Papillomavirus (HPV) infection is a serious problem for human immunodeficiency virus (HIV)-infected women, increases their risk of cervical lesions and cancer. In cervical carcinogenesis, mutations in the p53 gene occur most frequently within exons 5–8. To our knowledge, no previous studies have analyzed mutations in exons 5–8 of the p53 gene in HIV- and HPV-infected women. In our study, we verified these mutations in women with and without cervical abnormalities.Human p53 reactions). HPV-DNA was detected using polymerase chain reaction (PCR) and genotyping by PCR-restriction fragment length polymorphism analysis. Using primers for exons 5–8, the mutation of the p53 gene was verified by PCR-single strand conformational polymorphism. The total mutation of the p53 gene in exons 5–8 was not significantly associated with the HIV and control groups. The mutations in exon 7 were the highest in the HIV group (43.8%) and in exon 6 in the control group (57.2%) (p = 0.0793) suggesting a tendency toward differential mutation in exon 7 in the HIV group.The study included 160 women, divided into three groups: (1) 83 HPV- and HIV-infected women (HIV group); (2) 37 HPV-infected/HIV-uninfected (control group); and (3) 40 normal cytology/DNA-HPV negative/HIV-uninfected women  to observe the evolution of cervical lesions. Papillomavirus (HPV) infection is a particularly difficult problem for human immunodeficiency virus (HIV)-infected women because they are more vulnerable to infection and less likely to clear the virus, which increases their risk of developing cervical lesions and cancer[Human d cancer. Specifid cancer,2. The sd cancer.p53 and pRb, respectively. This process results in impaired tumor suppressor gene function, involving DNA repair, decreased apoptosis, deregulation of key controls in cell proliferation, and eventual cell immortalization[In cervical carcinogenesis, the integration of high-risk HPV (HR-HPV) into host-cell chromosomes is followed by the binding of HPV E6 and E7 oncoproteins with tumor suppressor proteins lization.p53 tumor suppressor gene specifically inhibits cell cycle progression and promotes DNA repair and/or apoptosis; its inactivation is correlated with a critical step in the development of many human cancers. Inactivation may result from a number of events, including mutation of the p53 gene  and binding of the p53 gene to cellular or viral proteins, such as the HPV E6 oncoprotein[p53 mutations are mostly missense in nature and are located predominantly within the DNA-binding domain, where a single mutation is sufficient to cause loss of normal p53 function[p53 function have yielded conflicting results[The  results,8.p53 gene occur most frequently within exons 5–8, which is the highly conserved DNA binding domain region[p53 that included all exons have suggested that mutations outside exons 5–8 are rare in tumors[p53 gene in cervical carcinogenesis are related to its protein expression by immunohisto/cytochemical or polymorphism in condon-72[p53 gene in HIV- and HPV-infected women. In our study, we verified these mutations in women with and without cervical abnormalities.Mutations in the n region. Studiesn tumors. Howeverp53 gene exons (negative control p53 reactions). The women included in the study were enrolled in the Specialized Assistance Service (SAE) for sexually transmitted diseases (STD)/AIDS in Maringá, Brazil, between April 1, 2011, and October 30, 2011. In these samples, HPV-DNA was previously detected (data not shown) in the Clinical Cytology Laboratory at the State University of Maringá (UEM), Brazil, and stored at -80°C. Stored samples from HIV-infected and HIV-uninfected women, both with and without cervical lesions, were included in the study. The samples for 1 and 2 groups were also HPV-DNA positive.The study included cervical samples of 160 women who were divided into three groups: (1) 83 HPV- and HIV-infected (HIV group); (2) 37 HPV-infected/HIV-uninfected (control group); and (3) 40 cervical samples from women with normal cytology/DNA-HPV negative/HIV-uninfected that were only used as a negative control for the mutation of the Cervical and endocervical samples were collected using a cytobrush and an Ayre’s spatula, transferred to 1.5 ml tubes with 1.0 ml of sterile 0.9% NaCl solution, and stored at -80°C. Genomic DNA was extracted using the AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit  according to the manufacturer’s instructions. The quality and quantity of purified DNA were measured by spectrophotometry. HPV polymerase chain reaction (PCR) amplification for HPV was conducted using primers MY09 (5′CGTCCMAARGGAWACTGATC-3′) and MY11 (5′-GCMCAGGGWCATAAYAATGG-3′); co-amplification of the human β-globin gene was performed as an internal control using primers GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′) and PC04 (5′-CAACTTCATCCACGTTCACC-3′) under the same conditions as for the HPV-PCR: normal,HpyCH4V[p53 gene was verified by PCR-Single Strand Conformational Polymorphism (PCR-SSCP), which detects molecular changes in single-stranded DNA that cause changes in electrophoretic mobility[p53[For HPV genotyping of selected samples, a new HPV-PCR amplification was performed, which was followed