The debate surrounding the optimal caesarean rate has been ongoing for several decades, with the WHO recommending an “acceptable” rate of 5–15% since 1997, despite a weak evidence base. Global expert opinion from obstetric care providers on the optimal caesarean rate has not been documented. The objective of this study was to examine providers’ opinions of the optimal caesarean rate worldwide, among all deliveries and within specific sub-groups of deliveries.A global online survey of medical doctors who had performed at least one caesarean in the last five years was conducted between August 2013 and January 2014. Respondents were asked to report their opinion of the optimal caesarean rate—defined as the caesarean rate that would minimise poor maternal and perinatal outcomes—at the population level and within specific sub-groups of deliveries . Median reported optimal rates and corresponding inter-quartile ranges (IQRs) were calculated for the sample, and stratified according to national caesarean rate, institutional caesarean rate, facility level, and respondent characteristics.Responses were collected from 1,057 medical doctors from 96 countries. The median reported optimal caesarean rate was 20% (IQR: 15–30%) for all deliveries. Providers in private for-profit facilities and in facilities with high institutional rates reported optimal rates of 30% or above, while those in Europe, in public facilities and in facilities with low institutional rates reported rates of 15% or less. Reported optimal rates were lowest among low-risk deliveries and highest for Absolute Maternal Indications (AMIs), with wide IQRs observed for most categories other than AMIs.Three-quarters of respondents reported an optimal caesarean rate above the WHO 15% upper threshold. There was substantial variation in responses, highlighting a lack of consensus around which women are in need of a caesarean among obstetric care providers worldwide. Large differences exist between national caesarean rates worldwide, from 1.4% of all deliveries in Niger in 2012 to 52.3% in Brazil in 2010 ) investiImportantly, these ecological studies do not take into account morbidity of the mother or baby, and no optimal caesarean rate minimising both mortality and morbidity has been identified. In the absence of an evidence-based optimal caesarean rate for maternal and perinatal outcomes, expert opinion may be helpful to assess to what extent obstetricians agree with the WHO recommendation. Obstetricians’ opinions have been recognised as an important driver of caesarean section rates , 18, andFurthermore, the question of optimal rates among specific sub-groups of deliveries is unresolved. While the Robson classification has become increasingly used to analyse patterns of “over-intervention” within health facilities, there is no agreement on what thresholds indicate unnecessary caesareans. Although there is likely to be little disagreement that most Absolute Maternal Indications (AMIs), such as major cephalo-pelvic disproportion and complete placenta praevia, require surgery to avert the death of the mother , for othPrevious studies have documented differences in obstetricians’ attitudes towards caesareans on maternal request –28 and pA cross-sectional online survey was designed to collect doctors’ opinions of the optimal caesarean rate. In this study, the optimal caesarean rate was defined as the “proportion of women who should receive a caesarean for optimal maternal and fetal outcomes”, including mortality and morbidity. The target population were medical doctors having performed at least one caesarean in the last five years, worldwide (collectively referred to as “obstetricians” throughout this paper).A questionnaire was developed to collect respondent characteristics , level and caesarean rate of the facility where they most recently provided obstetric care). Reported optimal caesarean rate for all deliveries was collected in free text format, whilst reported optimal rates among specified categories of deliveries was collected in 10% intervals . The wording of the question for reporting optimal caesarean rates was adapted from an unpublished survey asking experts for their opinion on “plausible” rates of emergency caesareans . The queRespondents were also asked to report the optimal caesarean rate for four groups of deliveries, which were organised from high to low hypothesised need for caesareans. “Absolute Maternal Indications” (AMIs) are complications for which surgery is thought necessary to avert the death of the mother , and whihttps://www.surveymonkey.com), and was accessible from 14th August 2013 to 31st January 2014. Respondents gave informed consent by checking a box stating they had understood the information and terms of participation before proceeding to the survey, and respondents were able to exit the survey at any stage. Ethical approval for this study was granted by the Research Ethics Committee of the London School of Hygiene & Tropical Medicine (no 6455).The final version of the questionnaire was translated into French, Portuguese and Spanish, and translated versions were checked and edited by native speakers with medical training. The survey was uploaded via the online platform SurveyMonkey  were invited to participate. The social networking sites Facebook and Twitter were also used, and snowball sampling was encouraged by enabling respondents to share the survey with their colleagues.It was not possible to achieve a probability sample since there is no sampling frame for this population and the response rate is unknown: an unknown proportion of obstetricians is unreachable online, and surveys of medical doctors in high-income countries tend to have low response rates , 29, 30.The main explanatory variable of interest in this study was the national caesarean rate in the respondents’ main country of practice, grouped into four categories . The list of national caesarean rates compiled by Gibbons et al. in 2012  was updaThe median reported optimal caesarean rate was calculated for all deliveries along with the corresponding interquartile range (IQR). Where respondents responded by providing an optimal range (e.g. 15–20%), the interval midpoint was used to calculate the median optimal caesarean rate for the sample (e.g. 17.5%). For clinical and reproductive categories of deliveries, the optimal caesarean rate was calculated as the median interval (e.g. 51–60%).The magnitude of variation in optimal rates was examined by stratifying the median optimal caesarean rate according to national caesarean rate and secondary explanatory variables . Associations between national caesarean rate and category-specific optimal rates were also examined by calculating the median optimal rate for each category of deliveries stratified by national caesarean rate. Kruskal-Wallis one-way analysis of variance tests were used to investigate differences in opinions of the optimal rate between strata, due to the skewness of responses. All analyses were conducted in Stata version 13.A total of 1,377 respondents accessed the link to the survey, of which 320 (23%) questionnaires were excluded as the respondent did not answer any question relating to optimal caesarean rates. The final sample included 1,057 medical doctors from 96 countries . The majThe median reported optimal caesarean rate for all deliveries was 20% . There was strong evidence of an association between optimal reported rates and all explanatory variables , except for age. Providers in countries with caesarean rates above 30% reported higher optimal rates than those in countries with caesarean rates below 30% , while respondents practicing in Europe reported lower optimal rates than elsewhere . Providers exclusively practicing in the private for-profit sector reported higher optimal rates than those practicing exclusively in the public sector . Median reported optimal rates increased consistently with reported facility caesarean rates, from 15% among providers who report an institutional caesarean rate of 0–14%, to 30% for institutional rates over 50%.th percentile in each subgroup was at least 15%; the only exceptions were providers in Europe and in facilities with institutional caesarean rates below 15%, where the lower quartile was 14% and 10%, respectively.For each of these stratifications, the 25The percentage of missing values for optimal rates ranged from 0.4% for complete placenta praevia to 6.7% for nulliparous women among reproductive categories. There was substantial variation in reported optimal rates in most of the other clinical categories. The two exceptions were eclampsia and cord prolapse, with high optimal rates  and relatively narrow IQRs. For the other categories included in this group, the median optimal rate varied between 21–30% for diabetes and premature labour, and 71–80% for breech delivery. The IQRs for these categories were very wide, reaching 50 percentage points for pre-eclampsia, twin delivery, breech delivery and prolonged labour.The reported optimal rate was lower for reproductive categories than for clinical categories, with medians ranging between 1–10% and 21–30%, and they also tended to show less variation than clinical categories (other than AMIs), with the exception of high birthweight which had an IQR of 60 percentage points. The reported optimal rate was lower for low-risk deliveries than for all other clinical and reproductive categories .This was the first global survey of obstetricians’ opinions of the optimal caesarean rate. The median optimal caesarean rate reported by obstetricians was 20% (IQR = 15–30%) for all deliveries. The lower quartile of 15% indicates that 75% of respondents consider the optimal caesarean rate at the population level to be higher than the WHO “acceptable” range of 5–15%. There remains substantial variation in opinions of the optimal rate, with one quarter of respondents believing it is above 30%. Factors affecting variation in reported optimal rates include global region of practice, country income level, facility level, and facility caesarean rate. Reported optimal caesarean rates across different categories of deliveries are consistent with clinical interpretation, with very high median rates for AMIs and the lowest reported rates among multipara with singleton, cephalic delivery and no known risk factors.Although recent evidence suggests that mortality may continue to decline with caesarean rates above the WHO 15% upper threshold , in lineThe median rates of 20% and 30% among public and private for-profit providers respectively are consistent with the public-private gap in “ideal” rates of 16% and 20% reported by South African obstetricians in a 1992 survey, although reported optimal rates in both groups were higher in our survey , and a nThree groups of respondents reported relatively low optimal caesarean rates (≤15%), including obstetricians no longer practicing medicine , perhaps because they are more aware of the debates surrounding the risks of excessive caesareans in the academic literature. The second group is obstetricians predominantly practicing in Europe. Of the 246 respondents in this group, the largest contributions came from Norway (n = 95), the UK (n = 47) and Switzerland (n = 25). Few respondents had practiced in Eastern or Southern Europe, where caesarean rates are higher, which may have biased this finding. Western European countries have historically favoured midwife-centred delivery models and public healthcare systems, whereas in the Americas and certain middle-income countries, private insurance systems, obstetrician-led models of care, and higher utilisation of private facilities are thought to incentivise surgical deliveries , 41, 42.The extent of variation in reported rates, even among clinical groups of indications with solid available evidence for planned caesarean delivery, raises questions about the validity of recommendations based on expert opinion, such as those emanating from Delphi consultations . For exaA number of limitations to our study are worth noting. First, our sample was not globally representative of doctors performing caesareans. The dissemination strategy achieved a large and geographically diverse sample, though there were relatively few respondents from Asia . It was not possible to calculate a response rate for this online survey since the number of people who received the invitation is unknown. Selection bias may have affected the study findings if those who answered the survey tend to report different answers than those who are not reachable online or who chose not to answer the survey. Notably, the lack of responses from southern and eastern Europe may have biased the median reported optimal rate in this region. Furthermore, respondents may have misreported their opinions of optimal caesarean rates through survey fatigue or bias from personal experience , potentially leading to information bias. The survey design attempted to minimise the likelihood of information bias by beginning the questionnaire with the most clear-cut categories (AMIs) and ending with the population-level rate.Second, the sample was restricted to medical doctors who perform caesareans. Though non-physician clinicians perform a substantial proportion of caesareans in several countries such as Malawi, Mozambique, and Tanzania (up to 93% in some facilities) , 46, theOverall, the median optimal caesarean rate reported by obstetricians lies above the “acceptable” range defined by the WHO, although the “true” optimal caesarean rate—if indeed one exists across populations—remains unknown. The wide range of reported optimal rates indicates that there is little consensus among obstetric care providers as to the optimal caesarean rate, with the exception of Absolute Maternal Indications (for which caesareans are considered necessary). The recent WHO statement recognised the pitfalls of issuing population-level recommendations for caesarean rates, urging to focus efforts on “provid[ing] caesarean sections to women in need, rather than striving to achieve a specific rate” . NonetheS1 Table(DOCX)Click here for additional data file.


