The primary underlying cause of cervical cancer is infection with one or more high-risk (HR) types of the human papilloma virus (HPV). Detection and typing of HPV have been commonly carried out by PCR-based assays, where HPV detection and typing are two separate procedures. Here, we present a multiplex PCR-based HPV typing assay that detects 20 HPV types  using type-specific primers and agarose gel electrophoresis. 46 cervical, urethral, and biopsy samples were analyzed by both Multiplex PCR and PGMY09/11 consensus PCR, and results were compared. 611 samples were further analyzed by Multiplex PCR, 282 were positive for HR HPV, and 101 showed multiple HR HPV infections. The relatively ease and economic accessibility of the method and its improved ability to detect high-risk HPV types in multiple HPV-infected samples make it an attractive option for HPV testing. Cervical cancer is the second most common cancer in women worldwide  and is tDetection and typing of HPV have been commonly carried out by PCR-based assays, where HPV DNA is amplified by consensus primers and then typed by restriction enzyme analysis (RFLP), hybridization with type-specific probes, or direct sequencing of the amplicons, among the most common methods . RecentlHere, we present a multiplex PCR-based HPV typing assay that detect 20 HPV types (15 HR), 3 probably HR and 2 low risk (LR) using type-specific primers and agarose gel electrophoresis.611 samples of cervical (232) and urethral (164) scrapes and paraffin-embedded tissue biopsies (215) submitted for HPV assessment were collected for Multiplex PCR HPV analysis. A subset of 46 cervical, 16 urethral, and 21 tissue biopsies were randomly selected for additional analysis with PGMY09/11 consensus primer PCR. DNA extraction of samples was performed using the Dneasy Tissue Kit , following manufacturer's instructions.http://www.ncbi.nih.gov/genbank/). Primers were designed for each HPV type, and unique specificity was confirmed by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/). Primer selection for each reaction tube mix was carried out in silico [β-globin was included as positive control [DNA sequence files for HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 were obtained from Genbank  was used in all Multiplex PCR reactions, following manufacturer instructions. Each PCR was carried out in a DNA thermal cycler  with the following conditions: initial denaturing step at 95°C for 15 min, 10 cycles of 30 s at 94°C, 90 s at 65°C, and 90 s at 72°C, followed by 30 cycles of 30 s at 94°C, 90 s at 63°C, and 90 s at 72°C, with a final extension at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide, band sizes were estimated by comparison with a 100 bp molecular weight marker , and gels were photographed in a UV transilluminator  with a Canon PowerShot A60 digital camera . HPV type was assigned based on the amplification pattern. In cases where band interpretation was not clear, an additional PCR amplification with specific primers was performed to confirm. Selected PCR amplified fragments were cloned into pGem-T vector , each cloned product was sequenced with universal forward and reverse primers to confirm fragment identity. Additionally, selected amplified fragments were digested with restriction enzymes AluI, HaeIII, RsaI, or MspI , and digestion patterns were observed in a 2% agarose gel to also confirm fragment identity.HPV consensus PCR was performed using primers PGMY09/PGMY11 designed to amplify a fragment of the HPV L1 gene of approximately 450 bp as previously described . HPV genPolymerase Chain reaction (PCR) with consensus primers can potentially detect most mucosal HPV types . There aIn this report, we present an assay based in HPV DNA amplification with type-specific primers in a Multiplex PCR format to detect and type single or multiple HR HPV infections in samples of different sources. Primers specific to each of 15 high-risk, 3 probably high-risk, and 2 low-risk HPV types were included in seven independent Multiplex PCR reactions. Typing was assigned based on the amplification pattern. As a result of having specific primers, stringent PCR conditions can be set to increase the clarity of results by reducing the presence of amplification artifacts, and all HPV types in a sample are amplified by their specific primer pair. Also, detection and HPV typing are accomplished at the same time, without the need of an additional protocol for typing after HPV detection  were analyzed with Multiplex PCR and PGMY09/11 consensus PCR. Positive high risk is determined when a HR or probable HR HPV type is detected, and negative high risk is determined when a LR HPV type or no HPV infection is detected . 47 sampβ-globin control gene and were reported as being not informative. 35% (101) of HR HPV positive samples had infections with two or more HR HPV types, representing 16% of the total number of samples analyzed. Detecting all HR HPV present in a sample is important in patient treatment to asses prevalence of infection and response to treatment.Particularly important is the capacity of detection of HPV multiple infections. A total of 611 samples  were analyzed by Multiplex PCR, including 83 samples mentioned above. 324 (53.03%) samples were negative for HR HPV, and 282 (46.15%) samples were positive for HR HPV. Only 5 (0.82%) samples  failed to amplify the Multiplex PCR HPV detection and typing are simple and potentially affordable. After DNA extraction and Multiplex PCR amplification, detection and typing of HPV are deduced from the amplification pattern observed in an agarose gel electrophoresis. This is particularly important in low-income countries. According to a study in Peru , simple,Multiplex PCR HPV can detect single or multiple HR HPV infections in cervical and urethral scrapes and paraffin-embedded tissue biopsies. The relatively ease and economic accessibility of the method can potentially have an impact in HPV screening in low-income countries like Mexico, and its improved ability to detect high-risk HPV types in multiple HPV-infected samples makes it an attractive option for HPV testing.


Due to the machismo environment that often pervades the military, and thus the patients at Veterans Affairs Medical Centers, we wanted to explore if a yoga intervention was feasible with this population.Focus groups were conducted after the completion of four waves of an 8-week yoga intervention. Twenty-six veterans participated in the focus groups. The focus groups ranged in length from 30 to 90 minutes, and were digitally recorded, and transcribed verbatim. Constant comparison analyses were used to determine initial and emerging themes.Four primary themes emerged from the data. Related to preconceived notions, veterans identified that they did not know what to expect, in part because stretching activities seemed too simple to help. While we called this “balance exercise,” veterans had mixed feelings about calling this yoga. All were more accepting of this by the end of the study. However, several, in addition to saying they wouldn’t know what to expect from yoga, said “men don’t do yoga” while another compared it to voodoo. The veterans identified that benefits from participation in a group post-stroke yoga intervention included: acknowledgement of the idea that while there was no pain and the yoga was gentle, it was still effective and powerful; the yoga activities could be done at home alone; and it filled the gap after rehabilitation. Finally, we learned that when working with veterans who had seen combat, there were some important considerations when designing an intervention. These included making sure the participants were not seated with their backs to the door, and introducing all helpers at the beginning of each session, and identifying their role.Yoga is a feasible intervention for veterans who have had a stroke. However, there are important considerations that need to be taken into account when planning a study for veterans.


