The HVP vaccine is a useful tool for preventing cervical cancer. The purpose of this study is to determine the most frequent HPV genotypes in Equatorial Guinea in order to develop future vaccination strategies to apply in this country.A campaign against cervical cancer was carried out in the area on a total of 1,680 women. 26 of the women, following cytological screening, were treated surgically with a loop electrosurgical excision procedure (LEEP). Cases were studied histologically and were genotyped from paraffin blocks by applying a commercial kit that recognized 35 HPV types.Cytological diagnoses included 17 HSIL, 1 LSIL, 5 ASC-H and 3 AGUS. Histological diagnosis resulted in 3 cases of microinvasive squamous cell carcinoma stage IA of FIGO, 9 CIN-3, 8 CIN-2, 2 CIN-1, 3 flat condylomas and mild dysplasia of the endocervical epithelium. Fifteen of twenty-five cases genotyped were positive for HPV (60%). HPV 16 and 33 were identified in four cases each, HPV 58 in two other cases, and HPV 18, 31, 52, and 82 in one case, with one HPV 16 and 58 coinfection.The frequency of HPV types in the African area varies in comparison to other regions, particularly in Europe and USA. Vaccination against the five most common HPV types  should be considered in the geographic region of West Africa and specifically in Equatorial Guinea. Cervical cancer is the second most common malignancy in women worldwide, and contributes to 9.8% of all female cancers . In sub-Human papillomaviruses (HPVs) are a group of viruses associated with benign and malignant neoplasms of cutaneous and mucosal epithelia . It is eMore than 100 HPV types have been identified, and about 40 can infect the genital tract . CurrentHPV 16, HPV 18, HPV 31, HPV 58 and HPV 52 are the five most common HPV types in cytologically normal women. They also cause 50% of all HPV infections [HPV has a strong association with cervical carcinoma and its precursor lesions. In particular, the presence of HPV precedes and predicts the development of a squamous intraepithelial lesion (SIL) . Among lMoreover, the progression of LSIL to a malignant lesion is clearly different depending on the type of HPV infection. Both LSIL HPV 18 positive or HPV 16 positive have a higher probability of progression to cervical cancer than those containing other types of HPV. As a result, it is very important to know the type of HPV infection in order to differentiate women with a high risk of developing cancer from those with lower risk (who could be monitored carefully over a long period of time) .Worldwide, HPV types 16 and/or 18 are detected in more than 50% of all squamous intraepithelial high-grade lesions (HSILs), 70% of infections are detected in invasive cancer and 81.5% of infections are detected in adenocarcinomas . These tAll of the above implies that an effective vaccine against the five most common HPV types  could prevent approximately 90% of cervical cancer cases that occur worldwide. However, regional variations in the distribution of certain types of HPV should be considered in the development of vaccines tailored to different geographic regions [Samples were obtained from a cervical cancer screening campaign carried out on the island of Bioko  between May and October 2006. The study included a total of 1680 women between 15 and 71 years of age. None of the women had been screened previously for cervical cytological abnormalities or HPV infection.Study procedures were approved by local authorities. An informed consent was obtained and a face-to-face interview was conducted, soliciting information on the women's demographic characteristics and reproductive history. A general physical and detailed gynecological examination were also carried out. Following a visual inspection of the neck of the uterus, exfoliated cervical cell samples were obtained using the Papanicolaou method. Cytological smears were analysed in Madrid (Spain). Abnormalities were classified with the Bethesda classification system as falling within normal limits or reactive cellular changes , atypical squamous cells of undetermined significance (ASCUS), low-grade SIL (LSIL), high-grade SIL (HSIL), or carcinoma.In the second phase, we performed surgery on those women cytologically diagnosed with ASCUS, high-grade SIL, and carcinoma. Twenty-six out of thirty women (between 22 and 68 years of age) were able to undergo surgery. The surgical treatment chosen was loop diathermy conization; that is, LEEP . The surgical samples were obtained, processed histologically and analyzed in Spain, showing a high positive correlation between the cytological and pathological final diagnosis.Samples used for the detection and genotyping of HPV were obtained from paraffin blocks. The lesion area of each paraffin block was selected microscopically and punches of the same size (1 mm × 7 mm). One to three different samples were used to extract DNA. Twenty five out of twenty six cases were HPV genotyped. The remaining case ended after being histopathologically studied.Samples were shipped to Genomica Laboratory. HPV-DNA was isolated with K proteinase.® Human Papillomavirus  kit was used for HPV DNA genotyping. HPV testing was carried out by personnel who had no knowledge of the subject's medical history or cytologic diagnosis.The ClartAll samples were analysed for the presence of the following HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89.HPV detection was conducted using PGMYO9/PGMY11 general consensus primers designed to amplify a 450 bp HPV L1 gene fragment and two different controls: genomic DNA and PCR to insure a feasible assay.The detection of the amplified PCR product was performed with a new technological platform using a low density Microarray, anchored in 2 ml tube-AT tube. It allows simultaneous detection of multiple molecular markers in the L1 fragment of 35 different HPV types and in the necessary controls to insure a feasible assay. Finally, for each specific probe the array was printed in triplicate, so each sample was identified in triplicate. The entire process was carried out according to the manufacturer's standard protocol.PCR products were marked with biotin and, after amplification, they hybridised with the respective probes in specific known AT tube areas. Biotin binds to streptavidin-peroxidase after incubation. The addition of the TMB substrate  generates an insoluble product after hybridization.The results were processed using a software program which makes detection, interpretation and reporting for each sample possible.The 10 negative samples for HPV detection were re-evaluated. The initial DNA extraction and new samples for each case were obtained, following the same procedures explained above. For the new paraffin embedded samples a commercially available tissue DNA extraction kit was used .125 ngm of nucleic acid was used for each 50 μl PCR reaction with Taq DNA Polymerase (Perkin-Elmer Hispania). Each PCR reaction contained primers for the intron E6 of the HPV-16 or Beta-globin, following the the protocol described by Shibata et al. (1988) . Beta-glThe 50 μl DNA amplification reactions for HPV-16 contained 2.5 μl of Buffer I  gelatin - Perkin Elmer Hispania), 0.15 mM of the dNTP mixture, 15 pmol of each primer, 2.5 units of Taq polymerase, and 1 μl of DNA.Before adding the enzyme, we did a hot start at 95°C for 10 min., followed by 35 cycles: 45 sec. at 96°C for denaturalization, 45 sec at 55°C for annealing, and 50 sec. at 72°C for extension, with a final extension step at 75°C for 10 min. A second PCR amplification was performed in the same conditions with 1 μl of the previous product as a template. The final product was detected in a 3% agarose gel electrophoresis stained with ethidium bromide. Each PCR reaction and the subsequent hybridization contained negative and positive controls. The last one was a CIN-III, positive for HPV-16 obtained by situ hybridization and PCR amplification of the L1 region.Cytologic diagnoses included 17 cases of HSIL, 1 case of LSIL, 5 cases of ASC-H and 3 cases of AGUS Table . The patHPV DNA was detected in 60% of samples Figures  and 2. TIn the histological diagnosis we have detected HPV DNA in all microinvasive squamous cell carcinomas. HPV 16, HPV 33 and HPV 58 were found in these samples.Cervical intraepithelial neoplasia  had HPV DNA in 63,2% of our cases. HPV 16 and 33 were the predominant types identified in each of the three samples. These rates were followed in frequency by the HPV 58, in two samples. Finally, each HPVs 18, 31, 52 and 82 were only found in one sample.Twenty-five percent of flat condyloma or mild dysplasia of the epithelium (one out of four samples) showed HPV 16. The cytologic diagnosis of this sample was ASC-H.We have not found any of the 35 HPV genotypes in a high percentage of samples. Since reactivity for the beta-globin gene excluded DNA absence, we have analysed the most prevalent HPV types and samples corresponding to different cytological and histological diagnosis. Therefore, the results could probably be due to the absence of HPV infection. However, presence of some other HPV type, not detected by our method, or an HPV X should not be rejected. The presence of an uncharacterized type (HPV X) in the African continent has already been suggested in another study in Kenya .In different African regions the prevalence of HPV in women with normal cytology and, therefore, prone to cervical cancer, is very high. In particular, the prevalence of HPV in northern Africa (Morocco) was 21.5%, 31.6% in East Africa , 17% in West Africa  and 15.5% in southern Africa (South Africa) [It has been shown that women with cervical cancer in Africa usually have the following HPV types, in descending order, 16, 18, 33, 45, 35, 31, 58, and 52. On the other hand, HPVs 16, 33, 31, 18, 52, 58, 35 and 56 are those most frequently present in women with HSIL .We have found six of these types in our study . Our results reinforced the prevalence of 16 and 33 HPV types and highlighted the high percentage of positive cases found for HPV 58 (12%). We also highlight the lack of detection of HPV 45, although its prevalence in cervical cancer has been considered above average in Africa as compared to other geographical areas.A number of other papers has been published, determining the different HPV types in each African region. Analysis of the data obtained is relevant for the design of effective vaccines against cervical cancer in African regions.In Kenya (East Africa), a study was carried out on women (with or without pathology) that were clients of a family planning clinic. The study shows that the three most common HPVs types in women with HSIL were: HPVs 16 (37.7% of positive samples), HPV 52 (25.0%) and HPV 35 (17.9%). These researchers also reported an uncharacterized HPV type X in 15.8% of the positive samples [In another study from Kenya (East Africa), aimed at women with invasive cervical carcinoma (ICC), the most frequently HPV type detected was 16 (43.8% in HIV-negative and 41.2% in HIV-positive women). The second most common type was HPV 18 (nearly 20% in HIV-negative and nearly 30% in HIV-positive). The most frequently found types of HPV in HIV-negative women were the following: HPV 45 (17.0%) and HPVs 35 and 52 (5.2% each). In HIV positive women, the most frequently found types of HPV were: HPV 52 (19.6%) and HPVs 35, 45 and 56 (7.8% each) . This stHPV 35 was found slightly more often than HPV 16 in Mozambique (southeast Africa), both in women with normal cytology and in those with HSIL or more severe neoplasms . In SoutIn Senegal (northwest Africa), while the rate of LSIL lesions were found to be associated with the detection of HPV types 58 and 16, the risk of HSIL/cancer was found to be associated mainly with the presence of infection with some high-risk HPV types and, in particular, with HPVs 16, 58, 33 and 52, and also with types 31 and 18 [In Ivory Coast (West Africa) women with LSIL, the most frequent HPVs were HPV 16 (12.2%), HPV 18 (6.9%), HPV 31 and 33 (6.1% each) and HPV 70 (5.3%). In women with HSIL from the same region, the most frequent HPVs were HPV 16 (30.6%), HPV 18 (10.2%) and HPVs 33, 58 and 70 (8.2% each) . In NigeTo sum up, HPV types in women with cervical pathology in Africa are somewhat different from those in Europe and the USA, and they also vary in different regions of Africa. The three most prevalent HPV types were 16, 58 and 33. Other HPVs have appeared quite frequently in our study and have also been persistently detected in previous studies: HPV 18, HPV 31 and HPV 52 Table .The prevalence of HPV 16 in cervical cancer is higher in Europe than in other continents. This may be due to the fact that HPV 16 infection appears to be less favoured by the deterioration of immune status. Therefore, defects in cellular immunity  could contribute to a different frequency of HPV types in certain populations, i.e., those in Africa as compared to other countries .We have concluded that our results are in agreement with studies published on this subject