Patients with human papillomavirus (HPV) infection are at risk of developing cancer later in their life. Current research estimates the prevalence of genital HPV infection and explores the factors that are associated with the infection.The National Health and Nutrition Examination Survey 2007–2010 was used in this research study. The study population included females in the United States aged 18–59 years. The weighted prevalence of HPV infection was 41.9%. An estimated 59.4% of non-Hispanic black females had HPV infection. In a multivariate analysis, number of sexual partners, race, age, education level, marital status, income, smoking, and insurance status were associated with HPV infection. HPV infection was 5.77 times more likely for women with >11 sexual partners compared to women with 0–1 partners. Non-Hispanic black females were 1.87 times more likely to have HPV infection compared to non-Hispanic white females. Participants with only a high school degree had a 58% increased prevalence compared to college-educated women. Uninsured women had a 39% increased prevalence compared to those with insurance.This study found that 41.9% of U.S. females aged 18–59 years tested positive for genital HPV infection. We determined that individuals with more sexual partners, with a lower education level, with non-Hispanic black race, and with no insurance were the populations at greatest risk. It is necessary to continue monitoring the prevalence of this infection in the general population to provide a basis for effective treatment and prevention in the target populations. Genital human papillomavirus (HPV) is the most common sexually transmitted infection in the United States. HPV includes a group of more than 150 related viruses. Many of these viruses can be easily spread through direct skin contact during sexual intercourse . Some paMore than 40 HPV types can infect the genital areas of men and women . HPV infThe primary goals of our current research are to identify the potential factors associated with HPV infection and to estimate the prevalence of infection for U.S. females from 2007 to 2010. Eventually, we aim to help create programs targeting high-prevalence populations to prevent HPV infection and lower their risk of getting cervical cancer.Data was obtained from the National Health and Nutrition Examination Survey (NHANES) 2007–2010 . The surThe NHANES used a multistage probability sample design to select the participants. Consenting participants completed a household interview followed by a physical examination and interviews at a Mobile Examination Center (MEC). Non-Hispanic (NH) black and low income groups were oversampled in the NHANES to allow for an accurate statistical estimation in these population groups . The proFrom 2007 to 2010, 10,010 females of all ages were interviewed. The combined unweighted household interview response rate for that period was 78.9%; the examination response rate was 76.3%. All females aged 18–59 years (n = 4242) who visited the MEC were asked to self-collect a cervicovaginal swab sample. Out of all samples collected, 3738 (88.1%) were reported as positive or negative and were used in the final analysis. Combining the data for years 2007 through 2010 was justified. There was no significant difference in HPV prevalence between 2007–2008 and 2009–2010 for all but 5 of 37 HPV types, as detected by the Linear Array assay (data not shown).Demographic information, including gender, age, race, education, marital status, and country of birth, was obtained from all participants during the household interviews. The poverty index was calculated according to the U.S. Census definition. This method divides total family income by the poverty threshold after adjusting for family size at the time of the interview.Sexual history information, including if the participant had ever had sex and the age of first sexual experience, was self-reported by participants using an audio computer-assisted self-interview. Respondents who reported having sex  were asked additional questions about their lifetime sexual history and about any sexual encounters in the prior 12 months. These additional questions addressed number of sexual partners, sexual orientation, and condom usage.As described by Dunne et al. , self-coAlphapapillomavirus genera. Samples were reported as HPV positive if any of the 37 HPV deoxyribonucleic acid (DNA) types were detected, including high-risk  and low-risk  types. If the strips were positive for any of the types, the sample was coded as positive. If the strips were negative for all of the types and beta-globin was detected, the sample was coded as negative. If there was no beta-globin present in the sample and no HPV type was detected, the sample was coded as inadequate [Multiplex polymerase chain reaction (PCR) was used for detection of 37 HPV types within the adequate .We estimated the overall prevalence of infection for any HPV type with respect to sociodemographic and sexual behavioral characteristics. Due to the complexity of the design, all estimates were measured using 4-year MEC sample weights provided by NCHS to account for the unequal probabilities of selection and adjustment for nonresponse. The weighting methodology has been described previously . Taylor -values for multiple comparisons. An unconditional logistic regression model was used to determine associations between genital HPV infection and these factors. Variables for adjustment in multivariate logistic regression models were selected based on bivariate associations with p-values <0.2. Goodness of fit for the final step of the model was assessed using the Hosmer-Lemeshow Satterthwaite adjusted F test.The Wald χ2 statistic was used to assess bivariate association between HPV and sociodemographic or behavioral characteristics. No adjustments were made to the pSAS software version 9.4 for Windows  and SAS callable SUDAAN 11.0.1  were useHPV types and species by oncogenic risk category are presented in Figure In a univariate analysis Table , the oveThere was a lower trend of HPV infection with increased age, higher education level, and decreased family poverty level. There was a bimodal pattern in the prevalence of HPV for age, with a high HPV prevalence of 56.1% in the 18–24 age group and with a second peak in the 40–44 age group. HPV prevalence was 48% for women with less than a high school education, whereas it was 31.4% for those with a college education. Participants living in a family with an income-to-poverty ratio (PIR) of <130% showed a 55.3% prevalence of HPV infection. In terms of marital status, prevalence was lowest (29.4%) for married women and highest (>47%) for women classified as single, divorced, or never married.The association of HPV infection with behavioral factors is presented in Table Females who reported never having sex were 71% less likely to have HPV infection compared to females who reported having sex. The prevalence of HPV infection increased 89% for females having sex before 16 years of age compared to after 16. There was a trend of increasing HPV infection in women with a higher number of sexual partners in their lifetime (or yearly). The prevalence of infection was 8.9 times higher for women with >11 sexual partners in their lifetime compared to women with 0–1 partners. This trend was noticed among all races. Overall, NH black females showed the highest prevalence of HPV infection regardless of their number of partners Figure . CompareIn a multivariate logistic regression analysis, adjusting for other factors, the number of lifetime sexual partners had a significant association with HPV infection. HPV infection was 5.77 times more likely for women with >11 partners compared to women with 0–1 partners. NH black females were 1.87 times more likely to have an HPV infection compared to NH white females. Age was negatively associated with the prevalence of HPV, except for an increased peak in the 44–49 age group. Having a college degree and being married were also associated with lower HPV prevalence. Family income-to-poverty ratio, a habit of smoking cigarettes, and insurance status remained significant factors associated with HPV infection.When the results were adjusted for other factors, country of birth, recreational drug usage, ever having sex, age at first sexual encounter, condom usage during sex, and sexual orientation were no longer associated with HPV infection.From the NHANES 2007–2010 data set, we found that the overall prevalence of genital HPV infection was 41.9% (95% CI: 39.6-44.2%) in females aged 18–59 in the United States. We compared this result with two previous NHANES analyses observing data for females in the United States. Our estimate was similar to the result of the first study, which found a 42.5% (95% CI: 40.3-44.7%) prevalence for females aged 14–59 in the 2003–2006 data set .In our study, there was a bimodal prevalence of genital HPV for age, consistent with the same two previous NHANES studies , 10. ThiHPV infection was associated with certain racial and ethnic groups. NH black females had an increased prevalence even when controlling for factors such as number of sexual partners in their lifetime Figure  or otherSome studies have shown an inconsistent association between education level and prevalence , 25. HowThe number of sexual partners played a statistically significant role in HPV infection. This finding mirrors those seen in other studies , 10. We In conclusion, 41.9% of U.S. females aged 18–59 years tested positive for genital HPV infection. This study found that an increased number of sexual partners, a lower level of education, non-Hispanic black race, and a lack of insurance were factors of concern with HPV infection. Continuing to monitor the prevalence of HPV in the general population can establish a basis for possible interventions focusing on at-risk groups.http://www.cdc.gov/nchs/nhanes/nhanes_questionnaires.htm.All data used in this research can be downloaded from the following website: Use of data from the NHANES 2007–2010 is approved by the National Center for Health Statistics (NCHS) Research Ethics Review Board (ERB) Approval for NHANES 2009–2010 (Continuation of Protocol #2005-06), NHANES 2007–2008 (Continuation of Protocol #2005-06) and NHANES 2005–2006 (Protocol #2005-06).


