Within 2021, Norway intends to complete implementation of HPV DNA-based primary screening for cervical cancer for women 34–69 years, while continue cytology-based screening for women 25–33 years. Over the recent years, the incidence of cervical cancer has increased by 30% among women younger than 40 years. In this subset of women, nearly 30% were diagnosed with a normal smear, as most recent smear, prior the cancer diagnosis. This observation demands quality control of normal smears. The aim of this study was to assess increase in program sensitivity of CIN2+ after follow-up of women with false negative Pap-smears testing positive for a 3-type  HPV mRNA test in a cohort design over one screening interval. 521 women, aged 23–39 years, and no prior history of CIN1+ or HSIL, with an ASC-US or worse smear (ASC-US+) and 1444 women with normal screening cytology comprised the study cohorts. The positivity rate for the 3-type HPV mRNA was 1.9% (28/1444). Rescreening revealed 23 women with ASC-US, two women with LSIL, two women with ASC-H, and one woman with AGUS. If the HPV mRNA-positivity rate and histology findings from samples rescreened were applied to all women with normal cytology, an estimated increase in screening sensitivity of 16.4% (95% CI:15.3–17.5) for CIN2+ and 17.3% (95% CI:16.2–18.4) for CIN3+ were achieved. By rescreening less than 2% of women with normal cytology positive for a 3-type HPV mRNA test, we achieved a significant increase in screening program sensitivity. Cytology screening for cervical cancer has been effective in reducing cervical cancer incidence in countries with high-coverage and high-quality screening programs –4. In thPrimary HPV-screening with DNA-based tests find high-grade cervical intraepithelial neoplasia grade 2 and 3 (CIN2+) at an earlier time point than cytology based screening do . HoweverWorldwide HPV 16 and 18 are the most prevalent oncogenic types detected from cervical cancer tissue –15. CancIn Norway, there has been 30% increase in cervical cancer in women younger than 40 years over the past 10 years . About hIn the present study, we assess the impact of quality control by co-testing cytology normal young women with an HPV mRNA test targeting HPV-16/-18/-45 on screening program sensitivity, and evaluate the subsequent workload for the screeners caused by re-screening smears from women who had a positive mRNA test.http://screening.iarc.fr/colpochap.php?chap=2). One experienced pathologist (BW) reviewed all biopsies. Biopsies with uncertain cellular changes were immunostained with p16 (INK4a) (Roche mtm laboratories AG). If there was a discrepancy between biopsy and treatment histology, the most severe histology determined the endpoint.The Department of Pathology, Ålesund Hospital, assesses approximately 12 000 cervical smears annually. The department uses the Bethesda system for classification of cervical cytology and the WHO histological classification of tumors in cervical biopsies  a diagnostic kit for the qualitative detection and direct typing of E6/E7 mRNA from HPV 16, 18 and 45 according manufacturer’s instructions. The kit contains an intrinsic sample control (ISC) targeting a human housekeeping gene to assess specimen quality and reveal possible factors that may inhibit amplification. PreTect SEE sequences correspond to a subset of genotypes in PreTect HPV-Proofer (PreTect AS) and make use of real-time NASBA technology, an enzymatic one-step amplification process able to amplify RNA under isothermal conditions (41°C).Following national guidelines in delayed triage, Ålesund Hospital utilized the HPV DNA test Cobas 4800 (Roche Molecular Diagnostics), which detects 14 HPV types. In addition, HPV mRNA was detected using PreTect HPV-Proofer , which detects E6/E7 mRNA from types-16, -18, -31, -33, -45.During the study period, the Norwegian Cervical Cancer Screening Programme (NCCSP) recommended delayed triage of women with minor cervical lesions with repeat cytology and HPV testing 6–12 months after the index diagnosis of atypical cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesion (LSIL). In order to increase sample size (power) without compromising validity of testing, and to mimic the real life of follow-up in the NCCSP, we expanded the triage follow-up window from 90 to 540 days after the index smear.Referred to colposcopy/biopsy were women with high-grade squamous intraepithelial lesions (HSIL) or repeated ASC-US/LSIL with a positive HPV test. Women with a normal smear and a positive HPV test were recommended a repeat HPV test within 12 months, whereas women with an ASC-US/LSIL/normal smear with a negative HPV test returned to regular screening at a three-year interval .We sampled cells for conventional cytology (Pap smear) with a wooden spatula and a brush from the cervix uteri, and placed the material directly onto a glass slide followed by fixation. The same brush was rinsed in ThinPrep , and sent for subsequent HPV mRNA testing. In Norway, many hospitals have switched from conventional Pap smears to liquid-based cytology (LBC), but Ålesund Hospital still used conventional Pap smears over the study years.th, 2013, the department started rescreening all normal smears among women aged 23–39 years with a concurrent positive HPV mRNA test. During the inclusion-window 4747 women, 23 through 39 years of age had a valid smear. After exclusion of women with a history of CIN1+ (n = 339) or HSIL without CIN (n = 42), the study population comprised 4366 women. During primary screening we defined four follow-up cohorts; a passive arm of normal smears not tested for HPV (control arm), normal smears testing positive with the 3-type mRNA test (mRNA positive arm), follow-up of ASC_US-LSIL as the ASC_US-LSIL arm, and the HSIL-arm as follow-up of women with HSIL. Follow-up continued for one screening interval through December 31, 2017.From April 4All analyses were done in SPSS, version 24.0, as differences between means (detection rates) at significance level p < 0.05.The Regional Committee for Medical and Health Research Ethics  has approved the protocol as a quality assurance study in laboratory work (2014/669/REK midt). Norwegian regulations exempt quality assurance studies from written informed consent from the patients.In this low risk population of women younger than 40 years index smear were normal in 88.1% (3845/4366) of the women, 8.5% (370/4366) had ASC-US, 1.9% (82/4366) had LSIL (ASC-US/LSIL-arm), and 1.6% (69/3466) had HSIL, ASC-H, AGUS or ACIS (HSIL-arm). In samples diagnosed as normal, 1444 of 3851 (37.5%) had liquid based cytology (LBC) available for HPV-testing. In this subset of women, the positivity rate for HPV mRNA was 1.9% (28/1444) (mRNA-positive arm). Rescreening samples revealed 23 women with ASC-US, two women with LSIL, two women with ASC-H and one woman with AGUS. Finally, the control arm comprised 2401 women not tested for mRNA HPV (3845–1444).At triage, eight of 28 women in the mRNA-positive arm and 199 out 452 women in the ASC-US/LSIL arm returned to screening after having a negative HPV DNA test result and a normal, ASC-US, or LSIL cytology reading. In the ASC-US/LSIL arm, 35 women had no cytology follow-up, whereas 18 women had incomplete cytology follow-up. In the HSIL arm, two women never met for biopsy, and two women had only cytology follow-up. In total 20 women in the mRNA positive arm, 139 women in the ASCUS-LSIL arm, and 65 women in the HSIL arm had biopsies collected .Among women in the mRNA-positive arm, 32.2% (9/28) were CIN2+, respective 18.8% (85/452) in the ASC-US/LSIL-arm, and 88.4% (61/69) in the HSIL-arm. In total 146 women were diagnosed with CIN2+ in the ASC-US/LSIL- and HSIL-arms as practiced in the routine screening program .The overall screening program detection rate were 3.3% (95% CI: 2.8%-3.9%) for CIN2+, respective 2.8 (95% CI: 2.3%-3.3%) for CIN3+ . By rescDuring follow-up we diagnosed 23 cases of CIN2+ among 1 700 out 2 401women with normal smear and no mRNA testing at baseline. In this subset of women 19 CIN2/3 cases were tested at time for biopsy with a mRNA- or a DNA-test, among which 15 cases were positive for HPV-16, -18 or -45 in one (n = 8) or both tests (n = 7). Only one case was HPV-negative in both tests, while two cases were positive for HPV-31, and one case for HPV-33. There was 100% concordance between the mRNA- and the DNA-test results among the seven cases tested by both tests.Norway has started, and will within 2021, implement national screening for cervical cancer with a primary DNA test for women 34 through 69 years of age, and continue cytology based screening for women 25 through 33 years. By restricting the analyses to women aged 23 to 33 years, the study population comprised 2770 women, with a HPV mRNA-positivity rate of 2.6%. The estimated increase in screening sensitivity for CIN2+ and CIN3+ in this subset of women were 21.3% (95% CI: 20.1–22.5) and 14.1% ((95% CI:13.1–15.1), respectively.We diagnosed four cases of cervical cancer during follow-up after a positive screening cytology outcome  . For thrBy rescreening less than 2% of cytological normal samples that were positive for HPV E6/E7 mRNA types 16, 18 and 45, we achieved a significant increase in program detection rate for CIN3+ of 17–18%. Cervical cytology has a high rate of false negative samples. Low sensitivity for cytology in detection of CIN3 or invasive cervical cancer has been an issue for decades . Case-seAn improvement in diagnostic accuracy in cytology is one element of quality assessment in cervical cancer screening programs. While cytology represents morphological cellular changes, the detection of HPV mRNA expresses oncogenic molecular activity. Traditionally rescreening of a proportion of slides are considered an acceptable method for quality assessment if concordance of readings are high. However, this method is time consuming and tedious for cyto-technicians, and human errors are easily repeated (10). A molecular method is complementary to cytology in validation of diagnostic accuracy. It is therefore important that the molecular method target the HPV types that comprise the highest risk for progression to cervical cancer.One way of expressing progression rate of precancerous lesions to cervical cancer is to study prevalence ratio of HPV types found in cancer tissue by types in tissue from CIN3. A ratio above 1.0 expresses an association, while an expression less than 1.0 weakens the potential oncogenic properties of the targeted HPV types. Worldwide, publications consistently report HPV 16, 18 and 45 with a prevalence ratio above 1.0, while most other HPV-types have prevalence ratios less than 0.4 . The PreTransient infections of many HPV-types are prevalent in the female genital tract. Most women have normal smear readings from cervix uteri in situations of transient infections ,29. In oThe strength of our study is the population-based study design in a country having had a well-organized screening program for over 20 years. We excluded women with a history of high-grade cytology and/or CIN1+ that would represent persistent HPV infections, thus leaving a subset of low-risk women being exposed or not exposed to a probable incident HPV infection with oncogenic expression.Our study reflects the “real life” situation, the way women participate in the screening program, and how women and their doctors adhere to the recommended follow-up algorithms. We tried to avoid observation and attrition bias by comparing follow-up outcomes within one screening interval. The follow-up of women with normal cytology and no mRNA testing (N = 1701) revealed 23 incident cases of CIN2/3, among which 15 out of 19 tested positive for HPV 16, 18 and 45. This indicates that the estimates for increase in screening sensitivity by rescreening all women with normal cytology with a positive HPV mRNA test may be an underestimate of what is possible to achieve in a real life among women less than 40 years.According to annual reports from the NCCSP 1058 of 2712 (39%) cervical cancers were diagnosed among women less than 40 years in Norway over the 10-year period 2007 through 2016 . Among wIn 2011 the costs of the NCCSP were NOK 730 millions when approximately 3500 CIN2+ were diagnosed . If we aIn order to prevent cervical cancer with screening in women less than 40 years, high quality diagnosis and treatment of precancerous lesions before age 34 will be important for many years. In Norway, we have to wait until 2037 before the fully vaccinated cohorts reach 40 years.By testing all women less than 40 years of age with normal cytology with a specific 3-type HPV mRNA test, we achieved a significant (17–18%) increase in screening program sensitivity for CIN3. The 3-type HPV mRNA is a high throughput assay, representing similar workload as any HPV DNA test commonly used in primary screening. The volume of rescreened smears (1.9%) represent a low workload. In addition, the study adds quality to educating the screeners by rescreening presumably false negative Pap-smears.


