefore, CCNA1 promoter methylation test may be used in the HPV DNA positive cases to classify the urgency of colposcopy and the colposcopist should pay more attention to CCNA1 positive patients because of their higher chance to identify the significant lesions.  Cervical cancer is the third most common gynecological cancer worldwide . The incidence of cervical cancer is 14 per 100,000-person year . It is the second most common cancer in Thai women. Atypical squamous cell of undetermined significant (ASC-US) cytology is the minor cytologic abnormality. Incidence of invasive cancer in ASC-US is low and varies from 0.1 to 1% . According to American Society for Colposcopy and Cervical Pathology (ASCCP) guideline 2012 , there are two options to manage ASC-US including follow up and reflect HPV testing. HPV testing is used to triage because the risk of cervical cancer in HPV negative patients is extremely low . Therefore, HPV negative patients should not be sent for colposcopy . However, HPV testing has low specificity and low positive predictive value (PPV)  . Use of HPV testing still has some problems because positive rate of HPV testing in ASC-US patients are as high as 31.5% while the high grade lesion in ASC-US is only 5-10%. Therefore, HPV testing results in unnecessary colposcopy in many cases.Some studies reported that CyclinA1gene which is a tumor suppressor gene that was activated after E6 and E7 protein of HPV embedded in the host cells leaded to progress into cervical cancer. Some studies reported that the CCNA1 methylation was found in high grade lesions and cervical cancer but no CCNA1 promoter methylation in normal cervixes . The previous study reported that CCNA1 methylation in normal cervixes, LSIL and HSIL are different  . Therefore, CCNA1 methylation may be used to triage atypical squamous cell of undetermined significance (ASC-US) . This study was designed to evaluate the diagnostic values of CCNA1 methylation in the patients with ASC-US group and compare to HPV DNA test.This cross sectional analytic study was conducted in the patients with ASC-US cytology. The ASC-US specimens were reviewed by cytopathologists at King Chulalongkorn Memorial Hospital (KCMH). This study protocol has been approved by the Institutional Review Board (IRB), Faculty of Medicine, Chulalongkorn University (IRB No.603/59). All female patients with ASC-US cytology who came to colposcopy clinic at KCMH between February 2017 and January 2018 were recruited in this study. The patients who were performed Pap test from other hospitals were excluded. The participants signed their inform consent forms voluntarily. The process of cytologic specimen collection was done in the routine cervical cancer screening program by the residents, fellows and staff at KCMH. The collected cytologic specimen from BD SurePath™ liquid-based Pap test was sent for HPV DNA and CCNA1 promoter methylation testing. The cases with ASC-US cytology were sent to the colposcopy clinic for colposcopic examination and biopsy was performed at the most severe lesions. In case that no lesion was detected, random biopsy was performed. HPV DNA TestHPV DNA test was performed with Cobas® HPV testing, fully automated test based on extraction of the HPV and cellular DNA then followed by real time PCR to detect 14 high risk HPVs which consisted of 16, 18 and other high risk types . After that, the specimens were sent to CCNA1 promoter methylation testing at our lab. DNA bisulfite modificationDNA concentration was measured by nanodrop and subsequently adjusted to 750 ng/µl. Bisulfite treatment to 20 µl of each sample was performed by using the EZ DNA Methylation Kit (Zymo Research). The converted DNA solutions were eluted in 20 µl of M-Elution Buffer and stored below -20°C for subsequent use.CCNA1 real-time PCR® 7500 Real-Time PCR System . The PCR conditions were first denaturation at 95°C for 2 minutes then go after with 40 cycles as follows: denaturation at 95°C 15 seconds, followed by annealing at 60°C for 30 seconds. Negative control (dH20) and positive control ) were included in each PCR. A melting curve was generated to determine the specificity of the primers. Later, the threshold cycle (Ct) of the amplified methylation products was detected. The results of all samples must have Beta actin products as internal control. Data analysis reported positive if present of CCNA1 methylation product and reported negative if no CCNA1 methylation.To detect CCNA1 promoter methylation, we designed two TaqMan™. probe real-time PCRs. The CCNA1 methylation set was composed of the forward primer (5’ GGTAGGAAGAGTAGGTGTGTG 3’), reverse primer (5’ ACAACCCCTAACAACCCCCTCTAA 3’) and probe (FAM-5’GGGTTAGAGTGGGTAG 3’-BHQ). The Beta actin set primers were designed in the area of no CpG island to serve as internal control. The Beta actin set consisted of forward primer, reverse primer and probe. Both PCR reactions were prepared in a volume of 20 µL containing 10 µL of 2X TaqMan GTXpress real-time PCR master mix, 0.4 µL of 10 µmoles of each primer as well as probes and 2 µL of bisulfite-treated DNA template; the remaining volume was adjusted by adding milliQ DNase-free sterile water. Real-time PCRs were performed in duplicates using Applied BiosystemGold standard The gold standard for diagnosis was the most severe pathologic results from colposcopic directed biopsy. Colposcopists in this study had to pass the colposcopic training course and had to pass the 50 validated cases before performed colposcopic examination in this study. Colposcopic examination reports were recorded. Colposcopic impression which meant the opinion of the colposcopist whether the lesion was high grade or low grade before biopsy. The biopsy results were reviewed by pathologists and recorded.Statistical analysisStatistical analysis was performed using SPSS software version 22.0 . Demographic data were analyzed by descriptive method. Diagnostic test was calculated both HPV test and CCNA1 methylation test for sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and 95% confidence interval. Sample size was calculated by infinite population proportion formula based on sensitivity of CCNA1 from the previous study which reported 83% . Therefore, 170 participants were required in this study. All of the 211 patients of ASC-US cytology who underwent colposcopic directed biopsy at colposcopy clinic at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand from February 2017 to January 2018. Forty-one cases were excluded because the cases had undergone Pap smear test from other hospitals. Accordingly, the remaining 170 cases were included to performed HPV DNA and CCNA1 methylation testing.Demographic data are shown in CCNA1 promoter methylation was test in 170 specimens and positive test was found in 5 cases. The positive cases consisted of CIN2 (2 cases) CIN3 (2 cases) and condyloma (1 case); details are shown in Results of HPV testing are shown in The final outcomes of ASC-US in this study had high grade histology (CIN2+) 12.4%. This prevalence of high grade lesion in ASC-US is higher than the previous study in our institution in 2008-2012 which was 6.71% . The different of prevalence may result from the widely use of reflect HPV based on ASCCP recommendation. Therefore, ASC-US with negative HPV was not sent to perform colposcopy. However, some of the patients insisted to perform colposcopy instead of HPV triage were also sent to colposcopic clinic. For those reason, the proportion of high grade histology in ASC-US in this study was higher than previous study. However, this prevalence concordance with the study in Northern part of Thailand that found high grade histology 10-20% in ASC-Us cytology . The colposcopic impression or the opinion of colposcopist in the ASC-US cytology was reported low-grade lesion 70.6% which concordance with the previous study which found low grade lesion in colposcopy in ASC-US cytology as 68.1% . The diagnostic value of HPV testing in this study had high sensitivity 90.5% and high NPV 95.9% which was the high performance of screening test. Nevertheless, the specificity was low as 31.5%, which was concordance with the previous studies . Previous studies showed HPV testing had high sensitivity, low specificity and PPV in ASC-US cytology range 89.4-93.4%, 25.1- 59.3% and 17.5-20.3%, respectively . CCNA1 promoter methylation testing in this study had high specificity 99.3% which was the characteristic of the good diagnostic test. However, this test shown low sensitivity 19% which was too low to be a screening test. The data from the previous studies Chujan et al., (2014) and Kitkumthorn et al., (2006) showed that CCNA1 promotor methylation had high discrimination power to differential high grade lesion and invasive cervical cancer from low grade lesion . The Likelihood ratio for the positive test of CCNA1 in our study was very high (28.4) which was represented a good diagnostic test. Moreover, positive predictive value of CCNA1 was also as high as 80.0 (37.5-96.4). High likelihood for the positive test result and PPV told us that if CCNA1 positive there was high chance to identify the high grade lesion in the patient. These results may be used in clinical application. For example, CCNA1 promoter methylation test may be use in the HPV DNA positive cases to classify the urgent of colposcopy in the long waiting time situation. In CCNA1 positive patients, urgent colposcopy may be required. In case that HPV and CCNA1 results were negative, the patients may be omitting colposcopy because of high NPV 94.7% in this test. From our study, colposcopic results showed that 5 cases of high grade lesions were underestimated from colposcopic impression. CCNA1 may help in this situation. In the patients who had CCNA1 positive, the colposcopist should pay more attention to identify the lesions because of the high likelihood ratio for positive test. Our strength of this study is the final pathological confirmation was performed in all cases which was the gold standard to evaluate the diagnostic values. There are some limitations in our study, however. First, this study had small amount of CIN2+ histopathology. Second, there was positive test of CCNA1 methylation only 3% which less than the other studies therefore the result still inconclusive to be used as triage model. For further study, we may increase the number of the patients with CIN2+ for evaluation. Hence, the result of diagnostic values and role of triage may be clearer.In conclusion, CCNA1 promoter methylation test may not be a good screening test but CCNA1 may be useful when combined with HPV DNA test to allocate the urgency of colposcopy in the test positive patients and used as alert sign for carefully find the lesion when the test is positive. This research was supported by a 2019 Research Chair Grant from the National Science and Technology Development Agency (NSTDA), Thailand, and the Anantara Siam Bangkok Hotel, Four Seasons Hotel Care for Cancer Fun Run in coordination with the Thai Red Cross Society.


