The aim of this study was to examine the prevalence and genetic variability of the capsid L1 gene of rare HPV genotypes that were found in the cervical lesions of women from North-East Brazil. A total number of 263 patients were included in this study. HPV detection was performed using PCR followed by direct sequencing of MY09/11, as well as type-specific PCR to detect the Alpha-9 species. Epitope prediction was performed to determine whether or not the genetic variants are inserted in B-cell and T-cell epitopes. The prevalence of rare HPV types in cervical lesions was found to be 9.47%. The rare HPV genotypes that were detected were HPV-53, 54, 56, 61, 62, 66, 70, and 81. The genetic variability in the L1 gene of rare HPV types involved thirty nucleotide changes, eight of which were detected for the first time in this study. Moreover, some of these variants are embedded in B-cell or T-cell epitope regions. The results of this research suggest that rare HPV types might be involved in cervical lesions and some of these variants can be found in B-cell and T-cell epitopes. Data on the prevalence and variability of rare HPV types will assist in clarifying the role of these viruses in carcinogenesis. Cervical cancer is ranked as the third major cause of female cancer worldwide, with an estimate of more than 529,000 new cases diagnosed and 275,000 deaths in 2008 . In deveAlphapapillomavirus (Alpha), Betapapillomavirus (Beta), Gammapapillomavirus (Gamma), Mupapillomavirus (Mu), and Nupapillomavirus (Nu) [Persistent infections caused by Human Papillomavirus (HPV) can result in cervical lesions and cervical cancer . HPV is rus (Nu) , 6. Amonrus (Nu) . The Intrus (Nu) . Moreoverus (Nu) .Recently, epidemiological data on the worldwide incidence of HPV have shown that Alpha-9 and Alpha-7 species cause 91% of invasive cervical cancer [There have been several studies that describe the prevalence and genetic variability of the HPV-16, HPV-18, HPV-31, and HPV-58 genotypes –21. HoweThus, the aim of this study was to investigate the prevalence of rare HPV types, as well as the genetic variability of the capsid L1 gene of these viruses, in cervical lesions of women from North-East Brazil.Cervical cells were collected from November 2010 to August 2011. The samples were obtained from women during their medical consultations at the Gynaecological Unit of the Integrated Medicine Center, in Sergipe State and the Oswaldo Cruz University Hospital, in Pernambuco State, North-East Brazil. A total number of 263 women agreed to participate in the study and signed the consent form. The study included patients with Cervical intraepithelial Neoplasia (CIN)  and cervical invasive cancer. All the patients were sexually active and their average age was 38.02 (with ages ranging from 16 to 77). This study was approved by the Ethics Committee of the University of Pernambuco (HUOC/PROCAPE 64/2010) and the Ethics Committee of the Federal University of Sergipe (CEP/CCS/UFPE N° 491/11). The collected cells were placed in polyethylene tubes containing phosphate-buffered saline and then stored at −20°C until analysis. DNA was extracted by using the DNeasy Blood and Tissue Kit 135 (Qiagen) in accordance with the manufacturer's instructions. μL containing 100 ng of DNA, 1.5 mM of MgCl2, 50 μM of each dNTP, 20 pmol of each primer, and 1 unit of Taq DNA Polymerase and 1x buffer. The PCR cycling conditions were as follows: initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 1 minute, elongation at 72°C for 2 minutes, and a final extension at 72°C for 10 minutes. The PCR products were run on the agarose gel (1%). HPV DNA detection was performed by using polymerase chain reaction (PCR) with degenerate primers MY09/11 , which aThe positive samples were purified with the Invisorb Fragment Cleanup (Invitek) kit and sequenced (in duplicate) by using ABI PRISM BigDye Terminator Cycle Sequencing v 3.1 Ready Reaction (Applied Biosystems) to obtain both the forward and reverse sequences. The sequences obtained were assembled into contigs using the Staden package . They weNew variants were submitted to GeneBank with the following accession numbers: HPV-53-JX912952; HPV-54-JX912948; HPV-56-JX912947; HPV-62-JX912951; HPV-70- JX912950 and HPV-81-JX912949.http://www.imtech.res.in/raghava/bcepred/. The prediction was carried out with the aid of physico-chemical parameters, such as hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface, turns, and antigenic propensity [The putative impact of variability in L1 gene of rare HPV types was estimated in silico by predicting the B-cell and T-cell epitopes. In this study, it was assumed that changes in amino acid sequences of L1 proteins within B-cell and T-cell epitope regions could affect the binding affinities of the neutralizing antibodies and did not initiate an epitope-specific immune response, respectively. Thus, the B-cell epitope of prototype sequences was predicted by using the BcePred server, which is available from URL: opensity . http://www.imtech.res.in/raghava/propred1/) was used to predict MHC Class-I binding regions [http://www.imtech.res.in/raghava/propred/) was used to predict MHC Class-II binding peptide [The T-cell epitope predictions were performed by using ProPred and ProPred I servers. ProPred I server . These samples were genotyped and the results showed the presence of HPV-6, 11, 16, 18, 31, 33, 45, and 58 genotypes. With regard to rare HPV types, the following genotypes were detected: HPV-53, 54, 56, 61, 62, 66, 70, and 81 . Among tTwenty-seven cervical samples were coinfected, out of which eleven were infected with the HPV-16/31 genotype, two infected with HPV-16/18, six samples with HPV-31/58, two samples with HPV-16/33, and the remaining samples infected with the following genotypes: HPV-16/58, HPV-16/6, HPV-16/33, HPV-31/33, HPV-16/54, and HPV-16/58/33. With regard to the Alpha species, it was found that 86.46% of the positive HPV DNA samples were infected with Alpha-9, followed by Alpha-3 (4.2%), Alpha-7 (3.4%), Alpha-10 (3.4%), Alpha-6 (2.5%), and Alpha-1 (0.8%) species . HPV-53 was obtained from patients with HSIL but without coinfection with HPV-16, 18, 31, 33, and 58 types. The analysis of the variability of the L1 gene revealed single nucleotide exchanges in six positions, when compared with the reference sequence. Among these, the mutation point C6942T led to amino acid changes involving P430S in the protein sequence of the L1 . FurtherThe patient infected with HPV-54 was coinfected with the HPV-16 type. The L1 genetic variability showed single nucleotide exchanges in five positions when compared with the reference sequence. The A6622T and T6625C mutations have never been described until now. These two mutation points lead to Q313H and S333T amino acid changes in the L1 protein sequence, respectively . An anal HPV-81 was detected in one sample with CIN I but without coinfection with HPV-16, 18, 31, 33, and 58 genotypes. An analysis of the genetic variability of L1 showed a new synonymous mutation at the A7148G position . The samThe analysis of the L1 of HPV-70 was detected in two cervical samples with histopathological findings of adenocarcinoma and CIN I, but without coinfection with HPV-16, 18, 31, 33, and 58 genotypes. The sequence variation of these viruses showed five mutation points, one of which was a nonsynonymous variant. A6886G variation leads to a change in the amino acid T433A). Furthermore, the T433A mutation is embedded in the T-cell and B-cell epitope binding regions  (Tables 33A. FurtThe genetic variability in L1 protein of HPV-62 showed nine nucleotide exchanges, five of which are nonsynonymous mutations. Among these, two new mutations were found at 6838C and A6921G, which led to E354D and Q382R changes in amino acid, respectively . With reThis study described the presence of rare HPV genotypes in CIN I, III and invasive cancer in women from North-East Brazil. The results of this study showed thirty nucleotide changes, nine of which were reported for the first time. Moreover, this study showed the predominance of Alpha-9 and a low frequency of Alpha-7 species in cervical lesions. These correspond to epidemiological data from the rest of the world, which have shown that Alpha-9 is the main species involved in cervical cancer [Recently, epidemiological data on the worldwide incidence of HPV have provided evidence that 2.25% of total invasive cervical cancer was caused by rare HPV types , 30. ThiThe use of PCR with MY09/11 degenerate primers followed by direct sequencing allows other Alpha species to be detected, apart from the Alpha-9 virus group. This study reported rare HPV types, including new variants that were occasionally characterized as single infections. Rare HPV types have also been detected as a single infection in invasive cancer in other studies [Some of the rare HPV types found in cervical lesions or cervical cancer in this study are not considered as carcinogenic viruses . Hence, In conclusion, this study found a predominance of Alpha-9 species in CIN I, III and invasive cancer in infected women from North-East Brazil. Moreover, genetic variability in L1 genes showed several synonymous and nonsynonymous mutations, which can lead to an altered immune response. Further investigation of the prevalence and variability of rare HPV types can clarify the role of this virus in infections and carcinogenesis.