To investigate the probability of marriage and divorce among Swedish military veterans deployed to Afghanistan relative to non-deployed matched comparators.Matched cohort study in Sweden.Military veterans were identified through Swedish military personnel registers regarding foreign deployments, and comparators from the Military Service Conscription Register (1969–2013). Of 1,882,411 eligible conscripts, 7041 had served in Afghanistan at some point in time between 2002 and 2013. To each military veteran, up to 5 non-deployed comparators who underwent conscription were matched by age, sex, psychological assessment, cognitive ability, psychiatric history and social characteristics. After matching there were 4896 (82%) unmarried and 1069 (18%) married deployed military veterans. The main outcome was marriage or divorce after deployment to Afghanistan. Data on marital status were retrieved from Statistics Sweden until December 31, 2014.During a median follow-up of 4.1 years after deployment of married individuals, 124 divorces were observed among deployed military veterans and 399 in the matched non-deployed comparator cohort . During a median follow-up of 4.7 years after deployment in the unmarried cohort, 827 new marriages were observed among deployed military veterans and 4363 in the matched non-deployed comparators cohort .Military veterans were more likely to divorce and less likely to marry after deployment compared with matched non-deployed comparators. There is a general belief that military veterans struggle with intimate relations and re-adjustment to family life after returning from foreign deployment.,2 While European veterans from the recent wars in Iraq and Afghanistan generally have lower levels of PTSD than their US counterparts, which could be at least partly explained by differences in combat exposure during deployment.–14 It isAccording to a systematic review by the RAND Corporation, there is a gap of research using longitudinal data investigating military deployment and risk of divorce, and studies with appropriate control groups. This is The purpose of this study was to investigate the effect of deployment to Afghanistan on the post-deployment incidence of marriage and divorce among Swedish military veterans.https://www.epn.se/stockholm/om-naemnden/), Sweden. Data were de-identified prior to delivery to the research group.This is a cohort study of Swedish military personnel deployed to Afghanistan (deployed military veterans) and matched comparators without deployment history. Matched comparators were identified from the Military Conscription Service Register containing individuals who had gone through military conscription tests but not necessarily completed military service . The cohIn Sweden, military conscription was mandatory for men until 2010. Women volunteers and men went through a 2-day test protocol at military conscription, including both physical and psychological examinations. Only 2–3% of all Swedish men were exempt from conscription testing until 2005, in most cases because of severe disabilities or congenital disorders. Approximately two thirds of those who underwent conscription were accepted for military service with 7–15 months of military training depending on position. After their training, the military servicemen and women went back to civilian life. After 2005, the number of conscripts who were tested dropped to about a third of each birth cohort, and approximately one third of tested men and women were accepted for military service. Due to political decisions, mandatory conscription and military service was abolished in 2010 when Sweden began a transition to professional armed forces.Men and women who completed their military service with sufficiently high military grades could apply for foreign military deployment at any time after their military service. The grades were set by the closest military officer in command and were approved by a ranking officer at the battalion level. The final decision as to who should be deployed was made by a military officer at the squadron level in the deployed military unit.In December 2001, the NATO-led security mission International Security Assistance Force (ISAF) was established by the United Nations Security Council to improve the security situation in Afghanistan. The Swedish Armed Forces sent troops to ISAF from 2002 until 2014, when ISAF was disbanded. With over 8000 deployments, ISAF was the most extensive military engagement for Swedish forces during the period.Data were collected from several government agencies. We used the following registers :2; categorized into underweight (BMI<18.5), normal weight (BMI 18.5 to <25), overweight (BMI 25 to <30), and obesity (BMI≥30)).The Military Service Conscription Register: The coverage and use of the Military Service Conscription Register has been described elsewhere. We retriPRIO database.Swedish Military Information Personnel (SWIP) Register: Deployment data were retrieved from the SWIP register for individuals deployed to Afghanistan at some point between 2002 and 2013. The SWIP register includes all types of service members, armed and unarmed, and contains information about country of deployment, date of deployment and return, sex, age, military rank, and regiment. Deployed military personnel have been registered in SWIP since 1965 except individuals serving in the Special Forces and classified personnel . Since 2012, the same deployment information has instead been entered into the Total Population Register: Date of emigration was retrieved from this register along with marital status, date of marriage, date of divorce, and date of death.National Patient Register: This register was started in 1964 and attained nationwide coverage for inpatient care in 1987.[Prescribed Drug Register: In July 2005, Sweden launched a nationwide complete prescribed drug register containing information regarding date and type of filled prescriptions.[The Register of Criminal Convictions: This register was started in 1973 and includes information based on convictions retrieved from all lower courts in Sweden. The register provides information on when a person was convicted of a felony and the type of sentence. We retrieved data on all types of violent crime convictions before deployment.Two cohorts were identified for this study from men and women in the Military Service Conscription Register.Deployed Military Veterans: Swedish military personnel who deployed to Afghanistan at some time between January 1, 2002, and December 31, 2013 were identified through the SWIP register and the PRIO database.Matched Non-Deployed Comparators: Up to 5 non-deployed comparators were selected by an exact matching procedure from individuals who underwent conscription. Matching factors included sex, deployment factors , conscription data , emigration, death, or end of follow-up, whichever came first .Pre-deployment and conscription characteristics were compared between deployed military veterans and non-deployed comparators using ANOVA for continuous variables and logistic regression for categorical variables to account for the matched design. This comparison and all other analyses were performed conditioned on the matching set consisting of one deployed military veteran and up to 5 non-deployed comparators sharing the same individual matching characteristics.Incidence rates and Kaplan-Meier failure functions were used to present absolute probabilities. Cox proportional hazards models were used to estimate hazard ratios for (a) the probability of divorce among married participants and (b) the probability of marriage among unmarried participants. AdditionSensitivity analyses were conducted excluding a) women, b) deployed military veterans with deployment duration <60 days, c) individuals who married the year before deployment (the divorce outcome) or individuals with a history of divorce (the marriage outcome), d) individuals who went through conscription after 2006 (when the Swedish conscription system changed) and e) military veterans deployed in unarmed service such as priests or chefs.Data were analyzed using SAS  and Stata . All tests were two-sided and P-values <0.05 were considered statistically significant.We identified 6072 military veterans with deployment to Afghanistan between January 1, 2002, and December 31, 2013. Of those, 107 were not eligible for matching due to missing data in the Military Service Conscription Register . After mCompared to age- and sex-matched non-deployed comparators, deployed military veterans had higher scores on cognitive ability and psychological assessment , lower pMarried vs. Unmarried Deployed Military Veterans Married military veterans had a higher average score on the cognitive ability test (72% vs. 60% scoring 6 or more) and the psychological assessment at conscription compared to their unmarried counterparts  or having no history of divorce (for the marriage outcome), individuals having performed conscription testing before 2006, and military veterans deployed in armed service .This study investigated the association of deployment to Afghanistan within ISAF and the post-deployment incidence of marriage and divorce among Swedish military veterans. We found that the military veterans who were unmarried at deployment (82%) were less likely to marry, and that the military veterans who were married (18%) were more likely to divorce after deployment in comparison to matched non-deployed comparators.Previous US studies have found mixed results for the effects of military deployment on divorce. US veterans serving in Vietnam, for example, do not seem to have an increased divorce rate than Americans in general, even though a strong association between PTSD and divorce has been reported in this population.,4 StudieA limitation of these US studies is that they did not take into account pre-deployment mental health, a factor that is likely to be linked both to the exposure (military deployment) and the outcome (marriage/divorce). In our study, individuals who went through the selection process for foreign military deployment had considerably better mental health  than theMeadows et al have suggested that higher divorce rates following foreign military deployment may be caused by traumas and unresolved mental health problems. For the Another possible explanation could be that several months of work-related absence from home hampers the possibility to nurture an ongoing marriage. Furthermore, some individuals could also decide to serve with the armed forces in order to distance themselves from an already failing marriage.We ruled out a number of potential mechanisms behind our findings