by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis using HpyCH4V. The mutmobility. The follity[p53. The womp53 exon mutations in the groups of women were compared using a non-parametric Z test. A p value < 0.05 was considered statistically significant.The statistical analysis was performed using STATISTICA 8.0 software, and all of the variables were expressed as absolute and relative frequencies. The rates of vs 35.7 ± 10.57 years old; p = 0.0254). The majority of the HIV group showed excellent control of the HIV infection, as determined by the correct use of highly active antiretroviral therapy (HAART) (79.2%), current CD4+ T lymphocyte count > 350 cells/mm3 (73.6%) and current viral load < minimum limit (58.4%) or between the minimum limit and 100 copies/mL (38.8%).On average, the HIV group was older than the control group (40.9 ± 11.23 p53 mutation occurred in only 1 sample of HPV-16 (9.1%) and in 6 samples of HPV-51 (54.5%). For the control group, HPV-16  and HPV-66  were the most common, but the p53 mutation was observed in only 1 sample each . HPV-58 was detected in 2 samples, and the mutation occurred in these 2 samples (100%). In regards to the cytological findings, the HIV group showed the following: 70 normal, 2 ASC-H, 10 LSIL and 1 HSIL. The findings in the control group were as follows: 21 normal, 6 ASC-H, 9 LSIL and 1 HSIL . Of these samples, a p53 gene. In total, 19.3% of the HIV group and 18.9% of the control group (p = 0.4896) showed mutations in the p53 gene and all had HR-HPV. As previously described, no mutations occurred in any of the negative control p53 reactions[Figure eactions which isIn the HIV group, a mutation in exon 7 occurred at the highest frequency in normal cytology 31.3%) followed by LSIL and HSIL (6.3% for both). For the control group, a mutation in exon 6 was most common in both normal cytology and LSIL (28.6% each) (Table .3% follop53 gene in different exons in HIV-infected and HIV-uninfected women, i.e., mainly in exons 7 and 6, respectively. A point mutation at the splice donor site at the 3′ end of exon 7 of the human p53 gene results in the retention of the intron 7 sequence in the mRNA, thereby inactivating the p53 protein[These findings suggested that the HPV infection appears to lead to a mutation in the  protein.p53 gene exons 5–8 was not significantly associated with the HIV and control groups, and in women of both groups with normal cytology or different grades of cervical abnormalities, similar to those described for p53 gene condon 72 polymorphism[p53 gene immunoexpression[p53 gene which deserves further studies. Overall, higher rates of cervical HPV infection and CC can be partly related to a more specific mutated exon in HIV-infected patients.We also showed that the total mutation in Through its effect on CD4 cells and regulation of immune responses to a variety of antigens, HIV infection may attenuate the systemic immune response to HPV. It is speculated that if there is a low number of circulating HPV-specific memory cells, then HPV-specific immunity may be particularly vulnerable to the effects of HIV. Possible due to this HIV-infected women are more vulnerable to infection and less likely to clear the virus, which increases their risk of developing cervical lesions and cancer. Accordip53 gene may act at later stages of carcinogenesis. Nevertheless, we believe that our study provides preliminary evidence that the mutation in exon 7 might be an important differentiating factor for cervical carcinogenesis in HIV-infected women. This aspect deserves an additional cross-sectional and longitudinal study using a larger sample size with a higher number of HSIL to observe the evolution of cervical lesions.In conclusion, we acknowledge that we did not study women with CC, and we studied few women with HSIL. However, our study cannot exclude the possibility that the mutations in the HPV: Human Papillomavirus; HIV: Human immunodeficiency virus; HR-HPV: High risk-HPV; p53: Related protein kinase; pRb: Phosphorylate the retinoblastoma; CC: Cervical cancer; SAE: Specialized assistance service; STD: Sexually transmitted diseases; AIDS: Acquired immuno-deficiency syndrome; PCR: Polymerase chain reaction; ASC-H: High-grade squamous intraepithelial lesion; LSIL: Low-grade squamous intraepithelial lesion; HSIL: High-grade squamous intraepithelial lesions; RFLP: Restriction fragment length polymorphisms; SSCP: Single strand conformational polymorphism; OpenEpi: Open Source Epidemiologic Statistics for Public Health; HARRT: Highly active antiretroviral therapyThe authors declare that they have no competing interests.All authors contribute to the manuscript. RPS, FG, ALPA and SCR-B searched the literature and manuscript preparation. SCR-B, MDBC and SMP collected the women biological samples. MELC, ALPA and FG wrote the manuscript. MELC, RPS, FG and ALPA participated in methodology design and execution. SCR-B, MDBC and SMP contribute to the statistical analysis and design of the study. MDBC, SMP and MELC had been involved in revising the manuscript critically for important intellectual content. MELC revised the final manuscript version, helped to provide information and suggestion. All the authors read and approved the final of the manuscript.