New commercially available Human Papillomavirus (HPV) assays need to be evaluated in a variety of cervical screening settings. Cobas HPV Test (cobas) is a real-time PCR-based assay allowing for separate detection of HPV genotypes 16 and 18 and a bulk of 12 other high-risk genotypes. The aim of the present study, Horizon, was to assess the prevalence of high-risk HPV infections in an area with a high background risk of cervical cancer, where women aged 23–65 years are targeted for cervical screening. We collected 6,258 consecutive cervical samples from the largest cervical screening laboratory in Denmark serving the whole of Copenhagen. All samples were stored in SurePath media. In total, 5,072 samples were tested with cobas, Hybrid Capture 2 High Risk HPV DNA test (HC2) and liquid-based cytology. Of these, 27% tested positive on cobas. This proportion decreased by age, being 43% in women aged 23–29 years and 10% in women aged 60–65 years. HC2 assay was positive in 20% of samples, and cytology was abnormal  for 7% samples. When only samples without recent abnormalities were taken into account, 24% tested positive on cobas, 19% on HC2, and 5% had abnormal cytology. The proportion of positive cobas samples was higher than in the ATHENA trial. The age-standardized cobas positivity vs. cytology abnormality was 3.9 in our study and 1.7 in ATHENA. If in Copenhagen the presently used cytology would be replaced by cobas in women above age 30 years, an extra 11% of women would based on historical data be expected to have a positive cobas test without an underlying cervical intraepithelial lesion grade 3 or worse. Countries with a high prevalence of HPV infections should therefore proceed to primary HPV-based cervical screening with caution. The higher sensitivity for high-grade cervical intraepithelial neoplasia (CIN) of Human Papillomavirus (HPV) testing compared with cytology digene Hybrid Capture 2® High Risk HPV DNA test . The HC2 assay has been extensively studied all over the world in randomized controlled trials and numerous split-sample studies For several years, HPV DNA testing has been synonymous with Qiagen’s The cobas® HPV Test , approved by the USA Food and Drug Administration in 2011, is a fully automated real-time polymerase chain reaction (PCR) assay. The assay batches up to 94 samples together with positive and negative controls. It allows for a differentiated positive result distinguishing HPV genotypes 16 and 18 separately next to the bulk of 11 other high-risk and one possibly high-risk HPV genotypes  In the present population-based split-sample study, we compared cobas testing with liquid-based cytology (LBC) and HC2 on consecutive samples from a large cervical cytology laboratory covering all of Copenhagen, Denmark. The population from this area is well-screened but has a high background risk of cervical cancer. In 1958–62, prior to the start of screening, the age-standardized incidence of cervical cancer in Copenhagen was 35 per 100,000 (world standard population) In Denmark, women aged between 23 and 49 years without a smear or a biopsy within the last three years are personally invited for cytology-based cervical screening, as are women aged 50–65 years if they had no screening in the last five years. In 2010, about 76% of women were screened within the recommended interval During the period from 10 June 2011 to 25 August 2011, a total of 12,138 routine cervical samples were received at the laboratory. On date of arrival, the samples were registered in the national Pathology Data Bank using the Danish personal identification number (CPR number) which includes the date of birth. Samples were placed in racks of 48, and labeled with the woman’s CPR number, a laboratory specimen identifier, and a barcode.The present study, Horizon, was nested into the routine laboratory practice. It utilized the residual material left in the vial after SurePath-based LBC had been completed, and after any postquot HC2 triage testing of samples with ASCUS diagnoses in women aged ≥30 years. A total of 6,258 samples (52%) were selected for the present study, taking the lowest rack numbers of samples on testing days equally from Monday to Friday. Based on capacity and processing considerations at the molecular biology laboratory, the target number of samples was set to 5,000. A maximum of 192 samples (four racks) were processed per day. Samples with insufficient residual volume for further HPV testing  were excluded from the study. An additional 21 samples were excluded for technical reasons due to human error. The final analysis of cobas testing could therefore be undertaken on 5,072 samples (81% of those selected for the study). In concordance with the protocol reviewed by the manufacturers prior to the study, 2 ml of SurePath media was added to the available residual material of approximately 2 ml (dilution factor roughly 1∶1), in order to obtain enough volume for additional testing which will be reported separately. All testing was done in the same laboratory.In total, 5,013 samples (98.8%) were from unique women, whereas the remaining 59 (1.2%) were collected from 29 women. Women’s screening history from 1 January 2000 onwards was ascertained from the nation-wide Danish Pathology Data Bank Cytology screening of LBC samples was performed routinely with a FocalPoint GS Imaging System . Cytological outcomes were reported according to the Bethesda 2001 classification. They were classified as ASCUS, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions or worse (≥HSIL) including atypical squamous cells – cannot exclude HSIL (ASC-H), atypical glandular cells (AGC), adenocarcinoma in situ (AIS) and carcinoma. Cytology was evaluated by cytoscreeners and pathologists without knowledge of the outcomes of HPV testing.For testing with cobas, 1 ml of the diluted material was aliquoted into a 13 ml round bottom test tube . All tubes were labeled with the CPR number, barcode and the laboratory specimen identifier used for cytology testing. Subsequently, the tubes were stored at 2–8°C until testing according to the manufacturer’s protocol. No pre-treatment of SurePath samples was required. Extraction of DNA was undertaken on the cobas x 480 instrument Additionally, we evaluated the intra-laboratory reproducibility of the cobas assay. For this study, we used samples tested as part of the pre-trial validation of the cobas equipment during which 10.8% of the cobas samples returned invalid. We selected 200 negative and 300 positive cobas samples from different batches, regardless of the women’s age, screening history or CT value. After the pre-trial validation, the company calibrated the instrumentation, resulting in a lower proportion of invalid samples (0.1%) for the duration of the trial.digene Hybrid Capture system version 2 software (DHCS v.2). Testing was done on cytology postquot material.With HC2, the detection of 13 high-risk genotypes is done in a bulk fashion with a “Positive” or “Negative” sample result readout. The test has no internal control for sufficiency of test material. Samples were either pretreated manually with DNA denatured prior to testing according to the manufacturer’s protocol, or DNA was isolated and purified using the DSP AXpH DNA kit on the QIASymphony SP platform 2 test. The outcomes of the cobas testing were tabulated by the age of the women, screening history (primary vs. follow-up samples), cytology outcome, and the HC2 outcome. The trends in HPV positivity by age were tested with the Mantel-Haenszel χ2 test for trend, an 95% confidence intervals (CI) for relative risks were calculated by assuming that their logarithms were approximately normally distributed.The outcomes of testing with cobas were reported hierarchically (HPV16>HPV18>other high-risk HPV>negative>inadequate). Positive cobas samples were defined in accordance with the manufacturer’s critical threshold (CT) values, being ≤40.5 for HPV16, ≤40.0 for HPV18, and ≤40.0 for other high-risk genotypes, and positive HC2 samples as those with a relative light unit per cut-off (rlu/co) value ≥1. Cytology was considered abnormal if ≥ASCUS was reported. Differences in the distributions of age, screening history, cytology, and HC2 outcomes between the included and excluded samples were tested with the χThis study was designed as a quality development study, utilizing only residual material that would otherwise have been anonymized and discarded. According to Danish regulations of biomedical research, an ethical approval is not necessary for such studies, in accordance with "Guidelines about Notification etc. of a Biomedical Research Project to the Committee System on Biomedical Research Ethics, No 9154, 5 May 2011, section 2.5″.2 = 11.2, df = 6, P = 0.084), cytology outcome , or HC2 outcome . There was a small but significant difference between the included and the excluded samples in terms of the women’s screening history. In total, 87.0% of the included samples, and 89.9% of the excluded samples were primary samples .Between the 5,072 samples available for testing with cobas, and the 1,186 samples that were excluded from the study, there were no significant differences in the distributions by age group . The mean number of days between storing and testing was 16 (range: 1–62). The mean age of the women was 37.3 years . In total, 162 (3.2%) samples were taken in women aged below the recommended start of screening (23 years), and 113 (2.2%) in women aged above the recommended ending of screening  had to be retested, with three (0.1%) remaining inadequate after retesting. Overall, 1,361 samples (26.8%) tested positive on cobas. Listing the HPV genotypes hierarchically based on oncogenicity for cervical cancer (HPV16>HVP18>12 other HR HPV genotypes>negative>inadequate), single or multiple infections with genotype 16 were detected in 365 (7.2%) samples; 130 (2.6%) samples showed single or multiple infections with HPV genotype 18 excluding any coinfections with HPV16; and the remaining 866 (17.1%) samples showed single or multiple infections with any of the remaining non-16/18 HPV genotypes targeted by the cobas assay.For women targeted by the organized screening program (23–65 years), the overall proportion testing positive on the cobas assay decreased with age from 43.1% in women aged 23–29 years to 10.3% in women aged 60–65 years (Only 373 (7.4%) samples were abnormal on cytology. Among women with normal cytology, 22.9% of the samples tested positive on cobas and 15.6% on HC2. Among women with ASCUS, these proportions were 63.4% for cobas and 64.2% for HC2; they were 79.7% and 89.5%, respectively, for LSIL; and 88.8% and 90.7%, respectively, for ≥HSIL.These results were fairly similar for the subgroup of 4,413 primary tests . In totaAmong the 300 samples that tested positive on the initial cobas run, 272 (90.7%) tested positive also in the second run. Among the 28 (9.3%) samples that changed from positive to negative, the average CT value was 38.0 (range: 16.3–40.1) in the first run, and 19 were uniquely positive for “other high-risk genotypes”. Among the 200 samples that tested negative on the initial cobas run, 196 (98.0%) tested negative in the second run, whereas 4 (2.0%) changed the outcome from negative to positive. The average CT value of the latter samples was 39.3 (range: 38.8–40.0) in the second run, suggesting weakly positive HPV samples.In Copenhagen, where the background risk of cervical cancer has been high, 27% of women tested positive for the bulk of 14 high-risk HPV genotypes included in the cobas assay. Even in samples without recent cervical abnormalities, 24% tested positive. As expected, this was considerably higher than the 7% and 5% of women, respectively, with abnormal cytology. The proportions of women testing positive on cobas decreased from 43% (41%) in women aged 23–29 years to 10% (9%) in women aged 60–65 years. About two-thirds of the positive cobas tests were due to genotypes other than HPV16 and HPV 18. This is not unusual, as infections with these HPV genotypes are frequent in women with normal cytology or with low-grade CIN In line with the higher prevalence of HPV genotypes 16 and 18 in high-grade lesions, 52% of cytological ≥HSIL in our study showed an infection with these two genotypes. However, we found that 11% of samples with ≥HSIL on cytology  tested negative on the cobas assay. In the Danish screening program, women with ≥HSIL are currently referred for colposcopy without HPV DNA triage. We will monitor them through linkage with the Pathology Data Bank to determine whether HPV triage could be used to reduce false-positive referrals for colposcopy.More surprising was the fact that the proportion of positive samples was 27% for cobas versus 20% for HC2. Some of this difference may be due to the differences in the designs of the assays. While the cobas was designed to detect 14 HPV types, HC2 was designed to detect 13 HPV types. Furthermore, cobas is a real-time PCR-based L1 DNA assay, whereas HC2 is an RNA to DNA hybridization assay.Although using samples from the pre-trial validation of the cobas instrumentation, the intra-laboratory reproducibility of negative cobas results was high. The 2.0% of the initially negative samples that tested positive in the second run showed very high average CT values. This is reassuring given the on-going international discussions on extending the screening interval. However, the 90.7% reproducibility of the initially positive cobas results suggests that the calibration of the probe for “other high-risk genotypes” is not highly robust. In a Dutch validation study using Universal Collection Medium, the intra-laboratory positive reproducibility of the cobas assay was 97.3% Our study was population-based and used consecutive samples from a large public laboratory. Nineteen percent of the selected samples had to be discarded because only small amounts of residual material were available for the study. There was no significant difference between the included and the excluded samples in terms of the age of the women, cytology interpretation, and the HC2 outcome. All samples were tested in one laboratory by the same staff that is involved in routine screening. The proportion of women aged 25–64 years without recent abnormalities who tested positive on the HC2 assay, 16%, was similar to that observed in an earlier study covering the same catchment area, approximately 17% The cobas assay, furthermore, functioned well on the diluted SurePath media, as only 3 (0.1%) out of 5,072 samples had an inadequate outcome. Our study is the first to report the cobas outcomes based on SurePath samples. SurePath has an estimated market share in England of about 55%, 70% in Denmark , and about 30% in the USA Following the standard recommendations within the Danish cervical screening program, women in our study population with abnormal cytology were referred for colposcopy or for repeated testing. As a follow-up to the current study, we will additionally invite women with positive HPV tests and normal cytology for repeated testing in 12 months. The standard measures of sensitivity and specificity of the screening tests will be reported once the histological follow-up of the study population has been completed.The cobas assay was FDA-approved based on the ATHENA study which included women presenting for routine screening me, and BD HPV test), two HPV mRNA assays , and one immunocytochemical assay (MTM CINtec p16INK4a) in 1,099 women Several studies evaluated the use of the cobas assay among women referred for colposcopy because of abnormal cytology. The Predictors 2 study compared the cobas assay to three other HPV DNA assays  of 16.2% of women aged 30–65 years tested positive on cobas, and 2.8% of women had both a positive cobas test and abnormal cytology . In contMuch work has been done to define optimal criteria for referral of HPV-positive women for colposcopy Reducing the extent of repeated testing is a priority issue for introducing HPV DNA-based primary screening. Countries with a high prevalence of HPV infection such as Denmark should therefore proceed to HPV-based primary cervical screening with caution because the extent of follow-up testing and diagnostics could be enormous whether or not triage is used to refer women for colposcopy.Our study showed a 27% high-risk HPV positivity proportion when unselected women from Copenhagen were tested with the cobas assay, and 20% when tested with HC2. These positivity rates were far above the 7% abnormality rate found in routine cytology screening. Even in women without recent abnormalities aged ≥30 years these percentages were 16% and 4%, respectively.


INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16 HPV infection is recognised as the necessary cause of cervical intraepithelial lesions (CIN) and invasive squamous cell carcinoma (SSC). However, only a minority of viral infections ever results in neoplastic lesions. It is well known that the majority of HPV infections may be cleared by the immune system, and that certain high-risk (HR) HPV types  are significantly more common among high-grade lesions and carcinomas [The most important factor in CIN progression is certainly the integration of HPV sequences into the host genome with the loss of E2 tumor suppressor gene. E2 physiologically regulates the expression of E6 and E7 oncogenes. There is consensus that integration is common in high-grade CIN and cancer, while it is infrequent or is lacking in low-grade CIN. HPV integration, disrupting cell-cycle control and escaping immune system surveillance, would induce stochastic accumulation of genetic aberrations, leading to CIN progression.Recently, a wide range of molecular techniques has been evaluated on cytological specimens, to improve cervical cancer screening strategies , 3. HPV-INK4a (henceforth p16) seems to represent one of the most widely investigated tool. On tissue specimens, the ideal test for the detection of HPV has not been established yet, although different assays have been analyzed . Potential useful marker should target viral genome or related proteins  or should identify host cell's products whose expression would be stimulated by HPV infection. In this context, immunohistochemical (IHC) localization of p16p16 is a tumor suppressor protein playing a crucial role in cell-cycle regulation. p16 prevents the phosphorylation of the retinoblastoma protein (pRb) by inhibiting cyclin-dependent kinases CDK4 and CDK6. Physiologically, non-phosphorylated pRb binds the transcription factor E2F, thereby preventing E2F stimulation of cell progression into S phase. The functional inactivation of pRb by HPV-E7 oncoprotein induces E2F factor release that becomes subsequently free to drive cell-cycle progression towards S phase.All the above mentioned markers and technologies are a matter of controversy, each having their advantages and drawbacks. PCR is considered the most effective method for HPV-DNA detection, but some problems still exist in routinely practice: DNA extraction compromises the preservation of tissue architecture ; moreovep16 demonstrated to be useful as surrogate biomarker of HPV integration and E7 overexpression. However, pitfalls such as positive staining by nondysplastic cells would limit its clinical accuracy. Recently, a novel concept of biomarker based on the combination of p16 and Ki-67 detection in cervical cytology specimens (p16/Ki-67 double stain) has been proposed. Under physiological conditions, the coexpression of these proteins does not occur, since they typically induce opposite effects , 10; allBasing on this background, in the first phase of this study, we aimed to analyze analytical and diagnostic accuracies of the novel CISH technology in detecting viral DNA and in identifying HPV physical status on formalin-fixed and paraffin-embedded tissue. To do that, CISH results were compared with results obtained from HPV-DNA test and HPV-mRNA test. In the second phase, we assessed the potential usefulness of CINtec PLUS p16/Ki-67 double-stain immunohistochemistry (IHC) on histological samples with different degrees of dysplasia, to detect cervical lesions expressing E6/E7 HPV oncogenes.This study was performed in agreement with the standards of the ethics review board of “SS Annunziata” Hospital and was approved by the Ethical Committees of “G. d'Annunzio” University, in accordance with the principles outlined in the Declaration of Helsinki of 1975.From the electronic files of Surgical Pathology Department of “SS Annunziata” Hospital of Chieti, 926 cases of biopsy-proven squamous cervical lesion, obtained from January 2010 to July 2012, were retrospectively retrieved. HPV-DNA test result by Hybrid Capture 2 (HC2), performed on liquid-based sample of exfoliated cells, collected from cervix immediately before colposcopy-directed biopsy of the lesion;result from mRNA testing, performed on residual cervical liquid-based cytological specimen.Among these casuistries, 154 cases met the following inclusion criteria: Cervical Intraepithelial Neoplasia grade 1, CIN1; CIN grade 2, CIN2;  CIN grade 3, CIN3; invasive squamous cell carcinoma (SSC). Two pathologists independently reviewed haematoxylin and eosin (H&E) stained slides and reported histological diagnosis according to the World Health Organization nomenclature and criteria as follows: Only cases reaching consensus in histological diagnosis were finally included in the study .A written informed consent was obtained from all the participants in the study, and corresponding FFPE specimens were subsequently taken. Identification codes were finally assigned to each case, in accordance with confidentiality standards.(i) Cervical Cytology. Cervicovaginal samples were collected from ecto-endocervix immediately before colposcopy-directed biopsy. Cervical specimens were then transferred into PreservCyt cytology medium  liquid and transported to Cytopathology Departments. Cytological vials were processed using ThinPrep 2000 . Slides were next stained with Papanicolaou procedure. (ii) HPV-DNA Test. After cytological slide preparation, an aliquot (4 mL) of each liquid-based cytological (LBC) sample, stored at RT, was removed to perform HPV-DNA testing by using the commercially available Hybrid Capture 2 system , in accordance to manufacturer's protocol. HC2 detects oncogenic HPV types . HC2 reactions were read by a luminometer, which provided a relative quantification of each individual sample in comparison to the mean of a series of positive controls containing 1 pg/mL of HPV DNA . The cut-off of 1 relative light unit (RLU) was used to classify a specimen as positive or negative. RLUs value in relation to control (RLU/CO) provided an estimation of the number of HPV-DNA copies of each sample . The RLU value of each individual sample was then recorded. According to RLU/CO values, HPV-DNA positive cases were arbitrarily categorized into three groups having “low viral load” (RLU/CO from 1.0 to 50.0 RLU/CO), “intermediate viral load” (RLU/CO from 50.1 to 100.00 RLU/CO), and “high viral load” (RLU/CO > 100).(iii) HPV-mRNA Test. A second aliquot (3 mL) from each residual LBC specimen was transferred into a fresh 10 mL tube for nucleic acids extraction. After centrifugation, the supernatant was removed and the sample was transferred into a tube containing 2 mL Nuclisens Lysis Buffer . Next, magnetized silica dioxide particles were added to the lysate to initiate the nucleic acids isolation process. Finally, nucleic acids were eluted from the solid phase in 55 μL of elution buffer and stored at −20°C if not further processed immediately after extraction.μL of nucleic acids was used to perform mRNA testing , in accordance with the manufacturer's instructions. 15 mRNA testing is based on real-time nucleic acid sequence based amplification (NASBA) procedure, which utilizes molecular beacon probes labelled with 5-carboxyfluorescein (FAM) and Texas Red fluorochromes, at an isothermal temperature of 41°C. The test identifies full-length E6/E7 mRNA from five high-risk carcinogenic HPV types . A fluorescent analyzer measured in real time the emission of the fluorescence from molecular beacon hybridized with amplified mRNA. As performance control, the human U1A mRNA from the small ribonucleoprotein-specific A protein has been used. Negative control reactions, consisting of all reagents except RNA, were performed at each run. mRNA testing was defined as positive if at least one of the five HPV genotypes detected by the test has been found . (iv) Chromogenic In Situ Hybridization. Two serial sections were cut to a thickness of 4 μm, one for CISH investigation and one for p16/Ki67 dual-stain IHC. The extra sections cut before and after each tissue section were stained with H&E and used to evaluate the adequacy of each FFPE for the subsequent investigations.Bond ready-to-use DNA CISH HPV protocol  able to detect 5 oncogenic HPV types  was optimized for reproducible sensitive and background free usage. Slides were then processed using the Bond-Max automated slide-staining system . Finally, CISH sections were counterstained with haematoxylin. HPV-positive controls consisted of FFPE sections containing two sets of cells: CaSki cervical cancer cell line (containing 200 to 400 copies of HPV-DNA types 16 per cell) and HeLa cervical cancer cell line (containing 10 to 50 copies of HPV-DNA types 18 per cell). Thyroid tissue has been used as negative control, since in the literature we could not find any evidence for the presence of HPV.Two pathologists independently evaluated CISH slides. CISH signals were determined for at least 10 high power fields. Nuclear peroxidase staining was considered a positive result for HPV-DNA. Positive CISH signal patterns were classified as follows: (1) diffuse (D), when nuclei were completely stained ; (2) punctated, when distinct dot-like intranuclear signals were noted (indicative of integrated HPV); (3) mixed, diffuses, and punctated (D/P) when both patterns are noted. Figures . (v) p16/Ki67 Dual Stain and p16 Stain. A commercial kit specifically designed for the simultaneous detection of p16 and Ki67  was used, accordingly to the supplier's instructions and adapting the protocols for the use on histological samples. One section for each case was stained with p16/Ki67 dual test. A red chromogen marked Ki-67 expression within the nucleus and a brown chromogen marked cytoplasmic/nuclear p16 expression. Sample was scored as positive when the simultaneous expressions of both markers were revealed within the same cells. Cases without any double-immunoreactive cell were called negative. Another section for each case was prepared for the immunohistochemical evaluation of p16 alone (clone