in recent years, and especially with studies in West Africa. We have found the same six most prevalent HPV types in Senegal and Ivory Coast Table . MoreoveIt is estimated that the currently available 16/18 HPVs vaccines could prevent some 350,000 cases of cervical cancer annually. An extension to the following six most common HPV types  could lead to an increase in protection of about 90%, the equivalent of 440,000 cases of preventable cervical cancer per year. It would be a good idea to take regional differences into account when preparing for the inclusion of the HPVs type , in caseAs a result, in the African regions, where the prevalence of different HPV types varies in comparison to what was found elsewhere in the world and particularly in Europe and the USA, the new vaccination strategies against HPV should take into account the most prevalent HPV types in a given population. In fact, other studies have suggested the need to address the HPV 58 during the development of the strategic approach for vaccination against cervical cancer in the geographical region of northwest Africa  . Thus, oOur study gives a valuable description of the most prevalent HPV types in West Africa and confirms other studies found in the same region. The results obtained are relevant due to the scarcity of similar studies conducted in the geographical area in which Equatorial Guinea is located. In fact, the present study is the only one that has been carried out in this country.The difficulty of obtaining cases is an ostensible weakness due to the number of cases we were, in the end, able to study, as well as the affect this has on the study's significance. It would be of interest to obtain more cases in the future for further study.HPV: Human papilloma virus; LEEP: Loop electrosurgical excision procedure; ASC-H: Atypical squamous cells-cannot exclude HSIL; AGUS: Atypical glandular cells of undetermined significance; LSIL: Low-grade squamous intraepithelial lesion; HSIL: High-grade squamous intraepithelial lesion; SCC/ICC: Squamous cell carcinoma; invasive cervical carcinoma; CIN: Cervical intraepithelial neoplasia; VIA: Visual inspection of the uterine cervix with acetic acid.® Human Papillomavirus kit related to the content of the manuscript.Genómica S.A.U.  hold the patent of The ClartBGE participated in the design of the study, DNA samples acquisition and analysis, global data analysis and preparation of the first draft. MPNB revised critically the first and final draft, data analysis. SR genotyping data adquisition, carried out the molecular genetic studies. FSS conceived the desing of the study, conceptual and design of the Equitorial Guinea campaign, clinical attendance and surgical performance. MCPM and PCC clinical attendance and surgical performance, data adquisition. LCC planning of the citological studies and data acquisition. EMR conceptual and planning of the design of the study, surgical pathology data acquisition and analysis, final editing. All authors read and approved the final manuscript.


While many studies have compared the efficacy of Pap cytology, visual inspection with acetic acid (VIA) and human papillomavirus (HPV) DNA assays for the detection cervical intraepithelial neoplasia and cancer, few have evaluated the program effectiveness.A population-based sample of 5603 women from Medchal Mandal in Andhra Pradesh, India were invited to participate in a study comparing Pap cytology, VIA, and HPV DNA screening for the detection of CIN3+. Participation in primary screening and all subsequent follow-up visits was rigorously tracked. A 20% random sample of all women screened, in addition to all women with a positive screening test result underwent colposcopy with directed biopsy for final diagnosis. Sensitivity, specificity, positive and negative predictive values were adjusted for verification bias. HPV testing had a higher sensitivity (100%) and specificity (90.6%) compared to Pap cytology  and VIA . Since 58% of the sample refused involvement and another 28% refused colposcopy or biopsy, we estimated that potentially 87.6% of the total underlying cases of CIN3 and cancer may have been missed due to program failures.We conclude that despite our use of available resources, infrastructure, and guidelines for cervical cancer screening implementation in resource limited areas, community participation and non-compliance remain the major obstacles to successful reduction in cervical cancer mortality in this Indian population. HPV DNA testing was both more sensitive and specific than Pap cytology and VIA. The use of a less invasive and more user-friendly primary screening strategy (such as self-collected swabs for HPV DNA testing) may be required to achieve the coverage necessary for effective reduction in cervical cancer mortality. Broad adoption of effective Pap smear screening programs is credited with a substantial reduction in cervical cancer incidence in many countries, but it has not been possible to implement this strategy in the developing world, which bears 80% of the global burden of cervical cancer. The reasons for this failure include a lack of infrastructure, requirement of specialized training, requirement of multiple visits by the woman for follow-up and treatment, the difficulties in implementing quality controls for the procedures, and lack of facilities to provide the needed treatment. Therefore, over the last decade, efforts to reduce the global cervical cancer burden through screening have focused on development and evaluation of alternative screening assays to the Pap smear. Two such assays have been widely promoted: visual inspection of the cervix following acetic acid application (VIA) and molecular tests for the presence of high risk human papillomavirus (HR-HPV) infection While both of these assays have been rigorously evaluated in controlled research settings The study protocol was approved by the Institutional Review Boards at SHARE India, MediCiti Institute for Medical Sciences and Johns Hopkins University. Written informed consent was obtained by signature or thumbprint.http://www.sharehealth.net/). The REACH project is centered in MediCiti Institute of Medical Sciences (MIMS) centrally located in the rural community it serves, 32.4 km north of Hyderabad. Combined use of ambulatory units staffed with a doctor, nurse and health supervisor and a team of community health volunteers (CHVs) facilitates frequent contact with the population in their communities. The REACH project is supported by intensive information technology (IT), with enumeration of the population in the targeted areas specifically, in Medchal Mandal in Ranga Reddy District, Andhra Pradesh. The census data are acquired by household surveys, and contact information as well as pertinent health indicators for the members of each household are maintained by computerized database. These data are updated annually. The combined resource of up-to-date census information and mobile health units afford a unique opportunity for evaluation of screening programs.REACH, or Rural Effective Affordable Comprehensive Healthcare, was designed by SHARE India, a non-governmental organization, to develop a replicable working model of heath care delivery that offers preventive, primary, and secondary health care to a rural population  and were then transported back to their villages. To obtain data for correction of verification bias, 20% of the enrolling women were randomized to receive colposcopy (immediate colposcopy arm) on the day of the enrollment screening exam regardless of screening test results. As soon as the results of all three tests were available, in about 3–4 weeks after the screening visit, the women were contacted at their homes and informed of their test results by a health supervisor. Women who were positive by any one or more of the screening tests  were asked to return to the hospital for a colposcopic examination. The screening and any required treatment were provided at no cost to the participant, who was also provided lunch and 2 kg of rice as an incentive for participation.Sarpanch) and the MediCiti CHV were contacted by the REACH project community liaison and the project health counselors to explain the project and obtain support to recruit in the village. Preparatory mass education programs on gynecologic health and cervical cancer prevention were delivered to the community prior to systematic house-to-house recruitment efforts. In the second phase, age-eligible women were recruited from their house by a team of health supervisors and health counselors. Additional education and detailed explanation of the screening goals and procedures was provided at that time. This ‘motivation’ phase was conducted in the evenings, and women who expressed interest and willingness to be screened were scheduled to be picked up by hospital vehicles the following morning for transportation to the screening clinic. Women came to the hospital in groups of 5-30 women per day (average 10 per day).Recruitment was conducted in two phases, systematically covering one village at a time. In the first preparatory phase, the elected village leader  ecto- and endo-cervical cells for Pap smear, exfoliated cervical cells for HPV DNA testing, and evaluated the cervix after acetic acid application (VIA). Any abnormalities found after examination of the vulva, vagina, and cervix  were recorded on a standardized pelvic exam form. This form also allowed systematic recording of any prescribed medication as a result of the pelvic exam . For the women randomized to receive colposcopy at enrollment, the colposcopic exam was performed after collection of specimens for Pap smear and HPV DNA testing and after conducting VIA.After removing any obscuring mucus from the cervix with a cotton swab, exfoliated ectocervical cells were collected and smeared onto a glass slide using an Ayres spatula. Endocervical cells were collected by endocervical brush and placed onto the same slide. Cells were fixed by placing the slides in ethanol. Slides were stained according to standard protocols, and reviewed by a trained local cytopathologist, who recorded the cytologic diagnosis on standardized forms according to the 2001 Bethesda System Following collection of the Pap smear, a Digene conical brush sampler was placed in the cervical os, rotated 360° three times, removed, and placed into 1 ml of Digene standard transport medium (STM). Samples were stored at 4°C for no more than 24 hours after collection before aliquoting and long term storage at −20°C. HPV DNA testing was performed locally using the hybrid capture 2 (hc2) test according to the manufacturer's instructions. Samples with an RLU/CO value ≥1.0 were considered to be positive for high risk HPV.Using standard interpretation guides Women requiring colposcopic examination because of a positive screening test result were contacted at their homes, and a hospital vehicle was provided for transportation to and from the clinic. The colposcopist was aware that referred patients had at least one positive screening test, but was masked to the specific VIA, Pap, and HPV DNA results. Even though the VIA test results were available immediately at the time of screening, referral to colposcopy was made only after all three test results were available. Biopsies were taken from any suspicious lesion. All biopsies were read locally, and were subsequently reviewed by an expert pathologist at Johns Hopkins Medical Institutions (JHMI). Women with histologically confirmed cervical intraepithelial neoplasia (CIN) grade 2 or 3 by either local or JHMI review were referred for treatment by LEEP/cold knife conization or hysterectomy. Women found to have operable invasive cancer were treated at MIMS. Women requiring radiotherapy were referred to the government cancer hospital. A release form was signed by any woman with CIN2+ who refused treatment.Differences in test positivity by age were assessed using Pearson's chi-square tests. Test agreement was measured using kappa statistics with 95% confidence intervals. The agreement between the JHMI and MIMS diagnosis of <CIN2 vs. CIN2+ was good . For this analysis, cases were defined based on the JHMI pathology diagnosis. Diagnostic accuracy statistics  were calculated for each of Pap, VIA, and HPV testing for each of CIN2+ and CIN3+ outcomes. Crude estimates of diagnostic accuracy were biased due to non-random exclusion of women who did not have the opportunity for full diagnostic verification  The degree of participation in the study and compliance with screening and diagnostic follow-up is summarized in shobhanam) as the best surrogate marker of sexual debut. These data were normally distributed with a mean age at shobhanam of 15.6 years (SD 2.8). A few women (13.1%) reported use of tobacco products, and the majority of this was pan (betel leaf) use (93.4%). A more substantial fraction reported passive tobacco smoke exposure (37.9%). Most women were parous (96.8%), with a median of 3 live births. Most women (95.5%) reported