by restricting the cohort of deployed military veterans under investigation. We did not see any material changes in the magnitude of the hazard ratios when excluding women, veterans with short deployment duration (<60 days), recently married individuals (the year before deployment), individuals with history of divorce, and unarmed service members. Also, duration of marriage was associated with divorce rate and there were statistically significant differences between deployed military veterans and matched comparators in our study. Adjusting for duration of marriage had, however, only a limited effect on the hazard ratio estimate.Finally, the differences in divorce and marriage rates found between the military veterans and the non-deployed matched comparators from the general population could be driven by personality differences. The individuals who volunteer to military service in Afghanistan, with all the associated risks and hardships, might have a more adventurous character and be less risk averse than the comparators who chose not to volunteer for foreign military service. Individuals who are more prone to risk taking may be less interested in as well as less suited for marriage. Hooper et al have proposed that voluntarily entering the military brings together individuals with high risk tolerance, which is desirable for certain military occupations.One major strength of this study was the possibility to compare deployed military veterans with appropriate individuals from the general population. A systematic review performed by the RAND Corporation (a nonprofit research organization) highlighted the need for new studies with suitable control groups in the research field. The presA limitation of this study was the lack of detailed information about divorce such as marriage quality prior to deployment, the reason for divorce, and the initiator of the divorce. Another limitation is that we only investigated marriages and divorces, but did not have data on cohabiting men and women who were not married or did not marry. In the deployment data, there was no information regarding combat exposure, neither at the individual nor at the aggregate level, precluding analyses stratified by a factor that has previously been shown to influence marriages. RegardinThe deployed military veterans and their families should be informed prior to deployment that divorce is more common after foreign military deployment than in comparable non-deployed individuals in the general population, at least in a Nordic context. Future research should try to shed light on the mechanisms behind the higher divorce rate among deployed military veterans, for example by collecting information on the quality of marriages before deployment, and, in the event of a divorce after deployment, information about which partner initiated the divorce and for what reason. Preemptively informing about where to find family therapists when soldiers return from deployment may also be warranted.Swedish military veterans who served in Afghanistan within ISAF were more likely to divorce and less likely to marry after deployment compared to non-deployed civilian comparators with similar demographic and pre-deployment mental health status. These findings stand in contrast to many studies with non-deployed comparators among US veterans who served in Vietnam, Afghanistan or Iraq.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


This cross-sectional study investigates the prevalence of human papillomavirus (HPV) infection among US women who received between 0 and 3 doses of HPV vaccine. Demographic characteristics and immunization history were self-reported and collected by trained interviewers during a home interview. Sexual behavior data were self-reported by participants in the Mobile Examination Center. Participants provided self-collected cervicovaginal swab specimens. The specimens were evaluated by polymerase chain reaction followed by type-specific hybridization. Details of the survey questionnaire, sample collection, and laboratory methods are available elsewhere.F test was used to examine the difference in the prevalence of HPV infection  by the number of doses received. The differences in predicted probability for HPV types 6, 11, 16, and 18 by number of vaccine doses and by the levels of risk factors were estimated using a multivariable logistic regression model. The model was adjusted simultaneously for age as a linear term, race/ethnicity, age at sexual debut, and lifetime number of male sexual partners. Statistical significance was tested at 2-sided P < .05. All analyses were performed with SAS software version 9.4 (SAS Institute) using SAS PROC SURVEY procedures, which included weight, cluster, and strata statements, to incorporate sampling weights and to adjust for the complex survey design.We identified women aged 18 to 26 years at the time of survey participation with nonmissing HPV vaccination and HPV test data. Nationally representative estimates for prevalence and the representative population counts were computed using NHANES sampling weights. The survey weight–adjusted Wald STROBE) reporting guideline.This study was deemed exempt from review and requirements for patient informed consent by the institutional review board of the University of Texas Health Science Center owing to the use of publicly available and anonymized data. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology , of whom 1004 were unvaccinated and 616 received at least 1 dose of HPV vaccine: 106 received 1 dose, 126 received 2 doses, and 384 received 3 doses.Compared with unvaccinated women , infection with HPV type 6, 11, 16, or 18 was significantly less prevalent among women who received 1 dose , 2 doses , or 3 doses  of HPV vaccine . There wIn adjusted analysis, the predicted probability of infection with HPV 6, 11, 16, or 18 was higher in unvaccinated women  compared with women who received 1 dose , 2 doses , or 3 doses  . Black w4 The limitations of our study include a cross-sectional design that precluded us from evaluating the timing of HPV vaccination compared with potential exposure. Furthermore, any conclusion regarding efficacy by individual number of doses cannot be drawn given the self-reported immunization history that may be prone to recall bias.6 If ongoing trials confirm sufficient efficacy and sustained duration of protection from a single-dose regimen, vaccine initiation (as opposed to the series completion) will become a more achievable metric of population coverage.Our study suggests that US women who received 1 dose of the HPV vaccine may have gained similar protection against vaccine-type infections compared with those who received additional doses. These findings support previous observational studies and post hoc analyses of vaccine trials that demonstrated comparable effectiveness of 1 dose to 2 or 3 doses.


Human papillomavirus (HPV) is the cause of a fraction of head and neck squamous cell carcinoma. Although this relation is well-known, it is still not clear the role of HPV in premalignant oral lesions such as oral lichen planus (OLP) and dysplasia. We aimed to evaluate the HPV-DNA prevalence and type distribution in a set of oral biopsies obtained from patients diagnosed with OLP and dysplasia, as well as the role of HPV in these lesions.th 1995 to May 21st 2014 at Hospital of Bellvitge and Odontological University Hospital of Bellvitge was identified and classified in four groups: OLP (groups 1 and 2) and dysplasias (groups 3 and 4) that progressed or not to invasive cancer during follow-up. A random selection targeting 25 cases was aimed to be performed for each group. All selected cases were subjected to pathological evaluation, DNA quality control and HPV-DNA detection. HPV-DNA positive samples were further subject to p16INK4a analysis.A retrospective cohort of all premalignant oral lesions consecutively diagnosed from March 30INK4a expression and none of the HPV positive cases progressed to invasive cancer during long-term follow-up.A total of 83 cases yielded a valid HPV-DNA result. From those, 7 and 34 cases were OLP that progressed or not to invasive cancer during follow-up, whereas 24 and 18 cases were displasias that progressed or not to invasive cancer during follow-up, respectively. HPV-DNA was detected in 4 samples (3 dysplastic lesions and 1 OLP). Two samples were HPV16 positive (2%), 1 sample HPV18 positive (1%) and 1 sample (1%) was HPV indeterminate. Two out of four HPV-DNA positive cases had high p16We found a low HPV-DNA attributable fraction in premalignant lesions of the oral cavity, suggesting that HPV is unlikely to play a significant role in oral carcinogenesis in our setting. Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC), with an estimated 200,000 new cases every year . OSCC maHPV attributable fractions in OSCC are estimated at 2.2%, substantially lower than those for oropharyngeal squamous cell carcinoma, estimated a 30,8% . The relPremalignant oral lesions include oral leucoplakia, oral erythroplakia, oral proliferative verrucous leukoplakia, oral submucous fibrosis, oral lichen planus (OLP) and actinic cheilitis ,9. In ouDysplasia is also considered a potentially malignant disorder, as some studies relate oral dysplasia with HNSCC ,23. It iINK4a and progressed to invasive cancer during follow-up or not.We aimed to assess the prevalence of HPV infection in a Spanish series of OLP and dysplasia, the type distribution of HPV-DNA positive cases and the role of the virus in these lesions, evaluating if they expressed p16th 1995 to May 21st 2014 with OLP and oral dysplasia at Bellvitge University Hospital and Odontological University Hospital of Barcelona (Spain) with available clinical data was conducted. All selected patients had to fulfil pre-established inclusion criteria: to be diagnosed with OLP or oral dysplasia in the pre-established anatomical locations  and not having had a previous oral cavity cancer or oropharyngeal cancer. Since the causal role of HPV in HNSCC is only consistently established in oropharyngeal cancer and moreover, the identification of HPV-related precursor lesions in the head and neck remains puzzling [A retrospective cohort study of all patients consecutively diagnosed from March 30puzzling . Data waIt was requested to provide samples using a common protocol from sample selection, retrieval, processing and shipping to Catalan Institute of Oncology (ICO) .the ICO according to a robust and well validated methodology [Pathology procedures were performed at Hospital of Bellvitge and ICO and laboratory procedures at hodology –29. Befohodology –29. FirsINK4a immunohistochemistry (IHC) [INK4a expression was evaluated on HPV-DNA positive cases using the CINtec histology kit , following the manufacturer’s protocol [INK4a staining has a high predictive value to identify HPV-related cancers when the pattern shows a strong and diffuse nuclear and cytoplasmic staining in at least 70% or more of the tumour [INK4a and/or HPV-mRNA in addition to HPV-DNA yielded the most accurate approximation to judge HPV carcinogenicity in HNSCC also in non-oropharyngeal sites.A sample was determined to be adequate for HPV testing if the lesion of interest was observed in both H&E-stained sections of the study specimen. Intermediate sections were used for HPV-DNA detection and genotyping and p16ry (IHC) . For HPVprotocol –29. It ie tumour . Howevere tumour  the methe tumour  where the tumour  that usiComitè Ètic d’Investigació Clínica de l’Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, Spain). Study implied the use of archival material only, and it did not envisage any contact with the patients. Adequate measures to ensure data protection, confidentiality, patients’ privacy and anonymization were taken into account. No informed consent was available due to the retrospective design of the study and the large proportion of deceased and untraceable patients. However, informed consent was obtained for a single patient diagnosed in 2014 (n = 1).This study had formal approval by the Ethical Committee for Clinical and Epidemiological research of our research centre, Hospital of Bellvitge, Catalan Institute of Oncology (ICO), Odontological University Hospital of Barcelona and Hospital of University of Barcelona . The final number of cases for each group was: 34 OLP that did not progressed to invasive cancer during follow up (group 1), 18 dysplasias that did not progressed to invasive cancer during follow up (group 2), 24 dysplasias that progressed to invasive cancer during follow up (group 3) and 7 OLP that progressed to invasive cancer during follow up (group 4). Two samples from group 2 required a second testing for technique problems and were finally included. All controls were HPV-negative and all HPV-DNA negative cases were tubulin positive.Characteristics of the samples are presented in INK4a with 25 to 50% of cells stained  was a 60-year-old woman, non-smoker and non-alcoholic, with no comorbidities, who developed an OLP in the gum that did not degenerate. The HPV18 positive-case (patient 3) was a 55-year-old smoker and drinker male with many comorbidities who had a moderate dysplasia in a leucoplakia at floor of the mouth with positive p16INK4a stain that healed during follow-up. The undetermined HPV case was a 67-year-old woman, non-smoker and non-alcoholic, with comorbidities presenting a leukoerythroplakia lesion in the tongue diagnosed as low-grade dysplasia that resolved within the next 4 years.One HPV16 positive-case was a 33-year-old male . When examining the positivity by type of lesion, we found one OPL that did not progressed to invasive cancer during follow up  and three dysplasias that did not progressed to invasive cancer during follow up . This low prevalence limited our ability to analyse factors associated with HPV infection. Likewise, HPV-DNA has been observed in a small proportion (6.4%) of dysplasias in preliminary results of a recent Canadian study . RegardiNoteworthy, the attributable fraction of HPV in oral cavity cancer has been estimated between 2.2%  and 4.4%None of our 4 HPV DNA positive cases progressed to invasive cancer during follow-up. To date, there is no clear understanding about HPV natural history at premalignant oral lesions  and it seems that its course depends on the HPV serotype and other associated factors such as immune status, smoking, alcohol, environmental factors, poor oral hygiene and eating habits .INK4a pattern, representing a 2.4% of the total sample. Preliminary results from the Canadian study showed a HPV-DNA positivity of 6.2% of the dysplasias, whereas 5.5% of all analysable cases demonstrated a p16INK4a diffuse pattern [INK4a expression had a significantly lower risk of lesion progression, whereas a diffuse p16INK4a expression did not represent a specific biomarker of HPV-related precancerous lesions in the oral cavity [Only two out of four HPV-DNA positive cases, which were positive for HPV16 and HPV18, showed a diffuse p16 pattern . The stul cavity . ResultsINK4a expression at positive cases, as it is known that the mere detection of HPV-DNA is not sufficient to establish causality in premalignant oral lesions and HNSCCs [INK4a and/or HPV-mRNA in addition to HPV-DNA yielded the most accurate approximation to judge HPV carcinogenicity also in non-oropharyngeal sites. We tested a small number of samples and we obtained low rates of HPV positivity. However, our population represents a specific geographic region, and our results are consistent with other studies performed in the same area. The low rate of HPV-positivity also deterred us to evaluate HPV infection as prognostic marker. The study was not population based, and thus, it is not possible to exclude some degree of referral or selection bias. For this reason, we tried to minimize this limitation selecting all cases consecutively. We neither have information about the size of the oral lesions in most of the cases nor specific protocol about the treatment of premalignant oral lesions.Our study has several strengths comprising the combined use of well-validated protocols with highly sensitive assays for HPV-DNA detection and a marker of HPV related transforming process such as p16d HNSCCs . Additiod HNSCCs ,41. Howed HNSCCs , where tAfter studying a cohort of patients with premalignant oral lesions using a highly sensitive PCR technique for paraffin-embedded tissues, we found a low HPV-DNA attributable fraction in premalignant lesions of the oral cavity, suggesting that HPV is unlikely to play a significant role in oral carcinogenesis in our setting.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


When administered as standard three-dose schedules, the licensed HPV prophylactic vaccines have demonstrated extraordinary immunogenicity and efficacy. We summarize the immunogenicity of these licensed vaccines and the most commonly used serology assays, with a focus on key considerations for one-dose vaccine schedules.Although immune correlates of protection against infection are not entirely clear, both preclinical and clinical evidence point to neutralizing antibodies as the principal mechanism of protection. Thus, immunogenicity assessments in vaccine trials have focused on measurements of antibody responses to the vaccine. Non-inferiority of antibody responses after two doses of HPV vaccines separated by 6 months has been demonstrated and this evidence supported the recent WHO recommendations for two-dose vaccination schedules in both boys and girls 9–14 years of age. There is also some evidence suggesting that one dose of HPV vaccines may provide protection similar to the currently recommended two-dose regimens but robust data on efficacy and immunogenicity of one-dose vaccine schedules are lacking. In addition, immunogenicity has been assessed and reported using different methods, precluding direct comparison of results between different studies and vaccines. New head-to-head vaccine trials evaluating one-dose immunogenicity and efficacy have been initiated and an increase in the number of trials relying on immunobridging is anticipated. Therefore, standardized measurement and reporting of immunogenicity for the up to nine HPV types targeted by the current vaccines is now critical. Building on previous HPV serology assay standardization and harmonization efforts initiated by the WHO HPV LabNet in 2006, new secondary standards, critical reference reagents and testing guidelines will be generated as part of a new partnership to facilitate harmonization of the immunogenicity testing in new HPV vaccine trials. All three vaccines are based on non-infectious recombinant type-specific L1 capsid proteins assembled into viral-like particles (VLPs) as immunogens. The expressed recombinant L1 capsids self-assemble in arrays of 72 pentamers that present an exterior surface closely mimicking HPV virions and it is this multiplicity of L1 domains that bestows the VLP antigen with significant immunogenicity, even in the absence of concomitant immune modulators (adjuvants) The vaccines differ in the antigen expression system used, antigen composition and adjuvants included  [12], [1The vaccines were originally licensed for use as a three-dose regimen, administered intramuscularly. The licensed HPV vaccines have demonstrated remarkable efficacy in phase III clinical trials in HPV-naïve young women, providing nearly complete protection against incident infection and cervical disease caused by the HPV types that they specifically target A degree of efficacy against some non-vaccine types (i.e. cross-protection) has been demonstrated in vaccine trials, particularly for HPV-31 and HPV-45 Correlates of protection against infection afforded by HPV vaccines have not been formally identified and are difficult to study because of the exceptional efficacy of the vaccines. There have been very few cases of breakthrough infection or disease with the current vaccines to identify the threshold of antibody level that confers immunity among vaccinated subjects. Most studies attempting to address this question have analyzed the protective levels of naturally acquired antibodies in the placebo arm of vaccine trials Immunogenicity testing of prophylactic HPV vaccines in clinical trials has focused primarily on antibody responses because neutralizing antibodies are thought to be the principal effectors of protection against HPV infection. This is primarily based on experimental evidence in animal models demonstrating that protection against papillomavirus infection can be passively transferred in serum or purified immunoglobulin (Ig) G from VLP-vaccinated animals 22.1+ T cell responses, as well as lower memory B cell responses, particularly for HPV-18 up to 24 months after vaccination A number of recent publications have thoroughly reviewed the immunogenicity and efficacy of licensed HPV vaccines in the context of the standard three-dose and reduced dose regimens High and durable HPV-16 and HPV-18 antibody levels, significantly above natural infection levels, have been reported after vaccination with Cervarix® for at least 9.4 years Gardasil®9 has been reported to elicit comparable HPV-16 and HPV-18 antibody responses as Gardasil®. HPV-18 antibody levels induced by three doses of Gardasil®9 measured with the cLIA also declined over