Furthermore, HPV-positive tumors had significantly lower expression of Cox-2 mRNA and higher expression of PD1 mRNA compared to HPV-negative tumors. The presence of a high level of intratumoral immune cell infiltrates might play a crucial role in the significantly better response of HPV-positive patients to standard therapy and their favorable clinical outcome. Furthermore, characterization of the HNSCC immune profile might be a valuable prognostic tool in addition to HPV status and might help identify novel targets for therapeutic strategies, including cancer immunotherapy.Human papillomavirus (HPV) infection is one of the most important etiologic causes of oropharyngeal head and neck squamous cell carcinoma (HNSCC). Patients with HPV-positive HNSCC were reported to have a better clinical outcome than patients with HPV-negative cancers. However, little is known about the possible causes of different clinical outcomes. In this study, we analyzed a detailed immune profile of tumor samples from HNSCC patients with respect to their HPV status. We analyzed the characteristics of immune cell infiltrates, including the frequency and distribution of antigen-presenting cells and naïve, regulatory and effector T cells and the cytokine and chemokine levels in tumor tissue. There was a profound difference in the extent and characteristics of intratumoral immune cell infiltrates in HNSCC patients based on their HPV status. In contrast to HPV-negative tumor tissues, HPV-positive tumor samples showed significantly higher numbers of infiltrating IFNγ HNSCC is a heterogeneous group of tumors located in the oral cavity, oropharynx, hypopharynx and larynx. Originally, tobacco and/or alcohol exposure were the main risk factors for HNSCC, but in an expanding subset of patients with oropharyngeal carcinoma, HPV infection has been described in the last two decades as a crucial etiologic agent.in vitro when compared to HPV-negative cells. However, in vivo, HPV-positive tumors were more sensitive to radio- and chemotherapy in immunocompetent mice. Importantly, neither radiotherapy nor cisplatin therapy cured immunocompromised mice, indicating an important role for the immune system in HPV-positive HNSCC outcome.Despite the improved response of HPV-positive HNSCC to conventional treatment involving a combination of surgery, radiation therapy and chemotherapy, HPV-positive cell lines were shown to be more resistant to radiation and cisplatin + T lymphocytes,+ T lymphocytes and the ratio of infiltrating CD8+/FoxP3+ T lymphocytesIndeed, characterization of the adaptive immune response has been shown to be an important prognostic tool in a wide range of carcinomas, potentially even more relevant than the current cancer staging system.+ and CD4+ T cell subsets, dendritic cell subsets (DCs) and monocytes/macrophages as well as the chemokine and cytokine profile in fresh HNSCC samples with regard to HPV status. Our findings confirm that HPV-positive tumor samples show a distinct immunologic profile compared to HPV-negative samples, with high levels of infiltrating IFNγ+ CD8+ T lymphocytes, IL-17+ CD8+ T lymphocytes (Tc17 lymphocytes), myeloid DCs and spontaneously produced proinflammatory chemokines and cytokines. Additionally, HPV-positive samples expressed significantly lower levels of Cox-2 mRNA and higher levels of PD-1 mRNA than HPV-negative samples.In this study, we analyzed the distribution and phenotype of CD8n = 20) of patients; 55.0% (n = 11) of the HPV-positive tumors were localized in the tonsils, 40.0% (n = 8) at the base of the tongue and 5.0% (n = 1) at the base of the oral cavity (+) in 90.5% of HPV-positive and 66.7% of HPV-negative patients, but this difference did not reach a statistical significance. The tumor grade and the stage were equivalent in patients with HPV-positive and HPV-negative HNSCC.As expected, only the HPV 16 type was detected in all of the samples that were positive for HPV DNA. The expression of HPV 16 E6 mRNA was detected in 45.5%  in the HPV-positive tumor samples (p < 0.1) (in vitro (p = 0.030)  .Additionally, we also analyzed subsets of antigen presenting cells, namely mDCs, pDCs and monocytes/macrophages in the tumor tissue. We observed an increased frequency of all of these cell populations in patients with HPV-positive tumors. The total numbers of mDCs in HPV-positive tumor samples showed a statistically significant increase compared to HPV-negative samples; however, the frequency of pDCs and monocytes/macrophages were not statistically significantly different . As expected, the levels of CXCL12 positively correlated with the lymph node status .In the supernatants from unstimulated cell cultures, high levels of CXCL9, CXCL10, CXCL12, CCL20, CCL21 and CCL22 were detected. Of the other chemokines measured, only CCL5, CCL17 and CCL19 were at low levels (p = 0.030) . On the contrary, we observed a significantly higher expression of PD-1 in HPV-positive tumor tissues compared to HPV-negative samples (p = 0.018). The mRNA expression level of Tim-3 was similar in both groups of patients . Surprisingly, the levels of CCL21, a ligand for CCR7, positively correlated only with the frequency of Th17 cells but not with the number or frequency of DCs and naïve T cells. Although tumor-derived cells of HPV-positive tumors also expressed higher levels of proinflammatory cytokines, namely IL-2, IL-17, IL-23 and IFNγ, these differences did not reach statistical significance due to high variability in the samples.In addition to differences in immune cell infiltrates, we also observed a markedly different chemokine profile in HPV-positive and HPV-negative tumor tissue-derived cell cultures. HPV-positive tumor tissue-derived cell cultures produced much higher levels of chemokines, namely CXCL9, CXCL10, CXCL12, CCL17 and CCL21. The chemokine CXCL12 (SDF-1) and its receptor CXCR4 were shown to play a key role in regulating the trafficking of cancer cells to sites of metastases.Cox-2, PD-1, PD-L1 and Tim-3, also showed differences associated with HPV status. The expression of Cox-2, which specifically catalyzes the production of prostaglandins, is undetectable in most healthy tissues but is usually overexpressed in inflammation, premalignant lesions and tumors. Functionally, Cox-2-derived prostaglandins were shown to promote angiogenesis and induce tumor invasion.Cox-2 expression was associated with an unfavorable outcome in breast cancer patients.Cox-2 in control lymph nodes and significant overexpression of Cox-2 in tumor tissues from our cohort of HNSCC patients  and positively correlated with the mRNA expression of Tim-3 . Interestingly, the HPV-positive tumor samples expressed significantly lower levels of Cox-2 mRNA compared to the HPV-negative samples, which is in accordance with a previous report stating an improved outcome in HPV-positive HNSCC patients.Analysis of the mRNA expression levels of four regulatory genes, PD-1 and Tim-3 belong to a group of immune checkpoint proteins that decrease T-cell reactivity and were identified, together with CTLA-4 and Lag-3, as benchmarks for exhausted T cells.Tim-3 and PD-1 were only found in tumor tissue but not in the peripheral blood.+ Tim-3+ PD-1+ cells exhibited a surface phenotype that is consistent with effector/memory T cells, indicating that exhausted T cells emerge from effector T cells.The surface receptors PD-1 mRNA in tumor tissue compared to control lymph nodes. To examine the capacity of Tim-3-PD-1-, Tim-3-PD-1+ and Tim-3+PD-1+ tumor-infiltrating T cells to produce IFNγ, we analyzed the phenotype of these cells after in vitro stimulation with PMA and ionomycin using flow cytometry. Although we detected IFNγ-producing cells in all of the subtypes of T cells tested, the proportions of IFNγ+ T cells were markedly lower in CD8+PD-1+Tim-3+ T cells compared to CD8+PD-1+Tim-3- and CD8+PD-1-Tim-3- T cells, as expected. These data indicate that Tim-3 together with PD-1 might be considered a better exhaustion marker in HNSCC-infiltrating CD8+ T cells than PD-1 alone.In agreement with published data, we only found high proportions of Tim-3+ PD-1+ T cells in tumor tissue but not in the peripheral blood and control lymph nodes of HNSCC patients. On the contrary, Tim-3-PD-1+ T cells were observed in all of the tissues analyzed. Consistent with the flow cytometry data, we detected significantly higher levels of Tim-3 but not PD-1 expression in HPV-positive tumor samples compared to HPV-negative tumors. In a preclinical model, Badoual et al.+ T cells. An anti-PD-L1 monoclonal antibody vaccine further enhanced this immune response. Here, we confirmed that PD-L1 was markedly more expressed in tumor tissue than in the control lymph nodes. As Tim-3 expression was also tumor tissue specific, these two molecules, instead of PD-1 alone, might be a very promising target for immunotherapy in HNSCC.Badoual et al.Taken together, our data show that HPV-positive tumor tissues have a distinct immune profile compared to HPV- negative tumors. Substantial infiltrates of immune cells are usually associated with a good prognosis and indicate a strong past antitumor immune response in HPV-positive tumors, which might be reactivated/reprogrammed by not only a targeted immunotherapy approach, but even during the standard therapy. Better understanding of targets of the immune response in HPV-positive vs. HPV-negative tumors and of mechanisms directing the recruitment of immune cells to the tumor will hopefully lead to the design of successful immunotherapeutic strategies.Blood samples, primary HNSCC specimens metastatic and control lymph nodes were obtained from 54 patients immediately after radical surgery at the Department of Otorhinolaryngology and Head and Neck Surgery, 1st Faculty of Medicine, Charles University and Motol University Hospital in Prague between April 2011 and November 2013. The patients enrolled in this study had not received any neoadjuvant chemo- or radiotherapy. All of the patients signed an informed consent approved by the Institutional Review Board of the University Motol. The clinical-pathological characteristics of the patients are summarized in The tumor tissues, metastatic and control lymph nodes were minced into small pieces with scissors and digested in RPMI 1640 containing 1 mg/mL of Collagenase D and 0.05 mg/mL of DNase I  for 30 min at 37°C under permanent gentle rocking motion. Afterwards, the specimens were passed through a 100-μm nylon cell strainer  and washed with PBS. The PBMCs were isolated from the peripheral blood by centrifugation on a Ficoll-Paque density gradient .