Men who have sex with men (MSM) are disproportionately affected by anal cancer, predominantly caused by high-risk (HR) human papillomavirus (HPV) infection. Currently, the nonavalent HPV vaccine provides coverage against nine HPV genotypes, including seven HR-HPV genotypes. Here, we characterize anal HR-HPV genotype distribution and associated risk factors in MSM from Toronto, Canada recruited between September 2010 and June 2012. Wilcoxon–Mann–Whitney test was used for continuous variables, Chi-square test was performed for categorical variables, and a multivariable model using logistic regression was created to assess for correlates of anal HR-HPV infection. A total of 442 MSM were recruited, with a median age of 45 (IQR 38–50) and an overall HPV prevalence of 82%. The prevalence of any HR-HPV infection was 65.3% and 50.7% in the HIV-positive and HIV-negative MSM, respectively. No participant tested positive for all genotypes covered by the nonavalent vaccine. HIV status , smoking  and the number of lifetime sexual partners  were independent risk factors for anal HR-HPV infection. Our findings will be useful to inform HPV vaccine rollout and HPV prevention strategies in Canadian MSM. Of the more than 200 HPV genotypes that have been identified to date, over 50 genotypes primarily target the transformation zone between the squamous and columnar epithelia of mucosal surfaces such as the ano-rectum3. Importantly, while most mucosal HPV infections have no symptoms and are spontaneously cleared by the host immune system, some cause persistent infection and can eventually lead to cancer of the affected area4. Mucosal HPV genotypes are classified as either low-risk (LR) or high-risk (HR), with LR genotypes sometimes causing benign warts but not cancerous lesions, while HR genotypes have oncogenic potential5.Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) globally7. The incidence of anal cancer has been increasing in high-income countries8, with particularly rapid increases in men who have sex with men (MSM) where the incidence is 40/100,000 in HIV-negative MSM and over 100/100,000 in HIV-positive MSM9, in comparison to a background rate of 2/100,000 in the general population10. Current treatment options for anal dysplasia include mechanical or chemical ablative techniques, but there are high recurrence rates regardless of clinical approach and it has not been formally demonstrated that any of these approaches prevents the subsequent development of cancer12. Moreover, recent evidence shows that the clearance of anal dysplasia in the absence of any intervention occurs at a fairly high rate of 22/100 person-years, further adding to the complexity of this clinical issue13.Anal cancer is an important consequence of HPV infection, with a meta-analysis finding over 80% of anal cancers to be linked to HPV infection, most strongly with HPV-16 at a rate of 82% in HIV-negative and 67% in HIV-positive men14. More recently the nonavalent Gardasil-9 vaccine, which adds protection against HPV 31, 33, 45, 52 and 58, has also shown safety and efficacy in MSM15. Notably, while the HPV vaccine is recommended up to the age of 45 for everyone in the United States, the indications in Canada are only up to the age of 26 for men16. Nonetheless, Canada’s National Advisory Committee on Immunization (NACI) still recommends that HPV vaccine be strongly considered in all MSM and people living with HIV regardless of their age18. As such, these guidelines lead to uncertainty and inconsistency in practices around HPV vaccine recommendations19. Furthermore, men generally have lower acquired immunity in response to natural HPV infection as compared to women21, and unlike cervical HPV infection which peaks shortly after sexual debut, anal HPV infection in men are found at relatively constant rates across the lifespan23. Taken together, these findings suggest that older MSM may also benefit from the HPV vaccine.Although clinical trials are underway to optimize screening and treatment protocols for anal HPV-associated disease, prophylactic vaccination is an important and validated method of HPV prevention. There are currently three commercially available vaccines in Canada, two of which are recommended for MSM. The quadrivalent Gardasil vaccine, which targets HPV 6, 11, 16 and 18, was shown to be effective in reducing the incidence of anal precancer24 to characterize genotype distribution of anal HPV infection, and risk factors associated with HR-HPV infection among HIV-positive and HIV-negative older MSM.Given this rationale for HPV vaccination among all MSM including older MSM, understanding the prevalence and risk factors for HR-HPV infection in this population may better inform vaccination policy, research priorities and future clinical implementation strategies. The current study expands on a prior analysis of HPV infection in MSM from TorontoA total of 442 MSM were enrolled in our study, consisting of 294 HIV-positive MSM and 148 HIV-negative MSM. The median age for the overall population was 45 (IQR 38–50), with the majority being white (74.1%). A third (31.0%) of the population had never smoked cigarettes, while 34.3% reported as former smokers and 34.7% as current smokers. Complete data including socio-demographic characteristics were available for 429 participants, of whom 267 (62.2%) tested positive for at least one high-risk (HR) HPV genotype versus 162 (37.8%) who tested negative for any HR-HPV genotype , any HR-HPV infection  and infection by multiple HR-HPV genotypes . Notably, HIV-positive MSM had an increased prevalence of both HPV-16  and HPV-18 , as well as the low-risk HPV genotype HPV-11 that is typically associated with anogenital warts , compared to 40.7% (59/145) among their HIV-uninfected peers. In the combined cohort analysis, HIV status , RAI in the last 6 months , current smoking  and reporting ≥ 50 lifetime sexual partners  all remained significantly associated with anal infection by multiple HPV genotypes  MSM may still obtain benefit from HPV vaccination.This cross-sectional study of MSM living in Toronto, Canada was performed to characterize the anal HPV genotype distribution and risk factors for anal HR-HPV infection and found a high prevalence of both HPV and HR-HPV infection in the anal canal. In particular, the rates of HR-HPV infection in both HIV-positive and HIV-negative MSM were broadly similar to rates seen in other countries such as Spain, Italy and China29, and we also found it to be a risk factor associated with both having any anal HR-HPV infection and having multiple HPV infections. We also found having ≥ 50 lifetime sexual partners to be a risk factor for HR-HPV infection only in HIV-negative MSM, which is in line with data from other groups. For example, a group in China found that sexual behavior such as condomless RAI was associated with anal HPV infection in HIV-negative but not HIV-positive MSM30. Likewise, researchers from the Netherlands found that having a higher number of lifetime male sex partners was significantly associated with anal HR-HPV infection in HIV-negative but not HIV-positive MSM31. Similarly, studies that largely (> 96%) included HIV-negative MSM have observed that number of recent sexual partners is associated with anal HPV infection33 or that a high number of lifetime sexual partners and recent RAI are associated with the persistence of anal HR-HPV infection34. The reasons for the role of sexual activity in increasing anal HR-HPV infection risk in HIV-negative but not HIV-positive MSM are unclear and merit further investigation.The risk factors for HR-HPV anal infection identified in our study are consistent with findings from other groups. For instance, cigarette smoking has consistently been associated with anal HPV infection35. Indeed, while HPV16 was the most frequently found anal HR-HPV genotype in both HIV-positive and HIV-negative MSM in our cohort, prevalence of other HR-HPV genotypes targeted by the nonavalent vaccine were found at high rates up to 16.3% and 8.1% in HIV-positive and HIV-negative MSM, respectively. Moreover, while others have