The divergent glacial histories of southern and northern Europe affect present-day species diversity at coarse-grained scales in these two regions, but do these effects also penetrate to the more fine-grained scales of local communities?We carried out a cross-scale analysis to address this question for vascular plants in two mountain regions, the Alps in southern Europe and the Scandes in northern Europe, using environmentally paired vegetation plots in the two regions (n = 403 in each region) to quantify four diversity components: (i) total number of species occurring in a region , (ii) number of species that could occur in a target plot after environmental filtering (habitat-specific γ-diversity), (iii) pair-wise species compositional turnover between plots (plot-to-plot β-diversity) and (iv) number of species present per plot (plot α-diversity). We found strong region effects on total γ-diversity, habitat-specific γ-diversity and plot-to-plot β-diversity, with a greater diversity in the Alps even towards distances smaller than 50 m between plots. In contrast, there was a slightly greater plot α-diversity in the Scandes, but with a tendency towards contrasting region effects on high and low soil-acidity plots.We conclude that there are strong regional differences between coarse-grained  diversity components of the flora in the Alps and the Scandes mountain ranges, but that these differences do not necessarily penetrate to the finest-grained  diversity component, at least not on acidic soils. Our findings are consistent with the contrasting regional Quaternary histories, but we also consider alternative explanatory models. Notably, ecological sorting and habitat connectivity may play a role in the unexpected limited or reversed region effect on plot α-diversity, and may also affect the larger-scale diversity components. For instance, plot connectivity and/or selection for high dispersal ability may increase plot α-diversity and compensate for low total γ-diversity. However, given the study's coarse grain, geographic variability in the current environment could not be excluded as a driver of this pattern. In particular, the habitats added with increasing topographic heterogeneity could simply be more species rich in southern than in northern Europe It is generally accepted that γ-diversity is determined not only by the current environment but also by long-term historical factors, i.e., ultimately immigration, speciation and extinction et al.et al.Substantial differences in β- and α-diversity between regions may also result from different historical filters 2) vegetation plot data from environmentally paired sites in the two regions. These two regions have experienced markedly different Quaternary histories. Notably, the Alps were never completely glaciated during the Pleistocene (reflecting their southern location and highly dissected topography) and were close to the main glacial refugia of the European flora in southern Europe and the peripheral alpine refugia around the margin of the Alps Here, we investigated the region effect on vascular plant diversity across spatial scales by comparing γ-, β- and α-diversity in the Alps (southern Europe) and the Scandes (northern Europe) mountain ranges using fine-grained , we used the original indicator values because of the close geographical proximity of the Alps to the region that was the focus of Ellenberg's indicator system. For the Scandes (n = 1203 plots), the indicator values were adjusted following Diekmann et al.Ellenberg's indicator system 2, restricting the study to fine-grained vegetation plots. A total of 11,249 plots in the Alps  and 481 plots in the Scandes  met these criteria.We then selected all plots that matched the following three criteria: (i) The plots were referenced in time (year) and space , were sampled during the period 1909–2009 and had unique geographical coordinates ; (ii) for each Ellenberg's indicator factor in each plot, at least five taxa with Ellenberg's indicator values were present to allow for a reliable estimation 2, median: 30 m2, third quartile: 100 m2) than in the Scandes . To account for these imperfections in the pairing, we used the six Ellenberg's indicator factors and plot size or transformations of these variables as covariates in the subsequent models of β- and α-diversity. The main vegetation types selected in the 403 Alps–Scandes pairs were subalpine-open-woodland, alpine-grassland and alpine-heath-like communities within which the most frequent species are respectively: Homogyne alpina, Leontodon pyrenaicus and Vaccinium myrtillus in the Alps and Deschampsia flexuosa, Polygonum viviparum and Empetrum nigrum in the Scandes.Before analysing the diversity components, we selected a subset of environmentally similar paired plots from the Alps and the Scandes using a principal component analysis (PCA) on all six of Ellenberg's indicator factors as well as plot size for all 11,249 plots in the Alps and then added the 481 plots from the Scandes see . Hereby,Having defined pairs of environmentally similar plots, we first compared total and habitat-specific γ-diversity in the Alps and the Scandes to evaluate our first hypothesis that total and habitat-specific γ-diversity are higher in the Alps than in the Scandes. Total γ-diversity values in the Alps and the Scandes were determined from the lists of all species occurring in the 403 Alps–Scandes pairs of plots.r rows (plots) and p columns (species):ijb is the estimated probability of species j to occur in plot i, is the number of species in plot i, jkm the number of joint occurrences of species j and k, kn the number of