+, CD11 c, CD14, CD16, CD19, CD20, CD45, CD45RA, CD45RO, CD56, CD62 L (Exbio), CD4+, CD123 , HLA-DR  and CCR7  for detection of myeloid DCs (mDCs characterized as CD45+LIN-HLA-DR+CD14-CD11c+), plasmacytoid DCs , monocytes/macrophages  and naïve T lymphocytes (defined as CD3+CD4+CD45RA+CD45RO-CCR7+CD62L+). The following anti-human mAbs were used for staining regulatory T cells (Tregs): anti-CD3 , anti-CD4+ , anti-CD8+ , anti-CD25 and anti-CD127  for surface labeling, which was followed by fixation and permeabilization using the FoxP3 staining buffer set (eBioscience) and intracellular staining with anti-FoxP3  and anti-Helios  antibodies. For analysis of Th17 and Th1 lymphocytes, cell suspensions were stimulated for 4 h with 50 ng/mL of PMA and 1 μg/mL of ionomycin  in the presence of Brefeldin A  before intracellular staining. Next, the cells were stained with anti-CD3 (Exbio), anti-CD4+ (eBioscience), anti-CD8+ (Exbio), anti-PD-1  and anti-Tim-3  antibodies, fixed, permeabilized and labeled with mAbs against IL-17  and IFNγ . The cells were analyzed on a BD FACSCanto II (BD Biosciences) and evaluated with FlowJo software (TreeStar).Single cell suspensions from peripheral blood, tumor tissue and control lymph nodes were used for cell surface labeling using monoclonal antibodies (mAbs) against CD3, CD86/mL in the presence or absence of PMA and ionomycin were cultured for 24 h in RPMI 1640 supplemented with 10% FCS, Glutamax and penicillin-streptomycin . The supernatants were then collected and stored at -80°C until use.For the analysis of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-21, IL-23, TNFα, IFNγ, CCL5, CCL17, CCL19, CCL20, CCL21, CCL22, CXCL9, CXCL10, and CXCL12 in culture supernatants harvested from tumor tissue-derived cell suspensions, a Quantibody Array Kit (Raybiotech) was used. Cell suspensions at the concentration of 1 × 106 tumor-tissue derived cells using an RNA Easy Mini Kit (Qiagen). RNA concentrations were determined with a NanoDrop© 2000 c UV-Vis spectrophotometer (Thermo Scientific), and the RNA integrity was assessed using an Experion automated system (BioRad). cDNA was synthesized from total RNA using the M-MLV reverse transcriptase (Invitrogen) and amplified by quantitative real time PCR in the presence of primers and TaqMan© probes specific for Cox-2, PD-1, PD-L1 and Tim-3, as well as the β-actin housekeeping gene, which was used as an internal reference. All primers and probes were commercially synthesized (TIB MOLBIOL). The identity of the qPCR products in each assay was verified by sequencing. The relative expression of the target genes was normalized to the expression of β-actin.Total RNA was extracted from 2 × 10The pathologist obtained two side-by-side sections of the tumor from the primary site. One of the paired sections from each anatomical location was labeled, snaps frozen in liquid nitrogen, and stored for future analysis. The other section from each pair was fixed in 10% neutral formalin and paraffin embedded. From each paraffin block, the first and last sections were histologically analyzed to confirm that the sections in between that were designated for the detection of viral nucleic acids and immuno-histochemical (IHC) analysis contained at least 10% of the tumor cells from the entire volume of the sample. Both DNA and RNA, were extracted from the tumor tissue using the Ambion Recover All TM Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Bioscience) as previously reported.+/GP6+) as described previously.All procedures have been described in detail previously.p16INK4 a  was used. The intensity of staining (graded + to +++) and the proportion of cells stained (scored in percentages) were evaluated. For p16 immunostaining, the location of the signal (cytoplasmic and/or nuclear) was also specified. A semi-quantitative evaluation was performed. The samples considered positive for p16 expression exhibited more than 50% positive cells and nuclear and/or cytoplasmic staining.IHC examination was performed as described previously.Statistical analyses were performed using Statistica® 10.0 software (StatSoft).p < 0.05.The parametric assumptions of the data were verified using the Kolmogorov–Smirnov test for normality. The homogeneity of the variances was tested by the Levene test. The differences between HPV-positive and HPV-negative tumor samples were analyzed using a general linear model (GLM), and age was added as a covariate. The differences between control lymph nodes and tumor tissue were analyzed using a paired t-test. The results were considered statistically significant when


In women aged ≥30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30–65 years in Copenhagen, Denmark. Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected more ≥CIN3 than cytology , although statistically the differences were not significant. Cobas and CLART detected significantly more ≥CIN2 than cytology . The proportion of women with false-positive test results (positive test results without ≥CIN3) varied between 3.3% with cytology and 14.9% with cobas. All HPV assays led to significantly more false-positive tests, whereas compared to HC2 cobas and CLART were associated with a significantly higher and APTIMA with a significantly lower proportion. Detection of CIN1 was particularly increased for the three DNA assays. With APTIMA combined with cytological triage, about 20% more women were referred for colposcopy than with cytology screening. With the three DNA assays, the increase was ≥50%. The number of women with repeated testing was twice as high with APTIMA and almost five times as high with cobas compared to cytology. To our knowledge, Horizon was the only study set in routine practice that compared more than two HPV assays in the same women while also ascertaining the histological status of women with normal cytology/HPV-positive test results. HPV-based screening of Danish women aged 30–65 detected more high-grade CIN but decreased the screening specificity, and increased the demand for additional testing. Detection of high-risk Human Papillomavirus (HPV) infections is a more sensitive cervical cancer screening method than the current standard of cytology . HoweverThe aim of our study was to compare four commercially available HPV assays with cytology in primary screening of women aged 30–65 years. We used data from the Danish Horizon study, which was nested into routine screening and compared Hybrid Capture 2 High-Risk HPV DNA Test , cobas HPV Test , CLART HPV2 Assay , APTIMA mRNA HPV Test , and cytology. In this study, women with normal cytology and positive HPV test results had repeated testing. Unlike other major studies comparing several HPV assays in primary screening which only assessed histological outcomes in women with abnormal cytology , in the The design of the Horizon study was described in detail previously –5,8,9. IWomen were recommended for cervical screening every three years until age 49, and every five years until age 65, and those included in the Horizon study were managed according to cytology and HPV test results. Women with abnormal cytology were followed according to the routine Danish guidelines, which recommended repeated testing in case of low-grade squamous intraepithelial lesions  or HPV-negative ASCUS, or a colposcopy in case of other cytological abnormalities. At this laboratory, HC2 was routinely used for HPV triage of ASCUS. Per study protocol, women with normal cytology and a positive test result on at least one of the four HPV assays at baseline were invited for repeated cytology and HPV testing in approximately 1.5 years, in November 2012. Additional information was provided on woman’s request by phone or e-mail by one of the investigators (JB) or the laboratory’s medical staff overseeing the screening program in the Capital Region (including CR). A reminder for follow-up was sent in March 2013. At follow-up, two SurePath samples were taken from these women; in the laboratory, they were pooled before the testing, which was undertaken on samples diluted 1:1 in SurePath. Women with abnormal cytology or a positive HC2 test result at follow-up in 18 months (corresponding to the routine in the laboratory at the time of the study) were recommended for colposcopy. The last follow-up sample was received in the laboratory in June 2013. Follow-up was ascertained by linkage to the national Patobank in December 2013, allowing all women with abnormal repeated tests at least six months to attend for colposcopy. Hence, all histology was ascertained in approximately 2.5 years from the baseline testing, which, for women below age 50 years, corresponded to an almost entire screening interval. CIN lesions detected in HPV-positive women with follow-up other than study samples were included in the analysis. All colposcopies were undertaken by private or hospital-based gynecologists following routine protocols recommending