reported a significantly higher prevalence of HPV31, 52 and 58 in HIV-positive compared to HIV-negative MSM36, they were found at comparable rates in our cohort; our results highlight the need to broaden our scope of understanding to include all HR-HPV genotypes in MSM regardless of HIV status.We decided to focus on the risk factors associated with anal HR-HPV genotypes, and not solely with anal HPV16 infection, given relatively high prevalence of several other HR-HPV genotypes. According to a recent meta-analysis, the prevalence of the following anal HR-HPV infections among HIV-positive MSM was above 5% and even exceeding 15% in some cases: HPV18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Similar trends with a generally lower prevalence were seen in HIV-negative MSM with rates above 5% for the following anal HR-HPV genotypes: HPV18, 39, 51 and 5238. However, given that recent sexual activity including having a new sexual partner or RAI in the past 6 months did not associate with anal HPV infection, it is likely that the majority of HPV infection from this study reflected a persistent, rather than an incident, infection. This is an important distinction as persistence of HR-HPV infection is a better surrogate marker of anal pre-cancer and/or cancer than the incidence/prevalence, which may reflect a transient infection. Data regarding participants’ HPV vaccination history were not available. However, at the time this study was performed, the HPV vaccine was not yet covered in Ontario for boys or gay men under the age of 26 years. Therefore, although it is unlikely that many of our participants would have received the HPV vaccine, it is not possible for us to completely discount the potential effect of HPV vaccination on the prevalence of HPV genotypes in MSM from Toronto.The cross-sectional design of our study is a limitation since it precludes us from making definitive conclusions about the time course of HPV infection detected in our cohort. Notably, several groups have reported a high incidence of HR-HPV infection in HIV-positive MSM with a history of recent sexual activityIn summary, the prevalence of anal HPV and HR-HPV infection was high in a cohort of older MSM in Toronto, Canada, but no participant tested positive for all genotypes targeted by the nonavalent vaccine. Risk factors associated with HR-HPV infection for the entire cohort were HIV serostatus, smoking and the number of lifetime sexual partners. Our findings will be useful to inform HPV vaccine rollout and HPV prevention strategies in Canadian MSM.The study was approved by the Research Ethics Board at the University of Toronto . Research was performed in accordance with the relevant guidelines and regulations. Informed, written consent was obtained from all participants.24.Self-identified MSM were recruited into a cross-sectional study of the epidemiology of HIV and other co-infections at the Maple Leaf Medical Clinic (MLMC) in downtown Toronto, Canada between September 2010 and June 2012. All self-identified MSM at or over the age of 16 receiving clinical care from MLMC were invited to participate in the study. A high-level overview of the prevalence of sexually transmitted infections in this cohort has been previously describedParticipants attended a single study visit. They completed a computerized, self-administered ACASI questionnaire  2.5, Nova Research Company, Bethesda, USA), which included information about socio-demographics, sexual and medical history. The sections on the ACASI questionnaire included: (1) interview information, (2) socio-economic characteristics, (3) sexual history and behavior, (4) STI, hepatitis and HIV testing, diagnosis and treatment, (5) other health behaviors, and (6) HIV and STI knowledge, beliefs and attitudes. Blood, first-void urine and a self-collected anal swab were collected to perform diagnostics tests against viral and bacterial infections as described in the next section. All specimens were stored at 4 °C until pick-up.N. gonorrhoeae and C. trachomatis as previously described24. For HBV testing, “active HBV infection” was defined as having a detectable HBV surface Ag and “ever infected with HBV” was defined as having undetectable HBV surface Ag but a positive HBV surface or core Ab with no vaccination history. With regards to syphilis testing, non-reactive CMIA test was considered as negative, “active syphilis” was defined by positive reactions from all three tests , and “previously treated syphilis” was defined by having reaction to CMIA and TPPA but not RPR.Participants were screened for HIV, herpes simplex virus (HSV)-1/2, cytomegalovirus (CMV), hepatitis C (HCV), hepatitis B (HBV) and syphilis with serology tests using 20 mL of blood collected in the serum separating tube and the acid citrate dextrose (ACD) tube. First-void urine was used to test for 39. Of note, 13 of the 46 genotypes were defined as being high-risk: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. All other genotypes were defined as being low-risk. DNA from anal samples were extracted using the Roche MagNA pure 96 automatic extractor. In brief, samples were spun down and subsequently eluted using MagNA Pure 96 DNA and Viral NA Large Volume Kit (Roche) and the Viral NA Universal Program. HPV DNA extracted from anal swabs was amplified by nested PCR using AmpliTaq Gold DNA polymerase (Perkin-Elmer) with the PGMY primers followed by the GP5+/GP6+ primers. HPV type-specific probes of 30 nucleotides in length located at the L1 gene were used to detect the PCR products by xMAP technology. Hybridization was analyzed using a Luminex Liquid Chip 200 flow cytometer (Qiagen) and the Luminex IS software (Luminex). A fluorescence signal of 100 fluorescent units or higher was determined as the threshold for positivity. Beta-globin negative specimens post-amplification were considered invalid and therefore excluded from our analyses.Participants were instructed to self-collect an anal swab by moistening the CultureSwab polyester-tipped swab (BD) tip with a saline solution, inserting the swab about 2–3 cm into the anus and rotating the swab 10 times in one direction and 10 times the other direction. Self-collected anal swab was collected in PreservCyt Transport Medium (Cytyc Corporation) and used to perform microsphere-based HPV genotyping  to test for 46 mucosal HPV genotypes at the Toronto Public Health LaboratoryData from the laboratory and the ACASI questionnaire were analyzed using SPSS, version 27 for Mac (SPSS). Wilcoxon–Mann–Whitney test was used for continuous variables being reported with medians and interquartile ranges (IQR), while Chi-square test was performed for categorical variables being reported as frequencies and proportions with 95% confidence intervals (CIs). Factors associated with anal HR-HPV infection were determined using logistic regression. A conceptual framework of anal HPV infection among MSM was constructed based on the literature, to help better understand the different risks and correlates, along with any potential interactions, and to guide the construction of our multivariable model. Multicollinearity was assessed by checking the ‘tolerance’ and ‘variance inflation factor’, and it was determined that none of the variables in the models constructed had ‘tolerance’ or ‘variance inflation factor’ values that indicated multicollinearity. A combination of a priori and a change-in-estimate inclusion of variables were used for our model. The following variables that have consistently been associated with HR-HPV infection were selected a priori regardless of the results from the univariate analyses: history of receptive anal intercourse in the last 6 months, HIV serostatus, lifetime number of male sexual partners and smoking status. The following variables were selected using a change-in-estimate approach, defined as changing the parameter estimate of the primary risk factor variable—receptive anal intercourse—by 10% or higher: number of sexual partners in the last 6 months, use of methamphetamine, ecstasy or MDMA in the last 6 months and drug use within two hours of sexual activity.


