Human papillomavirus (HPV) is an important etiologic factor in different cancers of anogenital region and also in a fraction of head and neck cancers (HNC) particularly oropharyngeal tumors. The HPV16 genotype associated with the majority of HPV-related head and neck carcinomas. Th incidence of oropharyngeal cancer is increasing in many countries, and the rate of HPV positive tumors is about 70% in Europe and North America. Little known about the prevalence of HPV in HNC in Central Asia.It’s a prospective analysis of patients with verified oral or oropharyngeal cancer. Sociodemographic and clinical data obtained on admission to treatment. The diagnosis of HPV positivity assessed by both the P16 expression on immunohistochemistry(IHC) and polymerase chain reaction (PCR)with HPV DNA detection and HR HPV type determination.Seventy six patients with oral and oropharyngeal cancer tested for HPV. Forteen cases were positive for HPV by PCR and 15 cases by P16 IHC. Of the 35 oropharyngeal tumors, nine were HPV DNA and p16 IHC positive, giving the rate of 25.7%. Of the 41 oral tumors, five were HPV DNA and six p16 IHC positive, giving the rate of 12.2%.It is the first study mapping prevalence of HPV positivity in oral and oropharyngeal cancer in the Central Asian region. The rate of HPV positivity was higher in oropharyngeal than in oral cancer, the nonsmokers were significantly more frequent in the HPV positive group and HPV 16 was the most frequent type. However, the HPV positivity rates are lower than referred in the western world. Oral cavity and oropharynx cancer is a major global health issue, they both constitute the majority of head and neck cancers. Globally 300,000 men and 130,000 women get diagnosed with these diseases annually, and the annual mortality is 160,000 and 68,000 respectively [Main predisposing factors are smoking and alcohol consumption . DespiteIt’s known that HPV is an important etiologic factor in oropharyngeal cancer, yet, its role in other head and neck sites is unclear. There are more than a dozen of high-risk HPV types that can cause cancer. The 13 most frequent types of high-risk HPV (HR HPV) that are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 . These tKazakhstan is a multi-ethnic country located in Central Asia with a population of 17 million people. Its population is well known for high level of diversity, due to forced translocation of many nations during the Soviet period and a mixed structure of Kazakh nation . The varThe role of HPV in cancerogenesis is now well documented, and the biological and clinical differences between HPV positive and negative tumors will probably very soon have consequences also in treatment approach . From thIt is a prospective analysis of patients with verified oral or oropharyngeal cancer referred to a single tertiary institution . Sociodemographic and clinical data obtained through a questionnaire on admission to treatment. Approval for the study received from the local institutional ethics committee, and informed consent obtained from all study participants.The study enrolled 76 patients with oral and oropharyngeal cancer treated between February 2015 and May 2017 in Kazakh Institute of Oncology and Radiology. All patients have squamous cell carcinoma of different grades. Out of 76 patients, 35 had oropharyngeal cancer and 41 oral cavity cancer.Part of the fresh tumor tissue from a biopsy specimen taken for PCR analysis and p16 IHC staining performed on formalin fixed paraffin embedded slices.RIBO-sorb nucleic acid extraction kit was used to extract and purify DNA from cancer tissue. RIBO-sorb nucleic acid extraction kit is reagents kit for rapid and efficient manual extraction and purification of DNA from various biological materials.AmpliSens HPV HCR genotype-FRT PCR kit is an in vitro nucleic acid amplification test for qualitative detection and differentiation of high risk (HR) human papillomaviruses (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 DNA in the clinical material by using real-time hybridization-fluorescence detection of amplified products [The test based on simultaneous PCR (multiplex-PCR) and real-time detection of three HPV types and b-globin gene DNA, used as internal control, in one tube. The analysis of 12 HPV types is carried out in four tubes. Each HPV type registered on its channel that allows not only to detect but also to differentiate the virus genotype. The DNA target selected as an endogenous internal control is a human genome fragment. AmpliSens HPV HCR genotype-FRT PCR kit uses “hot-start,” which significantly reduces the frequency of nonspecifically primed reactions.p16 IHC was carried out using the CINtec Histology kit  on a Ventana Benchmark Autostainer. p16 IHC was scored as positive if there was strong and diffuse nuclear and cytoplasmic staining present in greater than 70% of the malignant cells. All other staining patterns scored as negative. All samples were scored independently by two expert pathologists, and discordant cases were reviewed to come to a consensus score. Examinations were carried out at the Department of Pathology of Kazakh Institute of Oncology and Radiology.There were 76 patients with oral and oropharyngeal cancer enrolled in the study. Of them 35 had oropharyngeal and 41 oral cavity cancer, 50 were male and 26 female. Mean age was 57.2 years (standard deviation - 11.45 years), with youngest and oldest participant were 26 and 79 respectively. Patients characteristics presented in Table Of the 76 patients, 14 cases were positive for HPV by PCR and 15 by P16 IHC. One patient had positive p16 IHC and negative PCR test. Of the 35 oropharyngeal tumors, nine were HPV DNA and p16 IHC positive, giving the rate of 25.7%. Out of nine positive cases, four are tonsillar cancers and two base of tongue cancers, what corresponds with the distribution of the whole group of oropharyngeal tumors.Of the 41 oral tumors, five were HPV DNA positive and six patients were p16 IHC positive, giving the rate of 12.2% (excluding the P16 only positive case). Three of them are tongue cancer, one floor of the mouth and one alveolar ridge tumors. All three tongue cancers were located closer to posterior third of the tongue and even with extension to the base of the tongue.HPV 16 was the most prevalent type in both the oropharyngeal and oral cavity cases, other types found were 18 and 56, one case each Table .Table 2Hp = 0.0018 (Chi-squared test).Smoking was less prevalent in the group of HPV positive patients, two had a history of smoking out of 14, comparing 40 out of 62 in the HPV negative group of patients. The difference is statistically significant with In the oropharyngeal cancer group, 25 patients had advanced tumors stage III and IV. Patients with oropharyngeal cancer received concurrent chemoradiotherapy (CRT) according to the local standards. The local CRT standard includes delivering IMRT radiotherapy to tumor and neck with a concurrent infusion of platinum-based agent weekly or every three weeks.In the group of oral cancers, 29 out of 41 had advanced oral cancer stage III and IV. They were treated mostly by surgery and adjuvant RT or CRT for non-operable cases. Adjuvant CRT in the oral cavity cancer group is used only for cases with adverse features on permanent pathology.Cancers of the head and neck is a heterogeneous group of tumors with various anatomic sites with different etiologic factors. Tobacco exposure (either via active or passive smoking as well as consumption of smokeless tobacco) is the leading risk factor for head and neck cancers . But in Kazakhstan is a partly Eastern European, partially Asian state with a mixed population. The primary risk factor for the local population is the smoking, as there is no or very low level of smokeless tobacco consumption prevalent in other Asian countries. The incidence of HNC is low in Kazakhstan when compared with, e.g., Europe. However, we can follow similar trends. There is rising age-standardized incidence rate of oral and oropharyngeal cancer from 3.8 in 2004 to 4.7 in 2015 per 100,000. In the same time incidence of laryngeal cancer mostly attributed to smoking has decreased from 2.6 in 2004 to 2.3 in 2015 . SimilarIn summary in this first study in the Central Asian region the prevalence of HPV induced oral and oropharyngeal cancer is lower than in Europe and North America, but higher than in other Asian countries , 21. In


High risk human papillomavirus (hr-HPV) infection and the dual burden of HIV remains a huge challenge in some low-income countries (LICs) such as Swaziland with limited or no data. We estimated the prevalence and investigated determinants of hr-HPV, including HIV infection among sexually active women in Swaziland.A total of 655 women aged between 15 and 49 years from five health facilities were randomly enrolled using a cross-sectional study design. Cervical cells were tested for hr-HPV types using GeneXpert HPV Assays.The overall weighted hr-HPV prevalence was 46.2% (95%CI: 42.8–49.5). Of hr-HPV infected women, 12.4% (95%CI: 8.6–17.5) were HPV16-positive, 13.8% (95%CI:12.0–15.8) were positive for HPV18/45, 26.7% (95%CI: 24.2–29.3) for HPV31/33/35/52/58, 7.6% (95%CI: 7.6–11.9) for HPV51/59 and 11.0%, (95%CI: 7.9–15.3) for HPV39/56/66/68. Prevalence of hr-HPV decreased with increasing age. Overall HIV prevalence remained high . HIV infection was associated with hr-HPV infection . Overall hr-HPV/HIV co-infection was 24.4% (95%CI: 20.3–29.1) which was significantly higher among younger age groups (p<0.001). Prevalence of multiple group hr-HPV infection was significantly higher in HIV-positive versus -negative women . The presence, absence or unknown of history of STI with HIV did not appear to modify the relationship with hr-HPV .The prevalence of hr-HPV infection was high and significantly associated with HIV among sexually active women. Furthermore, the study has provided essential information about the HIV link with hr-HPV infections which may explain the high prevalence among HIV infected women. This can contribute to policy development and planning of prevention strategies incorporating HPV infection prevention especially among youth and HIV infected people. In low-income countries (LICs), the burden of cervical cancer is a serious health problem and predominantly affects women of reproductive age . CervicaThe high prevalence of HPV infection among women in several African countries may be explained by the presence of risk factors such as early age sexual debut, the number of sexual partners of women and of their partners, and other STIs, including HIV, as discussed above –13. PrevStudies estimating HPV infection are limited in African countries . The lacTherefore, the main aim of this study was to estimate prevalence and identify associated determinants of hr-HPV, including HIV infection among reproductive aged women in Swaziland.The study participants were women aged between 15 and 49 years, attending healthcare facilities for routine health care-related services in June–July 2015. All women with a history of previous or current sexual activity who provided written informed consent were included. The women were recruited from five healthcare facilities , Hlatsikhulu, Sithobela and Siteki Public health unit) within the four political regions of Swaziland that had fully functioning CC screening services such as VIA and cryotherapy .To estimated prevalence of the outcomes of