biopsies from all suspicious areas, and random biopsies from the four quadrants if lesions were not visible.Routine cytological evaluation of SurePath samples was undertaken first by FocalPoint Slide Profiler . Blinded to the outcomes of HPV testing, samples were thereafter evaluated by cytoscreeners using FocalPoint GS Imaging System (BD), and abnormal findings were adjudicated by pathologists. Cytology was reported using the Bethesda 2001 system.All assay testing was undertaken in strict accordance with the protocols agreed upon with all manufacturers prior to the study, as described in detail previously –5,8,9. HBaseline testing on the residual material was undertaken as a quality development study. In Denmark, such studies do not require ethical approval. Invitation to follow-up of HPV-positive/cytology-normal women was approved by the Ethical Committee of the Danish Capital Region (H-4-2012-120). It was not permitted to reveal the results of the baseline testing in the invitation letter, but women could obtain them from their general practitioner (GP), where they signed their consent forms. Women could also obtain their HPV test result without study participation. The study was notified to the Danish Data Inspection Agency (notification number 2010-41-5594).A positive HPV test was defined according to the manufacturers’ recommendations . CLART was considered positive if at least one of the 13 high-risk HPV genotypes was detected. Abnormal cytology was defined as ≥ASCUS.For each of the five screening tests, we determined the proportion of women with high-grade CIN (detected at any point during the study) having a positive test result at baseline. A positive screening test result indicates an increased risk of CIN lesions, but CIN is detectable only in a proportion of all screen-positive women. Hence, a false-positive screening test result was defined with respect to the histological outcome, i.e. as a positive test result at baseline that was not followed by a diagnosis of high-grade CIN. The number of women attending a colposcopy was determined as the number of women with a positive screening test result having at least one biopsy regardless of its diagnosis. The number of women with only repeated testing was determined as the number of women with a positive screening test result who had only cytology or HPV testing in their follow-up. We calculated the relative proportions of women with positive screening test results, with a colposcopy, with only repeated testing, with low-grade and high-grade CIN detected, and with false-positive test results for each screening test by comparing them to those of the reference screening test (cytology). To compare the screening tests to one another, two-sided p-values were calculated using McNemar test for discordant pairs. Persistent infection was defined as a (pooled) high-risk positive HPV test result at baseline and at follow-up on the same assay. We calculated the positive predictive value (PPV) of a persistent HPV infection as the proportion of women with a persistent infection having high-grade CIN. Positive test results on cobas were additionally analyzed by channel; in case of multiple positive channels, the results were reported hierarchically for the most “severe” channel (channel 16 > channel 18 > channel other high-risk). The effect of loss to follow-up on the detection of ≥CIN2 and ≥CIN3 was determined by assuming that the detection would have been the same in women without follow-up as it was observed in women who were followed up. For this calculation, cytology results were grouped as normal/ ASCUS/ LSIL/ ASC-H, AGC, HSIL, AIS.Of the 5,034 women included in the Horizon study, 2,869 (57.0%) were screened at 30–65 years , Fig 1, Of the 127 women with abnormal baseline cytology, 117 (92%) were followed up . Among 5The 214 women with cytology-normal/HPV-positive samples at baseline had repeated testing in on average 18.1 months . Less than half of the initially HPV-positive women had persistent infections at repeated testing, i.e. a positive (pooled) high-risk HPV test result followed by another (pooled) high-risk positive HPV test result in 1.5 year, using the same assay. This varied between 35% (52/147) for CLART and 49% (68/140) for cobas. Most women with persistent infections had the same genotypes detected at baseline and at follow-up in 18 months. On cobas, 96% (65/68) of women with persistent infections had genotypes detected on the same channel, and on CLART, 88% (46/52) of women with persistent infections had at least one high-risk genotype detected both at baseline and at follow-up testing. The PPV for ≥CIN2 of persistent HPV infections was between 13 and 25% for all four assays; for ≥CIN3, this was between 9 and 13%. Of the same 214 women , 18 (8%) had incident cytological abnormalities at follow-up. The PPV of abnormal incident cytology were 33% and 22%, respectively.Of the 39 ≥CIN3, HC2 detected 35 , cobas and CLART each detected 37 , APTIMA detected 34 , but not by cobas. Cobas returned a negative test result for this woman, with a CT value of the internal control of 28.9 indicating sufficiency of the sample for a valid analysis. In this woman, CLART detected an infection with HPV16.For all four assays, normal or CIN1 histology at colposcopy was observed predominantly among HPV-positive/cytology-normal women. The proportion of false-positive test results for ≥CIN3 varied between 8.3% for APTIMA and 14.9% for cobas. The differences between the assays were significant, with a higher proportion of false-positive test results for cobas and CLART, and lower for APTIMA, compared to HC2 .When HPV assays were compared to one another using ≥CIN2 as the endpoint, similar patterns were observed, with no statistically significant differences in the detection and a statistically significant gradient in the proportion of false-positive test results.Using the outcomes from the Horizon study, we can compare the performance of routine cytology-based screening combined with HC2 triage of ASCUS, and primary HPV-based screening combined with cytology triage. With cytology screening, 127 (4.4%) women were recommended for further follow-up . With HPIn total, ≤1.0% of screened women had CIN1 detected by any of the five screening tests, and this was the lowest with cytology and APTIMA. The three HPV DNA assays led to a statistically significant increase compared to cytology. The detection of ≥CIN3 was between 10% and 19% higher , and that of ≥CIN2 between 18% and 26% higher for the four HPV assays compared to cytology. The detection of ≥CIN2 was statistically significantly higher for cobas and CLART than it was for cytology, with the differences for HC2 and APTIMA approaching significance. With just over 1% of women having ≥CIN3 detected, 3.3% of all women screened with cytology had a false-positive test result. Using any of the four HPV assays, the proportions of women with false-positive test results were statistically significantly higher, and ranged from more than doubling with APTIMA to more than quadrupling with cobas.Had the follow-up of abnormal test results been complete for all five screening tests, the differences between cytology and HPV assays in the number of women with a colposcopy referral and repeated testing would have increased. The differences in the detection of high-grade CIN between the HPV assays would have remained relatively small, within a range of ca. 11% higher detection estimated for cobas vs. APTIMA and HC2. The differences between the HPV assays and cytology would have become more visible; for example, the largest observed relative proportion of women with detected ≥CIN3, being 1.19 for cobas vs. cytology, would have increased to 1.35 (data not tabulated).On cobas alone, approximately two-thirds of the colposcopy referrals (64%), repeated testing (66%) and false-positive test results (68%) were due to the results on the “other high-risk” channel alone (data not tabulated).In Danish women undergoing primary cervical screening at 30–65 years, the detection of high-grade CIN was similar for HC2, cobas, CLART, and APTIMA. With the total follow-up of about 2.5 years and additional testing of cytology-normal/HPV-positive women, these four HPV assays detected more high-grade CIN than cytology. Owing to small numbers, however, the differences were not always significant. HPV assays combined with cytological triage led to more referrals for colposcopy and biopsies with <CIN2, particularly in women with normal cytology. All four HPV assays referred more women to repeated testing compared to the current standard of cytology, and led to significantly more false-positive screening test results.To our knowledge, Horizon was the only study set in routine practice that compared more than two HPV assays in the same women while also ascertaining the histological status of women with cytology-normal/HPV-positive test results. Most previous studies compared two HPV assays at a time using ThinPrep samples, whereas this was the first multi-assay study using the other widely used LBC medium, SurePath. Unless women moved out of Denmark, all histology could be accounted for by the linkage to the nationwide Patobank. Histology was obtained and read under routine circumstances. Baseline samples were representative for the routine screening population . They weAt the time of the baseline study, testing protocols for the handling of SurePath samples were only available for HC2, whereas the processing protocols for cobas, CLART, and APTIMA were agreed upon with the manufacturers. The manufacturers have since changed certain aspects of these protocols to support the testing also on SurePath samples, reflecting accumulation of experience with the