E6/H4) using CINtec Histology Kit . After antigen retrieval, sections were incubated with mouse monoclonal anti-p16 , with EnVision+ System HRP anti-mouse . Afterwards, diaminobenzidine chromogen  was applied and counterstaining with haematoxylin was performed. p16 overexpression was visualized as a brown colour precipitate within nucleus and cytoplasm. Expression of p16 in more than 10% of epithelial cells was regarded as a positive result. For dual stain and p16 immunohistochemistry, positive and negative controls consisted of SCC of uterine cervix, with and without primary antibodies, respectively. All tissue slides plus controls for p16/Ki67 dual test were stained in a single session that was different from that of p16 alone. In both cases, Dako Autostainer  was used. Both slide sets were subjected to two pathologists, which evaluated all cases blindly to all study results. K values were divided into six scales of strength of agreement: poor (<0.00), slight (0.00–0.20), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), or almost perfect (0.81–1.00) [By standard method authors calculated the prevalence of HPV-DNA, E6/E7 mRNA, p16/Ki67, and CISH positivities. Chi square or Fisher's exact test was used to assess the association between variables. Concordances between histopathological diagnosis and DNA test, mRNA test, and CISH were calculated by Kappa statistics. According to the criteria of Lands and Koch, the 81–1.00) . Chi squAccuracy parameters (sensitivity and specificity) of each test separately as well as the comparison of accuracy parameters between tests were assessed by receiver operating characteristic analysis. Histological diagnosis was regarded as the gold standard and CIN2+ lesion was considered as the worse outcome. To do that, histological results were dichotomized into CIN2+  and less than CIN2 . Areas under the receiver operating characteristic (ROC) curves and 95% confidence intervals (CI) were estimated to assess differences between test performances  and McNeCorrelation between CISH signal patterns and HPV viral load categories was evaluated by Cochran-Armitage trend test. P less than 0.05 were regarded as significant.Statistical analyses were performed by using SPSS software , version 15.0. In all analyses, probability values A series of cervical FFPE from sixty-three patients  were included in the study. Among these cases, 25 were diagnosed as CIN1, 16 as CIN2, 21 as CIN3, and 1 as SCC. Summary of results from histological diagnosis, HPV-DNA and mRNA tests, HPV viral load, CISH, and p16/Ki67 dual stain from each case included in the study are reported in Molecular Tests. HPV-DNA positivity was detected in 60 of the 63 (95.2%) cytological samples. Among these, 65% (N = 39/60) showed CIN2+ lesions in histological specimens. A positive DNA test result conferred a ≥CIN2+ odds ratio (OR) risk of 3.2 (95% CI: 0.4–26). 4.8% (N = 3/63) of women resulted HPV-DNA negative. Overall percent agreement between DNA testing test and histological diagnosis was 61.9% . E6/E7 mRNA positivity was detected in 71.4% (N = 45/63) of cytological cases; among these, 36 (80%) were CIN2+. Within the 18 mRNA negative cases, 16 (88.9%) were confirmed as CIN2−. mRNA test results were associated to CIN2+ diagnosis with a OR = 32 (95% CI: 7–144). Overall percent agreement between mRNA testing and histological diagnosis was 82.5% .P < 0.01).Diagnostic performances of both DNA and mRNA tests are represented in CISH Results. CISH showed a clear signal in 95.2% (N = 60/63) of the specimens. Invalid result has been found in 4.8% (N = 3/63) of the cases, due to unclear and weak signal. The rate of positive results was 73% (N = 46/63). Among these, 30.4% (N = 14) were CIN1, 30.4% (N = 14) were CIN2, and 37% (N = 17) were CIN3. The unique case of SCC showed CISH positivity. Negativity has been found in 22.2% (N = 14/63) of the cases. P < 0.01). Dichotomizing histological diagnosis and considering only CISH-positive results, diffuse pattern has been found in 64.3% (N = 9/14) of CIN2− and 3.1% (N = 1/32) of CIN2+. All cases of punctated pattern have been found in CIN2+, as well as 68.8% of mixed patterns. The proportion of punctated pattern increased with the severity of cervical lesion (Cochran-Armitage test for trend P < 0.0001) ( 0.0001) .Sensitivity and specificity of CISH analysis were 86.5% (95% CI: 71.4–94.4) and 39.1% (95% CI: 22.2–59.3), respectively. A positive CISH result conferred a ≥CIN2+ risk (OR) of 4.11 (95% CI: 2–13.9).N = 46/60) were CISH positive. Within CISH-negative cases, 85.7% (N = 12/14) were HPV-DNA positive, while 14.3% (N = 2/14) were HPV-DNA negative (P = .001). Overall percent agreement between CISH and DNA test was 80% .CISH results were assessed against HPV-DNA test . All CISN = 35/45) were CISH positive. Of those, 2.9% (N = 1/35) showed a diffuse pattern, 71.4% (N = 25/35) a mixed pattern, and 25.7% (N = 9/35) a punctated pattern. Among the 11 mRNA/CISH+ cases, only 2 cases (18%) demonstrated a punctated pattern (P < 0.0001). Overall percent agreement between CISH and mRNA test was 70% .CISH results were also assessed against HPV E6/E7 mRNA expression. Among mRNA+ cases, 77.8% (P < 0.0001), while difference between RNA testing and CISH did not reach significance (P = 0.06).Since HPV-DNA test is currently considered the most reliable method to detect papillomavirus infection in both cytological and histological samples, the performances of CISH and mRNA test were compared to HPV-DNA test performance. DNA testing achieved an area under the curves (AUC) of 0.53  CISH and of 0.64  and mRNA testing of 0.79  . DiffereCISH Results and HPV Viral Load. Among cytological samples testing HPV-DNA positive, the mean of viral loads was 502.9 ± 620.5 RLU/CO, the median being 155.79 RLU/CO (range 1.45–2663.29 RLU/CO). N = 19/60) of the cases showed low viral load, 6.7% (N = 4/60) intermediate load, and 61.6% (N = 37/60) high viral load. The rate of CISH positivity has been found to be lower in cases with low viral load level (58%) than in those with intermediate (75%) and high (86.6%) load levels   .P = 0.05).Correlation between CISH punctate signal pattern and viral load categories showed that the rate of this pattern was higher in specimens with low viral loads than in those having intermediate or high loads . The lower agreement was observed for CIN1 diagnosis, the higher for SCC (P = 0.08) , including 8 CIN1, 11 CIN2, 12 CIN3, and 1 SCC ( = 0.08) . SensitiN = 14/16) cases  and in 100% of CIN3 cases (within the three-third of the epithelium). Expression level of p16 positively correlated with that of Ki67 (P < 0.01). In the unique case of SCC, strong dual-stain positivity has been also shown by neoplastic cells infiltrating the stroma.Considering p16/Ki67 dual stain immunohistochemistry , all 63 k = 1). Sensitivity and specificity of dual stain were 100% (95% CI: 88.8–100) and 84% (95% CI: 64.6–94.1), respectively.The use of p16/Ki67 IHC significantly improved consensus among pathologists, which reached 100% (N = 35/45) of mRNA-positive cases. Among mRNA-negative cases, p16 showed no immunoreactivity in 88.9% (N = 16/18) of patients. Concordance between p16 and E6/E7 mRNA test was 81% (k = 0.59).Since in cervical tissue p16 is considered a surrogate biomarker of HPV-E7 expression, we correlated both p16 and p16/Ki67 staining results with HPV-E6/E7 status, as determined by mRNA test . p16 expN = 40/45) of mRNA-positive patients, negativity being detected in 88.8% (N = 16/18) of mRNA-negative cases. Concordance between p16/Ki67 dual stain and mRNA test was 89% (k = 0.74). Dual stain positivity has been found in 88.9% (k = 0.64). Concordance between dual stain and CISH (punctated and mixed pattern) was 83.3% , since only cells simultaneously showing p16/Ki67 expression have been considered as positive, irrespective of morphology. Finally, in our series dual stain improved specificity of p16 alone. In this setting, 98% of CIN2+ stained mRNA positive, while 100% stained p16/Ki67-positive. The only invasive cancer showed dual stain and E6/E7 mRNA positivity. It seems likely that dual stain positive/mRNA positive CIN2+ could represent cervical lesions at higher risk of progression toward invasive cancer . This faHPV are recognized as a necessary cause of CIN, but only a minority of HPV infections even results in cervical lesions. Although the majority of infections may be cleared by immune system, integration of HPV sequence into the host genome may induce CIN progression. The detection of HPV genome within cervical lesions and the assessment of its physical status are then crucial in prognostic terms.To the authors' knowledge, this is the first study evaluating the novel HPV tyramide-based CISH technology and the innovative CINtec PLUS p16/Ki-67 double stain immunohistochemistry on histological tissues, as well as the first investigation comparing both methods to molecular tests actually considered as the gold standards for HPV detection.Molecular assays may be expensive and require a high level of expertise, which are often difficult to reach in routinely laboratory. Although larger studies are needed, our data demonstrate the usefulness of CISH and p167Ki67 immunostaining in surgical pathology settings. In particular, CISH could be a feasible method to localize HPV genome on paraffin-embedded specimens. This technology would help to distinguish episomal from integrated HPV, thus allowing conclusions regarding the prognosis of the lesion. Likewise, the genotype-independent p16/Ki67 dual staining approach, which demonstrated greater efficacy than p16 alone, would confer a higher level of standardization to the diagnostic procedure.Finally, due to their strong correlation with tests which are currently considered the standards for HPV detection in cytological specimens, both CISH and dual stain technologies would be considered a viable potential alternative to molecular assays in the evaluation of the biology of cervical lesions. Nevertheless, these preliminary data need to be confirmed in a larger clinical cohort.