interest, assuming 95% confidence and an acceptable margin of error of 5%, and maximum variability i.e. 50%  a sample size of 384 subjects was required were used. The sample was further increased by a margin of 10% to account for potential non-response and multiplied by a design effect (D) of 1.5. The final sample size of the study was 650 women.rd woman of those first selected) using the lottery method. The participants were selected from each site until the calculated sample size was achieved per site.The recruitment of participants was done in two sampling stages; firstly women were stratified by age (seven age-groups) and then sampled using systematic random sampling . Each participant was assigned a unique study identity number that was used to link the questionnaire with the specimens. Personal information blinded for researchers was kept on site for results feedback and validation of data.After visual inspection of the vulva, a non-lubricated sterile disposable speculum was inserted and cervical cells were collected using a sterile cyto-brush by rotating the brush 3-times 360° in the cervix . The head of the cervical brush was then eluted in a labeled vial containing Thin Prep Preservcyt Preservation solution to assess the presence of hr-HPV and for the Liquid-Based cytology . After HIV pre-counselling, 4ml of blood were collected in a vacutainer tube from participants consenting to HIV testing. All specimens were collected and transported daily to the national laboratory. All tests were performed at the Swaziland Government National Referral Laboratory, Mbabane.in vitro test for the detection of the E6/E7 region of the viral DNA genome from hr-HPV in patient specimens. The Xpert HPV test gives results from 6 separate channels: (i) sample adequacy control (SAC), (ii) P1-HPV16, (iii) P2-HPV18/45, (iv) P3-HPV 31/33/35/52/58, (v) P4-HPV51/59 and (vi) P5-HPV39/68/56/66. An individual specimen can be positive for more than one probe. For the purpose of our analysis infection, positivity on one probe only was classified as single group hr-HPV infection (though with the possibility of multiple HPV type infection with P2-P5). Multiple group hr-HPV infection was classified as positivity on two or more probes.The HPV-DNA testing was done using the GeneXpert HPV assay (Xpert HPV Assay)  according to the manufacturer’s protocol. The Xpert HPV Assay is a qualitative The Alere Determine HIV-1/2 Ag/Ab Combo test was used to detect both HIV-1/2 antibodies and free HIV-1 p24 antigen. Reactive specimens were confirmed by Trinity Biotech Uni-Gold Recombigen HIV Test. All participants who tested HIV-positive were post-counselled and referred to health units offering the necessary HIV treatment, care and support services.2) test. Odds ratios (unadjusted and adjusted) and 95%CIs for potential risk factors associated with hr-HPV were estimated using logistic regression model. Variables significant at significant cut-off of 0.2 in the bivariate regression analyses were selected for inclusion into the final multivariable model. An adjusted p-value of <0.05 was deemed statistically significant.Data were processed and analysed using Stata 13.0SE . Data were checked for possible errors and missing values prior to analysis. Age and region-weighted analyses were done to estimate the overall hr-HPV prevalence and co-infection with HIV infection given the stratified systematic random sampling design. Survey weighted Analysis was done to adjust the sample characteristic to match up with the target population (15–49) that they were selected to represent. It’s applied to bring the proportion of women in the sample in the alignment with the portion of women in the target population. Therefore all results reported as weighted. Survey weighted prevalence and 95% confidence interval (CI) were calculated. In addition, assuming that our study subjects are representative of the population of Swaziland, we used survey weights to extrapolate sample proportions to population totals (age-region weighted) to estimate burden counts based on the 2007–2030 population projections aligned to the 2014 population estimates total of 377169 women aged 15–49 years ) age for enrolled women was 32.2 (±8.7) years, and at menarche 14.4 (±1.7) years, at first intercourse 17.9 (±2.9) years, and at first pregnancy 19.4 (±3.9) years. Of the 644 participants, 571 (88.7%) had been pregnant previously, 542 (84.2%) had a history of contraceptive use, 272 (42.6%) had a history of STIs, 116 (18.0%) had STIs that had been treated in the past 12 months, 345 (53.6%) were married or cohabiting, 373(57.9%) had attended secondary/high school, 340(53.0%) were unemployed and 513(79.7%) reported one lifetime sexual partner .–19 years old, 54.5% in 20–24 years old and 55.8% in 25–29 years old) as compared to older women (aged between 30 and 49 years). The prevalence declined with increasing age  . Of hr-Hsing age . The higsing age .Based on sampling weights, the overall population burden of hr-HPV among women aged between 15 and 49 years was estimated at 174 046 (95%CI: 153 294–194 797). When stratified by types 46 642 (95%CI: 28 097–65 188) were estimated to have HPV16, 51 959 (95%CI: 45976–57942) HPV18/45, 100 598 (95%CI: 86 384–114 811) HPV31/33/35/52/58, 28 730 (95%CI: 16 672–40 787) HPV51/59 and 41 630 (95%CI: 29 163–54 097) HPV39/56/66/68 . MultiplThe overall HIV weighted prevalence was 42.7% (95%CI: 35.7–46.2). The HIV prevalence increased with age; from 24.5% (95%CI: 12.3–42.8) in the 15–19 year age group, to a peak of 58.1% (95%CI: 40.7–73.7) in the 30–34 year age group, and then declined in the 45–49 year age group  . The oveEducation status, parity, age at first sexual encounter and history of STIs treated in the past 12 months were not associated with hr-HPV after multivariable adjustment.This is the first study assessing prevalence of and risk factors for hr-HPV infection in Swaziland, a country severely affected by the HIV epidemic. The present study shows a high prevalence of hr-HPV infection (46.2%), corresponding to 174,046 women (aged between 15 and 49 years old) with hr-HPV in Swaziland. Differences were observed regarding the HPV types, whereHPV31/33/35/52/58 group types had the highest frequency (26.7%) among the study population. Among the same population, high prevalence of HIV (42.7%) was found. The age-specific prevalence of hr-HPV significantly decreased with age, yet HIV prevalence increased with age and slightly declined in the 45–49 year age group. The hr-HPV/HIV co-infection was 24.4%, with a high peak in the 30s and decline in older groups. The prevalence of multiple hr-HPV infections was statistically significantly higher among HIV-positive women (27.7%) when compared with HIV-negative women. In our sensitivity analysis HPV16 and HPV31/33/35/52/58 types were significantly higher among HIV-positive women when compared to HIV-negative women. Multivariate analysis showed that HIV status was the strongest risk for hr-HPV infection (OR = 4.9). Also the risk of being infected with hr-HPV infection decreased with increasing age (OR = 0.92), and being married (OR = 0.4).The key strength of our study was that primary data and new specimens were used to ascertain the study outcomes. Being the first hr-HPV study done in the country we were able to extrapolate population totals of the 15–49 age group from the 2007–2030 population projections aligned to the 2014 population estimates [Our study shows a high overall prevalence of hr-HPV infection, with the highest among the younger age groups. These findings are consistent with previous studies reporting high HPV prevalence in the population of sub-Saharan Africa, Asia and Latin America –27. A stHPV prevalence is highest among women <25 years old with a rapid fall among women in their 30s and 40s . As highIn relation to screening, the main problem is that if the country decided to do, for example, HPV and treat this group they would be treating 40% of the 30–34 age group and 30% of the 35–44 age group, which is much more than what has been done in other places. In this context the country would definitely require a triage method or a reduction in the number of hr-HPV types included for primary screening. The high frequency of types rarely seen in cancer is particularly important.In this study, HIV weighted prevalence was very high among the study population (42.7%) and there was a high prevalence in the 25–29, 30–34 and 35–39 age groups, . The increase in the prevalence of HIV as age increases has been reported in different surveys , 34. TheStudies have demonstrated unemployment as significant driver for the increased STIs among women and it’s associated with significantly higher odds of STIs , 43. HowA vital clinical and public health consequence of our findings is that HIV was the strongest risk factor associated with hr-HPV infection. HIV-positive women are approximately 5 times more likely to be infected with hr-HPV infection. Additional, overall infection and specifically HPV16 and HPV31/33/35/52/58 were significantly more common among HIV-positive women as compare to HIV-negative women in our study. The strong association between HIV and HPV infection has been demonstrated in several previous studies , 45, 46.We further assessed the interaction between HIV and history of STIs with hr-HPV. Our study found that the presence, absence or unknown of history of STI with HIV did not appear to modify the relationship with hr-HPV infection. To our knowledge no much studies that have assessed the interaction between HIV and history of STIs with hr-HPV. However, there is an evidence that STIs or having had STI 12months before increases the probability of HIV including HPV infection , 50.Factors such as educational status, history of pregnancy, number of pregnancies, age at first sexual encounter, age at menarche, number of sexual partners, contraceptives use, history of STIs and history of STIs treated in these past 12 months were not associated with HPV infection in our study, although in other studies, the factors were strongly associated with among population at risk HPV infection , 51, 52.In conclusion, the prevalence of hr-HPV is high among women of reproductive age in Swaziland. HIV prevalence among reproductive aged women remains high in the country. The study confirms the high HPV/HIV co-infection rate and the high correlation between hr-HPV and HIV infection observed in previous studies. In addition, HPV infection was significantly common among HIV-positive women as compared to HIV-negative women in all age groups. The prevalence of multiple hr-HPV infections was significantly high among HIV-positive women compared to HIV-negative women. Furthermore, the study has provided essential information about the HIV link with HPV infections which may explain the high incidence. This can contribute to policy development and planning of prevention strategies incorporating HPV infection prevention especially among the youth and HIV infected people. Furthermore, our findings support the suggestion of comprehensive screening approaches incorporating hr-HPV testing with the existing screening programme, must be considered with triage modalities other than cytology. Finally, our findings provide epidemiological knowledge about the distribution of hr-HPV. This is crucial to guide the future introduction of prophylactic vaccine in Swaziland.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