Indigenous women in Australia have a disproportionate burden of cervical cancer despite a national cervical screening program. Prior to introduction of a national human papilloma virus (HPV) vaccination program, we determined HPV genotype prevalence by Indigenous status and residence in remote areas.We recruited women aged 17 to 40 years presenting to community-based primary health services for routine Pap screening across Australia. A liquid-based cytology (LBC) cervical specimen was tested for HPV DNA using the AMPLICOR HPV-DNA test and a PGMY09/11-based HPV consensus PCR; positive specimens were typed by reverse hybridization. We calculated age-adjusted prevalence by weighting to relevant population data, and determined predictors of HPV-DNA positivity by age, Indigenous status and area of residence using logistic regression.P < 0.001), specifically HPV clades α5  and α7, excluding type 18 . In multivariate analysis, detection of any HPV genotype was strongly associated with smoking and Pap-test abnormalities, with both risk factors more common among Indigenous women.Of 2152 women (655 Indigenous), prevalence of the high-risk HPV genotypes was similar for Indigenous and non-Indigenous women , and did not differ by age group. In younger age groups, the prevalence of other genotypes also did not differ, but in those aged 31 to 40 years, HPV prevalence was higher for Indigenous women (35% versus 22.5%; Although we found no difference in the prevalence of HPV16/18 among Australian women by Indigenous status or, for Indigenous women, residence in remote regions, differences were found in the prevalence of risk factors and some other HPV genotypes. This reinforces the importance of cervical screening as a complement to vaccination for all women, and the value of baseline data on HPV genotype prevalence by Indigenous status and residence for the monitoring of vaccine impact. Since the introduction of a comprehensive, organized, cervical cytology (Pap) screening program in 1991, Australia has seen a marked reduction in incidence of cervical cancer from 13.2 per 100,000 in the early 1990s to a current stable rate of around 6.9 per 100,000 since the early 2000s. This has been matched by a mortality reduction from 4 to 1.9 per 100,000 for the respective time periods[Cervical cancer is caused by oncogenic human papillomavirus (HPV) types, with 70%Existing Australian data on cervical HPV prevalence in the general population are limited, with either none or, in one case, only small study datasets from subpopulations included in international meta-analyses of prevalence in women with low-grade or normal cytology-15. AlthIn April 2007, Australia commenced the most comprehensive government-funded national HPV vaccination program in the world, using the quadrivalent vaccine for all girls 12-13 years of age as an ongoing program, with a catch-up program delivered through school and community providers for women up to 26 years of age, which finished on 31 December 2009. The data from this study provide an important baseline for comparisons internationally by epidemiologic setting and ethnicity before and after the commencement of widespread programs of HPV vaccination.The aim of this study was to determine whether there were any differences in HPV type prevalence by area of residence or ethnicity, and this aim guided our choice of methods. Engagement and recruitment of Indigenous women in remote areas, by providing the opportunity to participate meaningfully in improving their own health and that of their communities, was recognized as crucial to the success of the study. Therefore, the sampling strategy was guided by the use of methods that would include these women in a culturally appropriate way.In recognition of the need for research to benefit Indigenous communities, and for Indigenous people to be involved in the design, planning, undertaking and dissemination of Indigenous research, we identified collaborative research partners involved in frontline community care and service provision in the area of cervical cancer prevention in Indigenous communities. Prior to initiation of the study, we undertook consultation with Indigenous communities (including the establishment of an Indigenous Steering Committee), medical services, healthcare workers, public-health practitioners and servicing cytology groups, commencing in January 2005, including on-site workshops and subsequent staff training. Protocols and communication materials (including flipcharts and brochures) were developed to support the study, with local input at different sites to tailor the materials so that they would be locally and culturally appropriate as needed. A pilot study was conducted in central Australia between April and July 2005 to examine feasibility, communication materials and protocols, and laboratory techniques: it was endorsed by the Central Australian Human Research Ethics Committee. The multisite study was approved by 34 site ethics committees. Women were enrolled into the study between July 2005 and February 2008.Women aged 18 to 40 years who were attending their usual healthcare provider for routine Pap smear cytology were invited to participate in the study. Some women aged under 18 years, who were assessed as mature minors and hence competent for giving consent without parental or guardian involvement and attending for a routine Pap test, were also enrolled by providers. Although most women in Australia see a general practitioner  for Pap-test screening, our primary focus was upon adequate recruitment of Indigenous women, and therefore we targeted Indigenous health providers and clinics, and equivalent service providers  for non-Indigenous women, to ensure comparability of Indigenous and non-Indigenous women in the study. The study sample size was determined based on the HPV prevalence estimates from a large community-based study in the USA of women aged 18-40 attending for a Pap smear and was powered  to detect an absolute difference in HPV16/18 of ≤ +/-5% between Indigenous and non-Indigenous women, and between Indigenous women residing in remote areas and other Indigenous women. We deteWe recruited women in all states and one territory (Northern Territory) of Australia from sixteen Indigenous health services, eight family-planning services and ten community clinics. Owing to the incorporation of the study into routine clinical care, response rates (number enrolled divided by number approached) were not accurately recorded at all sites. Where response rates were recorded or estimated from the number of eligible (by age) women who had a Pap test taken during the recruitment period, these ranged from 46% to 87%. Recruitment of Indigenous women in some sites was challenging, and required further strategies for community engagement with the study to be developed,21.Women were asked to allow the cervical samples collected for Pap cervical cytology to be tested for HPV DNA also. Once fully informed consent was obtained, Pap smear samples were collected in the routine clinic settings. In addition to routine pathology specimen details, information confirming Indigenous status, most recent previous Pap-test result, pregnancy status, smoking status and hormonal contraceptive use was requested from participants. Following the testing of specimens, identifying details were used to report Pap cytology and high-risk (HR) HPV DNA results back to the clinician.Most specimens were collected using a brush  and a spatula. The sample was then smeared onto a glass slide and fixed with ethanol ready for conventional Pap cytology, and the remainder was rinsed into vials containing fixative solution . In a minority of samples, LBC was standard, and hence an aliquot was removed using good microbiological practices within a biohazard cabinet, prior to processing for cytology. All samples were transported to the Regional WHO LabNet for HPV located at the Royal Women's Hospital in Melbourne, Australia, for HPV DNA detection and genotyping.Results of the HR HPV testing were returned to the woman's healthcare provider so that they could be conveyed back by trained staff to each participant in a culturally appropriate way. All women were managed according to National Health and Medical Research Council guidelines for the follow-up of abnormal Pap tests. Currentβ-globin gene as an internal control.DNA was extracted from stored specimens (in PreservCyt) using a commercial isolation and purification system  Genotyping Test; Roche Molecular Systems), using 50 μl of extracted DNA, and following the manufacturer's instructions with minor modifications as previously reported[All samples that tested positive by the AMPLICOR or PGMY09/11 PCR test were genotyped  according to the Australian Modified Bethesda classification, 2004 . For the2 tests (or Fisher's exact test when the expected count in any cell was < 5), and mean ages using t-test. Binary logistic regression was used for multivariate analysis. Interaction terms were created for predictor variables to check for effect modification. No significant interactions were identified. Because of the high correlation between living in a remote area and Indigenous status, the effect of area of residence was analyzed separately for the two groups of women, with the analysis of non-Indigenous women comparing residence in a major city versus non-major city.Age-adjusted prevalences were calculated by weighting the WHINURS sample to the relevant Australian Bureau of Statistics (ABS) population structure, by single year of age. Data were analyzed using SPSS software , except for weighted confidence intervals, which were calculated using STATA software . Univariate analysis compared proportions using Pearson's χIndigenous status and area of residence were variables of prior interest. Multivariate models also included other known determinants of HPV infection: age, smoking status, and use of hormonal contraception. We did not collect sexual history from participants. Although some women were pregnant, there was no association between pregnancy and HPV detection in univariate analysis, and hence this was not considered further.Of a total of 2156 samples from women age 15 to 40 years, four were found to have no ß-globin signal, indicating inadequate sample collection, and these were excluded from the analysis, leaving 2152 participants with HPV results available  (Table P = 0.02), and the prevalence of abnormal Pap-test results was higher. The prevalence of HSIL as predicted by cytology was higher for Indigenous than non-Indigenous women for both their most recent previous Pap test , and for the Pap test performed during this study (current Pap test) . Of the 45 women with a current high-grade abnormal Pap test, 25 (56%) were Indigenous, including one with squamous cell carcinoma and one with possible microinvasive cancer.Regarding demographic variables, Indigenous women were more likely to be younger, to smoke, to be pregnant and less likely to use hormonal contraception Table . RegardiP = 0.003)  Table . To evalThe age-adjusted prevalence of detection of any HPV DNA was higher for Indigenous than for non-Indigenous women when standardized to the general Australian population, although confidence intervals overlapped Table . HoweverP < 0.001), although caution is needed in interpreting this finding given the multiple comparisons of genotype groups performed.HPV16 was the most common genotype in both Indigenous and non-Indigenous women, with types 51, 52, 18 and 39 being the other four most common high-risk types in both groups , whereas the high-risk type HPV59 was found more frequently in women living in non-remote areas . The low-risk (LR) types HPV71 and HPV72 were found in women in remote areas . HPV81 was also more common among women in remote areas . Again, caution is needed in interpreting these findings, because of the multiple comparisons.Among Indigenous women, the high-risk types HPV56 and HPV58 were detected more frequently in women living in remote areas . Excluding women with an abnormality on the most recent previous or the current Pap test gave a similar result . The higher HPV prevalence for Indigenous women was partly explained by a higher prevalence of HPV risk factors. In this age group, Indigenous and non-Indigenous women had similar distribution of age by single year  and hormonal contraceptive use , but Indigenous women were