occurrences of species k in the matrix and ikx a binary value either equal to one or zero depending on the corresponding presence or absence of species k in plot i. The whole floristic matrix was built by merging all 806 plots from the two regions. For each plot and species in this matrix, we then calculated the probability ijb. Therefore, all plots were associated with a vector of ijp b values. Finally, to estimate the habitat-specific γ-diversity for each of the 806 plots we intersected the list of all species occurring in its region with the list of all species with a probability ijb exceeding a certain cut-off value for the plot ijb, we increased the strength of environmental filtering ijb values because many species often co-occur with highly frequent species, whereas ijb is usually low for rare species ijb, which are more likely to include only the most frequent species in the habitat-specific γ-diversity.To estimate the size of the habitat-specific γ-diversity, we intersected the total γ-diversity in a region with the lists of all species bound to a certain set of environmental conditions, following the approach proposed by Ewald ijb (0<ijb<1) relative to the overall frequency jf (0<jf<1) of species j in the whole matrix:Because only environmental filtering and not the relative overall frequency of each species should influence the size of habitat-specific γ-diversity, we re-computed the probability ijb' does not represent a probability value anymore and can reach values >1 and <0. The fundamental logic behind equation 2 is to give more weight to rare species: for a given ijb value the lower the denominator (jf), the larger the increase in ijb', i.e., emphasising rare species over frequent ones. Since we computed ijb' across the whole matrix , there was no bias in the comparison of the two regions with regard to species frequency. We estimated the size of the habitat-specific γ-diversity for each plot in the Alps and the Scandes by choosing the cut-off value for ijb' that maximised the strength of environmental filtering  without lowering the size of the habitat-specific γ-diversity below the observed species richness in any of the plots in the Alps and the Scandes. In other words, we iteratively increased the cut-off value for ijb' until the estimated size of the habitat-specific γ-diversity of at least one of the 806 plots fell below the observed species richness of that plot, and we chose the former iteration as a cut-off value for ijb'. We then tested whether the estimated size of the habitat-specific γ-diversity per plot was greater in the Alps than in the Scandes using a one-tailed paired-sample t-test.Note that jksim is Simpson's similarity index computed between plot pairs (j ≠ k), jka the number of species shared by both plots j and k, jb the number of species unique to plot j and kc the number of species unique to plot k. This approach yields a plot-to-plot measure of species turnover and corresponds to the pair-wise approach of compositional dissimilarities in the terminology of Tuomisto jkc values in each region. For each sampling plot, we also computed the average of all ΔjFc values between the focal sampling plot F and all other 402 j plots in a given region . For each of the two regions, we accounted for environmental distances between plots by first fitting the best model of the floristic similarity matrix (jksim) against the six Ellenberg's distance matrices and the plot size distance matrix. Then, we fitted the residuals of these models against geographical distance. To test the significance of the differences in the rate of distance decay in floristic similarity between different regions, Baselga P values of the respective null hypotheses, we used the proportion of the 1000 iterations for which the intercept in the Alps was larger or the slope smaller, respectively, than the equivalent coefficients in the Scandes. We note that the results were quite similar if we instead fitted for each of the two regions the full model of jksim against geographical distance as a main effect and all six Ellenberg's distance matrices and the plot size distance matrix as covariates in the model (results not shown).For each of the two regions, multiple linear regressions on distance matrices (MRM) To evaluate our third hypothesis that plot α-diversity is higher in the Alps than in the Scandes, we compared plot α-diversity between the two regions, measured as the number of vascular plant species per plot, using a one-sided paired t-test. In addition, we carried out an OLS analysis of covariance (ANCOVA) to explore plot α-diversity in the two regions, with region as a main effect and the independent environmental variables (the six Ellenberg's indicator factors and plot size) as covariates. We used backward elimination to retain only significant (P<0.05) covariates in the ANCOVA. To reduce positive skewness in plot α-diversity after pooling the Alps and the Scandes data (n = 806 plots), we log-transformed plot α-diversity before using it as a dependent variable in the ANCOVA. We tested for spatial autocorrelation in the ANCOVA residuals using a Moran's I correlogram We also tested for trends in the Alps–Scandes paired differences in plot α-diversity against environmental variables. To do so, we carried out an OLS model with “plot α-diversity in the Alps – plot α-diversity in the Scandes” as the dependent variable and the six Ellenberg's indicator factors and plot size for the Alps plots as independent variables .For each region, the significance of the relationship of plot α-diversity to habitat-specific γ-diversity was tested using a Monte Carlo test