A low cost and accurate method for detecting high-risk (HR) human papillomavirus (HPV) is important to permit HPV testing for cervical cancer prevention. We used a commercially available HPV method  which was documented to function accurately in a reduced volume of cervical specimen to determine the most prevalent HPV types and the distribution of HPV infections in over 1795 cancer-free women in Guatemala undergoing primary screening for cervical cancer by cytology.HR-HPV detection was attempted in cervical samples from 1795 cancer-free women receiving Pap smears using the Hybribio™ real-time PCR assay of 13 HR types. The test includes a globin gene internal control. HPV positive samples were sequenced to determine viral type. Age-specific prevalence of HPV was also assessed in the study population.A total of 13% (226/1717) of women tested HPV+, with 78 samples (4.3%) failing to amplify the internal control. The highest prevalence was found in younger women  and older ones . The six most common HR-HPV types among the 148 HPV+ typed were HPV16 (22%), HPV18 (11%), HPV39 (11%), HPV58 (10%), HPV52 (8%), and HPV45 (8%).In this sample of cancer free women in Guatemala, HPV16 was the most prevalent HR type in Guatemala and the age-specific prevalence curve peaked in younger ages. Women in the 30-59-year age groups had a prevalence of HR-HPV of 8%, however, larger studies to better describe the epidemiology of HPV in Guatemala are needed.The online version of this article (10.1186/s12885-018-4438-y) contains supplementary material, which is available to authorized users. Across the world, women living in poverty suffer disproportionally from cervical cancer (CC). In Guatemala, CC is a leading cause of cancer in women  31/100,000) resulting in an estimated 717 deaths) (Globocan 2012). In the Instituto de Cancerología (INCAN), the main adult oncology hospital in Guatemala, over 40% of women diagnosed with malignancies have advanced CC, requiring costly management that often has a poor outcome [There is overwhelming evidence that persistent infection with specific types of HPV is the main cause of CC , 3. HPV In most populations, HPV incidence peaks in women in their late teens or early 20s –9 followThis study sought to assess the performance of a validated low-cost HPV assay  in the setting of a low and middle income country . We deteTo explore the epidemiology of HPV in Guatemala using H13 , a total of 1795 samples were obtained from asymptomatic sexually active women undergoing routine primary screening with Pap smear at hospital-based screening clinics in Guatemala, after obtaining informed written consent. The participants were recruited from women attending cervical cytology screening programs at public hospitals in two urban areas , and were tested only one time. Most women live near the respective hospital and so this is a largely urban sample. A questionnaire on reproductive and socio-demographic characteristics was administered by trained personnel on the Guatemalan subjects . The samThe detection of HPV was performed using a Hybribio Assay that detects the 13 high-risk HPV types by real-time PCR. The performance of the assay was previously evaluated in a clinical validation study . A cell To determine HPV type by sequencing, HPV positive samples were subjected to Touchdown PCR and DNA sequencing Fig. . SamplesP < 0.05 was regarded as statistically significant. We performed analyses of association between HPV infection and age.Statistical analyses on the samples were performed to determine age-specific HPV prevalence, comparing the age groups with the Pearson Chi-square test using GraphPad Prism version 7 for Windows. To evaluate the minimum required assay volume of the H13 test we tested the standard 20 ul and 10ul and determined that a 10ul real-time PCR volume gave equivalent results  at an approximate cost of $11 per test, but many poor and rural areas remain unscreened. A method has been developed with support from donor and non-profit foundations, called careHPV (Qiagen). This system has been rigorously tested in China, India and in pilot programs in other areas  and is uWe sought to establish a method that would be cost effective, and use only equipment available in many molecular biology laboratories. We employed a validated storage buffer, mouthwash containing 15% ethanol, which costs $0.01 per sample with fewer shipping requirements than methanol based buffers . The HybOur study has several limitations that might affect our conclusions. We used a Hybribio assay that detects only 13 HR types. In addition, we attempted to sequence all positive samples to determine type. In a portion of samples (4.3%), we had a failure to detect the IC indicating a failure in sample collection, preservation or storage. Most of these samples were negative using both Hybribio and a touchdown PCR method (data not shown). This could indicate a limitation to using Scope mouthwash as a preservative. We have limited histopathology or other outcome data on the women and have not demonstrated that Hybribio Assay is effective in CC prevention in Guatemala. However, the manufacturer reports data on a comparison with Qiagen (with > 95% agreement with an FDA approved test), the test passed two WHO proficiency trials and has been used in a study of 48,559 women in China .In conclusion, we have used the Hybribio H13 test, an affordable alternative for HPV screening to determine that the high-risk HPV prevalence is 13% in a population of asymptomatic women in Guatemala. The distribution of HPV types is typical of other countries and the highest HPV prevalence is in youngest age groups (< 30). This low-cost approach to detect HPV could be employed in other countries planning to introduce HPV screening to reduce the burden of CC.Additional file 1:Figure S1. Amplification of HPV positive and negative cell line genomic DNA by Touchdown PCR using BS GP5+/6+ primers. A dilution series of an HPV positive cell line (HeLa), HPV negative (HEK293) and HPV-status unknown cell lines were amplified by touchdown PCR with both HPV primer sets and a globin primer pair, and run on 1.5% agarose gels. Figure S2. Touchdown PCR from swab cell lysate, (A): Lane 1.- Lane 4 HPV positive control with input DNA 1 ng, 0.1 ng, 0.01 ng and 0.001 ng; Lane 5. HPV negative control (cell line); Lane 6. no template control. Lane 7 to Lane 16 indicate unknown swab samples. M indicate the DNA ladder marker. Touchdown PCR from HPV positive swabs by Hybribio Assay (B): Lane 1. – Lane 18 are swab cell lysate; Lane 19. HPV positive control (cell line); Lane 20. HPV negative control (cell line); Lane 21. no template control. The samples were amplified with the BSGP5+/6+ primers. (DOCX 2548 kb)


A few studies previously suggested that human papillomavirus (HPV) E6 messenger RNA (mRNA) may exist uniformly in all grades of cervical intraepithelial neoplasia (CIN), whereas the detection rate of E7 mRNA may increase with disease progression from low-grade CIN to invasive carcinoma. The aim of this study was to clarify the different roles of E6 and E7 mRNAs in cervical carcinogenesis. The presence of each E6 and E7 mRNA was analyzed in 171 patients with pathologically-diagnosed CIN or cervical carcinoma. We utilized a RT-PCR assay based on consensus primers which could detect E6 mRNA (full-length E6/E7 transcript) and E7 mRNAs (spliced E6*/E7 transcripts) separately for various HPV types. E7 mRNAs were detected in 6% of CIN1, 12% of CIN2, 24% of CIN3, and 54% of cervical carcinoma. The presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with E6 mRNA or high-risk HPV (HR-HPV) DNA . The presence of both E6 and E7 mRNAs was significantly associated with HPV16/18 DNA but not with HR-HPV DNA (p = 0.0079 and 0.21), while the presence of E6 mRNA was significantly associated with HR-HPV DNA but not with HPV16/18 DNA (p = 0.036 and 0.089). The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity (100% and 19%) for detecting CIN2+ by contrast with the positivity for HR-HPV DNA showing low specificity and high sensitivity (19% and 89%). The positive predictive value for detecting CIN2+ was even higher by the presence of both E6 and E7 mRNAs than by the positivity for HR-HPV DNA (100% vs. 91%). In 31 patients followed up for CIN1-2, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in contrast with E6 mRNA, HR-HPV DNA, or HPV16/18 DNA . Our findings support previous studies according to which E7 mRNA is more closely involved in cervical carcinogenesis than E6 mRNA. Moreover, the separate analysis of E6 and E7 mRNAs may be more useful than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our findings. Cervical cancer is the fourth most commonly diagnosed cancer and the fourth most common cause of cancer death in women worldwide . Each yeThe current study comprised two parts: a cross-sectional study of analyzing E6/E7 mRNAs in cervical specimens from patients with CIN or invasive cervical carcinoma and an adjunctive longitudinal study of following up patients with CIN1-2 . Women wGenomic DNA was extracted using SepaGene kit  according to the manufacturer’s instruction. HPV genotyping was performed by L1-PCR and RFLP analyses as described previously  or at a ACC GAA AAC GGT TGA ACC GAA AAC GGT and GAG CTG TCG CTT AAT TGC TC; β2-microglobulin, TGT CTT TCA GCA AGG ACT GG and GAT GCT GCT TAC ATG TCT CG.Total RNA extraction and DNase treatment were performed as described previously . RT-PCR Differences in proportions were evaluated by the Fisher’s exact test. Diagnostic indices of sensitivities, specificities, positive predictive values, and negative predictive values with 95% confidence intervals were calculated for detecting CIN2+, CIN3+, and invasive cervical cancer. Disease progression of CIN1-2 was examined as a surrogate by the Kaplan-Meier method calculating the intervals from E6/E7 sample collection until patients showed upgraded results of Pap test compared with the cytology at E6/E7 sample collection or they were censored, and the difference was statistically evaluated by the log-rank test.We first analyzed the E6 and E7 mRNA expression patterns in human cervical cancer cell-lines CaSki and HeLa by RT-PCR , and conSubsequently we analyzed the E6/E7 mRNA expressions in liquid-based cytology samples from 171 patients. Next, we examined diagnostic accuracies for cervical neoplastic diseases by E6/E7 mRNA analyses. For detecting CIN2+, the presence of both E6 and E7 mRNAs showed high specificity and low sensitivity  –18, 23. In conclusion, our separate analyses of E6/E7 mRNAs demonstrated here that the presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with positive E6 mRNA or HR-HPV DNA. Besides, the presence of both E6 and E7 mRNAs was significantly associated with the positivity for HPV16/18 DNA, while the presence of E6 mRNA was significantly associated with the positivity for HR-HPV DNA. The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity for detecting CIN2+ by contrast with the positivity for HR-HPV DNA. Furthermore, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in the patients followed-up for CIN1-2 by contrast with positive E6 mRNA, HR-HPV DNA, or HPV16/18 DNA. Our findings suggest a closer involvement of E7 mRNAs than E6 mRNA in cervical carcinogenesis. Moreover, the separate analysis of E6 and E7 mRNAs may be a more useful tool than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our proposal.