more than twice as likely to smoke  and more than seven times as likely to have a current high-grade Pap-test result  or to be having their first ever Pap (n = 3 (3.2%) versus n = 1 (0.4%), P = 0.03). Adjusting in multivariate analyses for age, hormonal contraceptive use, smoking, low-grade Pap-test results and most recent previous Pap status reduced, but did not eliminate, the excess risk of HPV detection for Indigenous women . High-grade results were too infrequent to be included in this model.In the 36 to 40 years age group, 39.4% of Indigenous women had detectable HPV (any HPV genotype) compared with 22.1% of non-Indigenous women .Overall, 57.6% of HPV-positive non-Indigenous women and 55.6% of HPV-positive Indigenous women had multiple types detected. However, infection with multiple types was less prevalent with increasing age Figure . AlthougP < 0.001) and having a low-grade Pap abnormality on current Pap testing  were significantly associated with the detection of multiple HPV types.In a multivariate analysis of HPV-positive women , only younger age  and 85.9% LSIL . Having had an abnormality on the previous Pap test was also strongly associated with HPV DNA detection .)As expected, having a current abnormality on a Pap test was strongly associated with HPV DNA detection , which remained significant in multivariate analysis .All variables of interest were significantly associated with the detection of any HPV DNA in univariate analysis Table . In multP = 0.001); this variable remained significant in the adjusted analysis .In both univariate and multivariate analyses, age, smoking and hormonal contraceptive use were all associated with HPV16/18 detection  had more non-HPV 16/18 types detected. The dominance of HPV16 is consistent with worldwide prevalence studies, but HPV51, the second most common high-risk type we detected, does not have such a high prevalence globally .et al. previously found that lower education levels (as a proxy for socioeconomic status) are correlated with cervical cancer risk but not increased HPV prevalence, and may be mediated through risk factors such as earlier age at first intercourse, and higher and earlier parity [It is unclear why Indigenous women in their 30s had higher rates of non-HPV16/18 genotype carriage. Indigenous Australian women have a different demographic profile to non-Indigenous women, as also reflected in our recruited sample, in ways which could have an effect upon their likelihood of HPV infection, that is, younger age, more than twice as likely to smoke, and a higher fertility rate. Indigenr parity . Regardlr parity .The rates of absolute HPV prevalence, frequent occurrence of multiple HPV types and high rates of HPV DNA detected in young women are all consistent with other studies of screening populations in western countries using similar highly sensitive assays  ,35-37.There are three main limitations to our study. Firstly, we did not collect a sexual or reproductive history from participants. This means we cannot use this established predictor to determine whether women recruited were more or less likely than other Australian women to have HPV. Clearly, however, all women participating in the study were sexually active, and should have been so for at least two years, given that screening guidelines in Australia recommended Pap testing commence at the age of 18 years or 2 years after first sexual intercourse, whichever is later. Secondly, because our main focus was to recruit adequate numbers of women in remote area and of Indigenous women, our sample is not geographically or demographically representative of all Australian women. Non-Indigenous women attending free community health services are likely to be of lower socioeconomic status on average than other Australian women. However, the results were stratified by Indigenous status and ARIA status within the two groups of women, with similar recruitment of health services for both Indigenous and non-Indigenous women, allowing for comparison between the two groups. The non-Indigenous women we enrolled had smoking rates similar to those of the women in the general Australian population  , similarA truly population-based HPV DNA prevalence survey of women pre-vaccination in Australia was not feasible for several reasons. A random sample of women screened in the national program was not possible because the national program uses conventional dried slides rather than LBC. Under Australian legislation, the slide must be retained for a minimum of 10 years, but testing for HPV involves destruction of the slide. Women who choose to have LBC in Australia have to pay privately for the test, and are not therefore a representative sample. Australia currently has no national health survey with a biological sample collection component  which could otherwise form a sample frame for HPV testing of other suitable  sample types, as has been conducted in the UK (urine) and USA . AlthougTaken together with baseline HPV seroprevalence data for Australia, the WHINIn conclusion, we found no evidence that HPV16/18 prevalence differs significantly for Australian women according to their Indigenous status or, for Indigenous women, whether they lived in remote areas. However, we did identify differences in risk-factor prevalence and in the prevalence of some other HPV genotypes. Indigenous Australian women currently have higher rates of cervical cancer, probably due to factors such as smoking, higher fertility and lower rates of participation in cervical screening. This reinforces the importance of cervical screening as a complement to vaccination for all women, and the value of baseline data on HPV genotype prevalence by Indigenous status and residence for monitoring of the effect of a vaccine program.Other than the grant funding received for this study (see below), JC, ST, MS and DS have no competing interests. JB and PM were co-authors on an investigator-driven HPV serosurveillance study in which laboratory testing was funded by CSL Limited. JB and SG are partner investigators on an Australian Research Council linkage grant in which CSL is a partner investigator. SG has received advisory board fees and grant support from CSL and GlaxoSmithKline, and lecture and consultancy fees from Merck and Co. SG reports having previously owned stock in CSL. SG has received grant support through her institution from Merck and Co and GlaxoSmithKline (GSK) to carry out clinical trials for HPV/cervical cancer vaccines, and she is a member of the Merck global advisory and scientific advisory boards.SG, JB and PM designed the study with assistance from JC and DS. SG coordinated the study, and managed recruitment of participating sites and the study team, including performing study workshops at most sites, and was responsible for overall specimen and laboratory methods. JC coordinated site recruitment in the Northern Territory, and DS coordinated site recruitment in Western Australia. ST managed specimen coordination and HPV DNA typing which was performed by MS. JB analyzed the study data, with assistance from JC, PM and MS. Data interpretation was led by JB, JC, PM and SG. SG and JB drafted the initial manuscript with critical revisions by all authors. All authors approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/104/prepub


The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides. The Cellient Automated Cell Block System (Hologic), producing paraffin blocks could provide an ideal method for cytohistologists as it compares favorably with traditional cell block sectioning . AlthougIn a pilot study of 14 HIV-positive women with ASCUS or HSIL, the Cellient system for cytohistology was exploited to analyze p16 positive dyskeratocytes in HPV-positive cervical scrapes [From each sample six ThinPrep slides (Hologic) were prepared in the T3000. The remaining material in the vial was stored. The cytology ThinPrep slides were scored according to the Bethesda system [The BoonFixed samples were analyzed for 25 HPV genotypes using a highly sensitive PCR-reverse hybridization Line Probe Assay, the so-called INNO-LiPA HPV genotyping extra system . The SPF10 primer set amplifies a 65-bp region in the L1 open reading frame [All the remaining material in the vial was used for the Cellient method. This was collected into a cassette and loaded into the instrument. Eosin was applied and vacuum-drawn through the sample. Alcohol was similarly applied and drawn through the sample for dehydration. To clear the alcohol, the procedure was repeated with xylene. The sample was then embedded in paraffin and finally embedded in an additional layer of paraffin during processing in the finishing station.From the Cellient paraffin block eight serial sections were cut, two for the Papanicolaou staining, one for the hematoxylin eosin (HE) staining, and five for the p16 immunostainings.Metaplastic cells are defined as cells with a vesicular nucleus and a nuclear-cytoplasm ratio of around 0.5 . In the Koilocytotic cells display a clear zone well demarcated around the nucleus . ParticuSquamous pearls are whirls of squamous cells. In the ThinPrep slides these pearls display small pyknotic nuclei. The cytoplasmic staining is either turquoise or red. In the Papanicolaou-stained paraffin Cellient sections, the cytoplasm is either bright red or dark green; nuclei are not always in the section.In the ThinPrep slides, dyskeratotic cells have a pyknotic nucleus, a nuclear cytoplasmic ration between 2 and 6, and orange or green staining cytoplasm. In the Papanicolaou-stained Cellient paraffin sections, the cytoplasm is green or orange-red. In these sections, the pyknotic nuclei of the dyskeratotic cells can be relatively large. In the HE stain, dyskeratotic cells have bright red cytoplasm. In the p16 Cellient paraffin sections, dyskeratotic cells can display brown cytoplasm and brown-blue nuclei or intensely brown pyknotic nuclei in which the blue (hematoxylin) staining is hidden by the brown color.The boon fixative BoonFix can be used for cervical scrapes  and for In total, 11 HPV genotypes were found, two low risk (lr) (HPV44 and HPV70) and nine high risk . In In The Cellient Automated Cell Block System was able to provide high quality histology and outstanding images obtained with the biomarkers p16, all this on cervical scrapes on which 22 HPV genotypes were established by PCR.Nuclear and cytoplasmic p16 positivity was observed in metaplastic cells, koilocytes, and dyskeratotic cells. Of the CIN 1 epithelial fragments, 10–30% of the cells were p16 positive, whilst in CIN 3 epithelial fragments over 50% stained positive. This is in accordance with our experience of p16 in Shandon cytoblock paraffin sections. Note that p16 positivity can also be observed in the cytohistology of adenocarcinoma of the endocervix .INK4a protein in cervical premalignant and malignant lesions [It is well-known that HPV infection can induce a disturbance of the keratinization process leading to dyskeratosis. The Papanicolaou method (in which dye competition between light green and eosin is exploited) is well-suited to detect these dyskeratotic cells by their strikingly orange color, whilst others stain intensely green. It should be noted that the pyknotic nuclei of dyskeratotic cells can be rather large suggesting polyploidy. Such dyskeratotic cells can have relatively large nuclei and accordingly be designated as atypical. The biomarker p16 is used to detect overexpression of p16 lesions –15. Galgn = 41), Leiden BV intermediate , and Leiden BV positive . These findings indicate a low BV– prevalence in these African women.The BoonFixed samples can in addition be used for morphotyping the vaginal microbiome. In a study of South African women, the vaginal microbiome was assessed by the Leiden BV scoring system based on morphotyping the stained bacteria. The cases were identified as Leiden bacterial vaginosis (BV) negative , 90% of women with LSIL (n = 35), and 42% of women within normal limits (WNL) (n = 58) tested positive for hrHPV. Overall, the most prevalent hrHPV subtypes were HPV16 (26%) and HPV52 (30%).In BoonFixed cervical scrapes HPV testing can be combined with cytology on ThinPrep slides. The cross-sectional study of Dols et al.  was perfWe conclude that the Cellient system for paraffin histology can be successfully combined with HPV testing and morphotyping the vaginal microbiome thanks to BoonFixing.