The consistency of pathologists in the diagnosis of cervical intraepithelial neoplasia (CINs) is not ideal, especially between low- and high-grade squamous intraepithelial lesions (LSIL and HSIL). This study was aimed to explore efficient strategies for the grading of CINs.The medical records of patients with high risk human papillomavirus (HR-HPV) infections who had underwent cervical biopsy or conization from April 2018 to April 2019 in Beijing Chao-Yang Hospital were collected and examined. The HR-HPV E6/E7 mRNA in the tissues of patients with CINs was detected using RNAscope chromogenic in situ hybridization (RISH). Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a (P16) and Ki67.P < 0.05). According to the logistic regression analysis, merely E6/E7 mRNA positivity was significantly associated with CIN2/3 . In the detection of CIN 2/3, the sensitivity and specificity of HPV E6/E7 mRNA alone was not significantly inferior to that of its different combinations with Ki67, p16 and cytology .HR-HPV E6/E7 mRNA signals were detected in 3/27 (11.1 %) of CIN 1, and in 32/33 (97.0 %) of CIN 2/3. Most of the staining patterns  had a full-thickness epithelial layer staining with weak-to-strong nuclear and cytoplasmic dot-like signals in CIN 2/3, and there were also few special staining patterns that were significantly different from the others. A number of indicators were compared between LSIL and HSIL. There were statistically significant differences in E6/E7 mRNA, p16, Ki67 and cytology between the two groups (RISH is efficient in grading of CINs. The HPV E6/E7 mRNA expression might reflect the phase HPV infections, and its positive pattern might predict the development direction of CINs, providing the possibility to realize more accurate treatments for patients. Cervical cancer (CC) is one of the most common malignant tumors in women. Its incidence and mortality ranks fourth among malignant tumors in women, and approximately 300,000 women die due to this tumor every year . A largeP16, as a multi-tumor suppressor gene, is directly involved in the regulation of the cell cycle, inhibiting the activity of cyclin-dependent protein kinase CDK4/CDK6 . P16 oveThe overexpression of HR-HPV E6/E7 genes is the key causative factor for CINs and CC . The mRNThe medical records of patients with CINs detected by cervical biopsy or conization from April 2018 to April 2019 in Beijing Chao-Yang Hospital were retrospectively reviewed. All outpatients within 19–65 years old were routinely inquired whether they recently received CC screening examination. If not, cytology  and HPV detection  were performed under oral informed consent. The results for the cervical cytology were reported using the 2014 Bethesda (TBS) system [Patients with CIN1, CIN2 and CIN3 were compiled for further evaluation. Patients with concurrent invasive CC, which is a simultaneous or subsequent primary malignant tumor in other parts of the body, and incomplete medical records were excluded. Furthermore, patients with insufficient tissues for the ISH or IHC examination were also excluded.The patient clinical data, which included age at diagnosis, smoking history, drinking history, age at first intercourse, sex partners, education, fertility status, menopausal status, body mass index (BMI), monthly income, cytology, HR-HPV detection, surgical method, pathological diagnosis and screening history, was collected and evaluated. The BMI was calculated by dividing the weight in kilograms by the height in meters squared. Subjects who smoked at least one cigarette a day and continuously for more than six months were considered positive for smoking history. Drinking at least once a month, including social engagements, was considered positive for drinking history.The H&E slides of these patients were reviewed by two independent gynecologic pathologists, who were blind to the results. Any disagreements between these two reviewers were discussed and resolved. If a disagreement could not be resolved, a third review would be conducted until a consensus diagnosis (at least a two-way agreement) is reached. The histological classification criteria of CINs were referred to the World Health Organization (WHO) classification criteria for cervical intraepithelial lesions in 2014 . For CINFive 4-µm-thick sections were cut from the FFPE tissue. Hematoxylin and eosin (H&E), RISH and IHC staining were respectively performed.The RNAscope HR 18 HPV assay is designed to detect the E6/E7 RNA for 18 HR HPV genotypes . The RNAscope® 2.5HD probes and detection kit were purchased from Advanced Cell Diagnostics . The assay was performed according to supplier’s instructions (ACD). Pretreatment: After being de-paraffinized and dehydrated, these sections were serially treated with the Pre-Treatment 1 solution and Pre-Treatment. Overnight, Pre-Treatment 3 was performed. Hybridization: The sections were hybridized in the HR 18 HPV hybridization solution without a cover slip in a HybEZ Oven . Signal amplification: The hybridized probe was performed through the serial application of Amp 1–6. Visualization: Diaminobenzidine (DAB) was used to demonstrate the amplified signal. The sections were counterstained with H&E, dehydrated with graded ethanol and xylene, and mounted with Cytoseal.The staining data was recorded with the presence of dark brown, nuclear + and dot-like cytoplasmic+/−. The positivity pattern was recorded according to thickness of the epithelial staining, the presence and amount of diffuse, and/or the punctate nuclear staining and cytoplasmic staining, as well as the signal intensity. Morphologically, normal epithelia in the FFPE block were used as the internal negative control. A positive control was added to ensure that the dyeing process of the slides was successful. If the positive control did not show a positive staining, the batch of slides were considered as invalid.The RISH slides were scanned using an automatic digital pathological slide scanner at 20 × magnification. The expression intensity of the HR-HPV E6/E7 mRNA was analyzed using the StrataQuest software. The nuclei were identified and screened by H&E staining channels. The effective nucleus, which was a single effective nucleus that eliminated the identified oversized adherent nuclei and undersized cell fragments, was used as the core to identify the areas with DAB staining signals within the cell range. Then, the E6/E7 mRNA expression of a single cell was quantified using the software, according to the DAB staining intensity and number of cells in the lesion area.2O2, the sections were incubated with the antibodies. Then, the secondary antibody reagent was performed. The reaction was detected by DAB and counterstained with H&E. A positive control slide was used to ensure the validity of the staining procedure.IHC was performed using the antibodies of P16  and Ki67 , respectively, according to manufacturer’s instructions. Then, the sections were de-paraffinized and dehydrated. The antigen retrieval was performed by boiling the slides with the ethylene diamine tetraacetic acid (EDTA) Antigen Retrieval solution (pH 9.0) in a pressure cooker for 15 min. After blocking the endogenous peroxidase with HDefinition of P16 positivity: According to the Lower Anogenital Squamous Terminology (LAST) definition of p16 positivity , which cP-value of < 0.05 was considered statistically significant.SPSS (21.0) was used for the statistical analysis. The rate and percentage of expression was used to describe the general situation of the study subject. HSIL was used as the clinical endpoint of the disease to evaluate the diagnostic efficiencies of HPV E6/E7 mRNA ISH, P16 and P16/Ki67 on CINs, and calculate the sensitivity and specificity of these various detection methods. Chi-square was used to test for differences in expression rate, sensitivity and specificity. Binary logistic regression was used to calculate the OR and 95 % confidence interval (CI), which describes the risk factors associated