P value estimate for a slope difference between the two regions.We also tested the difference in the relationship of plot α-diversity to habitat-specific γ-diversity between the Alps and the Scandes by comparing the slope coefficients of plot α-diversity against habitat-specific γ-diversity in both areas. To test the significance of the difference in the slope coefficients between the Alps and the Scandes, we bootstrapped the coefficients of these two regressions using the ordinary non-parametric bootstrap with a case re-sampling approach based on 1000 randomisations. The proportion of bootstrapped slope estimates in the Alps overlapping with the distribution of bootstrapped slope estimates in the Scandes served as the All statistical analyses were carried out in R n = 565 species) was much greater than in the Scandes (n = 271 species), with 404 species unique to the Alps and 110 unique to the Scandes.In the 403 pairs of plots from the Alps and the Scandes, we found 675 species, 161 of which were present in both regions. Altogether for the environmentally paired plots, the total γ-diversity in the Alps (ijb'), declined precipitously with an increasing cut-off value  than in the Scandes  between all 403 plots within a whole region was clearly higher in the Alps  than in the Scandes . Moreover, compositional distinctness, as the average of the jFΔc values between each focal plot and all other 402 plots in a given region  in the Alps   for the jksim), included all six Ellenberg's distance matrices as well as the plot size distance matrix in both regions  and the Scandes . However, the regression of the residuals of the environmental models against geographical distance provided quite different coefficients in the two regions  than in the Scandes (−6.4×10−8±SE 1.1×10−9) and this difference was also significant  in the Alps  than in the Scandes . However, we found significant effects of soil pH (R) and plot size (A), as well as a marginal influence of light (L), on Alps–Scandes paired differences in plot α-diversity (n = 97), plot α-diversity was indeed significantly higher  in the Alps than in the Scandes, whereas for pairs with R<3 in the Alps , plot α-diversity was significantly lower  in the Alps than in the Scandes. Concerning plot size, for pairs with A≥50 m2 in the Alps , plot α-diversity was similar  between the Alps and the Scandes, whereas for pairs with A<5 m2 in the Alps , plot α-diversity was significantly lower  in the Alps than in the Scandes.Overall, plot α-diversity did not differ between environmentally paired plots in the Alps and the Scandes : plot α-iversity . Among tiversity , even afiversity . For paiAdditionally, we found a significant region effect on plot α-diversity in an unpaired ANCOVA of all 806 plots with light (L), temperature (T), soil humidity (F), soil pH (R) and plot size (A) as covariates in the ANCOVA . Correctr = 0.50, P = 0.0139) and the Alps . In contrast, there was no significant positive relationship  between plot α-diversity and habitat-specific γ-diversity for the Alps and the Scandes combined (pooling the data). Additionally, the slope of the regression between plot α-diversity and habitat-specific γ-diversity was lower in the Alps  than in the Scandes . Our results suggest that regional differences in diversity components at coarse scales  rather than similarity in environmental conditions. Therefore, Alps–Scandes pairs sharing a similar spectrum of Ellenberg's indicator values do not necessarily share a similar spectrum of species, but might rather show highly distinct species compositions . Hence, there is no bias towards selecting for similar plot α-diversity between Alps–Scandes pairs.In this paper, we also accounted for several of the most important processes affecting plot α-diversity by including both environmental variables and plot size as covariates in our models, thus decreasing the probability that the pattern of plot α-diversity differences between the two regions is due to environmental and area effects. Furthermore, in our analyses of plot α-diversity using a spatial model , we corrWhy did the region effect not leave strong differences in plot α-diversity in our environmentally similar Alps–Scandes plot pairs? We can point to at least two alternative and non-exclusive potential mechanisms that might override the predicted glacial history effects: a habitat-connectivity effect and the space-filling limitation. First, we note that most of the plot pairs in our study occurred on acidic soils because vegetation-plot data in the Scandes were largely restricted to the acidic end of the soil pH gradient covered by the Alps data . This paVaccinium myrtillus (second most frequent species in the Alps data) and Empetrum nigrum (third most frequent species in the Scandes data). It should also be noted that few species are physiologically capable of tolerating the high H+ concentration and the Al3+ toxicity of strongly acidic soils 2 towards higher plot α-diversity in the Scandes than in the Alps, any such space restrictions cannot be absolute at the scale of the plots studied.The second argument we suggest involves plot size as an important feature affecting plot α-diversity. At a given regional extent, substantial differences in plot α-diversity between two environmentally similar areas might be easier to detect at larger than at smaller plot sizes, simply because of the inevitable consequence of the physical limitations of the spatial unit considered and regardless of competition among species Plot α-diversity was positively related to habitat-specific γ-diversity in both regions , as prop2. We note that our