While HPV infection is the main cause of cervical cancer, genetic susceptibility to HPV infection is not well understood. Tumor necrosis factor alpha , involved in the defense against HPV infection, plays an important role in cervical cancer progression and regression. The aim of this study was to investigate the relationship between the TNF-alpha rs1800629 polymorphism and risk of HPV infection or cervical cancer.Three groups were involved in this study of Chinese women. Group 1 consisted of 285 high risk HPV positive cervical cancer patients, Group 2, 225 high risk HPV positive patients without cervical cancer, and Group 3, 318 HPV negative women with no cervical cancer. Blood samples were obtained from all patients and genotyped by PCR-RLFP. Fifty randomly selected samples were further sequenced.The allele and genotype distributions of the TNF-alpha rs1800629 polymorphism were not significantly different between each of the groups (P>0.05). There are no significant relationship between rs1800629 polymorphism and high risk HPV infection , cervical cancer , or cervical cancer with HPV infection .We demonstrated that there is no association between TNF rs1800629 polymorphism and the HPV infection, or cervical cancer with HPV infection. Cervical cancer remains the second most common cancer among women worldwide Several cytokines that modulate the immunologic response have been implicated in the development of cancer −/− mice are resistant to the development of benign and malignant skin tumors. TNF modulation may contribute to regulation of cell inflammation, and the subsequent development of malignant disease The role of TNF-α in cancer is not well understood. Deregulated TNF expression within the tumor microenvironment appears to favor malignant cell tissue invasion, migration, and ultimately metastasis formation TNF-α is regulated at the transcriptional level From 2008 to 2010, all patients treated at the Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University were evaluated for entry into this study. Patients were divided into 3 groups. Group 1 consisted of 285 patients diagnosed with cervical cancer and high risk HPV infection . Group 2 consisted of 225 patients diagnosed with high risk HPV infection, but without any abnormal cervical cytological or pathological change. Group 3 was composed of 318 healthy Chinese women presenting for routine healthy screening at our Health Check Center. Group 3 patients had no history of HPV infection, cervical neoplastic disease, or evidence of cervical pathology. Patients in Group 3 had a normal cervical cytology on at least two consecutive annual examinations. Women with any history of malignant disease were excluded from Group 3. All patients were Chinese. Informed written consent and 5 ml of peripheral blood were obtained from each participant. The study protocol was approved by the Ethics Committee of Shengjing hospital. HPV status was detected as previously described Genomic DNA was extracted from peripheral blood leukocytes (Tiangen Blood Genome Kit). Genotyping was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, which was based on the method of Cabrera et al. TNF-α rs1800629 polymorphism and HPV infection and cervical cancer was estimated using odds ratio analysis (ORs) and their 95% CIs calculated by multivariate logistic regression. All the ORs were adjusted for age and smoking status. Hardy-Weinberg equilibrium analysis was performed with the Chi-square test. The Chi-square test was used to compare variables. A p-value of 0.05 was used as the criterion of statistical significance. All analyses were conducted using SPSS software .The association between The basic clinical characteristics of the three groups are summarized in G1–G2 = 0.271, PG2–G3 = 0.287 and PG1–G3 = 0.916, G1–G2 = 0.551, PG2–G3 = 0.627 and PG1–G3 = 0.980, The PCR-RFLP analysis results of 50 random samples were all confirmed by sequencing . The genIn further multivariate regression analysis, no significant association was observed in the comparison of genotypes after adjusting for age, smoking, and HPV infection status. Comparing Groups 2 and 3, we found no significant relationship between TNF rs1800629 polymorphism and HPV infection , with high risk HPV and no cervical cancer (Group 2), and with neither high risk HPV or cervical cancer (Group 3) We evaluated the relationship between rs1800629 polymorphisms and HPV infection and cervical cancer. We did not observe a significant relationship between rs1800629 polymorphisms and cervical cancer or HPV infection.Although several studies have indicated that polymorphisms in the promoter region of rs1800629 may be a contributing factor to the development of cervical cancer Our findings are similar to those reported by Stanczuk et al. Kirkpatrick et al. The ethnic background in a population based genetic susceptibility study is also an important factor affecting the results. The genotype distribution of rs1800629 in our Chinese patients was similar to that reported in Indian Environmental exposures may also play an important role in these inconsistent results. Geographic variations in HPV type One limitation of the present study was that it was a hospital-based, case control study. Patients were selected from a single institution and thus may not have been representative of cervical cancer patients in the general population. Control subjects were selected from the same hospital, also possibly introducing a selection bias. However, the distribution of rs1800629 genotype frequencies in our patients was similar to other reports TNF-α rs1800629 allele A tend to increase the expression of TNF-α In our study, we found no association between TNF rs1800629 polymorphism and HPV infection, or cervical cancer with HPV infection.Figure S1PCR-RFLP analysis and sequencing of TNF-alpha rs1800629 genotype. (A) PCR-RFLP analysis results (electrophoresis on 3% Gel) (B) Sequencing results by ABI 3700.(TIF)Click here for additional data file.


The Eisenberg plot or hydrophobic moment plot methodology is one of the most frequently used methods of bioinformatics. Bioinformatics is more and more recognized as a helpful tool in Life Sciences in general, and recent developments in approaches recognizing lipid binding regions in proteins are promising in this respect. In this study a bioinformatics approach specialized in identifying lipid binding helical regions in proteins was used to obtain an Eisenberg plot. The validity of the Heliquest generated hydrophobic moment plot was checked and exemplified. This study indicates that the Eisenberg plot methodology can be transferred to another hydrophobicity scale and renders a user-friendly approach which can be utilized in routine checks in protein–lipid interaction and in protein and peptide lipid binding characterization studies. A combined approach seems to be advantageous and results in a powerful tool in the search of helical lipid-binding regions in proteins and peptides. The strength and limitations of the Eisenberg plot approach itself are discussed as well. The presented approach not only leads to a better understanding of the nature of the protein–lipid interactions but also provides a user-friendly tool for the search of lipid-binding regions in proteins and peptides. The Eisenberg plot or hydrophobic moment plot is one of the most beautiful examples of where bioinformatics really started off. In the search for methods to translate the primary sequence into more advanced structural information about the structure and folding of proteins, Eisenberg and co-workers developed their methodology ,2. Over E. coli SecA [Lipids and lipid–protein interactions play an increasingly appreciated and recognized role in many biological processes (see for reviews –11). Oneoli SecA  correspooli SecA –15.z), the mean hydrophobicity (<H>) and the mean hydrophobic moment (μH). In this study the Heliquest approach, though specialized in identifying lipid binding helical regions in proteins, was used to obtain the “original” Eisenberg plot. For this purpose the influence of using another hydrophobicity scale, the Fauchere and Pliska scale [This briefly exemplifies the potential power of the Heliquest-based bioinformatics method ,12. A clka scale  instead The Eisenberg plot methodology used an algorithm for detecting hydrophobic polypeptide sequence segments and discriminates between surface-seeking and transmembrane regions. This study checked whether the Heliquest data can give valid results according to the Eisenberg plot methodology ,2, and wThe results found with Heliquest generated data  correspoH> = 0.600 – 0.342 <H>. Secondly, potential transmembrane helices are assumed if the mean hydrophobicity <H> is greater than 0.51 and the mean hydrophobic moment is below the line as defined above [H> = 0.654 – 0.324<H> and <H> above 0.75 respectively.In the original Eisenberg plot methodology two features were extracted. First of all, a surface seeking propensity for surface helices are thought to exist for points close to the line <μIn order to check the validity of the newly obtained Eisenberg plot one step further, a number of more recent examples were checked which were not included in the Eisenberg databases ,2. In TaAll data confirmed the findings obtained with the original Eisenberg approach see , which sH> is in all these cases above 0.75. Additionally the lipid discrimination factor D identified a substantial amount of all depicted , the hydrophobic moment (μH) and the net charge (z) were calculated. In the analysis, 18-residue windows were used, and for each sequence under investigation the window with the highest discrimination factor was selected. The ultimate classification rule renders the discrimination factor (D):The lipid binding potential is performed as described before . In esseWhen this discrimination factor is above 0.68, the corresponding can be considered to be a  lipid-binding region. See  for detaH>) and the hydrophobic moment (μH) were extracted from the Heliquest program [The Eisenberg plot approach was essentially performed as described in the original study . Both th program  and subs program  instead  program . This st program ,2. The uThe data presented here indicates that Heliquest generated data can be utilized for a hydrophobic moment plot analysis. A comparison of both the original databases ,2 used bDuring the introduction of the Eisenberg plot ,2 there There is a growing perception that membrane proteins can also possess the so-called non-annular lipid-binding sites, where specific anionic phospholipids bind tightly to the protein and have been demonstrated to be involved in the formation of homo-oligomeric structures  and heteBased on all the sequences investigated in this study, a positive discrimination value above 80% was found for the Heliquest lipid binding discrimination factor, while the combined approach was able to identify all sequences as potential lipid binding. All sequences investigated were well reported examples of experimentally confirmed lipid-binding proteins or peptides. What the positive prediction value will be for not yet experimentally confirmed protein-lipid interacting proteins remains to be seen. This study clearly indicates however that the combined use of the Heliquest lipid binding discrimination factor and the Heliquest generated Eisenberg plot methodology provides a powerful tool for the search of possible lipid-binding regions in proteins. The presented bioinformatics approach might serve as a starting point for studying proteins which have not yet been characterized in detail when it comes to protein–lipid interactions.