time and 78% seropositivity was observed 5 years after vaccination Covariates of immunogenicity have been evaluated both for Gardasil® and Gardasil®9 in three dose recipients Critical questions that will need to be addressed by ongoing and planned trials include whether higher vaccine-induced antibody levels would predict longer duration of protection and how to improve sensitivity and specificity of antibody assays so that reliable correlations can be made between protection against infection and detection of antibodies.2.2Clinical studies evaluating reduced dose schedules and the interval between doses for both vaccines have demonstrated non-inferior antibody responses in girls younger than 15 years of age that received two doses, given six months apart, when compared with women who received the standard three doses of vaccine and had evidence of efficacy in clinical trials A head-to-head trial of two doses of Cervarix® or Gardasil® six months apart in 9–14 year-old girls demonstrated higher antibody levels for Cervarix® than Gardasil® at one year following initial vaccination Immunogenicity data for a single vaccine dose are limited and to date has been examined in clinical trial participants who did not complete protocol or in trials that ended prematurely. Although lower than the levels induced by two- and three-dose schedules, a single dose of Cervarix®, in 18–25 year-old women from the NCI-sponsored Costa Rican Vaccine Trial, induced detectable HPV-16 and HPV-18 ELISA antibody levels in all participants. These remained approximately 9-fold and 5-fold higher than natural infection levels, respectively, 4 years after initial vaccination With the increasing number of trials relying on immunogenicity data to support new vaccine recommendations, evaluation of one-dose trials, and approval of new follow-on vaccines, there is a need for the critical assessment of the sensitivity, specificity, reproducibility and comparability of HPV immunogenicity assays. These assays are briefly described in the next section along with the role for International Standards and steps being taken to address gaps in availability of these key reagents.3Measures of the type-specific immune response to HPV after vaccination include both cellular as well as humoral markers. The HPV vaccines induce potent T and B cell responses both in animal models and in humans Three main serological assays have been used to evaluate HPV L1 VLP antibody responses to HPV L1 VLP vaccines in clinical trials : (1) psein vitro neutralizing function . Since they are cell-based, neutralization assays tend to have a higher coefficient of variation than binding assays, are labor intensive, and costly. Generally, these have been difficult to adapt to a high-throughput format, although a 384-well format has been described The neutralization assays measure total immunoglobulins  that exhibit function A. This aThe cLIA assay requires labeled type-specific monoclonal antibodies and conformationally intact VLPs B. AntiboELISAs also require conformationally intact VLPs and they measure both neutralizing and non-neutralizing antibodies, recognizing both conformational and non-conformational epitopes C. VLPs aThe overall advantages and disadvantages of the three broad classes of serology assays are summarized in Because different laboratories have used different assays for evaluation of immunogenicity to the HPV vaccines and key reagents are prepared differently in each laboratory, inter-laboratory assay standardization has been quite challenging. In 2006, the Bill and Melinda Gates Foundation funded a WHO Global HPV Laboratory Network whose main activities were to work out systems for international comparability of HPV ELISA assays for HPV-16 and HPV-18 In addition to concentrations, antibody quality can be evaluated in terms of binding strength of antibodies to VLPs, or avidity. Only a few studies have examined avidity in samples from HPV vaccine trials Robust and standardized serological assays are critical for current and future HPV vaccine evaluation and may help identify immune correlate(s) of protection and provide early indications of potential efficacy of new vaccine products, such as biosimilars. For closely related vaccines, immunogenicity data may support regulatory approval, accelerating vaccine implementation.in vivo protection are estimated to be orders of magnitude lower than levels detected in vitro by ELISA or neutralization assays in vitro to match in vivo protection are warranted.Based on long-term efficacy data, HPV vaccines are expected to induce sufficiently high levels of immunity to protect against infection and subsequent disease. However, low levels of antibodies are expected to be induced by single dose regimens, in particularly for HPV-18 in Gardasil® and Gardasil®9 recipients as well as for HPV-45 in Gardasil®9 recipients, based on long-term antibody persistence data and the data published in reduced dose schedules It is clear that immunogenicity assessment in one-dose vaccine studies will be challenging. The importance of the immunologic data from these trials requires that the data from these assays be reliable and interpretable. This means that testing must be done with methods that allow reporting results in terms of directly comparable International Units. International Standards are still required for the additional 5 oncogenic types included in Gardasil®9. A newly launched HPV Serology Standardization project, co-funded by the Bill and Melinda Gates foundation and the US National Cancer Institute, is addressing critical needs for global harmonization of HPV serology testing in clinical trials of HPV prophylactic vaccines. The project plans to identify additional secondary standards for the types included in current vaccines, provide critical reference reagents (VLPs), and develop testing guidelines, all of which will be made available to the HPV serology community. This builds on previous efforts of the WHO HPV LabNet and will be done in parallel with ongoing WHO and NIBSC efforts for production of International Standards for the additional HPV types included in Gardasil®9.


Ureaplasma parvum being the most prevalent microrganism (39.0%). Co-infections between hrHPV and other STIs were demonstrated in 17.5% of women; no significant association was demonstrated between multiple infections and the colposcopy findings. This study provides new epidemiological data on the prevalence of cervical infections associated with HPV and seven other common sexually transmitted pathogens in a population of women presenting with an abnormal cervical cytology.Sexually transmitted infections (STIs) represent a major cause of morbidity in women and men worldwide. Human Papillomavirus (HPV) infections are among the most prevalent STIs and persistent infections with high-risk HPV (hrHPV) genotypes can cause cervical dysplasia and invasive cervical cancer. The association of other STIs with HPV cervical infection and/or dysplasia has however not yet been fully elucidated. The aim of this study was to assess the prevalence of HPV and other STIs among women presenting with an abnormal cervical cytology. Cervical infections with 28 HPV genotypes and seven other sexually transmitted pathogens were evaluated in 177 women referred for a colposcopy after an abnormal Pap smear. Positivity for at least one hrHPV genotype was shown in 87% of women; HPV 16 was the most prevalent (25.0%), followed by HPV 31 and HPV 51. The overall positivity for other STIs was 49.2%, with  Human papillomaviruses (HPVs) are viruses known to be the primary cause of cervical cancer (CC), responsible of approximately 311,000 women’s deaths in 2018 [To date, nearly 200 different HPV types have been identified, including twelve classified by the International Agency for Research on Cancer (IARC) as oncogenic or “high-risk” HPV types  [Despite HPV infections being widespread among the female population, only a small fraction of women with a hrHPV infection will progress to pre-cancer or cancer. This indicates that additional risk factors are important for carcinogenesis, potentially including viral factors such as persistent infection with specific hrHPV genotypes or the presence of concomitant pathogens able to infect the genital tract.It is well known that HPV 16 and 18 are the two most common genotypes related to cervical cancer development . In counChlamydia trachomatis (Ct) [Trichomonas vaginalis (Tv), Mycoplasma hominis (Mh) and Ureaplasma urealyticum (Uu) have also been associated with cervical inflammatory processes, a situation that may facilitate the entrance of HPV [There is growing evidence on the role of other Sexually Transmitted Infections (STIs) as co-factors for the development of cervical cancer in HPV-positive women ,9. Intertis (Ct) ,10. Ct itis (Ct) . Howevere of HPV .The purpose of this study was to assess the prevalence and distribution of HPV genotypes among Italian women referred for a colposcopy following a recent diagnosis of abnormal cervical cytology. Moreover, the second aim was to investigate in this group of women the presence of other concomitant STIs and to determine their possible correlation with the grade of cervical lesion.One hundred and seventy-seven women with a documented abnormal Pap smear were consecutively enrolled at the Gynaecology outpatient clinic of San Gerardo Hospital, Monza, Italy, between 2017 and 2019. The study protocol (Protocol: n. 305) was approved by the Ethics Committee of the University of Milano - Bicocca, Italy. A cervical sample was collected from all enrolled subjects for the molecular detection of HPV and STI pathogens. The cytological assessment was performed according to the 2001 Bethesda System .