environmentally similar plot pairs are embedded into landscapes with differing ranges of macroclimatic condition and differing degrees of environmental heterogeneity in the Alps and the Scandes which might contribute to these patterns. By contrast, regional differences in plot α-diversity were weak and inconsistent. Hence, region effects on diversity patterns obviously differ between local and regional/landscape scales. We suggest that the weak regional effect on plot α-diversity may be explained by: (i) the Scandes harbouring relatively more vascular plant species with high dispersal abilities selected after the retreat of the ice sheets and (ii) a higher connectivity between vascular plant communities in the Scandes because of less-complex altitudinal zonation of vegetation, greater continuity in geologic features and younger, less intensive history of agricultural land-use.In this study, we found evidence for strong regional effects on plant community assembly and species diversity patterns in European mountains, although these effects did not necessarily penetrate to affect diversity at the finest (plot) scale. Both total and habitat-specific γ-diversity as well as plot-to-plot β-diversity differed considerably between the Alps and the Scandes even with small distances between plots. The smaller total and habitat-specific γ-diversity, the lower plot-to-plot β-diversity and the reduced distance decay in floristic similarity in the Scandes than in the Alps are all consistent with the idea that divergent regional glacial histories have left strong legacies at scales larger than 1 kmText S1Details of the procedure to pair environmentally similar communities between the Alps and the Scandes.(DOC)Click here for additional data file.Figure S1Distribution of the Alps and the Scandes plots within the environmental space. Principal component analysis (PCA) of 11,249 plots in the Alps (gray dots) as active and 481 plots in the Scandes (black dots) plotted along the first three principal component (PC) axes. (A) PC axes 1 and 2 and (B) PC axes 2 and 3 are given, while (C) represents a 3D zoom of the first three PC axes showing the 403 Alps–Scandes pairs of plots used in the study. Arrows and their directions indicate increasing values for plot size (A), light (L), temperature (T), continentality (K), soil moisture (F), soil pH (R) and soil fertility (N).(TIF)Click here for additional data file.Figure S2Comparison of the environmental conditions between the 403 Alps–Scandes pairs. Boxplots of environmental conditions in both the Alps  and the Scandes  for the light (L), temperature (T), continentality (K), soil moisture (F), soil pH (R) and soil fertility (N) gradients. The line across the box indicates the median, box boundaries show the interquartile range and whiskers extend up to 1.5 times the interquartile range.(TIF)Click here for additional data file.Figure S3Spatial autocorrelation in the residuals of the non-spatial and spatial models of the region effect on plot α-diversity. Correlogram of residuals from the non-spatial model  and the simultaneous autoregressive model with a spatial error model (SARerr) . Both non-spatial and spatial models have the same relationship between the common logarithm of plot α-diversity and explanatory variables  whereas open symbols display non-significant values.(TIF)Click here for additional data file.Table S1Adjusted Ellenberg's indicator values of vascular plants in the Scandes.(DOC)Click here for additional data file.Table S2Non-spatial and spatial models of the region effect on plot α-diversity.(DOC)Click here for additional data file.


Prognosis of oropharyngeal squamous cell carcinoma depends on early diagnosis, despite advanced surgical techniques, the 5-year survival rate remains ~40-50%. Unfortunately, it is usually detected when it becomes symptomatic. This requires treatment which gives rise to a high rate of morbidity and mortality and, furthermore, early detection of oro-pharyngeal pre-malignant lesions is important to improve the survival rate and quality of life.Since dysplasia and in situ carcinoma contain much more DNA and RNA than the normal surrounding epithelium, the use of in vivo staining, by means of toluidine blue dye, is based on the fact that it is an acidophilic dye that selectively stains acidic tissue components such as DNA and RNA. Toluidine blue staining is considered to be sensitive in identifying early oro-pharyngeal premalignant and malignant lesions.In the present study, the use of toluidine blue staining was taken into consideration to identify clinically doubtful oro-pharyngeal lesions and to compare toluidine blue stain and with the histological evaluation. Key words:Early detection, improved survival, pre-cancer, toluidine blue, vital staining. Malignancies of the upper aero digestive tract are one of the common malignancies in the world . In IndiThe clinical examination is one of the best modalities in suspecting the pathology but the biggest disadvantage in the diagnosis lies in deciding the site of biopsy in early lesions and sometimes whether or not a biopsy is required in these lesions. Early stages are difficult to detect as the lesion may not be palpable and colour changes are not very different from the colour of the surrounding mucosa. Thus identifying clinically suspicious/undetectable lesions has gained importance whereby diagnosis can be confirmed by biopsy at an earlier stage .In developing countries such as India, where there is high prevalence of disease, the focus is on down staging oral cancer at diagnosis from advanced to earlier disease. It is assumed that if premalignant