assays’ performance on this cytology medium. In concordance with the protocol and by approval from the manufacturers, we diluted the original samples approximately 1:1 to obtain sufficient material for split sample testing by CLART, cobas and APTIMA. This can be seen as a weakness of the study, however, all three assays rely on testing aliquots of 0.5 to 1 ml out of the typically 10–20 ml available from liquid based cytology media or microbiology sampling liquids. Hence, the robustness of the assays should clearly be able to handle sampling variability in terms of cellularity.Study size was defined by the laboratory’s practical considerations, and in agreement with the manufacturers. The highest observed detection rate of ≥CIN3 for any of the evaluated HPV assays was 37/2869 (1.3/100), and the lowest was for cytology, 31/2869 (1.1/100). An ex-post power calculation for this difference, taking into account the paired study design, revealed that approximately twice as many women would need to be included in the study to detect this difference at 80% power and α = 0.05. To detect the maximal difference in the detection rate between HPV assays alone, 37/2869 (1.3/100) vs. 34/2869 (1.2/100), the study size would need to be more than four times as large.Of the women with cytology-normal/HPV-positive test results at baseline, 58% had follow-up. Very similar proportions of follow-up completeness were observed in randomized controlled trials comparing HPV testing with cytology in primary cervical screening . Had theCytology normal women with positive HPV test results were recommended for repeated testing in 18 months. Although the repeated testing was, as the baseline testing, undertaken with all five screening tests, the women were at this stage managed according to cytology and HC2 alone. This strategy may have missed high-grade CIN in women with normal cytology and a negative HC2 test result but a persistent infection on the remaining three HPV assays; their number was, most likely, modest.A number of studies have reported a direct comparison of cobas or APTIMA to HC2 or cytology. However, most often these studies were from referral populations where the observed differences between HPV assays were much smaller than in primary screening , thus emHorizon was the first study to evaluate CLART in primary screening. In previous studies including referral populations ,24, thisIn the Horizon study, the difference in the detection of high-grade CIN between HPV assays and cytology was not substantial . In Denmark, the quality of routine cytology may be relatively high. In our laboratory, cytopathology technicians undergo continued education by biennially participating in anonymous testing of adjudicated cytology samples. Upon reading the routine slides, the women’s complete screening histories as registered in the Patobank are available to cytopathology technicians as background information. The laboratory has defined standard operating procedures for cytology sample inadequacy. However, cases of cervical cancer with recent normal cytology are not uncommon in Denmark ,26. MoreThe expectedly better protection from cervical cancer will come at a cost. Our data, supported with data from the randomized trials ,28 and oOwing to the restrictions imposed by the Ethical Committee approval, women with normal cytology and positive HPV tests at baseline could only obtain the information on their HPV status through their GP. In routine HPV-based screening, women would be informed directly about their HPV status, so the loss to follow-up would probably be lower than observed in our study. Adequate follow-up of abnormal screening tests is one of the cornerstones of cervical cancer screening. There may be several reasons why this follow-up is often incomplete. One is that women might not be adequately informed about the abnormality. To this end, Danish women will from 2016 onwards receive their screening test results by email into the nationally mandated “e-box”. Another reason for the loss to follow-up might be that the abnormalities are not perceived as serious by the women themselves, or by their doctors. As an example, only 44% of Dutch women with borderline abnormal cytology were followed up on time when the definition of this diagnosis was broad and included inflammation and dysplastic changes . When thIn conclusion, differences in the detection of high-grade CIN, false-positive test results, and the number of follow-up procedures were observed between HPV assays  on the one hand, and cytology on the other hand in Danish women undergoing primary screening at age 30–65 years. The differences between cytology and HPV assays would have probably been larger had the follow-up of HPV-positive women been complete. The differences were small between the HPV assays alone, although HC2 and particularly APTIMA were associated with smaller increases in false-positive tests, number of needed colposcopies, and frequency of repeated testing than cobas and CLART.S1 Table(DOCX)Click here for additional data file.


Chlamydia trachomatis in HPV-induced cervical carcinogenesis has been reported for cervical cancer but studies on cervical adenocarcinoma are limited.A role of in situ  were tested for C. trachomatis and HPV DNA by a type-specific PCR bead-based multiplex genotyping (TS-MPG) assay.A total of 1,553 cervical smears taken up to 26 years before diagnosis in a large population-based nested case-control study of cervical adenocarcinoma , and adenocarcinoma C. trachomatis, with no significant differences between AC/AIS cases and controls. HPV-positivity was detected in 49.3% of C. trachomatis-negative and 65.4% C. trachomatis-positive samples, respectively.Only 1.7% of samples were positive for C. trachomatis infection.A large prospective study did not find any risk for cervical adenocarcinoma and/or AIS conferred by C. trachomatis appears not to be involved in cervical adenocarcinomas. Chlamydia trachomatis has been associated with an increased risk of cervical cancer [C. trachomatis primarily infects glandular cells [C. trachomatis DNA in 71 archived formalin-fixed paraffin-embedded tissues of cervical adenocarcinoma (5), but cross-sectional studies are less informative than longitudinal studies [C. trachomatis has only been detected in samples taken many years before the cancer, not close to the diagnosis [Persistent infection with human papillomavirus (HPV) is a virtually necessary cause of cervical cancer . One of l cancer –4. Whilel cancer ,5, whichar cells . Some star cells . One stuiagnosis .C. trachomatis and cervical adenocarcinoma.We previously reported a large prospective study of cervical adenocarcinoma that established a strong association with HPV present many years before cancer diagnosis (6). We now wished to use this large prospective study to investigate possible associations between in-situ  or invasive cervical adenocarcinoma  and matched controls  were tested for the presence of C. trachomatis and 19 mucosal HPV types , using a validated type-specific PCR bead-based multiplex genotyping  assay that combines multiplex polymerase chain reaction (PCR) and bead-based Luminex technology , as described elsewhere [Detailed characteristics of the study have been reported previously . A totalC. trachomatis  for the association between chlamydia infection in the first collected smear and subsequent adenocarcinoma were 1.25 for AIS (95% CI 0.34–4.65) and 3.0 for AC (95%CI 0.31–28.84); ORs associated with chlamydia infection in the last smear before diagnosis were not estimable due to the lack of exposed cases (AIS) and controls (AC), respectively  of samples were positive for chomatis . The 26 ectively . The fulectively .C. trachomatis-negative and 65.4% (n = 17) C. trachomatis-positive samples. Because of the overall low numbers, a further analysis of possible interaction between C. trachomatis and HPV status was not feasible.HPV-positivity was detected in 49.3% (n = 753) of C. trachomatis and cervical adenocarcinoma, with a follow-up period of up to 26 years, C. trachomatis was not associated with increased risks of subsequent invasive adenocarcinoma (AC) and its precursor, adenocarcinoma in situ (AIS). The average age of participants in this study was above 30 years, and it is possible that co-infections/interactions between C. trachomatis and HPV, or other STI, may be of more relevance at earlier age and this may require further studying. A higher prevalence of HPV infections have been found in younger Italian women affected with a C. trachomatis chronic infection from a STI centre than in the ones from an assisted reproductive technology clinic [C. trachomatis [C. trachomatis DNA and subsequent risk of CIN3+ among the ones who were HPV-positive or had a persistent HPV infection at baseline [C. trachomatis status, as assessed by DNA or IgG, and risk of cervical premalignancy, after controlling for carcinogenic HPV-positive status was found in a previous study from the United States [C. trachomatis and cervical premalignancy could have been caused, in part, by an increased susceptibility to HPV infection [C. trachomatis and HPV in squamous cell carcinoma of the cervix are mechanistically unexplained [In the current largest prospective study to date of y clinic . HPV genchomatis . Howeverbaseline . No assod States , which snfection . Howeverxplained  and furtS1 TableSubjectID–unique subject identifier, RisksetID–unique risk set identifier, CaseStatus–case-control status (1 = case), AgeAtDiag–age at the time of diagnosis, HPVpos–HPV-positivity (1 = HPV-positive), CTpos–Chlamydia trachomatis-positivity (1 = Chlamydia-positive), AgeAtSmear–age at the time of collecting cervial smear).(ODS)Click here for additional data file.