no previous pap screening.The baseline demographics of the enrolled population are presented in The population prevalence of positive results was similar by screening method . HPV prevalence did not vary significantly by age (p = 0.44), while Pap prevalence increased significantly with increasing age (p<0.001)) . VIA preA total of 733 (31.4%) women were positive by one or more tests, while only 16 (0.7%) were positive for all three tests. The agreement was better between Pap and HPV DNA tests compared to either of these assays and VIA, but agreement beyond that expected by chance was poor in all comparisons (kappa range 0.04–0.11). In order to better understand the disagreement between test results, we tested approximately 19% of VIA- and Pap-positive, but hc2 negative samples using consensus primer PCR We detected a total of 19 CIN2+ cases; 8 CIN2, 7 CIN3, and 4 invasive cancers. We first calculated the crude assay performance only among those women who had a colposcopic examination and did not refuse biopsy when indicated (N = 675); normal colposcopy results were considered to be negative for CIN2+. Our results show clear performance differences between the assays , with HPAdjusted test performance. Using inverse-probability weighting, we applied the CIN2+ and CIN3+ rates from women with colposcopy-biopsy results to the full cohort, as described in Methods. One case of CIN2 was detected among the 304 screen-negative women in the immediate colposcopy arm, for a disease prevalence of 0.33%. The verification bias adjusted estimates of test performance are shown in Using the data generated from applying the probability weights to the entire eligible cohort (N = 5603), we estimated a CIN2+ prevalence of 3.8% and a CIN3+ prevalence of 1.6%. This model predicts that 57.1 ‘true positive’ cases of CIN2+ (23 cases of CIN3+) would have been detected with complete follow-up (100% colposcopy and 100% biopsy where indicated), and an additional 137.3 ‘true positive’ cases of CIN2+ (55.4 cases of CIN3+) would have been detected if the entire eligible population had been screened. Of the 19 women who had CIN2+, we provided LEEP for 2, hysterectomy for 9, and referral to the cancer hospital for radiation therapy for 4 women who had invasive cancer. Four women refused treatment despite several direct visits by the study gynecologist for direct counseling.When evaluated as a complete program, the greatest threat to realizing a reduction in cervical cancer mortality was non-participation in the program and among those who participated, non-compliance with some aspects of the screening requirements, rather than the use of a less accurate screening test. Using standard population-weighted methods to correct for incomplete verification of disease status, we estimated that 86.2% of the potential underlying cases of CIN2+ may have been missed due to program failures; 27.8% as a result of incomplete compliance with follow-up procedures , and 58.4% as a result of non-participation in the screening program. We therefore conclude that despite our use of available resources, infrastructure, and guidelines for cervical cancer screening implementation in resource limited areas developed by the Alliance for Cervical Cancer Prevention (ACCP) Participation even in the primary screening phase in our study was low (38%). Evaluations of the screening test alternatives reporting coverage typically show participation rates at or above the 70% threshold thought to be required for effective reductions in cervical cancer mortality A detailed assessment of reasons for refusal will be described separately; in general, older women and women with lower household income were least likely to participate. We conducted focus groups to understand the attitudes of the women in our population toward participation in the screening program. Reluctance to participate was related to the perception that there was no need to go the clinic when they had no symptoms. When probed further, they cited fear/anxiety as a significant factor for reluctance to participate; fear of a cancer diagnosis, of pelvic examination and of community gossip and perception (unpublished data). We revised our educational material in response to the concerns elicited through the focus group. While we did not see any change in participation among women with the lowest reported income, participation improved in the middle- and high-income groups as a result of this intervention.The reasons for non-participation were very similar to those reported by Basu Among women screened, substantial differences in test performance for Pap smear, VIA, and HPV DNA tests were observed. As expected, adjustment for verification bias decreased sensitivity and increased specificity for all tests. The sensitivity estimates for HPV testing, particularly for detection of CIN3+, are generally consistent with prior studies When we removed CIN2 from the case definition, the performance estimates increased for all tests. Verification bias-adjusted sensitivity for CIN3+ was 100.0% for HPV, 78.2% for Pap, and 31.6% for VIA. The uncertainty of CIN2 diagnoses in detecting women with true precancerous lesions is well-described Our study has limitations. The low number of cases detected in our study likely reflects an under-ascertainment of disease. Our case definition was based on histologically confirmed CIN2+ from colposcopically-directed biopsy. This is a flawed reference standard Compliance with follow-up colposcopy and biopsy were also low in our study. Losses to follow-up are well-described barriers to cervical cancer screening in resource-limited populations, and therefore the colposcopic refusal results are not entirely unexpected. Our follow-up may have been lower than that reported in other studies Our study also has several strengths. Use of the census data from an entire Mandal allowed us to calculate population-based estimates of test performance as well as compliance with screening and diagnosis. In addition, each woman received all three screening tests, allowing for direct comparison of test performance, which was not possible in large community randomized trials. Use of PCR testing to confirm discrepant results also enhanced our understanding of the lack of correlation in the screening test results by providing a second objective measure.In conclusion, our results suggest that currently proposed algorithms for cervical cancer screening in resource limited regions are still plagued by infrastructural and compliance barriers. We have previously reported that participation rates may increase substantially if the primary screening is offered in the village by self-collection of samples for HPV testing


HPV burden is a predictor for high-grade cervical intraepithelial neoplasia and cancer. The natural history of HPV load in young women being recently exposed to HPV is described in this paper.A total of 636 female university students were followed for 2 years. Cervical specimens with HPV-16, -18, -31, or -45 DNA by consensus PCR were further evaluated with type-specific and β-globin real-time PCR assays. Proportional hazards regression was used to estimate hazard ratios (HR) of infection clearance. Generalized estimating equations assessed whether HPV loads was predictive of HPV infection at the subsequent visit.HPV loads were consistently higher among women <25 years old, and those who had multiple sex partners, multiple HPV type infections and smokers. HPV-16 integration was encountered only in one sample. Infection clearance was faster among women at lower tertiles of HPV-16 , HPV-18  or combined  DNA loads. The relationship between HPV-16 and HPV-18 DNA loads and infection clearance followed a clear dose-response pattern, after adjusting for age and number of sexual partners. GEE Odds Ratios for HPV persistence of the middle and upper tertiles relative to the lower tertile were 2.7 and 3.0 for HPV-16 and 3.8 and 39.1 for HPV-18, respectively. There was no association between HPV-31 or -45 DNA loads and persistence.The association between HPV load and persistence is not uniform across high-risk genital genotypes. HPV-16 integration was only rarely demonstrated in young women. Sexually active women are at risk for genital infection by human papillomavirus (HPV). Most genital HPV infections regress within two years and only a minority of women will develop persistent HPV infection that could eventually cause cervical intraepithelial neoplasia (CIN). High-grade CIN (grades 2 and 3) are precursors of invasive cervical cancer. Although measuring persistence has prognostic value in understanding the natural history of HPV infection and CIN, there is a need for studying additional virological endpoints to assist in risk prediction.HPV-16 DNA load seems to be independently associated with high-grade CIN and invasive cancer ,7-11. HPIntegration of high oncogenic risk HPV types (HR-HPV) is considered to be a key event in the progression of CIN to invasive cancer . Recent The focus of the current study was to assess prospectively, in this cohort of young women, the time course and association between HPV-16 integration, HPV-16, 18, 31 and 45 DNA loads, and type-specific viral outcomes.Female students attending either McGill University or Concordia University Health Clinics were invited to participate if they intended to remain in Montreal for the next 2 years and had not been treated for cervical disease in the last 12 months . A totalAmpliTaq gold  and with the Line blot assay for the detection of 27 genital HPV genotypes . Each assay consistently detected 10 HPV DNA copies (data not shown). HPV viral loads were expressed as the number of HPV DNA copies per cell.A total of 382 specimens collected from 183 participants were positive for HPV-16, -18, -31 or -45 DNA. Nineteen women were infected concurrently with two or more of these genotypes. Extracted DNA from these samples was first screened for the presence of PCR inhibitors by amplification of internal controls for HPV-16, HPV-18, HPV-31 or HPV-45, and for ί-globin DNA, as described previously . The preThe presence of integrated HPV-16 was investigated with real-time PCR assays targeting E6 and E2, as previously described . Since dHPV-16 integration was confirmed by restriction site PCR (RS-PCR), a sequencing technique that demonstrates the presence of HPV-16 and human DNA junctions in the same amplicon . BrieflyWe used Wilcoxon rank sum tests to compare type-specific viral loads among visits. The strength of the correlations among log-transformed viral load measurements at entry and follow-up visits for all four HPV types was measured using Pearson correlation coefficient (r). Geometric means of viral loads were calculated as a function of selected characteristics reported at the index visit of first positivity for a specific HPV type. The Kaplan-Meier technique was used to estimate the cumulative probability of infection clearance as a function of time since enrolment into the study. Proportional hazards regression was used to estimate the relative rate of clearance of type-specific infection, stratified by tertiles of the respective viral load distributions . LogistiThe 636 women enrolled in the McGill-Concordia cohort contributed 2694 completed visits (mean of 4.2 visits/subject) during an average of 21.5 months of follow-up. Nearly all cervical specimens  were suitable for HPV testing. Among those enrolled in the cohort, 68% completed all 5 visits, contributing a total of 13,353 woman-months of follow-up. The mean age of participants was 23 years . Four in five women reported that they were Caucasian, and one in four are smokers. Nearly half of participants had more than 4 lifetime sexual partners. The prevalence , cumulative incidence rates (cumulative incidence of any HPV = 36% and high risk HPV = 29% in 2 years) and mean duration of HPV infections have been reported elsewhere [Samples positive for HPV-16 , HPV-18 , HPV-31 , or HPV-45 , were further tested with type-specific quantitative real-time PCR assays. Nineteen samples contained more than one of the studied genotype. Descriptive statistics of log-transformed HPV loads stratified by baseline and follow-up time points are presented in Figure To investigate the consistency of type-specific viral load over time we calculated correlation coefficients for measurements taken in all possible pairs of visits Table . HPV loaTable Once detected, 30% of HPV-16, 44% of HPV-18, 63% of HPV-31 and 55% of HPV-45 infections cleared within 6 months. Table The association between HPV viral load and persistence of infection was investigated for each genotype by calculating crude and adjusted  GEE ORs Table . HPV perWe then investigated if persistent HPV-16 infection and/or high HPV-16 viral loads in young women could result in integration of HPV-16 DNA into the human genome. Forty-eight specimens were excluded from this analysis because they contained <15 copies of HPV-16 DNA per μl of extracted DNA. HPV-16 E6 and E2 DNA were thus