® PreservCyt® Solution .The cervical samples were collected using a L-shaped FloqSwab  and transported to the laboratory following resuspension in 20 mL of ThinPrep® easyMag® , an automated system for total nucleic acid extraction from clinical samples. All of the samples were processed using the “specific B” protocol, characterized by a higher final elution temperature (about 70 °C) and the use of silica beads diluted 1:2, in accordance with the manufacturer’s instructions. The nucleic acid extraction system was fully automated, except for adding the initial sample and subsequently the silica beads (125 μL), 10 min after sample lysis, reducing the potential risk of sample cross-contamination. The nucleic acid extracts were eluted in 100 μL of NucliSENS® easyMag® elution buffer (bioMérieux).The DNA extraction was performed starting from 1 mL of resuspended cervical sample using NucliSENSHPV detection and typing was performed using a commercial kit, Anyplex™II HPV28, , which can detect 28 HPV types  in 2 reaction tubes by means of real-time PCR assays on the CFX96 real-time PCR instrument .Chlamydia trachomatis (Ct), Neisseria gonorrhoeae (Ng), Trichomonas vaginalis (Tv), Mycoplasma hominis (Mh), Mycoplasma genitalium (Mg), Ureaplasma urealyticum (Uu), and Ureaplasma parvum (Up) was performed using Anyplex™ II STI-7 .The detection of 7 STI pathogens, The HPV and STI pathogen DNA detection was performed as recommended by the manufacturer using 5 μL of extracted DNA. The data recording and interpretation were done with the Seegene viewer software according to the manufacturer’s instructions.p-value less than 0.05 was considered statistically significant. The statistical software STATA version 15  was used for all statistical computations.The qualitative and quantitative variables were summarized with absolute and relative (percentage) frequencies and medians , respectively. A chi-squared or Fisher exact test was performed for the qualitative variables, when appropriate. A two-tailed n = 131) of whom showed a normal colposcopy evaluation and 25.1% (n = 44) identified as having a high-grade cervical abnormality. Those patients with a high-grade cervical abnormality underwent treatment with conization and/or biopsy which showed the following histological findings: 11.4% (n = 5/44) had cervical intraepithelial neoplasia grade 1 (CIN1), 18.2% had CIN2 (n = 6/44), 50.0% had CIN3 (n = 22/44), and 6.8% had cervical cancer (n = 3/44), two squamous cell carcinoma and one adenocarcinoma in situ.One hundred and seventy-seven women with an abnormal cervical cytology were recruited at colposcopy. Their median  age was 40 (31–46) years. Patients with LSIL constituted 42.9% of the study population, followed by those with ASCUS (24.3%) and HSIL (14.1%) . ColposcIn total, 132/177 (74.6%) patients were positive for at least one HPV genotype; 47% (62/132) were only positive for high-risk HPV (hrHPV) types, 40.2% (53/132) for co-infection with hrHPV and low-risk HPV (lrHPV) types, and 12.9% (17/132) for only lrHPV types . HPV 16 In total, 53.9% (62/115) of the women were infected with only one hrHPV type, whereas multiple hrHPV infections were detected in 46.1% (53/115) of patients .A total of 68.2% (15/22) of those infected with only HPV 16 had a high-grade cervical abnormality ; 91.7% , followed by Ureaplasma urealyticum  and Mycoplasma hominis . Chlamydia trachomatis was identified in 2.8% (5/177) of women, whereas Mycoplasma genitalium and Trichomonas vaginalis were found in three (1.7%) of the patients . patients .n = 87) were infected by a single pathogen , whereas 14.9% (13/87) and 11.5% (10/87) showed two or three or more STIs, respectively  was the most prevalent pathogen in co-infected patients, followed by Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.The majority of the women with a STI  with Ureaplasma urealyticum, and 15.4% had multiple STIs (6/39).Among the women with a positive colposcopy result, 54.5% (24/44) had STIs . In thosHPV infection is the necessary but not sufficient cause for cervical cancer. Other cofactors can play a role, influencing HPV persistence and cervical disease progression.The overall prevalence of both high-risk and low-risk HPVs in our study population of women referred for colposcopy after a recent abnormal Pap test was found to be 74.6%, with 65% 115/177) of them being positive for at least one hrHPV type. The high HPV positivity rates found in this selected population is in keeping with the range of HPV positivity previously reported among the patients with the same characteristics ,15,16,17/177 of tHPV 16, 31, 51 and 52 were found to be the most prevalent hrHPVs detected. Baasland et al.  reportedThe majority of the women with a HPV 16 single infection were found to have a high-grade cervical abnormality at colposcopy examination (68.2%), demonstrating again the principal role of this genotype in precancerous lesion progression.Ureaplasma parvum found to be the most frequently identified pathogen , followed by Ureaplasma urealyticum . Overall, positivity for one or more of the seven STI pathogens under study was demonstrated in 49.2% of women, with Chlamydia trachomatis was identified in 2.8% of women and was always in co-infection with other STI pathogens. This positivity rate was low compared to the 12.8% of Ct prevalence that our group found in a previous study conducted on women with a recent history of cervical dysplasia [ysplasia  and otheysplasia ,25,26,27Ureaplasma spp. infection in female genital tracts. A study conducted in Japan showed an Ureaplasma parvum prevalence of 41.7% in women attending their first prenatal visit [U. urealyticum (23.4%) compared to those aged above 25 years (9.2%), irrespective of symptoms [Ureaplasma parvum of almost 25% in vaginal specimens [Several previously published studies reported a very high percentage of al visit ; anothersymptoms . Other specimens  and 38.3pecimens .Ureaplasma spp. and Mycoplasma spp. detection in the genital tracts of sexually active individuals and their unclear clinical role in lower urogenital tract infections, a recent position statement from the European STI Guidelines Editorial Board has indicated that the routine testing and treatment of asymptomatic or symptomatic men and women for M. hominis, U. urealyticum and U. parvum are not recommended [As a result of the high prevalence of ommended .Ureaplasma spp. and other STIs in association with hrHPV, have supposed that these could be possible cofactors interacting with HPV in the development of precancerous and cancerous cervical lesions [However, several recent studies investigating the role of cervical co-infections of  lesions ,34,35,36Ureaplasma urealyticum and Ureaplasma parvum were associated with a significantly increased risk of overall HPV infection . Furthermore, infections with U. urealyticum, U. parvum, and Mycoplasma hominis were also found to be associated with a significantly increased risk of abnormal cervical cytopathology  [Moreover, a recent systematic review and meta-analysis aimed at investigating the association between female genital mycoplasmas and HPV infection, abnormal cervical cytopathology, and cervical cancer, concluded that ctively) .Ureaplasma parvum  was the most prevalent sexually transmitted pathogen, followed by Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis. In spite of the relatively high prevalence of hrHPV and STIs co-infections observed in the population studied, no statistically significant association was found between co-infections and positive colposcopy findings in these women.In the present study, infections with multiple hrHPV types were detected in 46.1% (53/115) of the HPV-positive patients; a total of 65 (36.7%) women were also found to be co-infected with HPV and STIs. In these co-infections, Ureaplasma spp. and/or other STIs are real cofactors or are just “followers”, taking advantage of the immune tolerance and abnormal regulation of the cell cycle control generated by HPV. Moreover, future longitudinal clinical studies in women with an abnormal cervical cytology will allow us to better evaluate the relationship between lower genital tract infections, co-infections, and high-risk human papillomavirus infections.Only in depth in vitro studies may confirm whether Ureaplasma parvum and other STIs. Although a statistically significant association between co-infections between hrHPV and other STIs with positive colposcopy findings could not be demonstrated in the studied population, the high prevalence of STIs found in hrHPV-positive women, who are at higher risk of developing cervical disease, indicate that screening for genital infections in this population may be important. This would allow not only to prevent other STDs and their sequelae, but also reduce any potential influence of concomitant microorganisms on HPV infection. Future longitudinal studies investigating the role of STIs as cofactors in HPV mediated cervical carcinogenesis are warranted.The results of this study have demonstrated, as expected, a high prevalence of hrHPV infections in women referred for colposcopy, but also high rates of co-infections with


AGT gene. The mutation is considered as pathogenic because it is cosegregated with ARRTD and detected in other unrelated ARRTD families. Our findings link the fetal ultrasound manifestations to the ARRTD, highlighting clues that are useful for prenatal diagnosis, which warrants confirmatory genotyping of the RAAS genes including oligohydramnios/anhydramnios, anuria , MCA-REDF, and a morphologically normal kidney.Autosomal recessive renal tubular dysgenesis (ARRTD) is a rare and lethal disorder that causes stillbirth or early neonatal death. Most of the reported cases are diagnosed postnatally by a histopathological hallmark of the absence or paucity of differentiated proximal tubules in kidneys. Prenatal diagnosis of ARRTD is challenging because only a few fetal features  are associated with this condition. In this study, we report a fetus with ARRTD, which showed anhydramnios and invisible urinary bladder since the second trimester, followed by growth restriction and reversed end diastolic flow in the middle cerebral artery (MCA-REDF). No morphological anomaly was detected on the fetal kidneys during an ultrasound scan. The baby died of refractory hypotension the day after their birth. Genetic analysis of genes that are involved in the renin-angiotensin-aldosterone system (RAAS), which are the known genetic causes of ARRTD, identified a novel, biparental-origin homozygous c.857-619_1269+243delinsTTGCCTTGC mutation in the  Renal tubular dysgenesis  is a rare and lethal disorder that affects renal development before birth and causes intrauterine fetal demise or early neonatal death. Most of the reported cases are diagnosed postnatally based on a histopathological hallmark of the absence or paucity of differentiated proximal tubules in kidneys . AdditioAGT , REN , ACE , and AGTR1  [RTD can be acquired during fetal development due to drugs or certain disorders ,10,11,12*106165) ,13,14. RAGT mutation that is associated with ARRTD.Further, ARRTD poses a dilemma for families and physicians during prenatal genetic counseling because almost all reported cases have resulted in fatal outcomes. Early recognition of fetal ARRTD on the basis of clinical and ultrasound analyses and the characterization of the genetic defects permits genetic counseling and early prenatal diagnosis. We report here the results of a clinical and genetic study of a prenatal case with ARRTD. We highlight the clues that may be useful for prenatal diagnosis and report a specific homozygous +6 weeks gestational age (wGA) because of unexplained severe oligohydramnios. Medical records showed that the pregnant woman received level II ultrasonographic screening at 22+2 wGA and the fetus was structurally normal and surrounded with amniotic fluid  and an invisible bladder were noted . Both tests showed that the fetus had a normal male karyotype 46,XY and genomic composition arr(1–22)x2,x1. After nondirective counselling, she continued pregnancy and regular antenatal care. Close surveillance of the pregnancy was advised.