Gardasil®, a quadrivalent vaccine targeting low-risk  and high-risk  human papillomaviruses (HPV), has been offered to 11–14 year-old schoolgirls in Switzerland since 2008. To evaluate its success and its potential impact on cervical cancer screening, HPV genotypes were examined in 18-year-old girls five years later (sub-study 1) and in outpatients participating to cervical cancer screening before and after vaccine implementation (sub-study 2).Chlamydia trachomatis PCR. Personal data were evaluated by univariable and multivariable statistics. In sub-study 2, the proportion of the vaccine-type HPV among anogenital HPV was examined with archived genotyping data of 8039 outpatients participating to cervical cancer screening from 1999 till 2015. The yearly evolution of this proportion was evaluated by segmented logistic regression.For sub-study 1, 3726 females aged 18 in 2013 were invited to fill a questionnaire on personal demographics and HPV risk factors and to provide a self-collected cervicovaginal sample for HPV genotyping and Chlamydia trachomatis (4.6%) and demographics confirmed that the subjects were representative of sexually-active Swiss young women. Vaccine (five-year coverage: 77.5%) was preferentially accepted by contraceptive-pill users (P = 0.001) and samples were mainly provided by sexually-active subjects (P < 0.001). The proportion (4%) of the vaccine-type HPV in this population was lower than in sub-study 2 outpatients  in the pre-vaccine era (25.7%). The proportion of the high-risk vaccine-type HPV decreased significantly  in the outpatients during the post-vaccine era, yet this decrease was restricted to those aged less than 26 years .690 (18.5%) women participated to sub-study 1 and 327 (8.8%) provided a self-collected sample. Prevalence of The low proportion of vaccine-type HPV in 18-year-old females and its rapid decrease in young women participating to cervical cancer screening extend the success of HPV vaccination to Switzerland. Our data suggest that cervical cancer screening is now entering a stage of reduced proportion of HPV16 and/or 18 in samples reported positive by cytology. In view of the high likelihood of reduced clinical specificity of cytology, primary screening modalities involving HPV testing and cytology should now be re-evaluated in Switzerland.The online version of this article (10.1186/s12879-017-2867-x) contains supplementary material, which is available to authorized users. Infection by high-risk (HR) anogenital human papillomaviruses (HPV) is a necessary cause of cervical cancer (CC) . HPV16 aVaccination against HR HPV targeting girls prior to sexual debut is expected to reduce CC burden, even in those who are not vaccinated thanks to herd immunity . Three HData obtained from large cohorts of women several years after implementation of Cervarix® , 10 or GGardasil® has been used in Switzerland and offered to schoolgirls aged 11–14 since 2008, yet its impact is not known in Switzerland. The aims of our work were to examine the proportion of vaccine-type HPV among anogenital HPV in sexually-active 18-year-old Swiss females five years after vaccine implementation, and the temporal impact of Gardasil® on this proportion in outpatients participating to CC screening from 1999 to 2015.This is an observational study divided into two sub-studies aimed at evaluating the distribution of HPV genotypes within two sets of individuals in the context of Gardasil® implementation in Switzerland.n = 3726) invited through an independent marketing organization  to participate in our study in 2013. BVA gets the residents’ records from all residents’ registration offices of the canton of Vaud. Sub-study-1 individuals corresponded to 82.2% of the 18-year-old women officially registered in the canton of Vaud as of December 31st 2013  since residents can oppose their being included in the BVA database. Women of that age had all been targeted by the vaccination campaign initiated in 2008 when they were 13 years old, and could have accepted the vaccine within this five-year period because of catch-up vaccination that was offered to older girls aged up to 19. This group served to establish the proportion of vaccine-type HPV among anogenital-HPV in sexually-active young women, up to five years after having been offered HPV vaccination. It served also to establish the demographics and uptake of vaccination in young women in our canton to assess whether our findings can be generalized to Switzerland. They were sent a questionnaire by BVA to address (1) personal data, (2) preventive aspects pertaining to HPV infection and cervical cancer and (3) risk factors of HPV acquisition and of sexually transmitted infections (STI)  testing, in which case women were sent a self-collection device to their home address that was encoded by BVA to ensure anonymity. CT prevalence was determined to evaluate the risk of STI independently of HPV vaccine acceptance because it had previously been assessed in sexually-active young women of the canton of Vaud to be used here as a comparator [n = 773′000 inhabitants) is one of 26 cantons of Switzerland (n = 8′327’000) and ranks third in terms of population after Zürich (n = 1′466’000) and Bern (n = 1′017’000). No financial incentive was used to encourage participation in the study. However free CT consultation was offered to those who were CT-positive provided they accepted to be contacted.Sub-study 1 comprised 18-year-old females (n = 8039) who visited the clinics of the Gynaecology and Obstetrics Service of our hospital centre and affiliated family planning centres from March 1999 to December 2015. Sub-study 2 served to assess the evolution of the proportion of vaccine-type HPV encountered in screened women before (1999–2007) and after the introduction of HPV vaccination until 2015. HPV-genotype distribution was retrospectively assessed with archived genotyping data from a total of 12′706 samples submitted to our laboratory as an adjunct to abnormal cytology (n = 11′576) or as follow-up  after Papanicolaou (PAP) testing. Abnormal cytology were atypical cells of undetermined significance , low grade squamous intraepithelial lesions , and high grade squamous intraepithelial lesions . HPV testing of FUP samples was requested despite normal cytology for patients who presented previously with persistent ASCUS or to monitor treatment.Sub-study 2 comprised all outpatients . Negative controls were tested in parallel throughout the procedures to monitor contaminations.Cervicovaginal sample (5 mL) was self-collected by sub-study-1 individuals with the Delphi Screener® device  and transferred in a coded tube prior to sending to our laboratory within 24 h. DNA from 200 μL cell suspension was isolated with the MagNaPure 96 Total Nucleic Acids kit according to the manufacturer  and eluted in 100 μL elution buffer. Five μL was used for time PCR  and for time PCR . HPV- orDNA extraction and HPV genotyping (PGMY-CHUV assay) for sub-study-2 individuals have been described , 17.CT infections. Multivariable logistic regression was used to investigate factors associated with vaccine acceptance and with self-sampling acceptance.Sub-study-1 questionnaires were reviewed to identify inconsistent responses. Together with empty fields, they were considered non informative (NI) and excluded from statistical analysis  to chart the yearly proportion of the vaccine-type HPV among anogenital HPV. This proportion was expected to diminish in the youngest outpatients some year after vaccine introduction in 2008, and its evolution was analysed by segmented logistic regression according to the following model: logit(p) = b0 + b1 * year + b2 * max, where p is the proportion of the vaccine HPV types among anogenital HPV and A is the breakpoint year at which the regression coefficient changed. The model before the breakpoint year A is logit(p) = b0 + b1 * year and the model after the breakpoint year A is logit(p) = (b0 − b2 * A) + (b1 + b2) * year. Hence the odds ratio associated with the yearly evolution of p before the breakpoint is exp(b1) while the odds ratio after the breakpoint is exp(b1 + b2) = exp(b1) * exp(b2). The value exp(b2) is the multiplicative coefficient at the breakpoint; it is a measure of the change in the odds ratio. A coefficient significantly different from 1 is indicative of a significant variation in the yearly evolution of p.The age limit of 26 was based on the assumption that the majority of vaccinated women would be younger than 26, seven years after vaccine implementation, taking into account catch-up vaccination offered to women aged up to 19. Genotypes were stratified by year of sample collection and patient’s age. For each patient, only the first occurrence of a genotype was considered to avoid overrepresentation of persistent viruses and to enrich the dataset in incident infections. Analysis was restricted to the 26 anogenital HPV  shared between PGMY-CHUV and Anyplex™ II HPV28 to facilitate comparison with the genotyping data of the 18-year-old sub-study-1 subjects. Version 1 of PGMY-CHUV was replaced mid 2013 by version 2 to improve detection of HPV68a . HPV68a P-values were double-sided. Raw data and R script are available as Additional files Statistical analyses were performed with R version 3.2.3  and GrapP = 0.001) and being in apprenticeship as opposed to the other two working status (student or other)  were associated with a higher odds ratio by multivariable analysis  responded to the questionnaire  of the 690 subjects had an intercourse at least once in the previous five years , two to five  and more than five . Usage of condom was systematic for 115 subjects (20.5%). The majority of subjects had not been treated for STI (>95%), nine (1.3%) reported suffering from an immune-related disease and 203 reported being smokers (29.4%).Most  and “being smoker” (P = 0.02) by multivariable analysis  by multivariable analysis  subjects accepted self-sampling for HPV and e P = 0.0 by multiCT. The proportion of subjects infected by any type of HPV was 29.5%  and did not differ significantly with the vaccinal status of the subjects .Of the 327 self-collected samples, 324 