with CINs. A A total of 67 patients, who underwent cervical biopsy or LEEP, were pathologically diagnosed with CINs during the study period. Five patients with concurrent invasive carcinoma were excluded due to invasive carcinoma. Two patients with insufficient tissue for ISH or IHC were excluded. Finally, a total of 60 patients, who met the inclusion criteria, were included for further evaluation. The clinicopathological characteristics of these patients are presented in Table P > 0.05). The analysis of the StrataQuest digital images revealed that there was no statistical difference in single-cell intensity or the percentage of positive cells between CIN2 and CIN3 (Table P > 0.05).The characteristics of the HPV E6/E7 mRNA staining of patients presented with an E6/E7 mRNA positive expression and LSIL disease, as presented in Fig. P = 0.000, 0.000, 0.000 and 0.024, respectively). Positive E6/E7mRNA was a risk factor for HSIL in the logistic regression analysis (P = 0.011), with an OR of 3.961, indicating that HSIL Positive RNA was 3.961 times more likely than negative RNA. For patients with positive E6/E7 mRNA expression, the risk rate of HSIL reached as high as 91.4 %, when compared to the rate of 4.0 % for those with negative E6/E7 mRNA expression.The potential predictors for HSIL disease were evaluated in Tables P > 0.05).The sensitivity and specificity for the HPV E6/E7 mRNA was 96.97 % and 88.89 %, respectively (Table The present study had three major findings: (1) HPV E6/E7 mRNA was highly expressed in HSIL, and its positive expression obviously increased with the grade of the lesion. (2) For the detection of HSIL, the sensitivity and specificity of HPV E6/E7 mRNA ISH were better than p16 and p16/Ki67, but were not significantly inferior to the other different combinations. (3) The HPV E6/E7mRNA positive expression might reflect the phase of HPV infection, and its expression pattern may predict the development direction of CINs.In the present study, HPV E6/E7 mRNA, Ki67, p16 and cytology were the predictors of HSIL in the univariate analysis. Cytology examination  has been widely used for the screening of CC. A large prospective multicenter clinical study revealed that the sensitivity and specificity of TCT is 73 %-94 % and 58 %-76 %, respectively, in the screening of CC. Compared with the traditional pap smear, the detection of TCT for LSIL and above could reach as high as 73 % . HoweverAs a nuclear proliferation antigen, ki67 is exclusively expressed in the proliferation phase of the cell cycle . Ki67 exP = 0.011,OR: 3.961) in the logistic regression analysis. The sensitivity and specificity of HPV E6/E7 mRNA alone were better than p16, abnormal cytology and Ki67, and were not significantly inferior to the different combinations. E6/E7 mRNA was the direct transcription production of the gene. The expression level of E6/E7 mRNA could accurately reflect the status of the HPV infection. Therefore, it was concluded that RISH can be used for the grading of cervical lesions, and that this has the potential to reflect the different stages of HPV infection.In the early stage of HPV infection, the virus mostly presents as a transient infection or latent infection. The HPV genome is in a free state in the nucleus, and maintains a low replication activity . HPV E6/The progressing risk of HSIL was significantly higher than that of LSIL. Furthermore, approximately a third of the patients could naturally regress, especially for CIN2 in young women, and this natural regression increased with the extension of time . Most yoAlthough the present study has some limitations, which included the relative small sample size and insufficient follow-up data, the present data provides an objective and convincing method to assist in the grading of cervical lesions.In summary, the data of the present study support the view that HPV E6/E7 mRNA ISH can be used to assist in the grading of CINs. HPV E6/E7 mRNA positive expression might reflect the phase of the HPV infection, and its positive pattern may predict the development direction of CINs, which could provide the possibility to realize a more accurate treatment approach for patients. There is a huge potential value in pathological diagnosis of CINs with HPV E6/E7 mRNA ISH, which is worthy of further investigation.


This study aims to screen the male human papillomavirus (HPV) infection status and genotyping in Qingcheng District, Qingyuan City, Guangdong Province, China to provide a reference basis for formulating prevention strategies for HPV infection.The present study collected urethral epithelium or scraped penile epidermis from high-risk male patients in Qingyuan People's Hospital during the last five years, extracted DNA fragments using the boiling method, and detected 23 types of HPV genotypes by PCR-reverse blot hybridization.The positive detection rate was 54.31% of 1044 males with high risk of HPV (567/1044). Among these males, the positive detection rate of HPV was the highest in patients initially diagnosed with warts, and the rate was 66.47%. Five main HPV types are identified as follows: HPV6 18.87% (197/1044), HPV11 10.25% (107/1044), HPV52 8.81% (92/1044), HPV16 6.90% (72/1044), and HPV51 5.08% (53/1044). Among these HPV-infected patients, single infection mainly by low-risk HPV6 and HPV11 accounted for 56.61% (321/567); high- and low-risk combined HPV co-infections accounted for 29.10% (165/567). The HPV infected patients was mainly between 21 and 40 years old, and the HPV infection rate was higher with increased age.The HPV infection rate in the Qingyuan area is higher than in other areas and the main infection is single infection. Furthermore, HPV52, HPV16, and HPV51 are the main high-risk infection types, while HPV6 and HPV11 are the main low-risk infection types. Papillomaviridae family [Human papillomavirus (HPV) is a circular double-strand DNA virus belonging to e family . It has e family . It has e family . Hence, Currently, comprehensive studies of HPV infection and genotypes in men are still rare. In order to improve the understanding of the male HPV infection and genotypes, here we screened the male human papillomavirus (HPV) infection status and genotyping in Qingyuan, China.This study was a retrospective trial. From January 2014 to September 2018, male patients with high-risk for HPV infection who were hospitalized and received a physical examination in Qingyuan People's Hospital were recruited in this study. These participants were hospitalized due to their disease. HPV infection can be divided into low-risk type and high-risk type according to the pathogenicity or carcinogenic risk. High-risk populations mainly include the following: (1) subjects with symptoms of urethritis, wrapping balanitis, etc.; (2) subjects with suspected condyloma acuminata, papules, and ulcers; and (3) subjects who are conscious of the symptoms of discomfort or have a physical examination. This study was conducted in accordance with the declaration of Helsinki and approved by the Ethics Committee of the Sixth Affiliated Hospital of Guangzhou Medical University and Qingyuan People’s Hospital. Informed consent was obtained from the patient or his guardian.Inclusion criteria: (1) male patients; (2) patients over 14 years old; (3) high-risk populations for HPV infection. Exclusion criteria: (1) patients with incomplete clinical data; (2) patients with non-conforming specimen collection.Urethral epithelium or scraped penile epidermis were collected. Urethral swab was used to sample the urethral epithelium . The swaThe HPV-DNA genotyping (23 types) detection reagent kit was purchased from Shenzhen Yaneng Biological Technology Co., Ltd, which includes 18 high-risk types  and five low-risk types . The main inspection instruments included the following: Life express PCR expander, which was purchased from Hangzhou Bioer Technology Co. Ltd.; YN-H18 semi-automatic nucleic acid molecular hybridometer, which was purchased from Yaneng BioSciences (Shenzhen).One ml of sample containing medium was transferred to a 1.5-ml centrifuge tube and centrifuged for 10 min at a speed of 13,000 rpm. The liquid supernatant was discarded to retain the cell precipitate at the bottom of the tube. Then, 50 μl of lytic solution was added to the suspended precipitate, centrifuged for 10 min at 13,000 rpm, and heated for 10 min to retain the liquid supernatant containing DNA sample for future use.2O. The controls  were included in the kit. The testing reagent was provided by Shenzhen Yaneng biological company.Five μl of the extracted DNA was taken for the PCR reaction under the following conditions: 50 °C for 15 min; 95 °C for 10 min; 94 °C for 10 s, 42 °C for 90 s, 72 °C for 30 s, 10 cycles; 94 °C for 10 s, 46 °C for 60 s, 72 °C for 20 s, 30 cycles; 72 °C for five minutes. After amplification, the membrane strip was marked with the patient number and 2 mL of liquid A and all the corresponding PCR products were lightly shaken and hybridized at 51 °C for 30 min. Liquid A was discarded, 2 ml of liquid B was added and shaken gently for 10 min. Liquid B was then discarded, an incubation solution was prepared according to liquid A:POD = 2000:1, 2 ml of incubation solution was added to each film strip and it was gently soaked at room temperature for 15 min. The incubation liquid was discarded, and each film strip was washed at room temperature with liquid A and liquid C. The film was soaked in 2 ml color-developing liquid for 15 min. Then, 2 ml of deionized water was added into the discoloration solution, it was shaken gently for 3 min, and the results were observed. Result interpretation: (1) the IC site on the membrane strip had a signal interpreted as genotype positive in the corresponding position; (2) blue spots were found at one or more HPV genotypic sites, which were judged to be positive for the genotypic markers at the corresponding position; (3) there was no signal at the IC site on the membrane strip, or at the HPV genotyping site. The human ACTB (β-actin) gene was used as an internal reference. The positive control was the HPV-16 type plasmid, while the negative control was HP < 0.05 means that the difference was statistically significant.In the present study, EXCEL 2007 was used to collate and draw the charts, SPSS 17.0 statistical software was used to process the data, and the measurement data were expressed as mean ± standard deviation. The enumeration data were expressed as percentages (%). It was considered that In the present study, a total of 1044 male patients were involved in the detection of HPV genotyping. The age of these subjects ranged from 15 years old to 83 years old and the infection rate of HPV in each age group was above 50%. The overall trend of the HPV infection rate increased with age. Among these positive infections, the majority of infected subjects were within 21–40 years old, accounting for more than 70% of the infected population. The specific results are presented in Table Among the 1044 male high-risk subjects, 567 infected subjects were detected and the infection rate was 54.31%. The HPV infection rate for high-risk types, low-risk types, and high- and low-risk types co-infections was 32.09%, 38.80%, and 29.10% respectively.There were 23 genotypes in the 567 HPV-infected subjects. Among these, HPV52 was the most high-risk subtype with the highest detection rate (8.81%), and this was followed by the HPV16 (6.90%), HPV51 (5.08%), HPV68 (4.12%), and HPV58 (3.26%) subtypes. The low-risk subtypes with high detection rates were HPV6 (18.87%), HPV11 (10.25%), HPV42 (5.36%), and HPV43 (4.78%)  were aged 21–30 years. HPV6, HP81 and HP11 were the most common types, with 20 patients (35.09%), 9 patients (15.79%) and 8 patients (14.04%), respectively. 54 patients were diagnosed with warts, 21 of them were the ages of 31–40 years (38.89%). HPV6, HPV11 and HPV16 were the three common types, account for 25 patients (46.3%), 15 patients (27.78%) and 8 (14.81%) patients, respectively. Compared with the physical examination population, patients with clinical diagnosis of warts had higher detection frequency of HPV16. The other diagnostic types included foreskin glanthitis, skin infection, dermatitis, urethritis in a total of 38 people  were clinically diagnosed with warts, of whom 28 patients (60.87%) were aged 21–30 years. Among them, 25 cases (54.35%) contained HPV6, 16 cases (34.78%) contained HPV11, and 7 cases (15.22%) combined with HPV6 and HP11. Physical examination screened 26 patients with 3 or mor types of HPV co-infections. Nine of them were aged 21–30 years, accounting for 39.13%. There were 14 patients (60.87%) contained HPV52 and 9 patients (39.13%) with HPV6. The remaining 23 cases were clinically diagnosed with skin infection, urethritis, paraphernalitis, skin rash, prostatitis and other related diseases, accounting for 24.21%  refers to prostate disease caused by a variety of complex causes, with urethral stimulation symptoms and chronic pelvic pain as the main clinical manifestations. The positive detection rate of patients with clinical manifestations was the highest, reaching 66.47% Table .Table 5DHPV is closely correlated to a variety of diseases and is one of the more frequent DNA oncoviruses at present. Furthermore, 5.5% of cancers worldwide are associated with HPV infection .. [HPV was epitheliophilic and preferentially infects squamous epithelium rather than columnar, cubed, or transitional epithelium. Therefore, the head of the penis, the body of the penis and the scrotum, the urethral orifice were common sites of HPV infection. A previous study by Ni et al.  identifi.  reported.  reportedAmong the 567 HPV-infected subjects, the single HPV-type infection was dominant (56.61%) and significantly higher than that of the double infection (26.63%) and multiple infection 16.75%). HPV6 and HPV11 were most common HPV subtypes.Although HPV6 and/or HPV11 are mostly latent infections in the genitalia and usually do not show clinical symptoms, these are the dominant genotypes that cause condyloma acuminata in countries around the world –13. The .75%. HPVIn the present study, it was found that the positive rate of HPV in vegetation was the highest, followed by prepuce glans and uremitis. It was reported that the initial clinical manifestations of HPV virus in Chinese men are often condyloma acuminata, which is vegetations on the genital surface. Condyloma acuminata are most common in the genitals and anus. The color and size of skin lesions vary according to the course of disease . At firsThe infection rate of HPV in male patients with venereal disease in the Qingyuan District was high and mainly consisted of low-risk HPV or single-type HPV infections. Patients who were positive for HPV were mainly in the age group of 21–50 years old, and the detection rate was higher in the age group of over 50 years old. HPV52, HPV16, and HPV51 were the three main high-risk HPV types in the region. HPV6, HPV11, and HPV42 were the three main low-risk HPV types in the region. Since there are regional differences in the distribution of male HPV infection types , 23, masThere remain limitations in the present study. First, the present study was a retrospective study, and not a randomized controlled trial, and no blinding method was established. Therefore, there is still a risk of bias due to causes such as the differing severity of the patient’s disease, the differing duration of the disease, and the previous use of various drugs. Second, the present study is a single-center clinical study. Hence, there is still a need to carry out multicenter clinical studies.HPV infection in the Qingyuan District was higher than in other areas, and single infection was the main infection type. HPV52, HPV16 and HPV51 were the major high-risk infection types, and HPV6 and HPV11 were the major low-risk infection types. Therefore, male patients need to regularly check the reproductive tract to reduce the incidence of genital tumors.