quantitated on the remaining 172 HPV-16-positive samples that had sufficiently high viral load to permit reliable measurement of integration. The mean HPV-16 E6 viral load on the selected samples was 57.5 ± 324.6 DNA copies per cell . The mean HPV-16 E6/E2 ratio was 0.97 ± 0.25 . Of the 172 specimens analyzed, 169 had a HPV-16 E6/E2 ratio <1.5, two samples had HPV-16 a E6/E2 ratio >1.5 and <2.0 (1.54 and 1.70) and one had a ratio above 2. Samples collected at consecutive visits from the two participants with HPV-16 ratios of 1.54 and 1.70 yielded HPV-16 E6/E2 ratios near or below 1.0 (data not shown). The only sample collected from a participant that generated a HPV-16 E6/E2 above 2 (ratio of 2.48) contained 0.94 copy/cell of HPV-16 E6 DNA and was obtained at the last visit. HPV-16 was detected only at the last of five visits attended by this participant. Normal cytology smears were obtained at the first four visits for this participant while a low-grade SIL smear was obtained at the fifth visit. RS-PCR was performed on the 18 samples that generated a HPV-16 E6/E2 ratio ≥1.2. Despite using several primer combinations, we could not demonstrate the presence of cellular and HPV junctions in any of the samples tested. A minimal amount of 35 μg of cellular DNA per test was analyzed for the only sample with a ratio >2, unsuccessfully, which could have limited our ability to sequence HPV-human DNA junctions.6 and from 104 SiHa cells, respectively (data not shown). One thousand copies of episomal HPV-16 DNA was detected without loss of signal when tested in a mixture of DNA extracted from up to 200,000 cells in the HPV-16 E2 assay and up to 40,000 cells in the HPV-16 E6 test  with real-time PCR on a set of samples collected prospectively in young women. These four genotypes are amongst the most frequently detected in cervical cancer. Contrary to cross-sectional studies of older women, the four HPV genotypes were detected at similar loads and were not substantially different between women with single and multiple type infections ,35.The quantitative real-time PCR assays we utilized to estimate HPV loads were specific and reproducible ,28. The As reported by others, younger woman (<25 years) harbored higher HPV loads than those >25 years of age . These wAlthough the regular use of condoms protects against most sexually transmitted infections, they are not as efficient against HPV infection . We founHPV-16 and -18 loads were good predictors of the duration of infection with these types but the same finding was not held for HPV-31 and -45. HPV-16 load has been reported by others to be a stronger predictor for persistence or lesions than HPV-18, 31 or 33 loads . There wHPV-16 integration often results in the disruption of the E2 gene, leading to uncontrolled expression of HPV-16 oncoproteins ,17. HPV-There are some limitations to our study. We cannot exclude the possibility that we may have underestimated HPV-16 integration due to HPV-16 disruption during integration in areas outside of the E2 hinge region. Nevertheless, HPV-16 E2 hinge is the most frequently disrupted site in studies conducted in North America [The analytical sensitivity of real-time PCR assays for quantitation of HPV-16 E6 and E2 DNA utilized in this study has been demonstrated previously and shown to be excellent ,28,26. TWe could not confirm the presence of HPV-16 integration with a standard technique identifying the presence of HPV-human DNA junctions in the only sample with a HPV-16 E6/E2 ratio above 2.0. However, the quantity of sample that could be analyzed was limited. A recent report using a similar technique to demonstrate the presence of HPV-human junctions did not find HPV-16 integration in specimens from women with low-grade SIL . Real-tiIn our study, few women had abnormal smears, reducing our power to test the associations between HPV load and lesion outcomes. The time interval between visits can influence the assessment of persistence and clearance. The majority of women in our cohort returned within 6 months of each visit and there was only a small proportion of women whose time interval between visits extended beyond one year . The assConsecutive detection of HPV DNA is due to either ongoing viral replication, reactivation of latent infections or new infections ,49. The In conclusion, this study demonstrates a clear association between HPV load and persistence of HPV-16 and 18 infections in young women at the early stages of their sexual life. The association between viral load and infection clearance seems to be type specific and, except for age, it is not substantially affected by sociodemographic characteristics and other risk factors for infection. More longitudinal studies are needed to clarify the onset of HPV integration and its relationship with disease progression.Financial competing interests: NoneNon-Financial competing interests: NoneThe authors declare that they have no competing interestsAVR performed the statistical analysis and drafted the first version of the manuscript. OG performed viral load measurements and integration studies, and interpreted their findings. HR participated in the design and coordination of the study and collection of epidemiologic data. PT participated in the design of the study, oversaw recruitment of subjects, and collected samples. AF participated in the design of the study, read Pap smears, and provided expert oversight. FC helped design the study, supervised HPV genotyping, viral load, and integration assays, participated in the analysis of results and helped interpreting the results and drafting of the manuscript. EF was the study's principal investigator, and participated in its design and coordination, supervision of analyses, and drafting. All authors read and approved the final manuscript and subsequent revisions.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/326/prepub


Endometrial carcinoma is the most common type of female genital tract malignancy. Although endometrial carcinoma is a low grade curable malignancy, the condition of the disease can range from excellent prognosis with high curability to aggressive disease with poor outcome. During the last 10 years many researches have provided some new valuable data of optimal treatments for endometrial carcinoma. Progression in diagnostic imaging, radiation delivery systems, and systemic therapies potentially can improve outcomes while minimizing morbidity. Firstly, total hysterectomy and bilateral salphingo-oophorectomy is the primary operative procedure. Pelvic lymhadenectomy is performed in most centers on therapeutic and prognostic grounds and to individualize adjuvant treatment. Women with endometrial carcinoma can be readily segregated intraoperatively into “low-risk” and “high-risk” groups to better identify those women who will most likely benefit from thorough lymphadenectomy. Secondly, adjuvant therapies have been proposed for women with endometrial carcinoma postoperatively. Postoperative irradiation is used to reduce pelvic and vaginal recurrences in high risk cases. Chemotherapy is emerging as an important treatment modality in advanced endometrial carcinoma. Meanwhile the availability of new hormonal and biological agents presents new opportunities for therapy. Endometrial carcinoma is the most common type of female genital tract malignancy. It is estimated that 42,160 cases of endometrial carcinoma were diagnosed in the United States in 2008 and 7780 women would die from the disease  progesterone receptor (PR)-A, which are estimated to contain potential value as prognostic factors for patients with endometrioid carcinoma –128. AddWith the progress of advanced gene techniques, it has become possible to identify potential molecular markers of endometrial carcinoma for its diagnosis, prognosis and therapy by global gene expression profiling. It may provide a foundation for the development of new diagnostic and prognostic markers and type-specific therapies against this common female genital malignant disease. Such procedure allowed us to give shape to preliminary gene expression profile typical for neoplastic tissue and to estimate protein expression of the most significant predictors of neoplastic transformation. Comparison of obtained data with tumor grade can reveal new markers of endometrial carcinoma useful in routine diagnostic procedures , 130. Genes related to the endometrial carcinoma progression and metastasis can be identified by differential gene expression profile with cDNA microarray and high-risk endometrial carcinoma may be distinguished before surgery by hierarchical cluster analysis . Similar With the applications of the target gene therapy, some valuable research had carried in advanced endometrial carcinoma. Since the year 2000, in advanced endometrial carcinoma, the GOG has conducted phase II trials with several molecular targeting agents including imatinib (Gleevec), trastuzumab (Herceptin), and gefitinib (Iressa) as single agents with negligible evidence of activity. The GOG does have active trials of chemotherapy with a molecular targeting agent such as bevacizumab (Avastin) in GOG 218, but there are no randomized molecular targeting agent trials in advanced endometrial carcinoma . Some geIn the follow-up of endometrial carcinoma patients, pain was the most common complaint in patients with recurrent disease, followed by vaginal bleeding, general malaise, loss of weight and intestinal complaints. With the evidence from randomized clinical trials we can conclude that a follow-up program in the first three years after primary treatment of endometrial carcinoma is helpful in detecting recurrent disease. In 2007, van Wijk et al., evaluated their clinical data of patients with recurrent endometrial carcinoma treated in the Erasmus Medical Centre in Rotterdam over a 20-year period . He repoTjalma et al. published an overview of 11 retrospective studies  on routine follow-up of endometrial carcinoma. In these studies symptomatic recurrences ranged between 41% and 81% (mean 65%) of all recurrences . Retrosp Tjalma et al. pointed out that because of a difference in survival between isolated vaginal recurrence and nonvaginal recurrences, 5-year survival, respectively 50% and 6%, it is important to identify isolated vaginal recurrences early. As the sensitivity of routine follow-up schemes appears very low, tailored follow-up protocols based on high risk and low risk for recurrence are suggested . Low ris Most endometrial carcinomas are sporadic, but approximately 10% of cases have a hereditary basis –153. TwoIn addition, risk-reducing surgery consisting of prophylactic hysterectomy and bilateral salpingooophorectomy should be offered to women aged 35 years or older who do not wish to preserve their fertility . SchmeleEndometrial carcinoma is a low-grade curable malignancy and most patients who present with early disease have excellent survival rate. Endometrial carcinoma remains a management challenge, presenting with a full spectrum of disease ranging from that with excellent prognosis and high curability to aggressive disease with poor outcome. There are many debates and controversies about optimal treatment for women with different staging endometrial carcinoma. How do we summarize the current recommendations and how do we proceed? Clinicians must balance delivering adequate therapy while attempting to minimize treatment morbidity and must always be weighed carefully.  Improved understanding of the mechanisms of carcinogenesis may help identify molecular signatures that could predict biologic behavior of individual disease presentations and discover potential molecular candidates for targeted therapies. Total hysterectomy and bilateral salphingo-oophorectomy is the primary operative procedure. Pelvic lymhadenectomy is performed in most centers on therapeutic and prognostic grounds and to individualize adjuvant treatment. Women with endometrial carcinoma can be readily segregated intraoperatively into “low-risk” and “high-risk” groups to better identify those women who will most likely benefit from thorough lymphadenectomy. Postoperative irradiation is used to reduce pelvic and vaginal recurrences in high risk cases. Treatment planning should be conservative in order to reduce patients' morbidity and overtreatment while maintaining acceptable recurrence and survival rates. Progression in diagnostic imaging, radiation delivery systems, and systemic therapies potentially can improve outcomes while minimizing morbidity. The availability of new hormonal and biological agents presents new opportunities for therapy. Novel strategies for screening and prevention also hold promise for reducing incidence and mortality of this disease. The current evidence suggests that there remain avenues to improve management and we need to continue rigorous investigation to identify and implement the best available practice. Research in the next ten years should provide valuable new strategies not only for treatment but also for prevention.