+5 wGA revealed bilateral anatomically normal kidneys and decreased signal intensity of her lungs and invisible bladder , and reversed end diastolic flow in the middle cerebral artery (MCA-REDF) were noted  for kidney and lung evaluation at 30 bladder A,B. Fetare noted C.+4 wGA because of antepartum hemorrhage. A second course of steroid administration was then provided and she underwent an emergent cesarean section at 33+5 wGA because of fetal distress. After birth, the Apgar score of the first and fifth minutes were 5 and 6, respectively. Poor spontaneous breathing with a low heart rate (<60 bpm) was noted. An endotracheal tube was then inserted and the heart rate of the baby recovered to 160 to 170 bpm. After the baby was transported to NICU, the first recorded blood pressure was 28/18 mmHg and an echocardiogram was then arranged and revealed pulmonary arterial hypertension (pulmonary artery systolic pressure was around 40 to 45 mmHg). Right side pneumothorax was noted by a chest X-ray. Despite inotropic agents and inhaled nitric oxide (iNO) treatment, the baby expired 22 h after birth.The pregnant woman was admitted at 33AGT, REN, ACE, and AGTR1) was carried out by polymerase chain reaction (PCR) and direct sequencing. Initially, no sequence variant was directly recognized. However, repeated PCR failures of exon 3 and exon 4 of the AGT gene suggested gross deletion(s) involving these two exons. Long range PCR was subsequently performed and a homozygous c.857-619_1269+243delinsTTGCCTTGC mutation was identified . This mutation was found in the heterozygous status in the unaffected mother and father, pointing to the biparental origin of the described mutation and further supporting a fetal case of ARRTD. To improve the diagnosis, an in-house designed PCR was provided for rapid detection of this specific c.857-619_1269+243delinsTTGCCTTGC mutation in the AGT gene (AGT mutation). The wild type allele was expected to yield a 343-bp amplicon and the mutant allele will yield a 250-bp amplicon under a definite PCR condition  the pregnant woman had no premature preterm rapture of the membrane, (2) no evidence of uteroplacental insufficiency, and (3) a normal fetal renal scan , then ARRTD should be enlisted in a differential diagnosis.For the prenatal sonographic findings, only a few reports mentioned the features of ARRTD because the disease was probably underdiagnosed ,14. OligAGT, REN, ACE, and AGTR1) have been reported to cause RAAS dysfunction, leading to ARRTD [ACE, REN, AGT, and AGTR1, respectively. AGT mutations are relatively rare and so far, only 27 AGT mutations have been recorded in The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). These mutations vary from missense, nonsense, splicing, and regulatory to small deletion. The c.857-619_1269+243delinsTTGCCTTGC mutation that we identified in the AGT gene is novel and is the first gross deletion that is associated with ARRTD. The deletion spanning across exon 3 and exon 4 of the AGT gene  resulted in partial loss of the Serpin domain of the matured AGT protein [AGT gene was also found in additional unrelated families of ARRTD in the Taiwanese population . This suggests that the homozygosity of the specific AGT mutation causes ARRTD and this specific allele may represent a result of the founder effect  or a combination of the founder effect and consanguinity. Further large-scale genetic studies may facilitate our understanding of ARRTD in the Taiwanese population. We provided an in-house designed PCR, allowing the rapid detection of this specific AGT mutation , and MCA-REDF without sonographic renal abnormality. Molecular genetic analysis can be used for rapid diagnosis of ARRTD when renal biopsy is not feasible. As was shown in this study, a novel homozygous


Kaplan–Meier univariate analysis showed significant associations between patients’ OS and age (p < 0.001), smoking (p = 0.02), and tumor stage (p < 0.001). A Cox proportional hazard multivariate analysis confirmed the significant associations with age, tumor stage, and also treatment (p < 0.01). In conclusion, HPV-DNA prevalence was significantly lower in our HNSCC patients than worldwide 32–36% estimates (p ≤ 0.001). Although infrequent, oropharyngeal cancer increased over years and showed 21% HPV-DNA positivity, which is close to the worldwide 36–46% estimates (p = 0.16). Besides age, smoking, tumor stage, and treatment, HPV/p16 status was an important determinant of patients’ survival. The HPV and/or p16 positivity patients had a better OS than HPV/p16 double-negative patients (p = 0.05). Thus, HPV/p16 status helps improve prognosis by distinguishing between the more favorable p16/HPV positive and the less favorable double-negative tumors.Head and neck squamous cell carcinoma (HNSCC) shows wide disparities, association with human papillomavirus (HPV) infection, and prognosis. We aimed at determining HPV prevalence, and its prognostic association with overall survival (OS) in Saudi HNSCC patients. The study included 285 oropharyngeal and oral-cavity HNSCC patients. HPV was detected using HPV Linear-Array and RealLine HPV-HCR. In addition, p16INK4a (p16) protein overexpression was evaluated in 50 representative cases. Oropharyngeal cancers were infrequent (10%) compared to oral-cavity cancers (90%) with no gender differences. Overall, HPV-DNA was positive in 10 HNSCC cases (3.5%), mostly oropharyngeal (21%). However, p16 expression was positive in 21 cases of the 50 studied (42%) and showed significantly higher OS ( Head and neck (H&N) cancer is the 9th most frequent malignancy worldwide, accounting for around 5% of all new cases and exhibits wide demographic variations ,2. The gSyrjanen et al.  were hisThe more prominent turning point is that HNSCC HPV-positive cancers appear to form a distinct tumor entity from smoking- and alcohol-related counterparts with distinguished epidemiology, genetics, characteristic histopathology, therapeutic response, and predictive clinical outcome to chemo-radiation treatment ,18,19. TThe epidemiology of HPV infection is known to have wide variations in human populations, remnant of socioeconomic, ethnic, and genetic predisposing factors ,24. Accop = 0.09; two-tailed Mann–Whitney Rank Sum test). By anatomical sites, 28 patients (10%) had oropharyngeal while 257 (90%) had oral cavity cancers. Interestingly, the number of oropharyngeal cancer cases increased with time: 8 cases were diagnosed in 2002–2008 (142 patients) compared to 20 cases in 2009–2016 (143 patients). The distribution of sub-anatomical sites of oral and oropharyngeal cancers by 5-year age groups is illustrated in The characteristics of the 285 H&N cancer patients included in the study are summarized in p < 0.001), and smoking (p = 0.02), while gender (p = 0.28) had no effect (p < 0.001) from T1 to T4 . By anatomical site, 21% (6/28) of the oropharyngeal and 2% (4/257) of the oral cavity cancers were positive for HPV infection. Most frequent HPV-positive cases were recorded from the tonsils (3/12), the base of the tongue (3/14), the retromolar (2/24), followed by the buccal (1/19) and the tongue (1/198). Thus, the highest HPV-DNA positivity was in oropharyngeal cancers (21%), which is not statistically different from the worldwide 36–46% estimates (p = 0.16). Overall, survival analyses showed a trend toward better OS for HPV-positive  compared to HPV-negative  patients but that did not reach statistical significance (p = 0.12), most probably due to the small number of HPV-positive cases (The Linear Array HPV Genotyping Test was first used to detect and genotype HPV infection. Results indicated that only 10 patients (3.5%) were HPV positive while 275 specimens (96.5%) proved to be negative after at least two separate tests and an independent concordant confirmation using the RealLine HPV HCR Genotype . By HPV ve cases A.p > 0.05). A survival analysis showed a statistically significant (p = 0.02) better OS for p16-positive  compared to p16-negative  patients (p = 0.05), whereby HPV and/or p16 positive patients displayed better survival  compared to HPV/p16 double-positive patients . However, there was no significant difference between double-positive and single positive patients (p = 0.85).In view of the small number of HPV-positive HNSCC patients found in this cohort, and the limited amount of pathological materials available, a subset of 50 representative specimens were processed for p16 protein IHC staining. These included all the 28 oropharyngeal cases, which are known as highly suspicious for HPV infection. In addition, 22 cases of oral cavity subsites were processed comprising 10 retromolar, 10 tongue and 2 buccal for which at least one cancer was positive for HPV. Thus, the 10 HPV-positive tumors along with 40 HPV-negative cases were included. Examples of p16 protein IHC strong (positive) and weak (negative) staining is given in patients B. In addpatients C, showedH&N cancer is an important health issue worldwide ,3,4. Thep ≤ 0.001) than the projected 32% computed from the estimated number of incident cases worldwide [http://gco.iarc.fr/today/home). Nevertheless, our data indicate an increase in the number of oropharyngeal cancer cases over time as it doubled in the last decade in the country. The subsites of H&N cancers that are most frequently associated with HPV infection are the tonsils, the soft palate, the base of the tongue (oropharynx) and the retromolar . These together formed 18% (52/285) of the cases in this cohort. The remaining 233 patients had other various oral cavity tumors including mostly tongue  decreased gradually from T1 to T4 (p < 0.001) and non-smokers (p = 0.02), but not for patients’ gender .As expected, tumor stage had independently major impact on patients’ OS which significantly (T1 to T4 A. This s’ gender . As for ’ gender . This inp = 0.16), indicating similar pathogenic association. As for oral cavity, only 2% were HPV-positive, mainly recorded from the retromolar (2 cases), the tongue, and the buccal cavity (one case each). This low prevalence is in agreement with a recent study by Vidal Loustau