The VA Measurement Based Care (MBC) in Mental Health (MH) Initiative supports implementing patient reported outcome measures (PROMs) for MH treatment planning and shared decision-making as a routine aspect of care. Using VHA administrative data, we identified Veterans initiating a new MH treatment episode (index encounter), i.e. prior 6-months without VHA MH encounters. We compare MH diagnoses, medications, and encounters during the 6-months from and including the index encounter by age  between Veterans receiving 1 or more measures (PROM) to those receiving none (noPROM). The percentage of PROM Veterans decreased with age: 26.7% (50-64); 18.5% (65-79); 12.5% (80+). Consistent across age, PROM Veterans had more encounters than noPROM Veterans. In the year before treatment initiation, a smaller percentage of PROM Veterans had multiple MH diagnoses (21.0% v. 29.1%). At treatment initiation, both groups were equally likely to have multiple diagnoses (20.7% v. 20.1%); a higher percentage of the noPROM group were diagnosed with schizophrenia (3.8% v. 1.0%), bipolar (4.5% v. 2.2%), or PTSD (29.2% v. 21.8%). Substance use disorder and major depression were more prevalent in the PROM group. These patterns held across age categories. A smaller percentage of PROM Veterans had been prescribed psychotropic medication during the index encounter (32.8% v. 42.8%). For PROM Veterans, an average of 3 measures were received 1.5 months apart. The number of measures declined and the interval between measures increased with age. Potential barriers and possible efforts to target the use of PROMs with older Veteran patients are discussed.


Finally, multiple infections had no effect on residual disease and did not influence the recurrence of high-grade CIN during a median follow-up of 25 months (IR 17–41). HPV16 interacted positively with HPV18 and negatively with HPV33, 51, 52, and 66 supporting the notion that HPV16 interacts mostly negatively with other HR-HPVs in CIN lesions. Among specimens coinfected with HPV16 and 18 or 52, there was an excess of CIN3+ although the impact on the prevalence of severe cervical lesions was limited.The aim is to evaluate the clinical consequences of coinfection between HPV 16 and other high-risk HPVs among women with a histological diagnosis of CIN or invasive cervical cancer. A total of 2985 women, with a diagnosis of either CIN or cancer (<IB) on cervical or cone biopsy, were included. HPV genotypes were identified using the INNO-LiPA HPV genotyping assay, version EXTRA, on cervical scraping, before the colposcopic evaluation and the colposcopic biopsies or conization. In the overall population, HPV16 interacted positively with HPV18  and negatively with HPV33, 51, 52, and 66, in log-linear analysis. There was an excess of CIN3 diagnoses among subjects coinfected with HPV16 and HPV18 or HPV52, although the absolute number of cases was relatively small. In a logistic model, the odds ratio of CIN3+ associated with coinfection of HPV16 and HPV18 (OR = 3.8, 95% CI 2.5–5.7,  Multiple simultaneous high-risk (HR) human papillomavirus (HPV) infections among women with abnormal cervical cytology or histology is common ranging from 20 to more than 50% of cases , 2. SeveMost of the published population data on the role of coinfection relating to the severity of CIN and to the recurrence after treatment are based on multiple infections as a whole rather than on the interaction of single HPV types. The purpose of the present study was to evaluate the rates and the prognostic significance of coinfection between HPV16 and other HR-HPV types in a population of women with biopsy-proven cervical intraepithelial neoplasia or invasive cervical cancer.μl final reaction volume for 40 cycles. Appropriate positive and negative controls were introduced for each set of reactions. Concurrent amplification of beta globin sequences was used as a control for DNA adequacy. HPV type-specific sequences were detected by the line probe assay INNO-LiPA HPV genotyping assay, version EXTRA , according to the manufacturer's instructions. The EXTRA version of the assay allows the simultaneous and separate detection of 18 high-risk (HR) HPV types  with proven carcinogenicity and Group I of the classification of International Agency for Research on Cancer (IARC), HPV26, 53, 66, 68, 73, and 82 with probable carcinogenicity and Group II IARC classification, seven low-risk HPV types , and two unclassified-risk HPV types (69/71 and 74) [Data for this retrospective cohort study were extracted from a database containing prospectively collected clinical, colposcopic, and virological details from subjects attending the colposcopic unit of our department because of an abnormal pap smear in the period 2010–2018. The database was composed by a series of anamnestic items gathered after a structured interview at entry and by clinical, colposcopic, and virological features compiled at entry and during the follow-up. Subjects (from 21 to 65 years of age) were referred to the cytologic screening unit of our department, from private practice and from screening services of external institutions. Exclusion criteria included pregnancy, HPV test or treatment for CIN in the year before enrollment, total hysterectomy, and the use of vaginal medication in the previous 3 days before screening. In the study, only subjects with a histological diagnosis of CIN or invasive cervical cancer (<stage IB) based on colposcopic biopsies or histological examination of specimens from loop electrosurgical procedures (LEEP) or cold-knife conizations were included. The study was approved by the local Ethical Committee of our hospital, and patients gave their informed written consent (RCXXX). All patients were treated according to an established protocol including HPV DNA detection and typing and colposcopy with targeted biopsies. Cervical samples for HPV typing were obtained immediately before colposcopy. After speculum examination, scrapes were taken with a cervical brush, suspended in Thin Prep-PreservCyt Solution , and stored at 4°C. DNA extraction was performed by lysis and digestion with proteinase K. HPV sequences from the L1 region were amplified by polymerase chain reaction (PCR) using SPF10 primers in a 50  and 74) . HybridiObservation, colposcopy, and/or cytology every 6–12 months for subjects with negative colposcopic impression and/or negative histological findings after an abnormal Pap smearObservation, colposcopy, and cytology every 6 months for CIN1 lesionsHPV testing coupled with colposcopy and cytology every 6 months for treated CIN2–3 or persistent CIN 1 lesions.A standardized colposcopic examination was performed immediately after cervical brushing for HPV typing by two gynecology residents certified by the Italian Society of Colposcopy. Multiple-targeted cervical biopsies were obtained in all cases where CIN was suspected on colposcopy and in all cases of high-grade squamous cervical lesions (HSIL) irrespective of colposcopic impression. Endocervical curettage was performed, according to the judgment of the clinician, when the extent of the lesion or the cervical squamocolumnar junction was not entirely visible or in the case of the presence of atypical glandular cells (AGC) on Pap smear. Histological diagnoses were based on consensus decision of two expert gynecological pathologists. In order to analyze the data, we either used the histological diagnosis obtained from punch biopsy or, in more severe cases, the diagnosis was made after cone biopsy obtained from LEEP (loop electroexcision procedure) or cold-knife excision. After diagnostic workup and treatment, patients referred to the cytologic screening unit of our department were enrolled in a follow-up program including the following:Univariate statistical analysis was carried out with the Kruskal–Wallis analysis of variance and chi-squared test to compare categorical variables, respectively. The Bonferroni method for multiple comparisons was used to evaluate partitioned chi-squared tests in multiway contingency tables. Spearman rank correlation coefficient was used to test for linear trend. To evaluate the likelihood of coinfection between HPV16 and other high-risk HPV types, we used log-linear analysis. An automated hierarchical stepwise method was used to select the model containing the least number of significant interactions necessary to fit the observed tables