Though cervical cancer is highly curable when detected early, it remains one of the leading causes of cancer death in women worldwide. Early detection is effective because the precursor lesions evolve slowly into invasive cancer, typically over a period of >10 years. These precursor lesions  are detected with cervical cytology screening, the Pap smear. In every country where a Pap smear screening program has been introduced, rates of cervical cancer have been substantially reduced. The discovery that human papillomaviruses (HPV) are etiologically linked with cervical cancer has led to efforts to apply this knowledge to improve cervical cancer screening and to potentially prevent cervical cancer through vaccination.HPV is not a single virus but a family of closely related viruses, each designated as a type, numbered in order of discovery. Typing is based on nucleic acid sequencing. More than 100 HPV types are known to exist, and at least 30 can be detected in the anogenital tract. No simple in vitro culture methods are available for identifying it, and serologic testing is insensitive. Techniques for identifying the virus are based on nucleic acid detection, either direct hybridization or after amplification. HPV types associated with malignancies are referred to as high-risk types, and those associated with warts (condylomas) are rarely found in cancers and are called low-risk types.Sexual transmission is the dominant mechanism for acquiring genital HPV. Infection is usually transient and not associated with symptoms. An estimated 80% of sexually active women have been exposed. Studies have detected HPV in >90% of cancers worldwide, and plausible biologic mechanisms can explain oncogenesis. The magnitude of the risk association between HPV and cervical cancer is greater than that for smoking and lung cancer. However, infection alone is insufficient to cause cancer, and additional factors are required for neoplasia.One investigational quadrivalent vaccine includes types 6, 11, 16, and 18. HPV-16 and HPV-18 (high-risk types) are found in 25% of all CIN I lesions and 70% of CIN II/III and anogenital cancers. HPV-6 and HPV-11 (low-risk types) are found in 25% of CIN I lesions and 90% of anogenital warts. Therefore a prophylactic vaccine against these four types would substantially reduce HPV-related disease.Vaccine candidates have been evaluated in animal models of papillomavirus infection. The L1 protein of HPV is the major capsid protein and self-assembles into viruslike particles (VLPs). Species-specific VLP vaccines provide protection against infection and disease. Protection was associated with the development of neutralizing antibodies. Serum from vaccinated animals conferred protection to unvaccinated animals.Saccharomyces cerevisiae (yeast), and yeast-derived vaccines have been given to millions of children and adults. The vaccine includes amorphous aluminum hydroxyphosphate sulfate adjuvant and is given in a 0-, 2-, 6-month dosing scheme. Phase I trials (300 participants) were performed to establish immunogenicity and tolerability of a range of doses of monovalent HPV L1 vaccines. Phase II trials  were performed to establish the immunogenicity and tolerability of a range of HPV L1 VLP vaccine dose formulations and provide preliminary proof of concept. Phase III trials  will determine the efficacy of the HPV L1 VLP vaccine by using prevention of type-related CIN I, genital warts, and CIN II/III as the endpoints.The HPV-6, HPV-11, HPV-16, and HPV-18 L1 VLP vaccine is manufactured in The results of the phase II trial of the HPV-16 VLP vaccine have been recently published  is ongoing to demonstrate vaccine immunogenicity and tolerability in boys and girls. In addition, a study with Nordic Cancer Registries is planned for long-term (>10 years) follow-up postlicensure to determine duration of efficacy, long-term safety, and replacement of vaccine types with other HPVs. Phase III programs will definitively evaluate clinical and public health impact of the HPV vaccine in adolescents and adult women.


HPV oncogenes mRNA detection gains momentum as an adjuvant for HPV-related cervical abnormalities diagnosis, but is based on costly detection assays not allowing viral type targeting.To assess detection rate of HPV oncogenes E6/E7 mRNA from cervical specimens using a manual, open technology, fully customizable protocol and determine whether HPV-related epidemiological features influence mRNA retrieval. We reviewed literature and compared our retrieval rate with automated technologies.We used 60 samples positive for HPV DNA types 16, 18, 31 and/or 45. We extracted mRNA with a TRizol-based protocol, and tested mRNA purity and concentration using light absorbance. We reverse-transcribed mRNA into cDNA for E6/7 detection.HPV oncogenes E6/E7 mRNA was retrieved from 36 (60%) out of 60 specimens. No HPV load-related clinical or epidemiological feature was significantly associated with mRNA retrieval. Presence of HPV-DNA 16/18 was associated with mRNA retrieval .The open-technology protocol yielded an mRNA detection rate similar to that of automated technologies. Advantages are lower costs and target HPV type customization. In the last two decades, the detection of Human papillomavirus (HPV) DNA in cervical samples has proven to be a good diagnostic and risk predictor tool for cervical intraepithelial neoplasia (CIN) and cervical cancer  and the NucliSENS Easy Q HPV (bioMérieux) are based on the same technology, and detect E6/E7 mRNA expression from the five most prevalent hrHPV types . Other two tests aim at an expanded HPV types set: the TaqMan real-time PCR assay, which targets 12 high-risk  and two low-risk (HPV 6 and 11) types using E6/E7 region primers and probes in a duplex format , Brazil, a public health institution dedicated to comprehensive care for women, was approved by the local Ethics Committee (Protocol: N° CEP 723/2009) in compliance with the recommendations of the “Declaration of Helsinki” for guiding medical doctors in biomedical research involving human subjects. The samples consisted of cervical specimens, collected from women who underwent cervical diathermic conization due to HPV-induced squamous lesions at Unicamp’s cervical pathology clinics. Women were invited to participate while awaiting the conization procedure. Every research subject signed a written informed consent stating their compliance with the study, and an interview was performed concerning clinical and epidemiological aspects. After that, and immediately before the conization procedure, a cervical specimen was obtained with an endocervical brush, and stored in a 1-mL tube containing Specimen Transport Medium .In order to obtain the sample for these experiments we examined 117 specimens between April and December 2010. We first performed PCR reactions in order to ascertain the presence of HPV DNA using standard protocols  and centrifuged at 13,000 g for 10 min. The supernatant was removed and 1 mL of the TRizol™ reagent  was added to the cellular pellet, and cells lysed by repeat pipetting and standing at room temperature for 5 min . Subsequently, 200 ul of chloroform was added and shaken vigorously, standing at room temperature for 3 min before centrifugation at 12,000 g for 15 min. The resultant aqueous layer was then transferred to a new tube. To the aqueous solution, a volume of 500 μl of isoamyl alcohol was added and mixed by vortex. The whole mixture stood at -80°C for 2 hours and 10 min at room temperature and centrifuged at 12,000 g for 10 min. Supernatant was then removed and 1 mL of iced ethanol was added and shaken vigorously. Finally, the mixture was centrifuged at 7,500 g for 5 min, the supernatant was removed and after the pellet of mRNA was dry, mRNA was eluted in 30 μl of mRNAase-free water (DEPC) and stored at -80°C pending analysis. The mRNA purity and concentration were determined by the absorbance at 260 nm (A260) and 280 nm (A280) using NanoDrop ND-1000 .PCR products were visualized using agarose gel electrophoresis. The reactions were performed separately according to the reagent mixture: five μl of DNA were added to a PCR mix containing 0.5 pmol of each primer Table , specifiThe mRNA was reverse-transcribed into first strand cDNA  according to the manufacturer’s guidelines. The cDNA PCR product was amplified as described in the type-specific PCR section, although with a final volume of 25 μl and 2 μl of cDNA.All statistical analyses were performed with the R Environment for Statistical Computing (R Project). Significance was set at 95% (p = 0.05) and 95% confidence intervals were used (95% CI). We constructed a multivariate regression model assessing HPV oncogenes E6 and E7 mRNA retrieval according to clinical features and presence of HPV DNA of types 16, 18, 31 and/or 45. Odds ratios were obtained by exponentiation of the regression coefficients.We retrieved oncogenes E6 and E7 mRNA from 36 (60%) of the 60 specimens previously found to be positive for the relevant HPV genotypes. None of the classically viral load - related clinical and epidemiological features that have been repeatedly reported in the medical literature was significantly associated with mRNA retrieval , we retrieved viable oncogenes E6 and E7 mRNA in almost 60% of the HPV DNA positive cases. The extraction technique was completely manual. There are plenty of reports on this subject using automated our semi-automated detection techniques, with manufacturer prespecified sets of HPV types included in the assay. Our mRNA detection rate was comparable to those reported by most authors using automated mRNA detection techniques , what could point out to an association of multiple genotypes and mRNA.However, scientific evidence accumulated from virological, molecular, clinical and epidemiological studies has unequivocally demonstrated that cervical cancer is in fact a sequel from a long term unresolved infection by certain HPV genotypes (Castellsagué Despite the substantial resources spent in cytology screening and follow-up, cervical cancer is still the 10th most common cause of cancer death in European women. In Brazil by contrast, although rapidly declining, cervical cancer is one of the leading causes of mortality among women, and the cervical cancer burden is even heavier in less developed countries. Because cervical cancer is the only cancer that is almost completely preventable through regular screening and thus early treatment, improvement and expansion of existing screening strategies and technologies constitutes a main target of the European Council Recommendation on Cancer Screening (Boulet et al. Current lines of evidence reinforce the biological reasons for detecting HPV oncogenes E6 and E7 mRNA. HPV screening cohort studies have shown that HPV DNA testing has a higher sensitivity than cytological testing for the detection of cervical lesions, although it has slightly lower specificity (Halfon et al. Given the fact that sustained viral oncogenes E6 and E7 expression is essential for the initiation and maintenance of the transformed phenotype induced by mucosal high-risk HPVs, several assays have been specifically developed for the detection of HPV E6⁄E7 mRNA (Snijders et al. DNA and mRNA testing may be employed together for screening to take advantage of the combined higher sensitivity and specificity these tests have shown; patients would e.g. then be referred for a biopsy when both tests were positive (Halfon et al.