were informative for HPV and 327 for P = 0.012) with a higher number of sex partners (P < 0.001) (Table P = 0.03), having been treated for an STI (P = 0.001), being examined with PAP (P = 0.001), and working status being neither a student nor an apprentice (P < 0.001) were also significantly associated with HPV positivity in contrast to tobacco smoking (P = 0.236). The working status was also associated with CT positivity . The other univariable analyses for CT acquisition were however inconclusive because of the low number of positive cases.Factors associated with HPV acquisition were being sexually active (1) Table . Condom n = 9) was 4% (9/217), with HPV16 (n = 6) ranking 14th, HPV6 (n = 3) 20th, HPV11 and HPV18 (n = 0) 23rd.The distribution of the 217 genotype-sample combinations in 18-year-old sub-study-1 subjects in 2013 is graphically displayed according to their vaccination status in Fig. n = 3619) observed before vaccine implementation (1999–2007) in the sub-study-2 outpatients were distributed by year and patients’ age , and ranked by decreasing occurrences within the youngest age group   was 25.7%, with HPV16 (n = 212) ranking first, HPV6 (n = 74) fifth together with HPV58, HPV18 (n = 64) 10th, and HPV11 (n = 19) 21st. Restricting the outpatients’ population to the youngest age (<26) with normal cytology  gave a similar proportion (25%) (n = 14 out of 56 genotype-sample combinations); HPV16 (n = 7) ranked first (data not shown). To test whether this proportion decreased after vaccine implementation, as suggested by the low proportion observed in the sub-study-1 subjects, the yearly proportion of the four vaccine-type HPV was calculated from 1999 to 2015 for each of the three age groups of outpatients, and its evolution examined by segmented logistic regression. Model adjustment was made to evaluate the year at which the regression coefficient of the model changed   in the post-vaccine era to 11.3% (95% CI: 10.2%–12.5%). Restricting the vaccine genotypes to HPV16 and HPV18, excluding HPV6 and HPV11 from the calculations, gave similar results; average yearly proportions of 21.0% (95% CI: 16.6%–25.3%) compared with 8.7% (95% CI: 6.7%–10.8%), 59% reduction (P = 0.0048).The HPV genotype-sample combinations n = 369 observeion 1999–007 in thion 1999–007 in thCT infections, consistent with HPV vaccination not influencing sexual behaviour as reported by others in England [CT prevalence rate (4.6 ± 1.2%) was similar (5.5 ± 0.9%) to that of a previous survey of sexually-active women aged <25 in the same area of Switzerland [This study is the first to investigate the impact of Gardasil® after its introduction in 2008 in Switzerland. We first assessed vaccination coverage and demographics as well as HPV genotypes in an 18-year-old women population targeted by the vaccine five years earlier in the canton of Vaud. The participation rate was low with 18.5% returned questionnaires and 8.8% self-collected samples. The low participation rate is a major weakness of this sub-study that necessitated to verify the representativeness of the participants. Reassuringly, our sub-study population was comparable to the Swiss women population of similar age based on risk factors for HPV such as hormonal contraception and use of condoms   and on d England  and in C England . In additzerland , consistIn the canton of Vaud, 69.5% of the schoolgirls aged 14 have been vaccinated (≥1 dose) against HPV during the 2010–2011 school year . The vacThe percentages of women who systematically used condoms 20.5%) and contraceptive pills (64%) are similar to those of young women in a stable relationship  (SMASH-02 report) .5% and c–26. In aA low percentage of returned samples (12%) was reported in a German study assessing HPV prevalence in women aged 20–25 years using the Delphi-Screener® . The eveGlobal HPV prevalence increased with number of sex partners and other variables linked to undergoing sexual activity, as well as with lower degree of education. These data confirm previously published data on identified risk factors for HPV infection . Four paIn view of the vaccination coverage well above 50%, the low proportion (4%) of the vaccine-type HPV was expected in our 18 year-old subject population compared with that (25%) in the youngest sub-study-2 outpatients during the pre-vaccination era according to a recent meta-analysis of the impact of HPV vaccination programmes and herd effect . Herd imThe lower proportion of the vaccine-type HPV in sub-study-1 subjects may have resulted from using different means of sample collection (Delphi screener self-collection vs. PAP smear) and HPV genotyping assays (Anyplex II™ HPV28 vs. PGMY-CHUV), hence for this reason was not tested statistically. The strength of our study however relied on our ability to assess without bias the evolution of the yearly proportion of the vaccine-type HPV with the sub-study-2 outpatients’ data because the cervical cancer screening algorithm, sample collection and HPV genotyping method remained identical over the years (1999–2015). The youngest sub-study-2 outpatients (<26) were most likely enriched in women to whom the vaccine was offered since 2008, while the older women 26–30 and >30) were gradually less likely to have received the vaccine or have benefitted from herd immunity. Consistent with this and with the known clearance time of HPV (90% within two years), a time wise significant reduction of the yearly proportion of the four vaccine-type HPV was observed for our outpatients aged <26. The 56% reduction of the four vaccine-type HPV and the 59% reduction of HPV16 and HPV18 achieved as of five years after vaccine implementation is reminiscent of the numbers published in the US  and in t and >30 The low proportion of vaccine-type HPV in 18-year-old females and its rapid decrease in young women participating to cervical cancer screening extend the success of HPV vaccination to Switzerland. Our data suggest that cervical cancer screening is now entering a stage of declining prevalence of precursor lesions associated with the two most carcinogenic HPV as proposed earlier by a modelisation study . There iAdditional file 1:Sub-study-1 flowchart and questionnaire results. (PDF 418 kb)Additional file 2:Incidence of HPV genotypes by age and age categories observed in sub-study-2 outpatients participating to cervical cancer screening between 1999 and 2015. This file should be renamed “HPV_1999–2015_evolution_raw_data.xlsx” to be used by the R script provided as Additional file Additional file 3:R script used to graph the yearly evolution of the vaccine-type HPV proportion recorded in the Additional file Additional file 4:Factors associated with self-sampling acceptance and providing a sample by sub-study-1 participants. (DOCX 16 kb)Additional file 5:Segmented logistic regression analysis of the evolution of the yearly proportion of vaccine-type HPV (vHPV) among all HPV in sub-study-2 outpatients. (DOCX 27 kb)


Human papillomavirus (HPV) vaccination rates fall far short of Healthy People 2020 objectives. A leading reason is that clinicians do not recommend the vaccine consistently and strongly to girls and boys in the age group recommended for vaccination. Although Web-based HPV vaccine educational interventions for clinicians have been created to promote vaccination recommendations, rigorous evaluations of these interventions have not been conducted. Such evaluations are important to maximize the efficacy of educational interventions in promoting clinician recommendations for HPV vaccination.The objectives of our study were (1) to expand previous research by systematically identifying HPV vaccine Web-based educational interventions developed for clinicians and (2) to evaluate the quality of these Web-based educational interventions as defined by access, content, design, user evaluation, interactivity, and use of theory or models to create the interventions.Current HPV vaccine Web-based educational interventions were identified from general search engines , continuing medical education search engines, health department websites, and professional organization websites. Web-based educational interventions were included if they were created for clinicians , delivered information about the HPV vaccine and how to increase vaccination rates, and provided continuing education credits. The interventions’ content and usability were analyzed using 6 key indicators: access, content, design, evaluation, interactivity, and use of theory or models.A total of 21 interventions were identified, out of which 7 (33%) were webinars, 7 (33%) were videos or lectures, and 7 (33%) were other . Of the 21 interventions, 17 (81%) identified the purpose of the intervention, 12 (57%) provided the date that the information had been updated (7 of these were updated within the last 6 months), 14 (67%) provided the participants with the opportunity to provide feedback on the intervention, and 5 (24%) provided an interactive component. None of the educational interventions explicitly stated that a theory or model was used to develop the intervention.This analysis demonstrates that a substantial proportion of Web-based HPV vaccine educational interventions has not been developed using established health education and design principles. Interventions designed using these principles may increase strong and consistent HPV vaccination recommendations by clinicians. The human papillomavirus (HPV) causes almost all cervical cancers, 50% of vulvar cancers, 65% of vaginal cancers, 90% of anal cancers, and 35% of penile cancers . Recent Missed opportunities for clinicians to recommend and administer the vaccine, as well as a lack of strong and consistent recommendations by clinicians who do recommend the vaccine, are primary reasons for low HPV vaccination rates in the United States . FactorsTo improve clinicians’ HPV vaccine recommendations, numerous Web-based HPV vaccine educational interventions for clinicians have been created. Web-based educational interventions have become a popular delivery method for health care professionals to obtain continuing education (CE) , as Web-Web-based CE interventions created