HPV-16, positive in 11 cases, was the most frequently detected HPV genotype determined by DNA sequencing. HPV-16 E2/E6 ratio, estimated from real-time PCR analysis, was much lower than the unity, suggesting that at least a partial HPV-16 genome was integrated in all but one HPV-16-positive SQCs. The remaining one case was suspected to have only episomal HPV-16. Although the viral load was low in most of the LCs, a case showed the HPV-16 copy number as high as 8479 per nanogram DNA, which was even a few times higher than the minimum viral load of seven cervical carcinomas . The expression of the HPV-16/18 E6 protein was found in only two HPV-16-positive SQCs (13%) but not in the case with the highest viral load. Although the viral load was in general very low and HPV E6 expression is none or weak, further studies seem warranted to examine aetiological involvement of high-risk HPV in lung carcinogenesis.The human papillomavirus (HPV) was detected in 20 (29%) out of 69 lung carcinomas (LCs) in Chile, by PCR and Southern blot, and was more frequently detected in squamous cell carcinoma (SQC) than in adenocarcinomas (46  The human papillomavirus (HPV) is an epitheliotropic double-stranded DNA virus, and its high-risk genotypes, including HPV-16 and -18, are associated with cancer of the uterine cervix and other genital cancers . Human pINK4a is known to be observed in cancers of the uterine cervix  showed that only 9% out of 35 HPV-16-positive specimens exclusively harboured the episomal form, whereas the remaining 91% contained either only the integrated form (6%) or a mixture of episomal and integrated forms of viral molecules (86%). Among the 30 cancer specimens carrying mixed integrated and episomal forms, 28 had E2/E6 ratios of less than 1, indicating the presence of an integrated form of viral genes in these lesions  for HPV presence. Using real-time PCR (N=37) and ACs (N=32) of the lung, diagnosed in the Hospital Salvador, Santiago, Chile during the period between 1998 and 2003. According to the 1982 WHO classification of lung tumours, LC can be classified into four broad categories: SQCs, ACs, small cell carcinomas (SCCs), and large cell carcinomas  of each formalin-fixed paraffin-embedded sample were prepared. We used 1–5 sections for each sample. The samples were added with 1 ml of xylene, and then with 1 ml of ethanol. After centrifugation, the pellet was resuspended in digestion buffer  containing 200 μg of proteinase K  and incubated during 24 h at 56°C. Then the solution was heated at 100°C for 10 min and centrifuged at 10 000 r.p.m. for 10 min. Phenol–chloroform extraction was made in all the samples and the DNA was precipitated with double volumes of ethanol. The DNA was quantified using ND-1000 Spectrophotometer . Since the quantity of tissue embedded in the paraffin blocks was variable between samples, β-globin amplification was made for all the samples to check the presence of PCR amplification inhibitors and of amplifiable DNAs. β-Globin amplification with a set of PCO3/PCO4 primers  was conducted under the following PCR conditions: initial denaturation at 95°C for 4 min, 40 cycles with the cycling profile of 95°C for 1 min, 52°C for 1 min and 72°C for 2 min and final extension for 5 min at 72°C. All the samples were positive for a fragment of the β-globin gene.Sections  in a total volume of 25 μl of reaction buffer . The conditions of amplification were as follows: initial denaturation at 95°C for 4 min; subsequent 45 cycles at 95°C for 1 min, 40°C for 2 min and 72°C for 1.5 min and final extension at 72°C for 5 min. HPV-6 and -18 full genomes  were used as the external positive control. The amplified products were confirmed through electrophoresis with 3.0% agarose gel. Previous to the sequencing, Southern blot was conducted to confirm the identity of amplified fragments and to increase the sensitivity of detection of amplified fragments. As for HPV probes, we used cloned PCR products of the HPV-positive DNAs, which were kindly given by Dr H zur Hausen, Germany, after purification of the PCR products from agarose gel by QIAEX II Extraction Kits . The ECL direct labelling and detection kit  was used according to the manufacturer's instructions. An ECL-labelled HPV18/GP5+/GP6+ fragment was used to detect high-risk HPVs and an ECL-labelled HPV6/GP5+/GP6+ fragment was used to detect low-risk HPVs. Because the generic HPV18 and -6 probes were used, low stringency conditions were used in the hybridizations and washes. The DNA was transferred onto a Hybond N+ nylon transfer membrane (Amersham) by capillary blotting using 0.4 N NaOH. The hybridization was made at 42°C overnight and then the membranes were washed at 42°C with solution containing 6 M urea, 0.4% SDS and 0.5 X SSC buffer. The samples that showed visible bands in the Southern blot procedure but not in the agarose gel electrophoresis were amplified again using 1/100 dilution of the first PCR product using GP5+/GP6+ protocol in 30 cycles. Negative controls were used to confirm no carryover of amplicons. After double PCR using GP5+/GP6+ primers, the obtained fragments were purified and sequenced to examine the genotype of additional HPV-positive samples by Southern blot analysis. In all the steps, technical considerations were taken, to avoid carryingover and contamination with previous amplicons in the tubes.Human papillomavirus amplification with GP5+/GP6+ primers (http://www.ncbi.nih.gov/BLAST/) and ClustalW  software servers.The HPV genotyping was made using DNA sequencing. Amplified PCR products that appeared as a visible band after ethidium bromide staining were purified using the QIAGEN PCR purification kit and were directly sequenced by fluorescent dye-labelled dideoxynucleotides and cycle sequencing methods using the Big DyeTerminator Cycle Sequencing Kit . Sequence analysis was performed on the ABI PRISM 310 Genetic Analyser (PE Applied Biosystems). The nucleotide sequences were aligned and compared with those of known HPV types available through the GenBank database  by using BLAST 2.2 . In order to block the nonspecific binding of the antibody, the slides were incubated for 30 min with 5% bovine seroalbumin (BSA) in PBS at room temperature. A 1/200 dilution in 5% BSA–PBS of monoclonal anti-p16INK4a antibody was used . The slides were incubated overnight at 4°C, washed with PBS, and then incubated with biotinylated horse anti-mouse Ig-G for 30 min followed by washing with PBS and incubation with 1 : 50 dilution of the avidin–biotin–peroxidase complex  for 30 min at room temperature. The reaction was visualized by adding diaminobenzidine  for 10 min. The sections were counterstained with hematoxylin and visualized. Negative reaction was considered when 0–9% of the cells were stained. Positive reaction was considered when 10–100% of the cells were stained, according to criteria reported previously  and HPV-16/18 E6 (1 : 50 dilutions) were used . The interpretation of the positive signal was the same as that used for p16INK4a immunostaining.The paraffin-embedded samples were cut in 2–3 μl volume containing TaqMan 2 × PCR Master Mix with 0.5 μM of E2- or E6-specific primers, 100 nM of dual-labelled E2 or E6 fluorogenic hybridization probe, and 1 μl of DNA template. For specificity and validity testing, DNAs from SiHa celll line . All but one the HPV-16-positive specimens confirmed by DNA sequencing were subjected to the analysis. The analysis of the remaining one sample was not possible because its paraffin-embedded tissue specimens were exhausted. The amplification conditions were 2 min at 50°C, 10 min at 95°C, and a two-step cycle of 95°C for 15 s and 60°C for 60 s for a total of 45 cycles (PCR amplifications were observed correctly when the target strains were used as templates. The primers and probes did not increase the fluorescence with the mismatched strains (data not shown).In order to determine the physical status of HPV-16, the ratio of E2 to E6 copy numbers was calculated  into celβ-globin (110 bp) to determine the genome equivalent present in each sample and to normalize samples for genomic DNA content. A 10-fold dilution series of a human DNA control  was used to generate the standard curve for β-globin. The amount of β-globin DNA present in each sample was divided by the weight of one genome equivalent (i.e. 6.6 pg cell−1) and a factor of 2  to obtain the number of genome equivalents in each sample. Viral load in each specimen was expressed as the number of HPV copies/genome equivalent or cell.We also conducted real-time PCR for P-values presented are two sided.Fisher's exact test was used to examine the statistical significance of the results. P=0.007), and also more common among smokers (P=0.013). The frequency of p16INK4a or p53 expression did not show any significant difference between SQCs and ACs. The expression of p16INK4a was positive in 24 out of 30 in well- or moderately differentiated SQCs but in only 1 out of 6 poorly differentiated SQCs (P=0.008). Such a difference was not observed in ACs. The expression of p53 was related to tumour differentiation in neither SQCs nor ACs.We examined 37 SQCs and 32 ACs of the lung diagnosed in Santiago, Chile. Their clinicopathological features are summarized in P=0.001). This difference was mainly accounted for by high-risk HPVs, which were detected in 16 SQCs and in 1 AC. Human papillomavirus presence was not related to gender, age, or smoking habits (data not shown). Even when age was divided into finer categories, we could not observe any evident relationship between age and HPV presence. The expression of p16INK4a or p53 showed no significant difference between HPV-positive and -negative LCs, either.The results of HPV detection and genotypes determined by DNA sequencing in LC are summarized inAs shown in INK4a and p53 expression in 10 and 70% of tumours, respectively. We also examined 7 out of 12 HPV-16-positive cervical carcinomas, and three of them had E6 copy numbers lower than that of the case LC-5. Viral load was not related to any clinicopathological features examined (data not shown).We conducted real-time PCR analysis to estimate viral load and to determine the integration status of HPV-16 . All but−6 ng DNA μl−1. Those six LC cases were considered to have only integrated form of HPV. On the other hand, there were two cases where E2/E6 ratio was 0.01–0.02 and considered to have both integrated and episomal forms of HPVs (integration status: mixed). The remaining one case had the E2/E6 ratio of about one, suggesting the episomal form.E2/E6 ratios are presented in We also examined the E6 expression among 14 HPV-16/18-positive cases by immunohistochemistry. There were only two SQC cases (13%) showing the E6 expression, and both of them were HPV-16 positive . Case LCP=0.001). Among HPV genotypes detected, 85% were high-risk HPV. The most frequently detected genotype in the present study was HPV-16, which accounted for 55% of HPV-positive cases, as was also the case in our previous study conducted in other Latin-American countries, including Mexico, Colombia, and Chile  examined in the present study. Since SiHa cell