Pterygium is a potentially vision-threatening fibrovascular lesion originating from the conjunctiva that often extends on the corneal surface. Although it has been extensively studied, its pathogenesis has yet to be fully elucidated. Recent evidence on molecular genetic abnormalities in pterygium suggested neoplastic changes of limbal stem cells potentially associated with exposure to ultraviolet (UV) light. Human papillomavirus (HPV) is an oncogenic virus, associated with squamo-proliferative lesions of the anogenital region, skin and oropharynx. Several studies have shown HPV involvement in the pathogenesis of conjunctival neoplastic lesions, including papilloma and squamous cell carcinoma. The involvement of HPV as a co-factor in the pathogenesis of pterygium, although suggested by several studies using PCR and immunohistochemical techniques, remains controversial. Moreover, a marked variation in the prevalence of HPV in ophthalmic pterygium has been reported by different studies. Ethnic susceptibility and methodological differences in the detection of HPV may account for this variation. Surgical excision, often using sophisticated techniques, is the standard current method of therapy for pterygium. However, recurrences are frequent and recurrent lesions tend to be more aggressive. If indeed HPV is involved in pterygium pathogenesis or recurrence, anti-viral medications or vaccination may be new options in pterygium therapy. Pterygium is a wing-shaped fibrovascular lesion of the ocular surface of unknown origin and pathogenesis. It grows at the inter-palpebral conjunctival area, more often nasally, and invades adjacent corneal epithelium . PterygiAlthough pterygium pathogenesis remains an enigma, epidemiologic studies suggest that it is a sun-related eye disease . In fact, the prevalence of pterygium seems to be associated with geographical latitude, with higher prevalences in areas located within 40º above and below the equator, suggesting that prolonged exposure to ultraviolet (UV) radiation may promote its development. In addition, repeated micro-trauma, mediated by exposure to dust, chronic conjunctival inflammation, genetic predisposition and ocular dryness have all been reported to be involved in pterygium, indicating a multifactorial pathogenesis ,3. EvidePterygium has been previously considered a degenerative condition. This hypothesis, however, has been challenged in recent years by the detection of important molecular genetic alterations in pterygium, including loss of heterozygosity (LOH) of microsatellite DNA or the overexpression of mutated versions of p53 with compromised function, which could promote tumor growth –14. Accoet al investigated the role of HSV in pterygium, where a prevalence of only 5% was reported  have failed to detect HPV or report very low prevalences of HPV in examined pterygia  ,40,41.Diagnosis of the viral infection is based on the detection of the HPV-DNA. However, the mode of sample collection, the quantity of the HPV-DNA of the isolated sample and the use of various HPV-DNA detection techniques with different sensitivity and specificity, are factors that may significantly affect the detection rates of HPV infections .in situ hybridization (ISH), Southern blotting and dot blot hybridization. However, these techniques are laborious, need a large quantity of purified DNA and their sensitivity is limited  is a non-radioactive signal amplification technique, accurate for mucosal lesions, that distinguishes high-risk from low-risk HPV-types, but is not appropriate for genotyping –42.Due to its high sensitivity, polymerase chain reaction (PCR) is frequently associated with a high rate of false-positive results . SoutherCurrent treatment of pterygium includes surgical excision and occasionally adjunctive therapy. Several surgical techniques have been described: bare sclera closure, sliding conjunctival flaps, use of autologous conjunctival and limbal grafts or amniotic membranes ,46 Fig.. Due to Interferons are a family of proteins with antiviral, antiproliferative, antiangiogenetic and immunomodulatory properties, produced from the organism in response to various stimuli . The recDespite controversies in the medical literature concerning HPV involvement in pterygium , most studies agree that HPV is detected in at least a sub-group of pterygia and that HPV infection may affect both pathogenesis and clinical behaviour (including recurrence) of these lesions. Accordingly, it would be interesting to explore the possibility of anti-viral medications or even vaccination, which may represent novel options in the therapy of selected, HPV-infected pterygia.