using strategic health communication design principles—communicating effectively with intended users and taking into account audience factors such as culture, race, ethnicity, language, access, functional needs , and expectations ,36—are lEvaluation of the leading Web-based HPV vaccine educational interventions is important in guiding efforts to promote clinician recommendations for the HPV vaccine ,46. EvalRigorous evaluation is a central component of developing successful health education interventions ,52 and eWe systematically identified current HPV vaccine Web-based educational interventions by examining general search engines , continuing medical education search engines , health department websites, and professional organization websites. The following search terms and variations of search terms were used within each of the search engines and websites: “clinicians,” “healthcare providers,” “HPV webinars,” “HPV vaccination webinars,” “HPV online education,” and “HPV continuing medical education.” Web-based educational interventions were included if they were (1) created for clinicians ; (2) delivered information about the HPV vaccine and how to increase vaccination rates; and (3) provided continuing education credits. Interventions were excluded if they were created for patients, parents, or adolescents, or if they focused on vaccines other than the HPV vaccine. We conducted the search from April 2016 to August 2017 and identified a total of 178 interventions. Of these, 21 interventions met all 3 research criteria for this study.The study team developed a quality evaluation framework based on criteria established in the literature for evaluating health-related websites -56. The Internal reliability of the subindicators was calculated using the Krippendorff’s alpha coefficient  . This coTo examine access of the educational interventions ,54, 2 suContent was evaluated using 7 subindicators ,55: idenThe design components of the interventions were evaluated by layout and graphics . The layEvaluation was assessed using 3 subindicators -55: whetThe indicator for interactivity included 2 subindicators ,55. The The theory and models indicator was assessed by examining whether there was an explicit statement that a theory or model was used to develop the intervention (score ranging from 0-1) -56. If aOnce interrater reliability was established for the evaluation tool with all indicator scores above .80, 2 independent coders  utilized the tool to evaluate the educational interventions identified. One of the coders, Mr Bishop is a health education doctoral student with expertise in sexuality education and evaluated the first 11 interventions. The other coder, Ms McDonald is a health education doctoral student with expertise in school health and evaluated the remaining 10 interventions. Frequency distributions were calculated for each of the subindicators to determine specific strength and weaknesses of the interventions. Because this study assessed access, content, and design aspects of interventions and did not include human subjects; this study is considered nonhuman subjects research and hence institutional review board approval was not required.A total of 21 interventions were identified out of which, 7 (33%) were webinars; 7 (33%) documentary, TV series, or videos; and 7 (33%) other . Medscape, a health information website, provided 10 (48%) interventions, Continuing Nursing Education University provided 2 (10%), CDC provided 3 (14%), professional organizations  provided 3 (14%), nonprofit organizations  provided 1 (5%), a federally-authorized regional organization  provided 1 (5%), and a university  provided 1 (5%). On the basis of the evaluation indicators, 13 (62%) interventions required registration, but all interventions were accessible without cost to the participant . Additionally, 17 (81%) educational interventions identified the purpose of the intervention , and 12 (57%) provided a date when the information had been updated: 7 (33%) were updated in the last 6 months . In assessing presentation of clear references, 18 (86%) interventions provided references , and 8 (38%) provided links to other sources . Most interventions  provided reliable references or sources . The references or sources included information from the CDC (n=16), published peer-reviewed literature (n=16), American Cancer Society (n=5), National Cancer Institute (n=4), Institutes of Medicine (n=4), WHO (n=2), and American Academy of Pediatrics (n=1). Of the 21 interventions, 14 (67%) had a statement of disclosures from the authors, sponsors, or developers .For the design subindicators, 2 interventions were documentary or videos that did not include text, and therefore, were not included in the total sample for these subindicators. All interventions (n=19) included easy-to-read font size, font style, color, and line spacing . For the subindicator “Graphics were clearly labeled,” only 13 interventions included graphics; thus, the sample for this subindicator is 13 interventions. Out of the 13 interventions, there were 10 (77%) interventions with a minimum of 75% of graphics labeled, 2 (15%) with a minimum of 50% of graphics labeled, and 1 (8%) intervention with a minimum of 25% of graphics labeled. No intervention had 0% of graphics labeled .Of the 21 interventions, 17 (81%) included an evaluation for participant outcomes: 17 (81%) assessed HPV and HPV vaccine knowledge, and none assessed attitudes toward HPV and the HPV vaccine. Furthermore, 14 (67%) interventions provided the participants the opportunity to evaluate or provide feedback . Five (24%) interventions included an interactive component . The most commonly used interactive component was a polling or knowledge check activity (n=4). No educational intervention explicitly stated a theory or model that was used to develop the intervention. This study provides a systematic, evidence-based assessment of the strengths and weaknesses of current HPV vaccine Web-based educational interventions. Strengths of the assessed Web-based educational interventions include: (1) being developed by experts in the field; (2) providing reliable references or sources; (3) providing clinicians with access to CEs for no cost; (4) following basic design principles with easy-to-read fonts, colors, and graphics; and (5) consistently providing evaluation opportunities for participant knowledge outcomes. Weaknesses of the educational interventions included lack of: (1) evaluation of outcomes including participants’ attitudes about HPV vaccination, intention to recommended vaccination, and recommendation of behaviors; (2) theory-based interventions; (3) opportunity for participants to provide feedback or evaluation of the intervention; (4) links to other sources or resources; and (5) interactivity throughout the intervention.HPV vaccination rates are well below the Healthy People 2020 objective , and cliWe found that none of the interventions examined included a statement that a theory was used to create the intervention. To improve outcome behaviors and increase clinician HPV vaccine recommendation behaviors, intervention developers should utilize science and evidence that supports effective medical education and behavior change . TheorieNone of the Web-based educational interventions included in this study evaluated HPV attitudes, intention to recommend vaccination, or actual recommendation behavior. This is concerning given that clinician attitudes are an important predictor of vaccine recommendations. Clinicians have reported concerns regarding HPV vaccine safety -70, a laAlthough face-to-face educational interventions have shown to improve clinicians’ willingness to provide immunizations and routinely screen immunization records at visits , evaluatWhile this study provides innovative insight into the quality of Web-based HPV vaccine educational interventions created for clinicians, there are limitations that should be considered. First, only Web-based educational interventions were evaluated, and these results cannot be generalized to other types of interventions such as face-to-face lectures, grand rounds, or seminars. There would be substantial benefit to conducting evaluations of face-to-face lectures and seminar materials to assess all venues of continuing education for clinicians regarding the HPV vaccine. Second, this quality evaluation did not assess participants’ experience of the intervention and therefore, cannot identify every area for improvement. Data were not collected from participants themselves regarding usability: this study identified only 7 indicators of usability. Third, this study was a quality evaluation and did not evaluate participant outcomes  after completing the intervention. Fourth, the evaluation was conducted using only the materials that were accessible at the time of the study, and there is the potential that a component  was not included in this evaluation. Despite these limitations, these findings provide valuable information for those who develop Web-based continuing education interventions regarding HPV vaccines, by providing a quantitative approach to identifying the design and usability strengths and weaknesses of HPV vaccine Web-based educational interventions.The data resulting from this study have the potential to help shift current research practice paradigms. The findings suggest that those who develop Web-based educational interventions to promote HPV vaccine recommendations utilize design science principles, a powerful approach and process that includes participatory action research to iteratively develop and evaluate health education interventions . AdditioThe quality evaluation of these interventions demonstrated that Web-based interventions were based on reliable sources, developed by experts, and were created with critical design aspects . However, there were limited outcome evaluations for users measuring attitudes, intentions, or behaviors, and lack of user interactivity. Results from this study suggest best practices for designing, refining, and implementing Web-based interventions to promote HPV vaccination within the clinician population.