Genital Human papilloma virus (HPV) is one of the most commonly diagnosed Sexually Transmitted Infection (STIs) in men and women. Knowledge about HPV infection among men is limited. This study aims to determine correlates of adequate knowledge of HPV infection among men who attend an STI clinic in Puerto Rico.A cross-sectional study of 206 men was conducted at an STI clinic in San Juan, PR. Adequate knowledge was defined as a score of at least 70% of correct responses among those men who reported having ever heard of HPV. Variables that achieved statistical significance in the bivariate analysis (p<0.05) were included in the multivariate logistic regression model.Although 52.5% of men reported having heard of HPV infection before the survey, only 29.3% of this sub-group had an adequate knowledge of HPV. Most men did not know that HPV is a risk factor for anal (38.7%), penile (50.0%) and oral (72.6%) cancer. Factors associated with adequate knowledge of HPV in age-adjusted models were being men who have sex with men (MSM) , self-report of genital warts  and herpes . MSM was marginally associated with adequate knowledge  and self-report of herpes remained significantly associated  in multivariate logistic regression analysis.Awareness and knowledge of HPV was very low in this group of men. Interventions to increase knowledge and awareness in this group are necessary to promote preventive practices for HPV-related cancers in high-risk groups. Human papilloma virus (HPV) is one of the most commonly diagnosed STIs, with over 630 million HPV infections worldwide and an overall prevalence between 9-13% . HoweverAlthough knowledge is not a direct predictor of health behavior , it is mIn Puerto Rico (PR), despite the HIV epidemic and documented high-risk sexual practices in population-based studies ,19, therThis study was part of an ongoing epidemiological core study among men and women attending the Latin American Center for Sexually Transmitted Diseases , located in San Juan, Puerto Rico A full description of the methodology used in the core study has been described elsewhere . BrieflyFollowing completion of the core survey interview, all male participants were informed of the opportunity to participate in an additional interview as part of an HPV study. Men expressing interest in the HPV study were required to provide additional written informed consent for a second interview, which included questions regarding HPV awareness, HPV knowledge, perceived susceptibility to HPV-related cancers, HPV vaccine awareness, and willingness to be vaccinated against HPV.Data collection procedures were approved by the University of Puerto Rico, Medical Sciences Campus Institutional Review Board (UPR-IRB).‘don’t know’ answer was given a score of 0. An example of items included in this scale was ‘only women can get infected with HPV’. Latter sets of questions were assessed only among those who reported having heard of HPV infection before the survey (n=106), or 52.5% of the total sample.Based on both existing measures  and addiExploratory factor analysis of the 21 item scale measuring HPV knowledge identified 4 subscales, in which one factor  was related to HPV knowledge. Chrombach alpha for the 15 item scale was 0.75.A mean knowledge score was computed by summing correct responses of the 15 item scale, with higher scores meaning a higher knowledge. Adequate knowledge was defined as a minimum score of 70% . Chi-squA total of 206 men participated in the study, of which 202 98.1%) completed the full assessment . Demographic and individual HPV risk-related characteristics are shown in Table % completMore than half reported recent (last 90 days) use of tobacco (52.5%) and alcohol (59.4%). More than a third (36.6%) reported recent illicit drug use.data not shown).12.4% of the men interviewed have had more than one sexual partner in the last 3 months. A significant proportion of study participants identified as being men who have sex with men (MSM) (29.2%). Among those reporting vaginal sex, more than a half (56.9%) reported condom use in their last sexual intercourse, and 33.7% of the participants were circumcised (data not shown). However, only 29.3% of these men demonstrated an adequate level of knowledge of HPV. Table More than half  of the sample had heard about HPV infection before the survey. Primary sources of information included television (24%), print media  (13%), and health care professionals (10.5%) . Men who reported a history of herpes  and genital warts  also were more likely to have adequate knowledge of HPV. In multivariate models (Table To our knowledge, this is the first study to attempt to examine awareness of HPV infection among men in PR. In a sample in which none of the study participants has being vaccinated and the awareness of the vaccine is low , resultsCommon sources of HPV information that were identified in this study were television, magazine and newspaper, followed by health care professionals and physicians. These sources of information are inconsistent with results from the population-based 2009 HINTS survey in PR, which showed that the Internet is the primary source for cancer information used by the general population of PR . A possiAfter age-adjusted logistic regression analysis, a marginal association between MSM and adequate knowledge of HPV was observed. Findings from an online survey in the US show similar results . In thisAlthough results from this clinic-based study are of great relevance for the development of cancer prevention and control efforts among men in PR, our findings need to be interpreted with caution. Research in an STI setting has limitations. Primary among these is that our findings might not generalize to the general population. Specificity and complexity of some of the items of our HPV knowledge score also needs to be further explored for future efforts. Despite its potential limitations, data collected from a highly selective population, such as a public STI clinic, may also offer an opportunity to reach targeted high-risk groups more efficiently.In summary, this study highlights the low prevalence of HPV awareness and of adequate knowledge of HPV infection in a sexually active group of men attending an STI clinic in Puerto Rico. Findings highlight the need to increase HPV awareness, primarily of HPV-related cancers such as oral and anal cancer. STI clinics might serve as venues for cancer prevention and control among high-risk men. In order to effectively moving forward strategies in these settings, further research which will help us understanding levels of health literacy, stratified by groups of interest, needs to be performed.The authors declare that they have no competing interests.VCL worked on the conceptualization of the study, study design, data analysis and interpretation and manuscript preparation; APO worked on the study design, data interpretation and manuscript preparation; LMDT worked on data analysis, interpretation and manuscript preparation; JP worked on conceptualization of the study, manuscript preparation and interpretation of data; MCC worked on the study design; HG worked on conceptualization of the study, study design and interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/346/prepub


Human papillomavirus (HPV) is closely associated with cervical cancer that the incidence of this tumor is regarded as a surrogate marker for HPV infection in countries lacking epidemiological studies. HPV is also implicated in subsets of anogenital and oropharyngeal cancers. Although cervical cancer is the third most common cancer in women worldwide, its reported incidence is low in Saudi Arabia, ranking number 12 between all cancers in females and accounts only for 2.4% of all new cases, despite the lack of national screening programs. However, the limited available studies from Saudi Arabia indicate that HPV prevalence and genotypes’ distribution in invasive cervical cancer show similar pattern as in the world. Cytology screening (Pap smear) and HPV vaccinations are the two preventive measures against cervical cancer. The two available vaccines are effective against the two most common HPV genotypes (HPV-16 and -18). Since 92% of cervical tumors in the Kingdom are infected with HPV of which 78% are HPV-16 and -18 genotypes, vaccination is expected to protect against more than two-third of cervical cancers in Saudi Arabia. Nevertheless, due to its low incidence , a proper cost-effectiveness analysis is required to justify the implementation of a costly vaccine bearing in mind that HPV could potentially be associated with about 3% of all cancers. However, further studies are needed to ascertain the real prevalence of HPV at the population level at large, its association with various types of cancers, and also the impact of local tradition and emerging behavioral trends that could affect HPV transmission and consequently the effectiveness of applying national vaccination program. Human papillomavirus (HPV) has been overwhelmingly associated with cervical cancer that the incidence of this malignancy is deemed as surrogate indicator of HPV infection in countries lacking epidemiological studies. This can easily be justified because the worldwide HPV prevalence in cervical cancer has been estimated between 85 and 99% , 2. AccoHuman papillomavirus infection is common particularly in sexually active period of age. Reported estimates of incident HPV infection among initially negative women have reached as high as 60% over a 5-year follow-up period –9. In adIn contrast to the global view, the incidence of cervical cancer is very low in Saudi Arabia, ranking number 12 between all cancers in females and accounts only for 2.4% of all new cases , despiteData concerning the prevalence of HPV infection, HPV genotypes, and its relationship with cervical cancer are globally scarce in Saudi Arabia. Studies profiling Pap smears examined at University Hospitals have revealed precursor lesions of cervical cancer , 18–20. The magnitude of the association between HPV infections and genotypes as causative agent of cervical cancer has been recently evaluated in limited number of patients in two independent studies from the same institute , 27. TheIn agreement with other studies, the most common HPV genotype was HPV-16  with a pThe second study included 90 patients with cervical cancer and had essentially reached similar results using polymerase chain reaction amplification methods with two common primers, MY09, MY11 and GP5+, GP6+ that amplify a wide range of HPVs of which isolates were genotyped using DNA sequencing and reverse line blot hybridization assay to identify the high-risk HPV genotypes . ResultsIn North America, it was noted that, although there has been significant reduction of the incidence of head and neck cancers as a result of the anti-smoking campaigns since the late 80s, there was a significant increase in the incidence of oropharyngeal squamous cell carcinomas in young (40–55 years) specifically in the tonsils and the base of the tongue where most of these patients are not alcohol or tobacco consumers . About 6So far, the prevalence of HPV infection in anal, penile, vaginal, vulvar, and oropharyngeal cancers has not yet been explored in Saudi Arabia. Nevertheless, it is important to include these forecasted potential when discussing the eventual impact of HPV vaccines in a society. In fact, the extrapolation of these rough estimated percentages of HPV-positive anogenital (96%) and head and neck (30%) cancers in Saudi Arabia is given in Figure in situ squamous cell carcinoma, in 55% of invasive squamous cell carcinoma, in 20% of climatic droplet keratopathy, in 35% of scarred corneas, and in 32% of normal conjunctival tissue obtained during routine cataract extractions. They concluded that HPV is detectable not only in epithelial neoplasms of the ocular mucous membrane but also in non-neoplastic lesions as well as in apparently healthy conjunctiva. Gazzaz and Mosli had explored the possibility of finding HPV infection in prostatic tissues of 56 patients presenting with benign prostatic hyperplasia (BPH) or prostate cancer  and comparable to the whole-world (85–99%) and that about 75% are HPV-16/18 genotypes Figure . In contAlthough cost-effectiveness criteria may vary between countries, a preliminary evaluation would suggest that there is no reason for public health to institute either screening or vaccination if Saudi Arabia truly has such a low incidence of cervical cancer. Therefore, theoretically, some