Although development of head and neck squamous cell carcinomas (HNSCCs) is commonly linked to the consumption of tobacco and alcohol, a link between human papillomavirus (HPV) infection and a subgroup of head and neck cancers has been established. These HPV-positive tumors represent a distinct biological entity with overexpression of viral oncoproteins E6 and E7. It has been shown in several clinical studies that HPV-positive HNSCCs have a more favorable outcome and greater response to radiotherapy. The reason for improved prognosis of HPV-related HNSCC remains speculative, but it could be owned to multiple factors. One hypothesis is that HPV-positive cells are intrinsically more sensitive to standard therapies and thus respond better to treatment. Another possibility is that HPV-positive tumors uniquely express viral proteins that induce an immune response during therapy that helps clear tumors and prevents recurrence. Here, we will review current evidence for the biological basis of increased radiosensitivity in HPV-positive HNSCC. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with an annual incidence of approximately 400.000 cases. Although tobacco and alcohol consumption are the main risk factors for development of HNSCC, a causal link between Human papillomavirus (HPV) infection and a subgroup of head and neck cancers has been established, mostly in the oropharynx –4. IncidRecent studies indicate that the expression of HPV-associated p16 (hereafter referred to as HPV/p16-positivity) in HNSCC is correlated with a better prognosis and improved response to conventional radiotherapy –12. WhilHuman papillomaviruses comprise a large group that has been subdivided in low-risk and high-risk viruses, the last ones being associated with cancer . HPVs ar ras to transform cells in vitro [E6 is coded at the 5′ early viral genome and is well conserved among viruses. E6 viral transcript can be spliced leading to two spliced versions of E6, namely, E6*I and E6*II mRNA. Unspliced E6 transcript gives rise to a 19 kDa protein that forms a complex with a ubiquitin protein ligase (E6AP) that will lead to ubiquitination of p53 tumor suppressor protein and its subsequent degradation . The funin vitro . E6 oncoin vitro . Maintenin vitro .High-risk HPV E7 oncoproteins have the ability to initiate DNA synthesis in differentiated epithelial cells mainly by binding and inactivating the Rb apoptosis/tumor suppressor gene and its associated pocket proteins p107 and p130 . InactivMany clinical studies have shown that patients with HPV/p16-positive tumors exhibit a far better prognosis compared to HPV/p16-negative ones when treated by primary radiochemotherapy (RCT) or RCT after surgery . DespiteRadiosensitivity is mainly due to ability of the cell to sense DNA damage and to control its repair, though tumor microenvironment  is also determinant for response to radiotherapy . The mosDespite the two previously cited papers demonstrating increased radiosensitivity of HPV/p16-positive cells , 35, theFinally, there are few studies indicating that HPV oncoproteins might compromise DNA damage repair mechanisms, a strategy used by viruses to facilitate viral genome integration in the host. In HPV type 1, 8 but also 16, E6 oncoprotein has been shown to bind to XRCC1, a protein required for the repair of DNA single strand breaks and genetic stability . In addiIn summary, although increased radiosensitivity of HPV/p16-positive HNSCC has been partially confirmed experimentally, further studies are still needed to identify molecular actors implicated in radioresponse of HPV/p16-positive HNSCC.As mentioned before, although radiosensitivity is mainly due to ability of the cell to sense DNA damage and to control its repair, oxygenation status of the tumor might also be determinant for response to radiotherapy . in vitro data comparing radioresponse of HPV/p16-positive and -negative cell lines under hypoxia indicated no difference in terms of regulated gene patterns by hypoxia, whereas HPV/p16-positive cells displayed resistance under hypoxia with an oxygen enhancement ratio (OER) similar to HPV/p16-negative ones [In this context, retrospective analysis of DAHANCA-5 trial showed that HPV/p16-positive tumors have a superior outcome after fractionated radiotherapy compared to patients with HPV/p16-negative tumors. Patients in this study also received the hypoxic cell radiosensitizer nimorazole and the use of this drug during radiotherapy improved locoregional tumor control only in the HPV/p16-negative group. Surprisingly, HPV/p16-positive tumors seemed to be insensitive to the hypoxic modification and showed no benefit from treatment with nimorazole. The authors suggested that HPV/p16-positive HNSCCs are less hypoxic than negative ones and that this apparent lack of radiobiological relevant hypoxia in HPV/p16-positive tumors could contribute to the superior prognosis observed . Howeverive ones . Therefoγ-producing T cells in cultures of PBMC from patients with HPV16+HNSCC [Another possibility is the implication of other tumor microenvironmental factors like components of the immune system. It is indeed suggested that HPV-positive tumors carry viral antigens that elicit T-cell responses that might participate in a tumor rejection process and carry out a long-term immunosurveillance. Along this line, most but not all HPV/p16-positive HNSCCs display a strong tumor infiltration by T cells . However16+HNSCC . In addi16+HNSCC , 55.However, if T-cell immunity detects HPV-positive HNSCC, it probably occurs early during tumor development meaning that clinically detectable tumors have probably been immunoselected, a process identified decades ago , 57 and  in vitro that radiotherapy induces calreticulin membrane exposure in some cancer cell lines [Most ongoing research is dedicated to new approaches for enhancing antitumor immune response either by increasing T-cell responses directly or by reducing various immunosuppressive mechanisms at work in the tumor microenvironment. However very little is known on the immunogenic potential of radiotherapy . Irradiall lines . The comll lines , 65. in vivo mouse model HPV-positive tumors were more sensitive to radiation and exhibited complete clearance at 20 Gy, compared to HPV-negative counterparts, which showed persistent growth. However, radiation therapy in immune-incompetent mice was enabled to cure tumors. Adoptive transfer of wild-type immune cells into immune-incompetent mice restored HPV-positive tumor clearance with cisplatin therapy [It is then possible that radio-/chemotherapy primes the immune system to target HPV positive cancer cells. In mouse models of HPV-induced cancers, cisplatin followed by vaccination resulted in stronger HPV-specific T-cell responses, increased T-cell infiltration of the tumor , and inc therapy . In addi therapy . CD47 is therapy .Although there is a growing amount of data supporting the hypothesis that HPV-related tumors have a better survival due to a higher sensitivity to chemo-/radiation therapy, it is difficult to conclude that the improved clinical outcome of HPV-related HNSCC is only attributable to the intrinsic radiosensitivity of the HPV-infected cells. More likely is a complex interaction among intrinsic mechanisms of radioresponse and the tumor microenvironment including cells of the immune system.In conclusion, HPV+ HNSCC is a distinct clinical entity with a favorable prognosis. These tumors respond better to radiotherapy even though there is little evidence for increased radiosensitivity. In other models radiotherapy cooperates with antitumor immunity, providing a rationale to investigate immune responses in HPV+ tumors after radiotherapy.


Endocavity ultrasound is seen as a harmless procedure and has become a common gynaecological procedure. However without correct disinfection, it may result in nosocomial transmission of genito-urinary pathogens, such as high-risk Human Papillomavirus (HR-HPV). We aimed to evaluate the currently recommended disinfection procedure for covered endocavity ultrasound probes, which consists of “Low Level Disinfection” (LLD) with “quaternary ammonium compounds” containing wipes.From May to October 2011 swabs were taken from endovaginal ultrasound probes at the Gynecology Department of the Lyon University Hospital. During the first phase (May–June 2011) samples were taken after the ultrasound examination and after the LLD procedure. In a second phase (July–October 2011) swab samples were collected just before the probe was used. All samples were tested for the presence of human DNA  and HPV DNA with the Genomica DNA microarray (35 different HPV genotypes).We collected 217 samples before and 200 samples after the ultrasound examination. The PCR was inhibited in two cases. Human DNA was detected in 36 (18%) post-examination samples and 61 (28%) pre-examination samples. After the ultrasound LLD procedure, 6 (3.0%) samples contained HR-HPV types . Similarly, HPV was detected in 6 pre-examination samples (2.7%). Amongst these 4 (1.9%) contained HR-HPV (types 53 and 70).Our study reveals that a considerable number of ultrasound probes are contaminated with human and HR-HPV DNA, despite LLD disinfection and probe cover. In all hospitals, where LLD is performed, the endovaginal ultrasound procedure must therefore be considered a source for nosocomial HR-HPV infections. We recommend the stringent use of high-level disinfectants, such as glutaraldehyde or hydrogen peroxide solutions. Human papillomavirus (HPV) is now recognized as the major etiological cause of invasive cervical cancer and cervical intraepithelial neoplasia worldwide HPV has the potential for nosocomial transmission due to high resistance and persistence in the environment. It has been shown that HPV retains 30% of its infectivity, even after dehydration for 7 days cervix uteri or vaginal wall may represent a potential vector for sexual transmitted infections, such as HPV. In 2010 Kac noticed HPV contamination of endovaginal and endorectal ultrasound probes, despite usage of specific probe covers Endovaginal cavity ultrasound, seen as a rapid and completely harmless procedure, has become a common medical diagnostic tool. The close contact between the probe and the nd of May to the 1stof July 2011, probe samples were obtained immediately after the endovaginal ultrasound was performed and the probe was disinfected. In the second sampling