The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis. Tumor progression involves well characterized cascades of events that enable metastasis formation. Members of the tetraspanin or transmembrane-4-superfamily (TM4SF) have been described as both, metastasis suppressors or promoters, due to their multiple functions in cancer cell biological processes .in vitro cancer cell migration/invasion and suppressed cancer metastasis in animal models [By genetic screening, KAI1 , one tetraspanin particularly relevant to tumor metastasis -7. KAI1 l models -24.So far, for KAI1, no intrinsic catalytic activity has been documented. Its functions rather target the regulation of membrane organization by its association with and lateral positioning of other membrane proteins within tetraspanin-enriched microdomains (TEM). Among these interaction partners are other tetraspanins, cell adhesion molecules, growth factor receptors, and G-protein-coupled receptors which are implicated in the regulation of a variety of cellular events, including cell signaling, transcription, cell adhesion, migration, survival, endo- and exocytosis, and cell differentiation , 24-26.Cellular activities of KAI1 are most probably mediated by its molecular crosstalk with integrin cell adhesion and signaling receptors, their expression levels, compartmentalization, internalization, and recycling , 3. So fMost interestingly, in metastatic gastric cancer, a splice variant of KAI1 (KAI1-SP) had been detected which lacks the complete exon 7 , 34. In in vitro tumor biological effects of KAI1-WT vs. KAI1-SP, human breast cancer cell lines MDA-MB 231 and MDA-MB-435, respectively, were stably transfected to overexpress either of the two KAI1 variants [Materials & Methods  Figure .Nested PCR primer pairs generating a 255 bp and a 171 bp fragments for KAI1-WT and KAI1-SP, respectively, were used to screen a small series of eight human breast cancer tissues for the presence of endogenous mRNA for KAI1-WT and KAI1-SP Figure , lane 9.We next wondered whether stable cellular expression of either of the two KAI1 proteins affects αvß3 expression levels. Indeed, by immunocytochemical staining and FACS analysis of MDA-MB-231 cells, we found that both KAI1 proteins decreased αvß3 expression levels, KAI1-WT by up to 50% and KAI1-SP by up to 30% when compared to vector-transfectants and wt cells. In contrast, in MDA-MB-435 cells, both KAI1 proteins induced αvß3 levels, KAI1-WT by up to 2.5-fold and KAI1-SP by up to 6.5-fold Figure  and 2B.Since functional crosstalk of membrane interaction partners may be facilitated by their close neighbourhood within the cell membrane, we performed co-localization studies by immunocytochemical double staining. On MDA-MB-231 cells, KAI1-WT significantly colocalized with αvß3, which was, however, not the case for KAI1-SP. The same was true for MDA-MB-435 cells, however, not as apparent as in MDA-MB-231 cells Figure .In the presence of vitronectin (VN) as underlying adhesive matrix, both wt breast cancer cell lines enhanced adhesion. In MDA-MB-231 cells, KAI1-WT provoked increased cell adhesion which was further enhanced by VN (up to 6-fold upon setting the adhesive strength of wt cells adherent to uncoated cell culture dishes to “1”). KAI1-SP strengthened the adhesive cell capacity even more: on uncoated cell culture dishes by up to 4.8-fold, onto VN by up to 8.3-fold. In MDA-MB-435 cells, which in the wt form harbor significantly lower endogenous αvß3 levels than MDA-MB-231 wt cells, the differential effects of KAI1-WT and KAI1-SP were not as obvious. KAI1-WT provoked here also increased cell adhesion, however, with no significant differences in the presence of VN, reaching an adhesive strength onto VN comparable to that of wt cells. In MDA-MB-435 KAI1-SP transfectants, concomitant with its induction of αvß3 expression Figure , the celIn addition, we monitored the adhesion profiles of the two wt breast cancer cell lines to the extracellular matrix (ECM) proteins VN, fibronectin (FN), collagen type I (Coll-I), or collagen type IV (Coll-IV) Figure . In bothMaterials & Methods. The closure rates of the wound gaps shown in Figure 2/h for MDA-MB-231 KAI1-WT transfectants, 6.7 μm2/h for MDA-MB-231 KAI1-SP transfectants as well as 6.9 μm2/h for MDA-MB-435 KAI1-WT- or MDA-MB-435 KAI1-SP transfectants, all cells being adherent to VN  and all ECM protein coatings tested here, both, KAI1-WT- and KAI1-SP-transfected MDA-MB-435 cells demonstrated higher motility than MDA-MB-231 cell transfectants Figure . To our nt to VN . For MDAnt to VN . The samnt to VN . Furthernt to VN . We obseImpact of KAI1-WT/KAI1-SP on the expression and activation of the focal adhesion kinaseMaterials & Methods. In addition, we analysed FAK and p-FAK expression, respectively, by Western blot analysis thereby confirming the regulatory effects of KAI1-WT and KAI1-SP on FAK activation as detected by immunocytochemical analysis , concomitant with strong focal adhesion formation. Total FAK expression levels were not altered in any of the KAI1 transfectants when compared to wt cells Figure . Althougs Figure .Cells were cultivated over 120 h and cell numbers counted at distinct time intervals. Cell numbers are given as `n-fold´ by setting the cell numbers of each cell transfectant at time point 3 h - when cell adhesion was fully established - to “1”. In MDA-MB-231 cells, KAI1-SP increased cell numbers by up to 8.1-fold, KAI1-WT by up to 3-fold, vector transfectants by up to 4.5-fold, which was comparable to wt cells (up to 4.3-fold). In MDA-MB-435 cells, we also noticed enhanced cell proliferation rates by KAI1-SP expression (up to 5.9-fold) as compared to KAI1-WT (2.9-fold), vector transfectants (3.8-fold), and wt cells (up to 4.1-fold) Figure .Materials & Methods. Moreover, we analysed EGF-R expression as a function of either KAI1-WT or KAI1-SP in human breast cancer cells by Western blot analysis and were able to confirm the results obtained by immunocytochemical analysis . These findings strengthen the idea of KAI1 as an important trafficking molecule. Because KAI1-SP provoked increases of cell proliferative activity and EGF-R levels, we wondered whether cell proliferation might be even stronger enhanced by the exogenous addition of EGF. Indeed, after 72 h of cell cultivation, we monitored an additional proliferative response to EGF in KAI1-SP expressers, whereas proliferation rates upon KAI1-WT expression were not further inducible by EGF Figure .The mechanisms and signaling events underlying the role of KAI1 as a metastasis suppressor are not fully understood yet. Previously it had been reported that KAI1-WT downregulated the activation of the Src kinase , which aTumor suppressors markedly affect tumor cell adhesion, migration/invasion, dissemination, proliferation, and survival thereby controlling cancer metastasis. Consequently, when tumor suppressors are lost, cancer progression is significantly facilitated . KAI1 waIn the present study, we explored differential effects exerted by the two KAI1 protein variants which impact on tumor biological events. To this end, we transfected human MDA-MB-231- and 435 breast cancer cells to express elevated levels of KAI1-WT or KAI1-SP. Because we previously identified for the first time integrin αvß3 as a novel interaction partner of KAI1 in human ovarian cancer cells, besides ß1-integrins, we studied KAI1-WT/KAI1-SP effects on αvß3 expression levels. In MDA-MB-231 cells, we demonstrated that both, KAI1-WT and KAI1-SP decreased αvß3 expression, whereas in MDA-MB-435 cells, KAI1-WT slightly increased αvß3 expression. These changes in αvß3 levels by KAI1-WT are contrasting to our previous findings in human ovarian cancer cells  as well Regarding the cell adhesive capacity, MDA-MB-231 KAI1-SP cell transfectants, with a slightly decreased αvß3 expression exhibited best adhesion; KAI1-WT transfectants, although exerting slightly reduced αvß3 levels, still improved cell adhesion when compared to wt cells which already harbor high endogenous αvß3 levels. These findings imply that here also other integrins/cell adhesion molecules might be affected by KAI1 proteins, since, in fact, several other integrins, e.g. of the ß1-subfamily, had been reported as KAI1 interaction partners . In MDA-Because a fine-tuned and appropriate cell type-specific adhesive strength is indispensable for an efficient cell migratory capacity, we next investigated whether KAI1-WT and KAI1-SP also exert here differential effects. In MDA-MB-231 KAI1-SP cell transfectants, we disclosed enhanced cellular motility as compared to KAI1-WT expressers. In wound scratch assays, the progress of wound gap closure was most obvious in the presence of VN. Interestingly, in MDA-MB-435 breast cancer cells, at least over a time frame of 11 h, neither an obvious difference in wound gap closure was visible between KAI1-WT and KAI1-SP transfectants, nor was the closure significantly affected by the underlying ECM proteins. In line with the observed accelerated wound gap closure in the presence of KAI1-SP in MDA-MB-231 cells, we also documented higher cell migration velocities along the wound scratch borders as compared to cells located within denser cell monolayer areas and/or those cells being attached more distant from the wound gap. Correspondingly, for KAI1-WT transfectants, we observed and calculated