line, having 1–2 HPV copies per cell, was found to have 144 copies per nanogram DNA in our analysis, this LC case is suspected to have HPV copies much more than 1–2 per cell.Although the viral loads of most LCs were low, one case showed the viral load of HPV-16 as high as 8479 ngIf a clonal expansion occurs after HPV infection in lung epithelial cells, HPV is expected to be found in all the carcinoma cells. However, our results suggest that this scenario is unlikely because only a very small proportion of the malignant cells are integrated. Regarding the role of HPV in LCs, INK4a is known to be upregulated by HPV , our method is considered to be more sensitive and enables us to detect HPV even when carcinoma cells have a small number of HPV copies. However, it is not possible for us to tell whether HPV is present in tumour or normal cells because most tissue specimens contained adjacent normal cells. Further studies using the microdissection technique are necessary to address this question.The DNA specimens extracted from paraffin-embedded blocks are known to be frequently degraded into 200 bp long or even shorter fragments (We detected HPV-18 in three SQC cases but not in ACs. In the uterine cervix, HPV-18 has been shown to have a higher risk of developing Acs, while HPV-16 is more strongly associated with SQCs (The transmission route of the HPV detected in LCs is as yet unclear. Studies on HPV infection and cancers of the oral cavity, oesophagus and lung suggested the possibility of sexual transmission (In summary, high-risk HPV, including HPV-16, was detected in Chilean SQC cases at a high frequency, and the real-time PCR analysis suggested the integration of HPV-16 into the cellular genome of SQC specimens. Although the viral load was in general very low and HPV E6 expression is none or weak, further studies seem warranted to examine aetiological involvement of high-risk HPV in lung carcinogenesis.


Cervical cancer incidence in western Africa is among the highest in the world.To investigate human papillomavirus (HPV) infection in Guinea, we obtained cervical specimens from 831 women aged 18–64 years from the general population of the capital Conakry and from 77 locally diagnosed invasive cervical cancers (ICC). Human papillomavirus was detected using a GP5+/6+ PCR-based assay.Among the general population, the prevalence of cervical abnormalities was 2.6% by visual inspection and 9.5% by liquid-based cytology. Fourteen of 15 high-grade squamous intraepithelial lesions were visual inspection-negative. Human papillomavirus prevalence was 50.8% (32.1% for high-risk types) and relatively constant across all age groups. Being single or reporting ⩾3 sexual partners was significantly associated with HPV positivity. HPV16 was the most common type, both among the general population (7.3%) and, notably in ICC (48.6%). HPV45 (18.6%) and HPV18 (14.3%), the next most common types in ICC, were also more common in ICC than in HPV-positive women with normal cytology from the general population.The heavy burden of HPV infection and severe cervical lesions in Guinean women calls for new effective interventions. Sixty-three per cent of cervical cancers are theoretically preventable by HPV16/18 vaccines in Guinea; perhaps more if some cross-protection exists with HPV45. The Republic of Guinea in western Africa has a population of approximately 9.4 million, of which 2 million live in the capital of Conakry. The country shows high birth rates (5.8 children per woman) and communicable diseases, notably malaria, which predominate as causes of death. Estimates of cervical cancer incidence and mortality rates in western Africa are among the highest in the world  vaccination  and HPV We report on HPV prevalence survey among a representative sample of the general female population in Conakry, Guinea, as well as in a corresponding sample of Guinean women with invasive cervical cancer (ICC).Study methods were similar to those used for earlier IARC HPV Prevalence Surveys (www.who.int]). In addition, among the 462 women below age 25 years who accepted the invitation to the study clinic, 213 (of whom 148 were self-declared virgins) were unwilling to undergo a pelvic examination, and opted to provide only a blood sample. Hence, no cervical cell specimen was collected for these 213 women; an additional 73 women aged 25 years or older did not undergo a pelvic examination mainly because of heavy menstrual bleeding.Of the 1725 invited women, 440 (25.5%) did not accept the invitation, mainly because they apparently did not understand the need for gynaecological examination in the absence of symptoms. Acceptance was similar in all the age groups, but owing to the age structure of the population, relatively few women over 44 years of age could be invited . The cervix was then inspected with acetic acid (VIA) and visual inspection with Lugol's iodine (VILI), with results reported according to the IARC criteria .Slides for liquid-based cytology were prepared using a Thin Prep 3000 processor (Cytyc-Hologic), stained according to manufacturer's instructions and read at the Department of Pathology at the Vrije University Medical Center, Amsterdam, the Netherlands. Cytological diagnosis was formulated according to CISOE-A standards and was translated into the Bethesda 2001 terminology system  or III (44 women). Biopsies were fixed with buffered formalin, embedded in paraffin (MH), then read (MK) in the Centre National d’Anatomie Pathologique, CHU de Donka. HIV testing was available for 41 women with ICC, of which eight (19.5%) were HIV-positive; among 19 women with ICC below age 45 years, six were HIV-positive.All participants, whether from the HPV prevalence survey or those with ICC, signed informed consent forms according to the recommendations of the IARC and the CHU de Donka ethical review committees, both of which approved the study.2 of tissue were pre-digested with Proteinase K after which DNA was extracted using magnetic beads .HPV testing was performed on exfoliated cervical cells and ICC biopsies in the Department of Pathology at the Vrije University Medical Center. DNA was extracted from the PreservCyt sample using magnetic beads  on a robotic system , according to the manufacturer's instructions. Invasive cervical cancer biopsies were sectioned using a ‘sandwich’ approach, whereby inner tumour sections were destined for HPV testing and outer sections for histological confirmation of tumour tissue. One or more five lM sections representing approximately 1 cmBeta-globin PCR analysis was performed first to confirm the presence of human DNA in all specimens (Odds ratios (ORs) for HPV positivity and corresponding 95% confidence intervals (CIs) were calculated by means of unconditional logistic regression equations, adjusted for age group , marital status (never- or ever-married) and lifetime number of sexual partners , as appropriate. The statistical significance of trends for ORs was assessed by considering the categorical variable as a continuous variable in the logistic model. Prevalence ratios and corresponding 95% CIs were used to compare the relative frequency of the most common HPV types in HPV-positive women with ICC with that among cytologically normal HPV-positive women from the HPV prevalence survey.β-globin-negative, seven missing), and an additional 81 had inadequate cytology, leaving 831 women with valid results for both HPV and cytology. Among them, 79 (9.5%) had abnormal cytological findings, including 48 (5.8%) atypical squamous cells of undetermined significance (ASCUS), 16 (1.9%) low-grade squamous intraepithelial lesions (LSIL) and 15 (1.8%) HSIL.Of 996 women who provided cervical cell samples, 84 had inadequate HPV DNA results .Twenty-one women had abnormalities suspected at VIA/VILI. The correlation between the results of VIA/VILI and those of liquid-based cytology and HPV testing was poor . VIA/VILOf the 21 women with suspected abnormalities at VIA/VILI, 17 accepted referral to colposcopy and 10 had colposcopy-directed biopsies, none of which showed lesions except the aforementioned ICC. Repeated attempts were also made after cytological findings became available  to recall the 14 women who had no abnormality detected at VIA/VILI but HSIL found at liquid-based cytology. Three women, however, had moved far from Conakry, one was not found at her address, one had died of a disease other than cervical cancer and one was in the third trimester of pregnancy. Of the eight biopsies taken, one was inadequate, three showed cervical intraepithelial neoplasia 1 and the other four showed no cervical lesion.vs <25 years=0.65; 95% CI: 0.44–0.95), marital status  and number of lifetime sexual partners . These three associations were slightly attenuated in the model additionally adjusted, as appropriate, for marital status and sexual partners.Education, number of births, age at first sexual intercourse, husbands’ polygamy or extra-marital sexual relationships and lifetime use of hormonal contraceptives were unrelated to HPV positivity in either adjustment model  as were vs cytologically normal women were 3.5 (95% CI: 2.2–5.5) for HPV16, 2.1 (95% CI: 0.9–4.6) for HPV18; 1.9 (95% CI: 0.9–3.7) for HPV45 and 0.8 (95% CI: 0.5–1.2) for high-risk types other than 16 or 18. Multiple-type infections were much less common in HPV-positive women with ICC than in HPV-positive women with normal cytology (Beta-globin-negative ICC biopsies (22) were excluded; among the remaining 77 ICC biopsies with valid HPV results, 8 were adeno- and the rest were squamous-cell carcinoma. The median age of women with ICC was 45 years (range: 23–80 years). Seven ICC biopsies, including one adenocarcinoma, were HPV-negative. Type-specific HPV prevalence in 70 HPV-positive ICC and 360 cytologically normal HPV-positive women from the HPV prevalence survey are compared in cytology . Among tOur large study, the first on HPV infection in Guinea, showed a very high (50.8%) prevalence of HPV, and nearly no history of cervical cancer screening. Approximately one-third of women in our survey were infected with high-risk HPV types and four prevalent ICC were discovered.The age-standardised HPV prevalence we found in Conakry (51.5%) was considerably higher than that observed in earlier studies performed using the same HPV testing protocol in areas at high risk for cervical cancer such as South America (12–18%) and India (17%) or even another western African population in Nigeria (27%)  . HIV infOne of the main aims of the IARC HPV Prevalence Surveys concerns the variations of age-specific and type-specific HPV prevalence by geographical region. The age-specific curve of HPV prevalence in Guinea resembled the age curves reported earlier in Nigeria (α-7 species, is usually less frequently detected than HPV18 in cytologically normal women (With respect to the relative importance of different HPV types, HPV16 was the commonest type overall, that is, 13.9%, 17.7% and 48.6%, respectively, in cytologically normal and abnormal women and in women with ICC (On account of the lack of experienced cytologists and HPV testing in Conakry, we relied on VIA/VILI for the immediate clinical management of study women from the HPV prevalence survey. Despite the substantial experience of study midwives in visual inspection (β-globin-negative samples in the HPV prevalence survey, and of HPV-negative findings in paraffin-embedded ICC biopsies. Most important, the lack of prompt histological confirmation of abnormalities and of random biopsies did not allow us to accurately estimate sensitivity and specificity of different methods. A prolonged curfew in 2007 delayed not only the shipment of cervical cell samples to Europe and, hence, the availability of cytological and HPV findings, but also the recall of women. Substantial population mobility and scarcity of pathology staff in Conakry also hampered the appropriate work up of screening positive women.Major strengths of our study include the large number of women, high participation, reliance on high-quality HPV testing and liquid-based cytology, and presence of a concurrent series of women with ICC drawn from the same study area. Limitations include the lack of information on HIV status and the relatively high proportion of The very heavy burden of HPV infection and severe cervical lesions in Guinea calls for new effective interventions. The quality of VIA/VILI should be greatly improved, or other types of screening used (e.g., rapid HPV test,