lesion is detected and treated, the lesion may not progress to cancer .In the past decades, adjunctive techniques have emerged with claims of enhancing the oral mucosal examinations and facilitating the detection of and distinctions between oral benign and oral premalignant and malignant lesions .Techniques that are promoted or assessed to improve earlier detection and diagnosis of oral malignancy include toluidine blue, vizilite plus with touidine blue, velscope brush biopsy, etc. The gold standard diagnostic test for oral mucosal lesions that are suggestive of pre malignancy or malignancy remains tissue biopsy and histopathological examination.Toluidine blue is a cationic metachromatic dye that may selectively bind to free anionic groups such as sulfate, phosphate, and carboxylate radicals of large molecules . It has When evaluated as a part of the clinical examination, toluidine blue staining may provide additional information. In vivo, toluidine blue stains deoxyribonucleic acid and/or may be retained in intracellular spaces of dysplastic epithelium and clinically appear as royal blue areas . ToluidiThe study was focused on 40 oral mucosal lesions in 32 patients, who reported to the Department of Oral Medicine and Radiology, Maharishi Markandeshwar College of Dental Sciences and research, Mullana. Ethical approval was taken from the university ethical committee according to Declaration of Helsinki. Patients who clinically presented with at least one oral mucosal lesion suspected of dysplastic changes and the patients who gave their consent for the study were included in the study. During history taking, information regarding habit of tobacco, its type, duration and frequency was recorded by asking the patient.Patient who had presence of frank malignancy or those who were suffering from any systemic disease that interfere with or are contraindications to biopsy procedure or undergoing any dental treatment such as orthodontic appliances or prosthesis that may interfere with the examination, were excluded from the study.All the lesions were first identified under visual examination. This was followed by an oral rinse with 1% acetic acid solution which was given to the patient to hold in the mouth for 20 seconds before expectorating. Toluidine blue (1% W/W) was applied as an oral rinse for 20 seconds and then 1% acetic acid was used for 20 seconds to eliminate mechanically retained stain. Only the dark staining lesions were taken as positive whereas equivocal stains (faint stains) and the lesions which did not retain the stain at all were taken as negative but all the lesions were subjected to histopathological examination.The biopsies were performed under local anaesthesia. The pathologists examining all the biopsies were not informed regarding the clinical or staining evaluation of each sample. Histopathologic diagnoses were referred as: non-neoplastic , mild dysplasia, moderate dysplasia, severe dysplasia, squamous cell carcinoma. For the statistical analysis, we used histopathologic assessment as the gold standard to which toluidine blue stains were compared.Out of 32 patients examined, 29 were males and 3 were females. Most common site where the lesion was present was buccal mucosa in 57.5% of the total cases (23 lesions) followed by labial vestibule 27.5% 11 lesions) and commissural area and buccal vestibule 7.5% each (3 lesions)  followed by smokeless tobacco in 7 patients 21%) and smoking and smokeless tobacco along with alcohol in 4 patients (13%)  out of the total 40 oral mucosal lesions stained positive while 11 (27.5) were negative Fig. .Histopathological examination of the 40 oral mucosal lesions showed 3 non-conclusive reports. Out of the 37 conclusive reports, 14 (37.83%) were benign lesions  and 23 were precancerous or cancerous lesions. Out of the 23 precancerous lesions, 13 (56.5%) were mild dysplasia and 10 (43.47%) were moderate dysplasia .The results of the toluidine blue staining and histological findings are outlined in The results of the toluidine blue test and histology, were compared in order to calculate the sensitivity (true-positivity) and specificity (true-negatives) . The senThe toluidine blue literature shows that it is a practical, rapid, inexpensive, and effective adjunct diagnostic tool . ToluidiIn all fairness its ability to detect malignant oral cavity lesions has been well documented in clinical settings ,9. StudiEpstein et al.  while scThe negative and the positive predictive value came out to be 90.9% and 84.6% respectively which was in accordance with the study conducted by Allegra et al. , indicating that the greater sensitivity of tolonium chloride was not associated with an excessive number of unnecessary biopsies  . TherefoFurthermore, it was observed that while comparing the dysplatic changes occurring at various sites with the histopathology, no statistically significant differences were observed (p=0.277), which depicted that it could not be used as a tool for site specific dysplastic changes.Toluidine blue is useful in raising or confirming clinical suspicion of malignancy or pre-malignancy and has the capability to reduce the number of biopsies being done. It has been proved in our study that when the lesion stained faintly, it came out to be histopathologically negative in most cases. Though our findings suggest that toluidine blue staining may be considered as an adjunctive diagnostic tool for detecting dysplastic changes in the epithelium but larger studies are required to assertively and definitively answer the questions related to the screening use of toludine blue stains as early detection tool.