period, from the 2nd of July to the 10th of October 2011, we assessed a potential risk of HPV transmission to the next patient, with samples being collected just before the probe was used on a new patient. The mean time interval to the previous disinfection procedure was 71 minutes (range: 16 minutes to 8 hours).We performed a prospective study, conducted during two periods in one ward of the gynaecology department of the Lyon University Hospital “Femme Mère Enfant”. Permission to enter the wards and collect the samples has been granted by the head-chief of the gyneco-obstetric department. No patient information of any kind has been gathered and no human samples were tested in this study, therefore no patient consent was required by the local ethical committee. During the first period, from the 2The disinfection of the probes was performed by nurses under supervision of the technician in charge of the sampling. Before the study all participating nurses were specifically trained on how to handle the probe in order to avoid contamination via hands or the cover itself. Probes were used with a disposable probe cover (93/42/EEC CE mark) in compliance with national health recommendations Samples were taken from two endocavity ultrasound probes used on two ultrasound machines . Probe samples were collected by a specifically trained technician, no more than 15 minutes after the end of the disinfection process. The sample was taken with a dry swab, applied lengthwise across the surface of the probe. The swab was then immediately suspended in a viral transport medium (EMEM) and send to the microbiological lab. Delay between sampling and lab processing did not exceed four hours.The samples were sent to the virology laboratory within less than four hours and stored at −20°C. All samples were extracted using a NucliSens easyMAG instrument  according to the manufacturer's recommended procedures. HPV amplification and genotyping were conducted using the Genomica SAU  microarray test Extractions and PCRs for all samples were run in specific series to avoid any risk of contamination from clinical samples. Each series included a negative control in order to test for contamination during the extraction procedure. One swab of each batch, as well as the EMEM medium batch was tested for the absence of HPV contamination.Statistical analysis was performed with EpiInfo software (V 3.5.1 CDC). Data were expressed as the percentages of positive sample. Inhibited samples were excluded from the calculation.In our study, no break of the probe covers was detected in any case, and visual inspection did not reveal any presence of blood or body fluids on the probe.nd to July 1st 200 probes samples were obtained after endovaginal use and standard disinfection. The PCR was inhibited in two cases. Of the remaining 198 samples, 7 samples (3.5%) were HPV positive. 1 sample showed low risk HPV and 6 samples (3.0%) were positive for at least one high risk HPV type. The detected HPV types include HPV 53, HPV 16, HPV 58 and HPV 31. Two samples revealed more than one HPV type, with a maximum of 4 different HPV types per sample being detected  samples. Four samples were positive with the HR-HPV types HPV 53 and HPV 70 and two samples revealed LR-HPV. We did not detect more than one HPV per sample , based on a human housekeeping gene, detected the trace of human DNA in 63/216 (29.2%) and 39/198 (19.7%) samples, before and after the probe use.This study shows a probe contamination with high risk oncogenic HPV DNA after low level disinfection, despite the use of probe covers. We observed a 2.2% rate of HR-HPV contamination of probes (1.8% before use and 2.5% after use). This result is in agreement with the results observed by Kac G et al The high rate of human DNA on the probes most likely represents human DNA from cells of the genital tract. We tried to avoid any hand contact with the probe during the sampling and the ultrasound examination, but it cannot be ruled out completely. The presence of human DNA from the genital tract on the ultrasound probe can be seen as a marker for a contamination with potential pathogens, attached to cells of the genital tract.This study may have several limitations. The HPV detection method is based on nucleic acid detection and does not detect the presence of infectious virus particles. The HPV diagnostic faces the same problem, as no HPV cell culture is available up to date. Therefore it is not possible to assess the infectious potential of HPV. Although we are not able to conclude on the infectivity of the detected HPV, considering the potential for HPV resistance on fomites This risk of swab or probe contamination, due to hand contact was a main concern during the study. Necessary precautions  were taken to avoid this identified risk, therefore direct HPV contamination of the swab is very unlikely. However, despite the use of gloves, we cannot definitely exclude manual contamination of the probe during probe handling. The main HPV type detected in our study is HPV 53. This high detection rate of HPV 53 is a main feature of the genital HPV genotype distribution of our population in Lyon, as reported in a previous study (12). On the other hand HPV 16, the most prevalent HR-HPV genotype, is detected less than expected. Given the limited number of HPV detected in the study, the HPV genotype distribution should be interpreted cautiously.The use of dry swabs for sampling could have resulted in a loss of sensitivity. We tried to avoid the problem, by suspending the swab into a viral transport medium (EMEM) and transport the samples into the lab within four hours.This study was carried out in an almost standard disinfection situation. The staff was aware of the study and this may have strengthened the compliance to the disinfection procedures. Under different circumstances, such as emergency units or wards with a high frequency ultrasound use, compliance may be lower and HPV detection therefore likely to be higher.The study focused on the detection of HPV on the ultrasound probe. Patients were not sampled in the study, as the main objective was to evaluate the disinfection procedure for covered endocavity ultrasound probes and not routes of transmission.Presently the French disinfection guidelines In conclusion the study shows a considerable number of endovaginal ultrasound probes being contaminated with HPV after the use of low-level disinfection. Low-level disinfection seems to be inefficient for preventing contamination of endocavity ultrasound probes with HR-HPV and pose a risk of nosocomial transmission of HR-HPV during an ultrasound procedure. We therefore recommend the use of high-level disinfection procedures after each endovaginal ultrasound examinations.


In Italy, cervical cancer screening programmes actively invite women aged 25–64 years. Programmes are hindered by low participation.A sample of non-responder women aged 35–64 years, belonging to three different programmes , was randomly split into four arms: two control groups received standard recall letters to perform either Pap-test (first group) or human papillomavirus (HPV) test (second group) at the clinic. A third arm was sent letters offering a self-sampler for HPV testing, to be requested by phone, whereas a fourth group was directly sent the self-samplers home.Compliance with standard recall was 13.9% (N619). Offering HPV test at the clinic had a nonsignificant effect on compliance =1.08; 95% CI=0.82–1.41). Self-sampler at request had the poorest performance, 8.7% , whereas direct mailing of the self-sampler registered the highest compliance: 19.6% . This effect on compliance was observed only in urban areas, Florence and Rome , but not in Abruzzo , a prevalently rural area.Mailing self-samplers to non-responders may increase compliance as compared with delivering standard recall letters. Nevertheless, effectiveness is context specific and the strategy costs should be carefully considered. Testing for human papillomavirus (HPV) DNA in the cervix has been shown to be an effective mean for cervical cancer prevention in large population trials carried out in different countries with huge differences in social, cultural and economical background , giving Even if an increase in test sensitivity is welcome, this is not the main problem in cervical cancer prevention. In fact, in most industrialised countries, the majority of invasive cancers occurs in never-screened or under-screened women , Rome southern city (Lazio) and Teramo (Abruzzo). In these areas, programmes actively invite all the resident female population aged 25–64 years for a Pap-test every 3 years.Women aged 35–65 years who had been invited by the screening programme in the previous months and had failed to respond were eligible for mail recall. Each programme has a different strategy for recall: in Florence, recalls are sent to all non-responding women after invitations for a whole district are completed, which is to say after a period of at least 3 months and not more than 5 months. In Rome and Teramo, recalls are automatically mailed to non-responders 3 months after the invitation.two control arms with a standard invitation letter to perform either a Pap-test (a) or an HPV test (b) at the clinic on a pre-fixed date;a group was offered the opportunity to receive the self-sampler device  . Random sampling and arm assignment were performed centrally by the coordinating centre (Rome) using STATA 8.2  and putting as seed for random number generator the first number drawn by the most recent National Lottery: four consecutive samples of the pre-determined size of independent statistical units were drawn and assigned to the corresponding arm. Couples of women resident in the same address were randomised as a single statistical unit.The planned sample size was 200 women per arm per centre. This would allow for 80% power to detect, with 95% confidence, a 7% increase in compliance, in the hypothesis of 15% compliance in the control arm. Sampling started in Rome, with the very sample size as previously planned (800). As a result, extra self-sampler boxes were available for the study in Florence, in which the sample size was slightly increased . In Abruzzo, we later planned an additional fifth arm, slightly decreasing the number of women in the four arms (180 per arm). Actually the fifth arm was implemented as a separate trial, a few months after this study, owing to