might argue that implementing a national vaccination program may not sensibly decrease the incidence of cervical cancer at the population level because it is already very low. In addition, if all 10–14 years old girls are to be vaccinated in Saudi Arabia , this would be a burden on health system and may not be cost-effective in comparison with other health priorities. Nevertheless, there may be specific individual benefits that screening or vaccination may offer for selected women, who would be identified upon careful analysis of the frequency of cervical cancer in groups at risk for HPV infection or women well-informed about the risk and voluntarily wish to be vaccinated. However, taking into consideration the projection of all potentially HPV-associated tumors outlined in Figure Currently, the vaccines are available in major hospitals in Saudi Arabia and are offered with or without fee to requesting girls upon physician’s prescription. Family physicians should also be provided with objective information regarding the HPV vaccine so they would recommend the vaccine to their patients . ParentsBeside the salient difference of having very low incidence of cervical cancer in Saudi Arabia, the involvement of HPV infection in this malignancy is comparable to the rest of the world with HPV-16 and -18 being the two most common genotypes and account together for three-quarters of HPV infection. Although vaccination against HPV would protect three-quarters of cervical cancer patients, the currently reported low incidence and the high cost of the vaccine would make it not-cost-effective in the Kingdom of Saudi Arabia. However, to ascertain or refute these conclusions, further studies are needed to find out the real prevalence of HPV at the population level at large, its association with various types of cancers, and also the impact of local tradition and emerging behavioral trends that could affect HPV transmission.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Among veterans, having been selected into the military and having easy access to medical care during and after military service may reduce premature mortality but not morbidity from mental distress and may not improve health-related quality of life. The objective of this study was to determine whether veterans in different racial/ethnic groups differ in their health-related quality of life from each other and from their civilian counterparts.Among 800,000 respondents to the 2007–2009 Behavioral Risk Factor Surveillance System surveys, approximately 110,000 identified themselves as veterans and answered questions about their sociodemographic characteristics, self-rated health, and recent health-related quality of life. Nonoverlapping 95% confidence intervals of means distinguished veterans and civilians of different racial/ethnic groups.Veteran and civilian American Indians/Alaska Natives reported more physically unhealthy days, mentally unhealthy days, and recent activity limitation days than their veteran and civilian counterparts in other racial/ethnic groups. Non-Hispanic white veterans and Hispanic veterans reported more physically unhealthy days, mentally unhealthy days, and recent activity limitation days than their civilian counterparts.Unlike findings in other studies, our findings show that veterans’ health-related quality of life differs from that of civilians both within the same racial/ethnic group and among different racial/ethnic groups. Because once-healthy soldiers may not be as healthy when they return to civilian life, assessing their health-related quality of life over time may identify those who need help to regain their health. Each soldier’s experience in the military is unique, whether the soldier volunteered or was drafted into military service . After bThis benefit of reduced premature mortality for soldiers may not carry over to reduced morbidity from mental distress and improved health-related quality of life (HRQOL) -5. OveraPrevious studies have examined the HRQOL of veterans with mixed results -23. SomeThis study is a descriptive analysis of cross-sectional data from respondents to the 2007–2009 surveys of the Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is an annual random-digit–dialed telephone survey in all 50 US states, the District of Columbia, Puerto Rico, the Virgin Islands, and the US Pacific territories . EligiblOf 1,278,028 participants in the 2007–2009 BRFSS, 801,862 (63%) answered a question about their status as a veteran (see definition below) and identified themselves as either non-Hispanic whites, non-Hispanic blacks, American Indians/Alaska Natives, or Hispanics. Twelve percent  of these reported being a veteran, 100,829 (92%) men and 9,536 (8%) women . We compared veterans and their civilian counterparts within racial/ethnic groups by age, marital status, educational level, employment status, annual income, and HRQOL.The HRQOL items used for this study were self-rated health , physically unhealthy days , mentally unhealthy days , and recent activity limitation days . The question about veteran status remained the same during the 2007–2009 BRFSS surveys: “Have you ever served on active duty in the United States Armed Forces, either in the regular military or in a National Guard or military reserve unit? Active duty does not include training for the Reserves or National Guard, but DOES include activation, for example, for the Persian Gulf War.” However, the response choices differed in the 2009 questionnaires from those in the 2008 and 2007 questionnaires. In 2009, participants chose from 7 responses: 1) yes, now on active duty; 2) yes, on active duty during the last 12 months, but not now; 3) yes, on active duty in the past, but not during the last 12 months; 4) no, training for Reserves or National Guard only; 5) no, never served in the military; 6) don’t know/not sure; and 7) refused. In the 2007 and 2008 BRFSS, there were 4 choices: yes, no, don’t know/not sure, and refused. For this study, we defined veterans as those answering yes to these questions on any of the 3 surveys and civilians as those answering no to these questions. We excluded from the analysis those answering don’t know/not sure and those refusing to answer these questions.The demographic characteristics analyzed were the following: race/ethnicity ; age group , marital status (currently married or not), educational level , employment status , and annual household income .To account for the BRFSS complex sample design and sampling weights, we used SAS-callable SUDAAN version 9.2  to estimate demographic characteristics and self-rated health and mean unhealthy days by veteran status and race/ethnicity, both unadjusted and adjusted for sex, age group, marital status, educational level, employment status, and annual household income. Nonoverlapping 95% confidence intervals of means statistically distinguished veterans and civilians of different racial/ethnic groups.Women were more likely than men to be civilians, although non-Hispanic black and Hispanic women were more likely than non-Hispanic white women to be veterans . HispaniAmerican Indian/Alaska Native veterans reported more physically unhealthy days and recent activity limitation days than veterans in other racial/ethnic groups . AmericaAfter adjusting for sex, age, marital status, educational level, employment status, and annual household income, American Indian/Alaska Native veterans still reported more physically unhealthy days and recent activity limitation days than veterans in other racial/ethnic groups . AmericaThis study explored differences in the associations between the HRQOL of veterans and civilians by racial/ethnic group. Despite the “healthy soldier” effect, other studies have documented poorer mental and physical health in some veterans, which might be expected to affect their health perceptions or HRQOL -5,25-27.Compared with these other studies, our study had several strengths. It analyzed HRQOL in different racial/ethnic groups and had sizable numbers of respondents in these groups, allowing for the adjustment of potentially confounding sociodemographic characteristics. The BRFSS questions on HRQOL have acceptable validity and reliability ,9. Becauwww.cdc.gov/brfss/), we could not tell whether exposure to combat and duration of military service affected the observed differences in HRQOL among the different racial/ethnic groups of veterans. Moreover, because BRFSS is cross-sectional, we could not tell whether the observed differences in HRQOL between veterans and civilians occurred because of events during military service or afterward. The small number of women veterans in some racial/ethnic groups precluded comparison of their HRQOL with that of their civilian counterparts. Until recently, BRFSS has been based on landline residential telephones and excludes US adults who use only cell phones and whose sociodemographic characteristics and responses to BRFSS may differ from those who use landline residential telephones.This study also had several limitations. Because BRFSS depends on self-reported experiences, we could not corroborate reports of veteran status, although respondents would not benefit from falsely reporting themselves as veterans or denying they were veterans. Moreover, HRQOL is inherently subjective, and we could not corroborate differences in HRQOL with objective indicators of health and functional status  that affect HRQOL. Because the questions in the 2007–2009 BRFSS do not distinguish between participation in the military and exposure to combat and do not ask about duration of military service (The HRQOL differences in this study between veterans and their civilian counterparts and among veterans in different racial/ethnic groups may indicate persistent health problems associated with military service, persistent discrimination against certain racial/ethnic groups despite their military service, or both. Because once-healthy soldiers may not be as healthy when they return to civilian life, assessing their HRQOL over time may identify those who need help to regain their health.This project was supported in part by appointment to the Research Participation Program for CDC administered by the Oak Ridge Institute for Science and Education through an agreement between the Department of Energy and CDC. This research received no specific grant from any funding agency in the public, commercial, or nonprofit sectors.


Understanding ubiquitous research-to-practice gaps in uptake of adult vaccines, particularly those for sexually transmitted infections , may provide evidence to support the successful dissemination of future HIV vaccines. To this end, we assessed rates of HPV vaccine acceptability and factors correlated with HPV vaccine acceptability among men.We used a comprehensive search strategy across multiple electronic databases to locate studies that examined rates and/or correlates of HPV vaccine acceptability. The search strategy had no date or language restrictions. Search keywords included vaccine, acceptability and all terms for human papillomavirus. We calculated mean HPV vaccine acceptability scores across studies. We conducted meta-analysis using a random effects model on cross-sectional studies reporting correlates of HPV vaccine acceptability. All studies were assessed for risk of bias.Of 301 identified studies, 22 met inclusion criteria. Across 18 studies (n=7787), weighted mean HPV vaccine acceptability = 52.3 (SD 17.5) . HPV vaccine acceptability was significantly higher among gay/bisexual/MSM  versus heterosexual men . Among 11 studies (n=4064) included in meta-analyses, perceived HPV vaccine benefits and doctor recommendation had medium effect sizes, and the following factors had small effect sizes on HPV vaccine acceptability: anticipatory regret, perceived effectiveness, fear of side effects, perceived partner support for vaccination, perceived susceptibility to HPV, number of lifetime sexual partners, having a current sex partner, Hepatitis B vaccine uptake, smoking cigarettes, HPV awareness, knowledge and non-white ethnicity.Public health campaigns tailored for men that promote positive HPV vaccine attitudes, HPV knowledge and risk awareness, and healthcare provider education, may support HPV vaccine acceptability for men; these factors may be instructive in planning for future HIV vaccine dissemination. Future investigations employing rigorous designs, including intervention studies, are needed to support effective vaccine promotion among men.