P = 0.003). The majority (75.4%) indicated that HPV vaccination should be given before their daughters are mature enough to understand about sex, and 80.3% reported that they will discuss matters related to sex with their daughters if their daughters want to know about the vaccine. Those who did not want to vaccinate their daughters highlighted that they want more information regarding safety of the vaccine which might change their decision towards HPV vaccination. A health education information method can increase the vaccination acceptance rate in South Africa.Cervical cancer is a preventable public health problem. The two new human papillomavirus (HPV) vaccines are available but not accessible to everyone in South Africa, as they are very expensive. This study aimed to investigate educated peoples acceptability regarding HPV vaccination. This was a cross-sectional survey conducted among 146 master of business administration students by self-administered, anonymous questionnaire. The majority (74%) of the participants ever heard of cervical cancer, but only 26.2% heard about HPV. After reading the fact information regarding cervical cancer and HPV, the intention to vaccinate their daughters increased from 88% to 97.2% ( Cervical cancer is a preventable disease, but it accounts for around 275,100 deaths, and more than half a million women are diagnosed each year in the world , 4. CervThanks to the new development of a HPV vaccine with high efficacy, cervical cancer incidence can significantly be reduced with 70%) if a comprehensive vaccination program could be implemented 0% if a c. Many coThe two vaccines, Cervarix  and Gardasil , are currently available in South Africa. But they are very expensive. The majority of the population cannot afford these vaccines for their daughters. The national Department of Health of South Africa is planning to implement HPV vaccination countrywide. Successful vaccine implementation will depend on how different population groups are aware of these vaccines.This study investigates the acceptability of HPV vaccination among educated people like master of business administration (MBA) students in South Africa. These groups of people are holding managerial positions/owing small to medium businesses in the country. They have the capacity and capability to develop new projects towards reducing cervical cancer incidence as well as they can disseminate the information regarding HPV vaccination and cervical cancer to their colleagues or employees. Moreover, in South Africa, corporate social responsibility (CSR) is a very serious topic for discussion in the business sector. CSR means voluntary involvement, or investment, of companies in social projects that help or uplift the community in which they operate in areas such as health care, housing, education, and basic services. CSR can also be defined as the commitment by business contributing to economic development and at the same time improving the quality of life of the workforce, their families and the local community, and society at large.This was a cross-sectional survey study, including a fact sheet, conducted among MBA students who are currently registered at the University of KwaZulu-Natal. A total of 245 students were enrolled for the MBA program. All these students were given the possibility to participate in the study.A self-administered questionnaire with closed and a few open-ended questions was used based on a study conducted among educated parents in India . The queUniversity of KwaZulu-Natal research and ethics committee approved the study prior to data collection. Participation in the study was voluntary. Anonymity and confidentiality were maintained at all times. Prior to data collection, the main researcher communicated with the corresponding lecturers about the data collection procedure. Once the lecturers agreed, the researcher went to the corresponding lecturers' lecture room during the lecture break. The researcher then explained the aims and objectives of the study. Each participant signed a consent document before completing the questionnaire. Students who were present in the class during that day and voluntarily participated were included in the study.P value < 0.05 was considered as statistically significant.Data were checked for completeness and then entered into a Microsoft Excel 2003 spread sheet and imported into SPSS 21.0. for Windows for analysis. The results were summarized using descriptive statistics: expressed as mean (SD) for continuous variables and percentages for categorical variables. Comparisons of categorical responses were evaluated using the Chi-square or Fisher's exact test. A A total of 146 students participated in the survey. Only these students were present on the day of data collection. Those who were not present had similar background characteristics because of the same admission criteria. n = 5) or their physician did not advise them (n = 4) to have their children vaccinated.Participants, who had children, were asked if their children were vaccinated. Results showed that almost all the participants (90%) had given regular vaccination to their children. The most important reason to vaccinate was that vaccines prevent certain diseases (86.1%) and the main concern to vaccinate was the seriousness of the disease that the vaccine prevents. Reasons and concerns for vaccinations are shown in P = 0.003)  . ParticiMore than half (58.7%) of the participants had indicated that it is necessary to explain to their daughters before vaccination that it protects against sexually transmitted infection, whereas about two-thirds (66.0%) did not think that vaccination will send no-objection message to start sexual relationship . The majAfter reading the fact sheet information, four participants still did not want to give HPV vaccination to their daughters. Amongst them, three participants reasoned that the vaccine is new and they are not sure if it will be safe for their children and one participant did not give a reason. When asked what can change their mind regarding accepting the vaccine, two participants wanted a detailed report on the safety and effectiveness of the vaccine, one participant was willing to have his daughter vaccinated if it is recommended from the child's school, and the last participant did not indicate anything (data not shown).As far as we know, this is the first study to investigate the acceptability of HPV vaccination among educated, urban people in South Africa. Awareness regarding cervical cancer varies. This study found that a majority of the participants ever heard of cervical cancer, but only 26.2% heard about HPV. An Indian study conducted among urban, educated couples found that about a third of the participants heard about cervical cancer . A crossIt is well known that vaccination prevents serious diseases. Globally as well as in South Africa, general vaccination coverage has increased over the last few decades. So, it was expected that all the educated people like our surveyed population vaccinated their children for their general, regular vaccination. This study found that about 90% vaccinated their daughters for general vaccination. This finding is in line with South African vaccination coverage. In South Africa, full vaccination coverage is 80% or more for one-year-old children in eight of its nine provinces . ConsideGlobally, vaccination acceptability is high among different population groups. Studies have found high acceptability for HPV vaccination in all the countries that investigated HPV vaccination acceptability among women as well as for their daughters. Our study found that 88.8% of the participants were willing to vaccinate their daughters against cervical cancer. A recent South African study conducted among female undergraduate students found that 77.3% of the participants were willing to take HPV vaccination for themselves . A studyThe effectiveness of health education on secondary prevention of cervical cancer is well documented. After reading the fact sheet information regarding cervical cancer and HPV, the respondents' intention to have their daughters vaccinated increased from 88% to 97.2%. This finding is similar to other studies conducted elsewhere. A qualitative study conducted among women aged 18–45 years in Malawi highlighted that before the study none of the participants heard of a HPV vaccine, but after given basic information, all the participants were willing to accept HPV vaccination . AnotherThe appropriate age to vaccinate HPV vaccines varies across different population groups. HPV vaccines are effective if they are given before the girl is not yet sexually active. In the present study, a majority mentioned that HPV vaccination should be given to their daughters before children understand about sex. A South African study reported that because of early sexual activity by the adolescents, health care providers and community respondents suggested that HPV vaccination should commence around the age of nine . The preSince HPV vaccines are new, it will face challenges to be accepted by the general population. Researchers have reported that potential side effects, health need, and benefit of the vaccine are the most imported challenges faced by the vaccine to get accepted by the general population . In thisThis study only sampled people from one university. As they are educated people, the results cannot be generalized. One important aspect of this study is that this group of people, who can significantly impact the society at large regarding HPV vaccination, has never been investigated before. The findings will be helpful for nationwide vaccination coverage as the business community plays a significant role in the community upliftment.The present study found a high acceptability rate of HPV vaccination for their daughters in an educated group of MBA students. Educational information increased the vaccine acceptability rate. These findings are important, because due to their position in the South African society, this group of educated people can play an important role in disseminating the knowledge and attitude regarding HPV vaccination among a large part of the South African population.