of associations between HPV16 and other HR-HPVs. In this model, nonsignificant interactions between variables are progressively eliminated (backward elimination) from the saturated model based on the likelihood ratio of chi-square. An initial model contained 18 HR-HPV types . The model fit was assessed by deviance modification. We used both ordered and multinomial/binomial logistic regression analysis with the aim of testing the association of HPV coinfection on the incidence and severity of cervical intraepithelial neoplasia. In ordered logistic regression equations, the outcome was modeled as a 4-level categorical variable , whereas exposure variables included age (continuous), HIV status , parity , cigarette smoking at entry , contraceptive use , and HPV status in 6 categories ; multiple infections associated with HPV interacting (HPV 16 negative)) We fitted the model estimating partial proportional odds using the gologit2 algorithm for the equations with variables violating the parallel regression assumption of ordered data . MultinoDuring the period of the study, a total of 3601 subjects between 21 and 65 years of age, with an abnormal pap smear who attended our department for colposcopic evaluation, were initially included in the study. Targeted biopsies were not taken in 373 (10.4%) women  and 48 with low grade SIL) with entirely negative colposcopic examination; these women were excluded from the analysis. In addition, from our study, we excluded 231 (6.4%) subjects with negative results on biopsies  and 12 (0.3%) subjects with macroscopic invasive cervical cancer (>Stage IB) who did not necessitate conization for evaluation of invasion which left us with 2985 subjects for our final analysis . Overall, after adjustment for age, parity, HIV infection, and type of contraceptive used, the ORs of CIN2, CIN3, and invasive cancer were 2.1 (95% CI = 1.5–2.8), 3.4 (2.51–4.7), and 4.8 (21.7–10.8) for single and 2.3 (95% CI = 1.6–3.2), 5.4 , and 5.7 (2.5–13.2) for multiple infections, respectively.The sociodemographic, cytologic, and virological data of the population under investigation is reported in p < 0.001 for two-way interactions, p=0.4 for three-way interactions). The results of modeling are reported in To test the significance of simultaneous associations of HPV16 with other HR-HPVs, we used hierarchical log-linear models. HR-HPVs were inserted in the model and backtested for simultaneous associations. The K-way and higher order effects test suggested that two-way interactions fitted the model adequately  or HPV16 and HPV52 , there was a statistically significant excess of CIN3 diagnoses, whereas the rates of coinfection with HPV16 and HPV51 or HPV66 were similar in the four groups of severity of cervical disease .p < 0.001 compared to multiple infection). Comparing odds ratios (p=0.011 and p=0.04) or multiple HPV16 infections without HPV 18 (p=0.003) or HPV 52 (p=0.001).To evaluate the effect of coinfection of HPV16 with HPV18 and HPV52 on the prevalence of CIN correcting for potential confounders, we used both ordinal and multinomial logistic analyses. In ordinal equations, outcome was inserted as a 4-level  ordinal variable . Overalls ratios , coinfecp=0.008 and p=0.03, respectively) or multiple HPV16 without HPV18 (p=0.01) or HPV52 (p=0.028) (p=0.01 compared to single HPV16 infections) and 5.73  among multiple HPV16 infections. When the outcome was modeled as CIN3+ subjects vs others, the odds ratio of CIN3+ was higher among women coinfected with HPV16 and HPV18 p=0.004 compared to single HPV16 infection) or coinfected with HPV16 and HPV52 (p=0.009) than women infected only with HPV16 . The likelihood of high-grade CIN associated with HPV16/18 and HPV16/52 coinfection was higher than those associated with single HPV16 (p=0.028) . The oveth HPV16 .Of the 2985 enrolled subjects, a conization procedure (1259 LEEP and 42 cold-knife) was performed in 1301 (43.6%) of the cases . Indicatp=0.015 compared to low-risk negative HPV). In stratified analysis, the coinfection between HPV16 and either HPV18, 33, 51, 52, or 66 did not influence the rates of residual disease on endocervical and ectocervical margins.The rates of low- and high-grade residual disease after conization on either ectocervical and endocervical margins are reported in p=0.08), respectively. Persistence/recurrence rate was also uninfluenced by coinfection between HPV16 and HPV18. The recurrence rate was 1.3 cases per woman/year in subjects with single HPV16 infection (7/197) and 2 cases (2 cases out of 41) per woman/year among subjects coinfected by HPV16 and HPV18. Finally HPV 16 persistence  was 52.3% (103/197) among single and 44.2% (46/104) among multiple HR-HPV16 infection at entry (p=0.22).Out of the 1301 women who received conization, follow-up data (follow-up of at least 6 months) were available in 1090 subjects (83.8%) mainly enrolled in the cytologic screening unit of our department. Subjects with invasive cervical cancer were excluded from the follow-up analysis. The median follow-up time for the entire cohort was 25 months (IQR 17–41 months) with a median of 3 colposcopies (IQR 1–5). A biopsy-confirmed low-grade and high-grade CIN after the first 6 months of follow-up was diagnosed in 94 (8.6%) and 46 (4.2%) of the subjects, respectively. The persistence/recurrence rate of high-grade CIN (CIN2+) was uninfluenced by multiple HR-HPVs upon enrollment. In fact, the recurrence rate was 0.1 cases per woman/year (4 cases out of 151) among subjects with low-risk/negative HPV, 1.7 cases per woman/year (25 cases out of 580), and 1.8 cases per woman/year (17 cases out of 355) among HPV16 single and multiple infections  interacted with α-7 (HPV18), α-5 (HPV51), and α-6 (HPV66) species. The rates of HPV coinfection of different α-species can be influenced by the type of population studied and geographic differences derived from genetic mutation of viral genome consequent to human migration and evolution [The rate of simultaneous multiple HR-HPVs of the present study is comparable to the 20–56% rate of multiple infections reported in the literature either in cytologic or histological series –7. In mugene DNA . We usedvolution , age, anvolution , 6, 7, 9volution . Howeverh α-7 HPV, α-5  and HPV16/52 (n = 28) represent 7.5% of all cases of CIN 3 in this series. These results were confirmed in logistic analyses corrected for potential confounders and by the use of both an ordinal model which captures the relationship between explanatory variables and increasing severity of the outcome, and multinomial models which in turn evaluate the effect of explanatory variables on distinct outcome categories. The main differences between our study and those of Wu et al. [Although by and large HR multiple infections seem to be associated with an increasing severity of HPV-related lesions, the role of HPV type interactions within multiple infections is less studied. A recent study from China  suggestsu et al.  are the u et al. . In our /52 n = 2 represenCompared to other investigations evaluating the occurrence and clinical significance of multiple HR-HPVs, the main strengths of this study were the homogeneity of population and the methods used, the histological diagnoses of CIN, and the evaluation of the effect of multiple HR-HPVs on the adequacy of treatment  and subsequent recurrence of high-risk disease. On the other hand, the observational nature of the study, a single hospital recruitment center, and the lack of a negative control population are the main limitations of this series.In conclusion, the results of this study suggest that, overall, coinfection with multiple HPV types is associated with an excess number of histologically confirmed CIN3 cases. In log-linear analysis, HPV16 interacted positively with HPV18 and negatively with HPV33, 51, 52, and 66 supporting the notion that HPV16 interacts mostly negatively with other HR-HPVs in CIN lesions. On the other hand, there was an excess (though modest in absolute numbers) of CIN3 cases among women coinfected with HPV16 and HPV18 or HPV52 suggesting that some interactions, albeit uncommon, can increase the risk of CIN. Finally, multiple infections did not influence the rates of residual disease on cone examination or the rate of recurrence of high-grade CIN during follow-up confirming the fact that the effect of HR-HPV interaction is limited on the natural history of CIN.