This increase paralleled the increases in mammographic screening. BreastScreen NSW, an organised mammographic screening programme, detected 65% of all DCIS. High-grade lesions were 54% of all lesions and were more likely to be 2+ cm in diameter  than low-grade lesions. In all, 40% of DCIS in women younger than 40 years was 2+ cm in diameter compared with 21% in women 40 years and older. Young age, high grade, mixed architecture and multifocality were significant and independent predictors of 2+ cm DCIS.In a population-based series of 2109 women with ductal carcinoma  While mammographic screening has undoubtedly caused increasing diagnosis of DCIS  Australia in 1995–2000.in situ has been notifiable in NSW since 1993, and by 1997 all but 4% of DCIS cases diagnosed by pathology laboratories were notified to the Cancer Registry (unpublished data).NSW women with a first diagnosis of DCIS in 1995–2000 and notified to the NSW Central Cancer Registry were eligible for the study; those with a previous or simultaneous (same month) diagnosis of invasive breast cancer were excluded. Ductal carcinoma Two experienced Cancer Registry personnel extracted information on the type of specimen, size, grade, architecture, presence or absence of necrosis and multifocality of DCIS, and clearance and width of the margins from pathology reports . These mammograms would include an unknown but not high proportion of mammograms that were primarily diagnostic. BreastScreen and Medicare account for most of the screening mammography in Australia.The frequency of mammography was obtained from reports of an organised screening programme, BreastScreen NSW , which bIncidence rates and 95% confidence intervals (CIs)  in logistic regression models that included year of diagnosis; the additional effects of urban or rural residence and SES of the women were also examined.The heterogeneity of DCIS distributions by size and grade among age groups and type of architecture among years of diagnosis was evaluated by standard In 1995–2000, 2109 NSW women were notified with DCIS. More than half (54%) were 50–69 years of age, the target age group for breast cancer screening in NSW, and had the highest incidence (32.3 per 100 000)  to 2000 (8.9 per 100 000) , mostly between 1996 and 1997 (39% increase) and in women 50–69 years of age (48% increase 1996–1997)  and increased strongly with increasing socio-economic status in Sydney . BreastScreen NSW detected 65% of incident DCIS, with higher proportions in women older than 50 years.The incidence of DCIS was about 25% higher in urban than rural areas of NSW  as women 40–49 (122.4) and 70+ years (120.1) . There was a weaker correlation between Medicare reimbursed mammograms and DCIS not detected by BreastScreen in 1995–1999 .The numbers of DCIS detected were strongly correlated with the numbers of women screened by BreastScreen in 1995–2000  and mastectomies (24%). Size was reported in 76%, grade in 89% (both in 69%), presence or absence of necrosis in 63%, architecture in 82% and clearance of the margins in 80%; 37% had all these items. The width of margins was reported in only 41%. All these items except the width of margins were appreciably more complete in 1998–2000 than in 1995–97.P=0.02) and a higher proportion were 2+ cm at 20–39 (53%) than 40 years and older (38%) (P=0.01). Size, however, was not stated for more DCIS at 20–39 years (34%) than other ages (24%).More than half (54%) the DCIS diagnosed in 1995–2000 were high grade and 39% were 2 cm or larger (P for difference between the two periods <0.001). High-grade DCIS were distributed across all types . Most of the 2+ cm DCIS were mixed (45%) or other specific types (27%) and few were comedocarcinoma (18%) or no specific type (9%).Most (65%) of the DCIS were described as cribriform or solid, or a mixture in which these two types predominated. Fewer were identified as comedocarcinoma (10%) in 1998–2000 than in 1995–1997 (26%) and more as a mixture of types , without which we cannot fully describe the contribution of screening to the occurrence and outcomes of DCIS.Other countries have observed increasing incidence of DCIS, sometimes three- to four-fold, with increasing mammographic screening  or resurWe observed a higher proportion of high-grade lesions (54%) than in Sweden (43%) and Switzerland (46%) in the early 1990s, but similar to that, 55%, in an Australia-wide sample survey  than older women (average 18 mm) is probably due to their lower mammography rate. That high-grade lesions were larger probably reflects a correlation with higher growth rate (1.8 mm per year low grade, 4.2 mm intermediate and 7.1 mm high grade; The pathology reports from 1998 to 2000 reported DCIS substantially more completely than did earlier Australian reports  and 2001Increasing incidence of DCIS is an outcome of mammographic screening for breast cancer. The high proportion of high-grade lesions we have observed suggests that its detection could contribute to reducing breast cancer mortality. If it does not, the high frequency of DCIS in association with screening may be source of unnecessary morbidity and cost. More research is needed into the costs and benefits of detection of DCIS in breast cancer screening.


Human Papillomavirus (HPV) infection is now well-established as a necessary cause of invasive cervical cancer (ICC) development. Only a few studies on HPV prevalence and type-specific distribution in ICC have been conducted in Italy.Cervical cancer represents an important global public health problem. It is the 2To describe the prevalence of HPV and the HPV type-specific distribution in ICC cases identified in Rome, Italy.25 (version 1).140 paraffin embedded tissue blocks of primary ICC diagnosed between 2001 and 2006 were identified at the Regina Elena Cancer Institute in Rome . HPV was detected through amplification of HPV DNA using SPF-10 HPV broad-spectrum primers followed by DEIA and then genotyping by LiPA134 cases were considered suitable for HPV DNA detection after histological evaluation; and overall, 90.3% (121/134) HPV prevalence was detected. 111 cases had a single HPV type, 4 cases had an uncharacterized type (HPVX) and 6 cases had multiple HPV infections. The five most common single HPV types among positive cases were: HPV16 , HPV18 , HPV31, HPV45 and HPV58 . 2 (1.5%) of the single infections and 2 (1.5%) of the multiple infections contained low risk types. Statistically significant differences in the relative contribution of HPV18 were found when comparing squamous cell carcinomas with adenocarcinomas.HPV16 and HPV18 accounted for almost 70% of all the HPV positive ICC cases. The study provides baseline information for further evaluation on the impact of recently introduced HPV vaccines in Italy. Around 493,000 new cases and 274,000 deaths from this disease were estimated in 2002 [th most frequent cancer in women, however, the 3rd most frequent cancer in women between 15 and 44 years old [Cervical cancer is a serious public health problem representing the 2 in 2002 . In this in 2002 . CurrentHPV infection is now well-established as necessary but not sufficient cause of ICC development -5. Only Very few studies on HPV type prevalence and type-specific distribution in ICC have been conducted in Italy -17. ThusA cross sectional period-prevalence study on archival paraffin embedded ICC specimens was performed. 140 consecutively selected paraffin embedded tissue blocks with ICC from women diagnosed between 2001 and 2006 were identified and retrieved from the Regina Elena Cancer Institute in Rome, Italy. Age at diagnosis and year of diagnosis were collected from medical records.Selecting the paraffin blocks and microtome sectioning was performed in Rome, under strict conditions to avoid potential contamination and following a protocol previously developed at ICO. Briefly, at least four paraffin sections were obtained for each block ("sandwich" method). First and last microtome sections were stained with Haematoxylin and Eosin (H&E) for histopathological evaluation. The sections that were cut in-between the two H&E slides were collected in two eppendorf type tubes for HPV DNA testing . A tissue-free paraffin block was cut after each study block to avoid HPV carry-over from block to block. A new blade was used for each study block, and the microtome was cleaned with Histoclear II, a xylene substitute, and 70% alcohol. Paraffin blocks containing non HPV-related lesions, such as, non HPV-related tumours or non-tumour biopsies were blindly included and analyzed as controls in the process for further quality control of laboratory results.The pathology classification of the histopathological diagnosis was done at ICO following the consensus criteria established by an expert panel of collaborating pathologists. These criteria were based on the World Health Organization (WHO) classification of uterine cervix tumours . The pat25 version 1. , that can detect 25 different high-risk and low-risk HPV types . The sequence variation within the SPF-10 primers allows the recognition of these different HPV genotypes, except for types 68 and 73, as their interprimer regions are identical and cannot be distinguished by this test. Positive hybridization on the strips is visualized as a purple band by means of a precipitating colour substrate on the probe site. Specimens that were identified as HPV DNA positive, but did not hybridize with any of the 28 LiPA probes patterns, were coded as HPV type X (uncharacterized type). SPF-10 PCR, detection and typing were performed at ICO.DNA isolation was performed by treating the cut sections of paraffin embedded tissues with 250 μl of freshly prepared 0.1% proteinase K solution . The samples were incubated at 56°C for 18 hours to overnight. Proteinase K was then