the earthquake that destroyed part of the Abruzzo region in April 2009.In the two control arms, the intervention was only the recall letter inviting for a Pap smear (arm (a)) or an HPV test (arm (b)) at a prefixed date at the clinic.In the arm with the self-sampler at request, we mailed a letter inviting women to dial a free-toll number to receive the self-sampling device and an information leaflet on HPV test and cervical cancer. We offered women two opportunities: to either have the device shipped home or pick it up by themselves at the clinic. The self-sampler box contained the following items:a presentation letter;a leaflet about HPV test and cervical cancer prevention;the informed consent form to perform the test and to be contacted by phone in case of positivity;the self-sampler device;the instructions for the device use;a tube and a pre-paid envelope to mail of the sample; anda short questionnaire asking the approximate date of the latest Pap-test, questions about the sampling  and the date of sampling.In the arm with the direct mailing, we sent an alerting letter explaining that the local Public Screening Program would provide women with a box containing a self-sampler device completely free. After 1 week, we shipped the box, containing the same items listed above.Questionnaires slightly differed among centres owing to length constrains and local priorities. In this study, we analyse only the questions that were almost identical in the three versions: date of women's latest Pap-test, reason for non-compliance with the screening programme (only Rome and Florence), questions about the self-sampling performance , what was mostly appreciated in the self-sampling , which kind of sampling was preferred .All women who resulted in being HPV positive were contacted by phone and letter to propose a counselling on HPV and cervical cancer risk and a colposcopy. If the colposcopy was positive, a biopsy was taken, and if colposcopy was negative, women were suggested to undergo control with Pap-test within a year's time.The main outcome we took into consideration was women's participation in screening: all women who accepted to perform a test in the screening programme within 3 months from the first letter mailing were considered a success.A secondary outcome was the impact on cervical cancer screening coverage, measured as the number of women never-covered or under-covered (latest test performed longer than 3 years earlier) for whom we obtained a cervical sample.Other secondary outcomes were HPV positivity rate and detection rate for CIN2+ in the intervention arm.g for 10 min. Supernatants were discarded and pellets suspended in 1 ml of STM . Before performing Hr-Hybrid Capture 2 (HC2), 200 μl aliquots were stored at −80°C.When samples arrived at the laboratory, they were centrifuged at 500 −1  was used as a cutoff control value, and all samples with an RLU/Control ratio ⩾1.00 were considered HR-HPV positive.Highrisk (HR) HPV was evaluated by HC (Qiagen), using only the B probe mix, which is specific for 12 HR HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and one probably carcinogenic HPV type: 68 (μl of un-denaturated STM samples with QIAamp DNAMini kit (Qiagen) according to the manufacturer's instruction. Amplification of the human β-globin gene was performed using GH20-PC04 primers (268-bp amplicon length) , in any setting (home or screening clinic).We report the relative risk (RR) of having a test comparing each intervention arm with the two controls. Participating centres samples were pooled if there was no heterogeneity.We used the information collected with questionnaires to calculate the impact on population coverage of each strategy according to the following formula: Nine hundred and fifty-one women were randomised in Florence, 800 in Rome and 729 in Abruzzo. Six women were excluded after randomisation in the direct mailing arm because the kit was not mailed owing to a logistical mistake and one was excluded because she had had a Pap-test in the screening programme before randomisation, but it had been notified to the coordinating centre just after the study started.The compliance with standard recall was 13.9%, similar to that obtained with a standard letter inviting women to perform HPV test at the clinic (14.9%). The group which received the self-sampler on demand had a poorer performance as compared with the standard recall letter arm, RR 0.62 (95% CI=0.45–0.86) with no differences among centres. The self-sampler direct mailing strategy had, on average, a higher performance and increased compliance by 40%  with respect to standard recall, but it showed a border line significant heterogeneity among centres. In fact, the positive effect was detectable only in urban areas, Rome and Florence, whereas in Abruzzo, a prevalently rural, small town area, no positive effect was detected.In the direct mailing arm, according to the dates reported in the questionnaires, the median time elapsed from the sampling and the laboratory check in for the specimen was 4 days and only one sample took longer than 1 week (8 days). Nevertheless, in Abruzzo, where the kits were mailed in July with very high environmental temperatures, some specimens looked contaminated by a strong bacterial growth and two samples were unsatisfactory.P=0.006).The overall positivity rate among the self-collected samples in the direct mail arm was 21.8% (22 out of 101). All but two women accepted to perform a colposcopy (91%) and no CIN2+ was found. The positivity rate in the HPV control arm was 6.5% (5 out of 77), and the difference was statistically significant . The woman had a colposcopy and no CIN2+ was found.We collected 147 questionnaires. The most frequent reason for failing to comply with previous screening invitation was recent Pap-test (40.6%). No woman declared that Pap-test is not important and only two of them declared that they did not perform the test because they were embarrassed . Table 2In the open answer questions, some women expressed concerns about the quality of self-collected samples, and six of them complained about broken devices . No woman reported concerns about having the HPV viral test instead of the cytological test, nor about the way of infection.Only six women (4.1%) referred to never having had a Pap-test before and five did not remember (3.4%). Among the others, 12 (8.2%) had had a test longer than 5 years before, and 45 (30.8%) had had it between 3 and 5 years before.The impact on coverage was computed only for Florence and Rome, the very places where a definite gain in coverage has been proved; and for the arm in which an increase in compliance as compared with standard recall has been observed: the direct mailing arm. Out of 91 women responding to the direct mailing of the kit in Florence and Rome, four had never had a Pap-test before and 30 were under-covered, that is, they had undergone a test longer than 3 years earlier, whereas the rest of women had had a Pap-test within 3 years. The proportion of not- or under-covered women on the total randomised sample is 34 out of 438 (7.8%). This proportion must be applied to the population that had not responded to first invitation, that is, the eligible population for the trial. This proportion in Florence and Rome was 65%. According to the formula proposed in the Population and Methods section, the impact of direct mailing on population coverage would be: (34/438) × 0.65=+5.1% (95% CI=3.6–7.1). If we consider under-covered those who had had a Pap-test over 5 years earlier, the impact on compliance would then be 2.2% (95% CI=1.2–3.7).Our results show that delivering self-samplers may increase compliance with cervical cancer screening programmes. Even if the observed heterogeneity was not statistically significant, we must highlight that a positive effect on compliance was observed only in Florence and Rome. Many context factors might explain this difference: there could be cultural and behavioural differences between urban and rural areas, or there may be more logistical problems in shipping self-sampler boxes in rural areas owing to different shape or location of the mail boxes. Finally, in Abruzzo the trial was performed in mid-July, when compliance with screening programmes is usually low and decreases as we get nearer August, when most Italian families are on holidays. In this period, the additional week needed in the direct mailing arm may have had a strong negative influence on compliance. This delay in performing our analysis was owing to the above-mentioned earthquake hitting the Abruzzo region in April 2009, which halted all studies for weeks.We only found one randomised controlled trial comparing the effect of using the self-sampler instead of the standard recall letter for conventional cytology on compliance with screening invitation  is relevant in terms of public health. An increase in population Pap-test coverage of 1 or 2% is what has been obtained by most trials that measured the impact and not only the RR of the intervention If the absolute relevance of such a gain in population health is not questionable, the costs to obtain it must be carefully considered. In our trial, we had to send out five self-samplers to get one sample back to be tested. This number goes up to 13 if we consider the number needed to gain uncovered women to screening. Low response rate and high population coverage owing to overlapping of organised and opportunistic screening are context factors that minimise the advantages and maximise the costs of direct mailing: we must ship many self-samplers to non-responders, but only a few under-covered women are the real target of the intervention. We tried to reduce costs sending the samplers only to women who were interested and requested it, but this strategy had a worse participation rate than standard recall letters. In this trial, we did not investigate self-sampling strategies that might strongly reduce the workload for midwives, that is, not targeted only to non-responders, but also to all the target population.Self-sampling for HPV testing is acceptable to women and mailing a device for HPV DNA self-collection to non-responders to cervical cancer screening may increase compliance as compared with standard recall letters. Nevertheless, effectiveness is context specific. On top of this, the strategy is very expensive in terms of number of self-samplers needed per screened woman.