R²) infections could be subdivided in a latency group  and a regressing group . In (≥3) serial viral load measurements, serial transient infections with latency is characterized by a nonlinear limited difference in decrease or increase of type-specific viral load (R² < 0.85 and slopes between 2 measurements 0.0010 and −0.0010 HPV copies/cell per day) over a longer period of time (1553 days), whereas regression of a clonal cell population is characterized by a linear (R² ≥ 0.85) decrease (−0.0033 HPV copies/cell per day) over a shorter period of time . Using serial HPV type-specific viral load measurements we could for the first time identify regressing CIN2 and CIN3 lesions. Evolution of the viral load is an objective measurable indicator of the natural history of HPV infections and could be used for future triage in HPV-based cervical screening programs.Persistent high-risk human papillomavirus (HPV) infection is strongly associated with the development of high-grade cervical intraepithelial neoplasia (CIN) or cancer. Not all persistent infections lead to cancer. Viral load measured at a single time-point is a poor predictor of the natural history of HPV infections. However the profile of viral load evolution over time could distinguish nonprogressive from progressive (carcinogenic) infections. A retrospective natural history study was set up using a Belgian laboratory database including more than 800,000 liquid cytology specimens. All samples were submitted to qPCR identifying E6/E7 genes of 18 HPV types. Viral load changes over time were assessed by the linear regression slope. Database search identified 261 untreated women with persistent type-specific HPV DNA detected (270 infections) in at least three of the last smears for a average period of 3.2 years. Using the coefficient of determination ( Although effective cytology screening methods were introduced in the previous century, today more than halve a million women are still diagnosed with cervical cancer each year and about 275,000 women die from it Randomized trials have demonstrated that a HPV-based screening leads to a significant decrease in the incidence of CIN3 and cancer y-axis against time on a linear scale expressed in days on x-axis) in serial measurements (R² > 0.9 and slope = 0.003 HPV copies/cell per day) Ultimately, the implementation of primary HPV screening will result in earlier detection of cervical cancer and later on of its precursors, increasing the clinical sensitivity. The implementation of HPV screening and increased coverage of HPV vaccination will lower the background risk of CIN3 and cancer, leading to longer screening intervals and a requirement for more specific tests In this study, we wanted to investigate if serial viral load measurements can differentiate between regressing lesions and serial virion producing transient infections in women with persistent type-specific HPV infections.We retrospectively calculated slopes between viral load measurements in untreated women with long-term type-specific HPV DNA positivity to try to define regressing lesions and serial transient infections with latency based on linearity of measurements and differences in slopes, and to see if these findings are in agreement with the proposed asymmetric epithelial cell replacement model.n = 781,737 samples from 359,410 women) was subjected to HPV measurements to enable HPV-guided cytology R² > 0.9; +0.003 HPV copies/cell per day) were excluded This retrospective natural history study was set up using the RIATOL cervical cancer screening and diagnostic follow-up database. The laboratory for clinical and molecular pathology (RIATOL), Sonic Healthcare, Antwerp, Belgium is processing more than 100,000 liquid-based cytology (LBC) samples annually, representing approximately one quarter of women participating in cervical cancer screening in Belgium. Since June 2006, all LBC samples were subjected to a density gradient to remove free virions  and the enriched cell pellet  against time on a linear scale expressed in days (x-axis). In six women were multiple HPV types were detected simultaneously each HPV infection (n = 15) was analyzed separately.For each subject, the successive viral loads (HPV copies/cell) of each individual HPV type were plotted on a logarithmic scale (The slopes between two successive measurements were defined as log10(type-specific HPV load on date 1) −log10(type-specific HPV load on date 2) divided by the number of days occurring between date 1 and date 2 y = a + bx, where y is the predicted log10 , a is the intercept, x is the time interval, b is the slope ]. For each regression, the coefficient of determination R² was calculated, which is a measure of deviation between the regression line and the observed points. We considered a R² of ≥0.85 as a linear increase or decrease, representing the doubling or halving of a clonal population with a constant amount of HPV DNA per clonal cell. For regressing infections the viral halving time (VHT) in days was calculated for each HPV type by (ln 2)/slope. The latency boundary was only calculated when serial measurements were nonlinear (R² < 0.85) by selecting the highest and lowest measured viral load and adding or subtracting 0.5 log HPV copies/cell.For three or more consecutive measurements, the slope was calculated between the consecutive viral load measurements using a simple linear regression model [VDT) calculated in clonal populations that evolved to CIN3+ VDT period as a proxy for the time needed for asymmetric basal cell division resulting in latency and serial virion productions. To calculate the number of VPEs in the latency group (R² < 0.85), the number of days a specific HPV type is detected was divided by the number of days of the corresponding type-specific VDT. For HPV 11, 53, and 68 an average VDT of 286.3 days was used to calculate the number of VPE. For all other HPV types, the type-specific VDT was used The type-specific viral doubling time  U test was used.The calculated A database search identified 261 untreated women with a smear between June and December 2013, which had a persistent type-specific HPV DNA detected (270 infections) in at least three of their last smears for a mean period of 1156 days (95% CI 1074–1238 days). The median age of women in the whole group was 39.0 years (95% CI 37.0–41.0 years). There was no statistical difference in duration in which the different HPV types were detected. On average the untreated women had five smears within this period  (n = 127) and a group with a nonlinear viral load course (R² ≤ 0.8340) (n = 143) , the median slope over three or more measurements was always decreasing . For the group with a nonlinear course, only slopes between two consecutive measurements were calculated. In this group, only a limited increase or decrease was measured with slopes between 0.0010 and −0.0010 HPV copies/cell per day (latency group). The HPV type-specific characteristics in the latency and regression groups are shown in TableP < 0.0001).In the group with a linear viral load course . There were significantly more CIN 2 cases detected in follow-up in the latency group 18.9% (14/74) than in the regression group 3.0% (2/67) (P = 0.0068). In the latency group, one case of invasive cancer was detected, and there were significantly more CIN2+ cases 23% (17/74) compared to the regression group 6.0% .Follow-up data (from January 2014 until October 2014) were available for 199 cases (73.7%), of which 110 from the latency group and 89 from the regression group. An overview of the follow-up outcomes is given in TableThe representative cases of the different serial viral load patterns observed in serial transient infections with latency and regression are shown in FigureThe aim of this study was to define regressing lesions and serial transient infections with latency based on serial type-specific viral load measurements.R², using the type-specific viral load in the last three consecutive measurements. In the group with R² ≥ 0.85 the viral load decreased with 0.0033 HPV copies/cell per day (slope = −0.0033). This negative slope clearly represents a halving of a clonal population of basal cells with a constant number of HPV DNA copies per clonal cell. So far, in the available follow-up data (10 months), the majority  of these women had a normal cytology with negative HPV test after a regressing viral load period. This might suggest that the treating physicians decided to wait and see as the type-specific viral load was dropping in three consecutive smears, independently of the cytology results. In the group with R² < 0.85, however, the differences in slopes was only minimal and probably reflects a serial production of a constant virion amount. A serial virion production would imply that these women are probably cyclically infectious over a longer period of time. This is in contrast with women with regressing lesions who are not infectious as infected basal cells do not allow production of new virions. Furthermore, during serial transient infections with latency, the remaining latent basal cell can still divide, and each new generation of basal cells have more risk to be transformed under the influence of the sustained oncogenic E6/E7 protein production. This increased risk for development of CIN in serial transient infections with latency is illustrated in the more severe histology outcome of 23.0% CIN2+ in this group (P = 0.0095). Delayed action by clinicians was probably induced by a combined lack of persistent abnormal cytology and absence of elevated or increasing type-specific viral loads. During serial virion production effects on cytology would only be minimal as desquamating cells have a limited time span, and the accumulation of newly formed virions within the cell cytoplasm is cytologically well recognizable as low-grade squamous intraepithelial lesions (L-SIL).The extensive data presented in this large retrospective study are in agreement with the proposed asymmetric epithelial replacement model. This model also allows simultaneous occurrence of regression of lesions as well as serial VPEs. We could clearly subdivide the group of untreated women with persistent type-specific HPV DNA detected over a longer period of time in two subgroups based on Clearing of the HPV infection then becomes possible in two different scenarios.VDT) of this underlying clonal population In exit scenario 1, depletion of parabasal cells occurs. When the last HPV-infected basal cell division occurs double the amount of parabasal cells (two) begin differentiation and start an ultimate virion production. This results in double the number of virion producing cells compared to the latency period in which asymmetric division only produces one parabasal cell, and results in a higher measured load compared to the latency period. In some of the cases with serial transient infections with latency a higher load is seen before clearing HPV Fig.. HoweverIn the second exit scenario 2, a depletion of basal stem cells occurs Fig. C and D Using serial HPV type-specific viral load measurements, we could for the first time identify regressing CIN2 and CIN3 lesions. Regression of the clonal population of HPV-infected basal cells could be due to cytotoxic T-cell response. Not surprisingly, regression is a slow process because HPV infection causes no viremia, evades innate immune response and delays activation of adaptive immunity R² and slope each type-specific HPV infection in HPV positive women can be categorized in one of four HPV courses , the slope indicates if the measured process is progressing (+0.003 HPV copies/cell per day) or regressing (−0.003 HPV copies/cell per day). Because the majority of clonal processes regress, triaging HPV infections by R² and slope could substantially reduce biopsy taking and overtreatment of regressing lesions. Women with single or with multiple HPV infections in which no clonal progressing process was detected would then need a less stringent follow-up. Noninfectiousness of regressing lesions would be another argument to the wait and see option. Prospective studies are needed to confirm the efficacy of triaging HPV infections by R² and slope in cervical cancer screening.Serial measurements may have the disadvantage that at least three sampling points are needed to be able to calculate linearity of viral load measurements, but allows measurement of regressing lesions and calculation which of the two HPV induced pathways is occurring. It is thus not a risk assessment but rather a mathematical representation of the virion producing or basal cell transforming pathway. The advantage of using HPV type-specific serial viral load measurements is that it can be used to triage each separate HPV infection into a clonal or nonclonal process, irrespective of the age of the woman, cytology result and number of simultaneously occurring HPV infections. Furthermore, by calculating We conclude that serial type-specific viral load measurements allows discrimination between serial transient infections with latency or regressing lesions in women who have prolonged type-specific HPV persistence, and that the evolution of the viral load is an objective measurable indicator of the natural history of HPV infections and could be used for future triage in HPV-based cervical screening programs.