heat inactivated at 95°C for 10 minutes. Short Polymerase Chain Reaction Fragment - using biotin labelled SPF-10 primers ,20 was pData analysis was performed with the Statistical Package SPSS 14.0. Overall and type-specific HPV prevalence  and type-specific relative contribution (within the HPV positive cases) were analyzed and 95% confidence intervals (95% CIs) for proportions were calculated. To estimate the HPV type distribution, single and multiple HPV infections were considered either separately or combined. Stratified analyses of overall HPV and type-specific distributions by age at diagnosis and histological characteristics were performed. Age was recoded as a categorical variable . Statistical significance for two sided chi-squared and linear trend tests were set at 0.05 levels .Specimens were received at the reference laboratory in Barcelona (ICO) under an anonymous manner . All protocols were approved by local and ICO ethical committees. The study's progress was overseen by an international steering committee specifically created for the supervision and to act as an advisory committee for handing critical ethics as well as scientific issues of the project.After histological evaluation, 134 ICC specimens from the original 140 cases were considered suitable for HPV testing; 6 specimens were excluded because they were classified as non-invasive cervical cancers. Mean age of HPV analyzed cases was 52.4 years . Squamous cell carcinoma was the most frequent histological type (83.6%), followed by adenocarcinoma (12.7%) and adenosquamous cell carcinoma (0.8%). Other histopathological types accounted for 3.0%, and were classified as 2 undifferentiated carcinomas, 1 poorly differentiated large cell carcinoma and 1 basal adenoid carcinoma. Microscope slides were systematically reviewed by the pathologists to identify characteristics that could affect detection such as grade of necrosis, tumour infiltration, concomitant lesions or if mucosa was associated. Table Data on HPV testing and typing is summarized in Table HPV16 relative contribution was higher in squamous cell carcinoma (62.4%) than in adenocarcinoma (43.8%) with HPV16 as the most frequent type identified in both (p > 0.05). HPV18 was more prevalent in adenocarcinomas (31.3%) than in squamous cell carcinomas (5.0%) (p < 0.05). HPV31, HPV45 and HPV58 single infections were as frequent as HPV18 in squamous cell carcinoma (5.0%) , 57% in the 40-49 years group and 54% in women older than 49 years; although these differences were not statistically significant, p-trend test >0.05.In the present study a high proportion (90.3%) of the ICC specimens analyzed was HPV DNA positive. HPV16 and HPV18 together accounted for 69.4% of the HPV positive cases. Types HPV31, HPV45, HPV58 and HPV33 also were identified with no major distinction between them in terms of relative importance. Collectively, these types accounted for an additional 15.6% of single infections.Several meta-analyses on HPV detection among ICC have been published by IARC ,11 prese3,607 women with incident and histological confirmed cervical cancer recruited in 25 countries were used for a pooled analysis of data in an international survey of cervical cancer HPV types and a multicenter case-control study, both co-coordinated by the IARC . The stuTenti et al. reported a slightly higher prevalence of HPV18 (29.7%) than HPV16 (28.3%) in a study conducted in Italy on 138 adenocarcinomas of the cervix [The association between HPV type and histological type has been described in a large number of studies ,11. Tente cervix . Similar25), our study is not without some limitations. Sample collection was available from only one hospital in Italy . Although the number of samples analyzed is limited and obviously not representative of the whole country, they were originated from a systematic collection at a national cancer reference centre providing a potential good representation of all cases in the country.Even though a high-sensitive HPV DNA PCR based technology was used for detection and HPV typing , from the Agència de Gestió d' Ajuts Universitaris i de Recerca (AGAUR 2005SGR 00695), the Marató de TV3 Foundation (051530), and from GlaxoSmithKline Biologicals, Sanofi Pasteur MSD & Merck & Co, Inc., who had no role in the data collection, analysis, or interpretation of the results.LM, EV, FM, AV and MB were responsible for the specimens' selection. BLL was responsible for the pathology evaluation. JK and WQ were responsible for the HPV DNA detection and quality control analysis. BQ, LA, and NM were responsible for the data analysis. NM, LA and SdS contributed to the writing of the manuscript and LM, NM and LA were responsible for the preparation of the manuscript for submission. XFB and SdS planned the international collaborative project on HPV in invasive cervical cancer (ICO survey-RIS HPV TT). All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/259/prepub


In human cervical neoplasia human papillomavirus (HPV) type 18 has a higher cancer/cervical intraepithelial neoplasia (CIN) prevalence ratio than HPV 16. Fibrosarcomas derived from rat fibroblasts transfected with HPV 16 or 18 genomes showed increased apoptosis compared with controls. However, HPV 18 was associated with significantly less apoptosis than HPV 16, affording one possible explanation for the more rapidly progressive cervical neoplasia associated with HPV 18.


Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.A green ciliate  Nitella and Chara, embryos of Caenorhabditis elegans, amoeba and ciliates C. elegans. Especially, in plant and amoeboid cells, successful biochemical and biophysical approaches to studying actin/myosin system have extensively promoted the understanding of the molecular mechanism underlying cytoplasmic streaming.Active movement of organelles called as cytoplasmic streaming has been observed in various kinds of cells such as green algae Paramecium bursaria harbors several hundreds of endosymbionts like Chlorella in the cytoplasm P. bursaria demonstrates rotational cytoplasmic streaming A ciliate Here, we found that no cytoplasmic streaming was observed in dividing paramecia, while endosymbionts were proliferating in host cells. Several experiments revealed that arrest of cytoplasmic streaming induced proliferation of endosymbionts in interphase paramecia. These data suggest that cytoplasmic streaming controls algal proliferation in green paramecia. This is the first report to show that cytoplasmic streaming works as a signal to control proliferation of eukaryotic cells.Paramecium bursaria syngen 1  were used in this study. Stocks BWK-4 and KN-21 were collected from the Lake Biwa  and the river Kino-kawa , respectively. A symbiotic algae-free strain (BWKw-4) was produced by a herbicide paraquat Klebsiella pneumoniae as food under a LD cycle (12 h light/12 h dark) at ca. 1500 lux of natural white fluorescent light and 23°C P. bursaria were obtained as described previously ca. 1500 lux of natural white fluorescent light and 24°C Cloned exsymbiotic algae (SA-1 and SA-3) isolated from P. bursaria, carmine powder was added into a paramecium culture and the flow of carmine particles in a paramecium cell was observed with video-microscopy. In this study, paramecia showing cytoplasmic streaming and without streaming were expressed as CS+ and CS-, respectively.To observe cytoplasmic streaming in A paramecium was squashed and the total number of the endosymbionts was counted with light microscopy P. bursaria at interphase was artificially arrested for 2 hr in a moisture chamber by pressure with a cover slip-glass slide spacing of 17 µm as described previously Cytoplasmic streaming in P. bursaria is arrested by nocodazole P. bursaria. To investigate roles of cytoplasmic streaming in P. bursaria, nocodazole in DMSO was added into a paramecium culture  It has been reported that cytoplasmic streaming in Each dividing paramecium was harvested using a pipette and placed on a coverslip. The cells were fixed with methanol for 6 hr at −20°C. The fixed paramecia were washed twice with PBS. The cells were mounted in FluoroGuard™ (Bio-Rad) containing 4′, 6-diamidino-2-phenylindole  for DNA staining. The specimen prepared was observed under both a Nomarski differential interference contrast (DIC) and a fluorescence microscope  equipped with a digital camera  and a confocal laser scanning microscope . In LSM observation, serial fluorescence sections of 1 µm were obtained and final images were compiled with Adobe Photoshop software.Paramecium bursaria  was observed in vivo. P. bursaria at interphase demonstrates dynamically rotational microtubule-based cytoplasmic streaming Cytoplasmic streaming in P. bursaria is arrested both by pressure with glass coverslip spacing and by addition of microtubule drug nocodazole P. bursaria, we determined the number of endosymbionts under pressure with a coverslip. As shown in P. bursaria at interphase, although the contractile vacuole (Cv) kept moving. Interestingly, endosymbionts started increasing within 2 hrs after initiating arrest of cytoplasmic streaming . The increase of number of endosymbionts was also observed in the paramecia pressed with glass coverslip, which show no cytoplasmic streaming  were pre-incubated in CA medium for 48 hr under constant light condition. After pre-incubation of endosymbionts (arrow), nocodazole was added into the algal culture. 24 hr and 48 hr after treatment with nocodazole, the number of exsymbiotic algae was determined with a hemocytometer. The result obtained shows that nocodazole was ineffective in the growth of exsymbiotic algae.Effects of microtubule drug nocodazole on exsymbiotic algae isolated from (0.71 MB PDF)Click here for additional data file.Movie S1P. bursaria having carmine particles. Cytoplasmic streaming is arrested in a dividing paramecium cell and particles locally oscillate. Note that the movie changes from a interphase paramecium (former movie: until about 30 seconds) to a dividing paramecium (latter movie).Cytoplasmic streaming in (1.98 MB MOV)Click here for additional data file.Movie S2P. bursaria at interphase was arrested in the presence of 6 µg/ml nocodazole.Cytoplasmic streaming in (0.36 MB MOV)Click here for additional data file.Movie S3P. bursaria  at dividing phase. Although dividing cells arrested dynamically rotational cytoplasmic streaming, the carmine particles near the division plane slightly moved. The particles have never penetrated the division plane. Note that the movie is played at 4-fold speed.Movements of carmine particles near a division plane in (0.96 MB MOV)Click here for additional data file.Movie S4P. bursaria. After arrest of cytoplasmic streaming in a dividing paramecium cell, endosymbionts were eliminated from the division plane. The division plane looks like a white line due to the exclusion of symbiotic green algae. Note that the movie changes from an interphase paramecium (former movie: until about 15 seconds) to a dividing paramecium (latter movie).The arrest of cytoplasmic streaming and the gap near a division plane in (0.95 MB MOV)Click here for additional data file.