Invasive cervical cancer is the second most common malignant tumor affecting Brazilian women. Knowledge on Human Papillomavirus (HPV) genotypes in invasive cervical cancer cases is crucial to guide the introduction and further evaluate the impact of new preventive strategies based on HPV. We aimed to provide updated comprehensive data about the HPV types’ distribution in patients with invasive cervical cancer.Fresh tumor tissue samples of histologically confirmed invasive cervical cancer were collected from 175 women attending two cancer reference hospitals from São Paulo State: ICESP and Hospital de Câncer de Barretos. HPV detection and genotyping were performed by the Linear Array HPV Genotyping Test .170 out of 172 valid samples (99%) were HPV DNA positive. The most frequent types were HPV16 (77.6%), HPV18 (12.3%), HPV31 (8.8%), HPV33 (7.1%) and HPV35 (5.9%). Most infections (75%) were caused by individual HPV types. Women with adenocarcinoma were not younger than those with squamous cell carcinoma, as well, as women infected with HPV33 were older than those infected by other HPV types. Some differences between results obtained in the two hospitals were observed: higher overall prevalence of HPV16, absence of single infection by HPV31 and HPV45 was verified in HC-Barretos in comparison to ICESP patients.To our knowledge, this is one of the largest studies made with fresh tumor tissues of invasive cervical cancer cases in Brazil. This study depicted a distinct HPV genotype distribution between two centers that may reflect the local epidemiology of HPV transmission among these populations. Due to the impact of these findings on cervical cancer preventive strategies, extension of this investigation to routine screening populations is warranted. Invasive cervical cancer (ICC) is the third most common neoplasia among women worldwide, representing 8.8% of all cancers. Worldwide 607,402 new cases of ICC are predicted to occur by 2015[condylomata acuminata, and malignant lesions, especially in the uterine cervix[Anogenital infection by HPV is associated with benign lesions, like e cervix. More the cervix,5, 40 ofe cervix and 12 ae cervix. Virtuale cervix,9. HPV16e cervix-12. Sevee cervix-16, folle cervix,14, withTwo prophylactic vaccines against high-risk HPV16 and 18 were licensed, a bivalent (HPV16 and 18) and a quadrivalent one . In Brazil, these two HPV vaccines are available only in private clinics and due to their high cost are not accessible to most of the population. Its inclusion in the national programme of immunization remains under debate. The preBetween November 2009 and July 2011 women with a diagnosis of invasive cervical cancer were recruited from two reference cancer hospitals in São Paulo State: Instituto do Câncer do Estado de São Paulo (ICESP) and Hospital de Câncer de Barretos – Fundação Pio XII (HC-Barretos). ICESP is a tertiary public hospital located in São Paulo city, the largest city in South America, with approximately 11 million inhabitants. HC-Barritu (n = 11) and sample with normal endocervix and lack of ectocervix (n = 1). Therefore, the study analyzed specimens from 175 patients, between 21 and 86 years of age. The histopathological confirmation was made by the local Pathology laboratories. Stage of the disease was coded according to the International Federation of Gynecology and Obstetrics (FIGO) criteria[At the moment of study enrollment and sample collection all patients were naïve to cancer treatment. One hundred and ninety four patients were initially included in the study and had two biopsies collected: one for the histopathological diagnosis and another for the study. Nineteen patients were further excluded due to absence of confirmation of invasive cervical cancer by histopathology. Reasons for exclusion were: absence of invasive cervical cancer (n = 1), sample in which confirmation of cervical origin was not possible (n = 1), CIN I (n = 1), carcinosarcoma (n = 1), endometrial adenocarcinoma (n = 2), cervicitis (n = 1), carcinoma in scriteria. All cliThe study and the written informed consent signed by all patients or their legal representative were approved by the Research Ethical Committees of Hospital das Clínicas-Faculdade de Medicina da Universidade de São Paulo, Instituto do Câncer do Estado de São Paulo (CAPPesq n° 0451/09) and Hospital de Câncer de Barretos – Fundação Pio XII (protocol n°405/2010).At ICESP, fresh tumor tissue was collected and immediately introduced into a tube containing Specimen Transport Medium – STM  and transported to the Virology Laboratory of the Instituto de Medicina Tropical – Universidade de São Paulo (IMT-USP), where they were stored at −20°C. Tumor tissues collected at HC-Barretos were immediately introduced into a cryopreservation tube and sent to the hospital Tumor Bank, where they were stored in liquid nitrogen, until DNA extraction. After DNA extraction, samples were sent to the Virology Laboratory of IMT-USP where all molecular tests were carried out.Tumor tissue DNA was extracted using QIAamp DNA Mini Kit  or NucleoSpin Tissue kit , according to manufacturer’s instructions.DNA extracted from the 175 samples were submitted to Linear Array HPV Genotyping Test  for HPV detection and genotyping. This commercial available assay is able to detect 37 HPV types , 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39 and CP6108).http://www.cdc.gov/epiinfo). ANOVA, Yates corrected chi square test or Fisher exact test, as appropriate, assuming two-sided tests and a level of significance of 0.05.Statistical analyses were performed with EpiInfo6 software  patients were recruited at ICESP and 92 (53.5%) at HC-Barretos. Patient’s age ranged from 21 to 86 with a mean of 51.9 years (95% CI 49.7-54.1). Patients from ICESP  had similar mean age of the HC-Barretos’ patients  (p = 0.85). Five patients from ICESP (6.2%) were less than 30 years old. After histological assessment, 144 (83.7%) tumors were classified as squamous cell carcinoma (SCC) and 28 (16.3%) as adenocarcinoma (ADC). The two hospitals had similar frequencies of ADC (ICESP 16.2% versus HC-Barretos 16.3%) (p = 0.99). Women with ADC had a similar age of women with SCC (p = 0.91) , 170 (98.8%) were HPV positive. One HPV negative sample was from ICESP and the other one from HC-Barretos. Further investigation with additional amplification methods verified the presence of HPV DNA in those as well (data not shown). Most infections were identified as single infections (75.3%).The frequency of HPV types is illustrated in Figure The mean age at diagnosis of invasive cervical cancer of patients infected only by HPV33 are higher than the overall study population and that from patients infected only by HPV16 Figure .Differences in the relative contributions of HPV types by histological diagnosis were identified. ADC was correlated to HPV59 (p = 0.04) in the study population, but when the analysis was made for each hospital separately no correlation between HPV type and histological diagnosis was observed Table .The present study evaluated geographic differences in HPV genotypes among Brazilian women with invasive cervical cancer recruited at two cancer reference hospitals located at the same region of the country. Some differences between the results obtained in the two hospitals were observed and could be attributable to the fact that these hospitals attend populations with different origins and characteristics, as patterns of sexual behavior .Previous studies that evaluated ICC cases from Brazilian women were conducted in all five regions of the country: Eluf-Neto and colleagues 1994) evaluated 199 ICC cases from São Paulo city (Southeast region), Noronha94 evaluaIn our study, 98.8% of the samples were HPV positive, corroborating literature data stating that HPV infection is a necessary cause of ICC[The higher HPV prevalence obtained in our study in comparison to others Brazilian studies that obtained rates of 70.3% to 92%,14,16,19In the present study the observed rate of single infections (75.3%) is lower than reported by others,16,20,22Our study confirms the universal contribution of the eight most common HPV types  in ICC samples. Studies conducted in Brazil show that HPV16 is the most common type followed by HPV18,14,16,20Our data shows that patients from both hospitals had similar mean age, around 50 years old that is in agreement with an international study that evaluated 10,575 ICC cases and reported a mean age of 51.4 years. BraziliThe majority of samples were histologically classified as SCC cases, while ADC was found in 16.3% of cases, a ratio similar to that described by other studies,12, but Papillomaviridae family alpha-7 species)[We did not observe the correlation of HPV18 and HPV45 with ADC as reported by others-13,16. Ospecies).The current series confirms that the detection of exclusive low-risk HPV type in ICC is a rare event (one sample). This case, occurring in an HIV-infected heavily immunosuppressed patient, was described in more details elsewhere and remains unclear if and how a low-risk HPV type may induce ICC. HPV16 aThe women infected by HPV18 and 45 in our study had similar age at ICC diagnosis compared to the global study population, in disagreement with the lower average age of women ascribed to these HPV types reported in other studies,22. ThisTo our knowledge, this is one of the largest studies employing fresh tumor tissue of invasive cervical cancer cases made in Brazil. The present study confirms the continuing major role of HPV16 and 18 in invasive cervical cancer on recent cases in Brazil, especially at HC-Barretos. Fortunately, available vaccines do include these HPV genotypes, predicting a significant decrease in ICC future incidence over vaccinated women, as only 10.0% of cases would not be target by the current vaccines. Also, ICC screening by existing molecular assays would not require the inclusion of additional HPV genotypes, as they don’t seem to contribute significantly to ICC epidemiology in the Brazilian population.HPV: Human papillomavirus; ICC: Invasive cervical câncer; ADC: Adenocarcinoma; SCC: Squamous cell carcinoma; ICESP: Instituto do Câncer do Estado de São Paulo; HC-Barretos: Hospital de Câncer de Barretos – Fundação Pio XII; INCA: Instituto Nacional de Câncer; FIGO: International Federation of Gynecology and Obstetrics; STM: Specimen transport medium; PCR: Polymerase chain reaction; LA: Linear Array HPV Genotyping Test.The authors declare that they have no competing interests.CMO was involved in the design of the study, helped in the collection of clinical data, performed the molecular tests and analysis and drafted the manuscript. JHTGF enrolled the study patients at HC-Barretos, collected the biological samples, collected the clinical data and made a critical review of the manuscript. JPC enrolled the study patients at ICESP, collected the biological samples and clinical data and made a critical review of the manuscript. ADF collected the clinical data and made a critical review of the manuscript. JEL conceived the study and was involved in its design, supervised the study, helped to analyze the data and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/357/prepub