et al. [In agreement with published data, the highest prevalence of HPV was observed in oropharyngeal cancers (21%), mostly in tonsils (3/12) followed by the base of the tongue (3/14). Although this 21% prevalence remains below the compiled worldwide estimates of 36–46%, it was not statistically different  due to, most probably, the small number of HPV-positive cases (10/285). In addition, those patients had mainly T1–2 tumor stages with basically a favorable survival prognosis. Nonetheless, in a subset of 50 cases, a statistically significant (p = 0.02) better survival was observed for p16-positive compared to p16-negative patients (p = 0.006) association with OS. It also shows that surgery, which mainly underlies early stage tumors, result in higher survival compared to any other combined treatment. In other word, this result essentially captures that of the tumor stage since the treatment was stage standardized with some subtle adaptation to each individual case. In addition, a trend toward association with OS was apparent for HPV/p16 and smoking status but they did not reach statistical significance in the multivariate analysis. Nevertheless, our results are in overall agreement with published data with the overwhelming belief that p16-positive HNSCC have improved locoregional tumor control and survival with conventional therapy [The relationship between HPV and OS after the treatment showed a clear trend toward a longer survival of HPV-positive patients A as descpatients B. Althoupatients . In addi therapy ,27,41. P therapy , and inv therapy ,43, espe therapy .p = 0.05) where HPV/p16-positive cases showed substantially better OS than double-negative patients. Although double-positive cases showed slightly better survival than single-positive patients, the difference was not statistically significant, most probably due to the small number of patients who tested positive for HPV and/or p16. Nonetheless, taken together, these results highlight the importance of performing independent HPV and p16 testing when predicting individual patient’s prognosis [While the study points out toward the need of systematic testing of p16 overexpression, results obtained in a subset of patients, the results are also in line with a recent study evaluating the 8th TNM classification that integrates p16 status (as independent or surrogate markers for HPV infection) in oropharyngeal cancer ,45. The rognosis ,46. Thesrognosis . The finThe study was carried out using archival pathologic materials of H&N cancer obtained during routine diagnostic procedures. The samples were anonymized and processed with no patients’ identifiable characters. The study was reviewed by the institutional review board and approved by the Research Centre Ethics Committee at the King Faisal Specialist Hospital and Research Centre (KFSHRC) under the number RAC#2130 025.2. Patients having resectable tumors with poor prognosis were treated with combined concomitant CRT 66–70 Gy in 33–35 fractions with 3 weekly cisplatin 100 mg/m2. A combined concomitant CRT was also the standard treatment in oropharyngeal and non-resectable oral cavity cancer patients. Cetuximab was used for patients who were not fit for cisplatin chemotherapy. Radiotherapy modalities included 3D conformal that was gradually replaced with intensity-modulated radiation therapy (IMRT) in 2006, and also RapidArc in 2010 and TomoTherapy in 2012. Although some HPV-related histopathologic features were available for few cases, the treatment followed the same guideline for all patients with no difference between positive and negative HPV.Medical records of 1633 H&N cancer patients diagnosed between 2002 and 2016 at the KFSHRC tertiary care hospital were screened. The main eligibility criteria were adult patients with squamous cell carcinoma in anatomical location potentially associated with HPV infection. Following the exclusion of palliative cases and cancer sites that had not been proven to be HPV-driven , only 330 patients with oropharyngeal and oral cavity tumors remained for possible inclusion. After the examination of the histopathological slides, 285 patients’ samples were included due to the limited amount of pathological blocks available for this study. Patients’ treatment with curative intent followed timely standard clinical guidelines that depends on primary tumor location and extension . BrieflyFormalin-fixed, paraffin-embedded (FFPE) tissues proven to contain tumor sections of the 285 patients were obtained from the pathology department’s archive. For each case, 3–6 sections of 10 μm thickness were taken from the block for the extraction of DNA using the QIAamp DNA FFPE tissue kit , using the manufacturer’s recommended instructions. Briefly, the FFPE sections were deparaffinized using xylene followed by ethanol to extract residual xylene. The specimens are covered with ATL lysis buffer with 20 μL proteinase K  and incubated at 56 °C and 90 °C for 1 h each. Then, 2 μL 100 mg/mL DNase-free RNase A (Qiagen) was added, mixed and incubated at room temperature for 2 min. After the lysis and heating, followed by binding and washing steps, DNA was eluted in 50 μL of ATE buffer and quantified using a NanoDrop 2000c Spectrophotometer .Two different methods were consecutively used to detect and genotype HPV infection in all the H&N samples along with HPV negative (HTB-31) and HPV-16 positive (HTB-35) external controls:1 The Linear Array HPV Genotyping Test . This PCR-based test detects and genotypes the 37 most common anogenital HPVs , 81, 82 (MM4), 83 (MM7), 84 (MM8), 89 (CP6108) and IS39). Procedures followed the manufacture’s instruction described in detail previously . Briefly2 RealLine HPV HCR Genotype Fla-Format . This Real-Time PCR test allows the differential determination of the 12 most frequent high-risk HPV-DNA genotypes, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59, isolated from clinical specimens. It is based on the detection of the unquenched fluorescence produced by a specific reporter molecule that intensifies as PCR reaction cycles increased. The reporter molecule is a fluorophore-quencher dual-labeled DNA-probe designed to bind exclusively to the HPV-DNA target region. Fluorescent signal increases as a result of the cleavage of the probe by Taq DNA-polymerase exonuclease activity, which separates the fluorescent dye from the quencher during the repeated cycles of hybridization and amplification. The threshold cycle value (Ct) is defined as the cycle number at which the generated fluorescence crosses a set threshold within the reaction where the signal increases significantly above the background fluorescence of the procedure. Ct depends on the initial quantity of the HPV-DNA template present. A positive HPV control is run with the samples and an internal control (IC) detecting the content of human DNA (β-actin) is used to validate the quality of sampling and improve the reliability of results by preventing generation of false negatives which can be caused by the possible loss of a DNA template during sample preparation.3 Procedures followed the manufacture’s recommended methodology. Briefly, to analyze each sample for the detection of the 12 HPV-DNA genotypes, 4 tubes containing Master Mix  in 0.2 mL 96-well plates were used. The amplification is carried out on the CFX96 Touch Real-Time PCR Detection System  using the recommended cycling program. The sample is flagged as positive  when the Ct value via the fluorescent dyes, FAM, HEX, and ROX channels, for this sample (in any of MM 1–4 tubes) is less than or equal to 35 for HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or is less than or equal to 40 for HPV types 16 and 18. The HPV genotype is determined using a reference table provided by the manufacturer, which correlates each MM with an individual dye channel to one of the 12 specific high-risk HPV types.Procedures examining the expression of p16 protein were carried out using a Bond-III Automated IHC/ISH Stainer  according to manufacturer’s instruction and reagents. Briefly, where available, 4 μm FFPE sections were mounted on glass microscope slides coated with Poly-L-Lysine. They were deparaffinized using Bond Dewax Solution (Leica Biosystem), rehydrated, and washed with Bond Wash Solution. The slides were incubated with Bond Epitope Retrieval Solution and heated at 100 °C for 20 min, washed, and Peroxide Wash Solution applied for 5 min. The p16 primary antibody , Ventana, Tucson, AZ, USA) was added on the slides for 15 min, followed by the anti-mouse secondary antibody  for 8 min and the Bond Polymer Refine Detection solutions with intermittent washing. Slides were counterstained with Hematoxylin, and then dehydrated and mounted with DPX by using a Tissue-Tek film coverslipper . Negative controls were obtained by excluding the primary antibody. Scoring of p16 IHC cytoplasmic and nucleic staining were evaluated by an experienced pathologist, based on defined characteristics whereby p16 was scored as positive if it was strong and diffuse (>70% of tumor cells), and negative if absent, weak, or focal .p-value < 0.05 was considered statistically significant. Statistical analyses were done using the SigmaPlot platform , and MedCalc, Ostend, Belgium .A one sample z-test was used to detect differences in proportions when the referenced proportion was deemed constant. The non-parametric Mann–Whitney Rank Sum test was used to assess differences between groups. A univariate Kaplan–Meier Log-Rank survival analysis was used to evaluate the relationship between various risk factors and overall survival (OS) represented by the length of patients’ follow-up. A multivariate Cox proportional hazards model was used to test the effects of multiple covariates on patients’ OS. All statistical tests conducted were two-sided. A This study indicates an overall low prevalence of HPV infection in our HNSCC patients. Although oropharyngeal cancer cases were infrequent, they increased over years and 21% were associated with HPV infection. Age, smoking, tumor stage, and treatment had important effect on survival. Although all HPV-positive cases were p16-positive (double-positive), the p16 positivity is not exclusive and could be positive in HPV-DNA negative tumors. HPV and/or p16 positivity had better prognosis of survival than HPV and/or p16 negative patients. An important clinical application is in the stratification of patients according to HPV and p16 status. These tests could improve survival predictions by distinguishing between the more favorable HPV-positive/p16-positive group, and the less favorable double-negative HPV/p16 group of HNSCC patients who have the worst prognosis.