significantly lower velocities for cells located close to the edges of the wound gaps. A similar correlation was observed for the average motion: in the case of MDA-MB-231 cells, in the full assay time period, the average motion amplitude of KAI1-SP cell transfectants was much higher than that of KAI1-WT expressers. Differently, for MDA-MB-435 cells, we observed roughly the same average motion for all cells/ECM proteins studied. This average motion for all MDA-MB-435 cell transfectants/ECM coatings was even slightly higher than the highest average motion of KAI1-SP in MDA-MB-231 cells. Others also reported from human breast cancer cells and various other cancer cell entities, that cell adhesion and migration/invasion prominently decreased by elevated KAI1-WT levels , 46-48. The signaling events contributing to the tumor suppressive actions of KAI1 are yet not fully unraveled. Several mechanisms have been proposed so far, including KAI1-dependent control over integrin-mediated signal transduction and biological events arising thereof . Indeed,in vivo metastasis models, inoculation of KAI1-WT-transfected breast cancer cells into the mammary fat pads or tail veins of athymic nude mice led to a significant suppression of their metastatic potential and tumor burden [in vivo tumor growth after subcutaneous injection of KAI1-transfected cells into BALB/c mice, all of the parental and KAI1-SP-transfected cells formed tumors, whereas only 70% of the mice inoculated with KAI1-WT expressing cells developed tumors [Dysregulation of tumor cell proliferation also represents an oncogenic hallmark involving specific integrins . In brear burden . Moreover burden . All of d tumors . Furtherd tumors . Still, d tumors , 51. Thevice versa. Moreover, they interconnect in shared signaling pathways, leading to efficient and synergistic signaling even in the absence of their respective ligands. Dysregulated integrin and EGF-R expression and activity thus profoundly affect cancer progression and metastases [Both breast cancer cell lines studied here - while being triple-negative - do express the EGF-R which is also well known to crucially contribute to cell proliferation in concert with integrins. In fact, integrins induce changes in the expression and activity of growth factor receptors and, tastases , 59. Thetastases  and thattastases . This futastases  and the tastases .In summary, on the protein and mRNA level, KAI1-WT and KAI1-SP were recognized as valuable biomarkers in tumor tissues for the metastatic/invasive potential of cancer cells, serving to predict cancer patient prognosis. Based on accumulated evidence from clinical and basic research, the exploration of novel cancer therapies related to KAI1 had been inspired in recent years. So far, several approaches in (pre-)clinical trials aimed at reintroducing KAI1´s tumor suppressive functions in tumor cells by direct administration of KAI1 protein or, alternatively, via its adenoviral restoration .The data of the present study together with those of our previous investigations in human ovarian cancer cells, strongly support the notion that KAI1-SP does not only counteract the tumor suppressive function of KAI1-WT, but - beyond this - drastically promotes tumor cell biological activities in favor of tumor progression. Thus, in order to design successful KAI1-based therapeutic approaches, it will be indispensable to unravel the mechanistic events underlying the tumor biological functions of KAI1-WT and KAI1-SP and its differential functional crosstalk with integrins, growth factor receptors, and signaling pathways.Cloned AMV First-Strand cDNA Synthesis Kit, and the assay for the house keeper ALAS (assay ID: Hs00167441_m1) were from Thermo Fisher Scientific, Carlsbad, CA, USA. RNA was isolated by using the kit RNeasy by Qiagen . Vitronectin (VN), monoclonal antibodies (mAb) raised against the EGF-R, mAb directed to FAK or p-FAK, the dual luciferase reporter gene assay kit, and the renilla luciferase reporter gene vector pRL-SV40 plasmid were obtained from Beckton-Dickinson Biosciences, Franklin Lakes, NJ, USA. Fibronectin was from Collaborative Research, Bedford, MA, USA. Para-nitrophenyl-N-acetyl-β-D-glucosaminide, collagen type I and IV were from Sigma-Aldrich, St. Louis, MO, USA. The mAb directed to integrin αvß3 (clone # 23C6) and the polyclonal Ab (pAb) directed to the integrin subunit αv (clone #AB1930) were purchased from Millipore, Schwalbach, Germany. The mAb directed to KAI1 (clone # TS82b) was from Diaclone, Stamford, CT, USA. The mAb to EGF-R were ordered from Stressgen, Victoria, Canada. The antibodies directed to Src and p-Src were purchased from Cell Signaling Technology. Microchamber cell culture slides were obtained from Nunc Lab-Tek, Naperville, IL, USA. Cell culture inserts with two chambers for wound scratch assays were bought from ibidi GmbH, Planegg, Germany. The EGF-R promoter luciferase reporter gene plasmid was a kind gift by Prof. Dr. Ester Gonzalez, Saint Louis University, Division of Nephrology, St. Louis, USA. Materials for TaqMan Realtime PCR Analysis were obtained from Applied Biosystems, Darmstadt, Germany.Dulbecco's modified eagle medium (DMEM), fetal calf serum (FCS), Lipofectin®, the plasmid pcDNA3.1/Hygro, geneticin, hygromycin, Alexa-488- and Alexa 568-labeled goat-anti-mouse, goat-anti-rabbit IgG, the Preparation of expression vectors for KAI1-WT or KAI1-SP were generated as described earlier , 29. StaRNeasy-kit according of the manufacturers´ protocols. RNA (1 μg) was reversely transcribed into cDNA by applying the Cloned AMV First-Strand cDNA Synthesis-kit according to the manufacturers´ recommendations.RNA was isolated by using the 2, 0.4 μM of each primer, 0.2 mM dNTPs and 0.02 units of Taq polymerase. After initial denaturation for 5 min at 95°C, amplification was carried out as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles for each 15 sec at 95°C and 1 min at 60°C for annealing and extension. As housekeeper gene, ALAS was determined by using the assay by Thermo Fisher Scientific (ID: Hs00167441_m1) according to the manufacturers` protocol. The qPCR analysis was performed by using the Taqman ABI PRISM 7000 system .For the detection of elevated gene expression after stable KAI1-WT or KAI1-SP cell transfection, we performed PCR by using the following primer pairs: KAI1-WT-forward: 5’-CTGGGCATCATCCTC GG-3’; KAI1-SP-forward: 5’-GCCTGTGTACCAGGAGCT CC-3’; KAI1-WT/KAI1-SP reverse: 5’-GCAGGGAGATGGGG ATAGC-3’, and the probe FAM-CCTTGCTGTAGTC TTCGGAATGGACGT-BBQ. Amplification of cDNA and part of intron 10 was carried out in 20 μl reaction mixtures containing 20 mM Tris/HCl, pH 8.4, 50 mM KCl, 1.5 mM MgClNested primer pairs generating 255 bp and 171 bp fragments were used to detect endogenous KAI1-WT and KAI1-SP mRNA expression, respectively, in breast cancer tissues as previously described . As contHuman breast cancer cell lines were obtained from ATCC  and cultivated according to the providers´ recommendations: MDA-MB-231 cells are derived from the metastatic site of a triple-negative breast adenocarcinoma; MDA-MB-453 cells are basal B-like breast cancer cells. Authenticity of both cell lines was proven by short tandem repeat (STR)-analysis according to published recommendations .Human breast cancer cell transfectants expressing either KAI1-WT or KAI1-SP were grown on microchamber cell culture slides and stained as described , 29. TheKAI1 expression was measured by Western blot analysis as described earlier .Integrin αvß3 expression was detected by immunocytochemical staining and FACS analysis as described , 59.Breast cancer cell adhesion as a function of KAI1-WT/KAI1-SP was assessed as previously described , 29.Proliferative activity of KAI1-transfected breast cancer cells was determined by cell counting using a Neubauer hemocytometer , 29.Cell Motion Imaging System SI8000 (Sony Corporation), equipped with video/image-based analysis software capable of motion vector determination for motion analysis and data quantification as well as visualization functions. Herewith, cell movement parameters, such as velocity, acceleration, and frequency, may be quantified over time periods from hundredths of seconds to tens of days [For wound scratch cell migration assays, cells were cultivated on VN-, FN-, or uncoated cell culture dishes until cell monolayers reached an appr. 90% cell confluency, followed by the setting of a wound scratch using chamber inserts. Phase contrast optical microscopy images were taken using the  of days , 66, by  of days , 68. ForThe magnitudes of the velocity for all individual migrating cells were calculated in the corresponding images during the wound gap closure process  were determined by immunocytochemical staining and Western blot analysis as previously published , 69.Preparation of cell lysates, electrophoresis, and Western blot analysis were conducted as described earlier . After bSignificance of differences and p-values were calculated by employing the non-parametric Mann-Whitney-U test. A p-value ≤ 0.05 was considered to be statistically significant.The averaging of values from ROIs (for CLSM images or cell motion data) was the arithmetic mean of the values; the standard deviation was found by taking the square root of the average of the squared deviations of the values from their average value, with Bessel´s correction.