The incidence of tonsil cancers has increased in several countries. French data on HPV prevalence in tonsil cancers are scarce. The objective of this study was thus to assess the overall and type specific HPV prevalence in tonsil histological samples.This French retrospective multicenter study involved 12 centres located throughout the country. Were included 185 histological samples collected from year 2000 to 2009 with a validated diagnosis of tonsil invasive carcinomas. HPV prevalence was studied according to gender, age and histological type of cancer.Overall HPV prevalence was 57% in tonsil cancers. Mean age of diagnosis was comparable in HPV positive tonsils cases (60 ± 11.2) and HPV negative tonsil cases (59 ± 9.6). HPV prevalence was significantly higher in female than in male cases . About 53% of tonsil cases were infected by a single HPV type. Only eight (4%) samples were infected by more than one HPV type. Among HPV positive samples, HPV 16 was found in 89% of tonsil cases. All other HPV types had prevalence below 5%.Our results indicate that HPV is common in tonsil carcinomas and emphasize the predominant role of HPV 16. Head and neck cancers (HNC) are the fifth most common cancer worldwide with more than 600,000 cases diagnosed each year . France The overall incidence of HNC has fallen in recent years. This seems to be consistent with the decrease in tobacco and alcohol consumption. By contrast, the incidence of cancers in particular anatomical sites has increased . The incWell known main risk factors for developing oropharyngeal cancer are tobacco usage and alcohol consumption . Socio-eFrench data on HPV prevalence in tonsil cancers are scarce. The objective of this study was thus to assess the overall and type specific HPV prevalence in tonsil histological samples.The overall study which involved oropharyngeal and oral cavity invasive cancers is described elsewhere. We present here results of a substudy focused on tonsil cancers. This French retrospective multicenter study involved 12 centres located throughout the country. Selection of the centres was mainly based on their willingness to participate trying to get most French geographical areas represented. To be included, histological samples had to be collected after year 2000. Each sample with a validated diagnosis of tonsil cancers and fixed in 4% buffered formalin before paraffin embedding were retrospectively included. Cases with diagnosis other than SCC were excluded. Each centre decided whether its own cases could be included. Patient data such as age at diagnosis, gender, area of residence, year of sample collection were collected for each case when available.According to the French legislation  and since this study only involved data extracted from medical records and stored histological specimens, no informed consent from the patients was necessary. Data collected from participating pathology centres were strictly anonymous.HPV genotyping was centrally performed using the INNO-LiPA HPV Genotyping extra test . This kit allows the detection of 28 HPV types, 15 high-risk (HR) and 13 low-risk (LR) . In ordeThe HPV genotype-specific prevalence was expressed as the proportion of HPV-positive samples among all carcinoma cases and among HPV positive cases. HPV prevalence was studied according to gender, age and histological type of cancer. Prevalence of HR and LR HPV was also calculated. Categorical variables were studied using 2-sided Chi-square or Fisher exact test when necessary. All statistical analyses were performed using SPSS v.11.5 for Windows. A p-value below 0.05 was considered as statistically significant.A total of 220 of histological samples of tonsil invasive SCC were retrospectively collected from 12 French pathology centres scattered throughout the country. Patients were recruited throughout France: 28% in Paris area, 8% in the West area, 35% in the North and North East, and 28% in the South of France. 84% (185/220) were included in the study while 16% (35/220) were excluded from the analysis because neither HPV nor cellular DNA could have been amplified. Mean age at diagnosis was 60 ± 10.5 years. There were 81% (150/185) of males.We found an overall HPV prevalence of 57% 106/185). Mean age of diagnosis was comparable in HPV positive tonsil cancer cases (60 ± 11.2) and HPV negative tonsil cancer cases (59 ± 9.6). HPV prevalence was higher in female than in male cases (80% (28/35) versus 52% (78/150) respectively, p = 0.003). About 53% (98/185) of tonsil cancer cases were infected by a single HPV type. Only 4% (8/185) of tonsil samples were infected by more than one HPV type, with at least one HPV HR. HPV 16 was found in 89% (94/106) of HPV positive tonsil cases. All other HPV types had prevalence below 5%. HPV 16 and/or 18 was found in 91% (96/106) of HPV-positive tonsil cases.  in the 1970s, 29% (12/42) in the 1980s, 57% (48/84) in the 1990s and 68% (32/47) in 2000-2002 /30 in th. We did HPV 16 was the genotype the most identified in the literature, 93% (77/83) of HPV-positive cases in the series of Nasman et al 2003-2007 .In our series, HPV prevalence was higher in female than in male cases (80% versus 52%) even if men accounted for the majority (81%) of the total tonsil cancers, which was concordant with some studies. Nasman et al found that HPV was present in 95% 21/22) of women cases of tonsil cancers and in 82% 62/76) of men cases of tonsil cancers 2/76 of m. Chien e of womenDifferent authors found that HPV positive carcinoma was associated with younger patients. Nasman et al observed that patients with HPV positive tonsillar cancer were younger than patients with HPV negative cases (59 years versus 68 years) [Our study has limitations. First since the recruitment was rather heterogeneous between centres and centres were not evenly scattered on French territory despite patients were recruited throughout France, the study sample can not be considered as fully representative of the French tonsil cancer population. Secondly, we used DNA detection only; in this retrospective study it was not possible to determine the presence of transcripts. However, Nasman et al found that HPV 16 E6 or E7 RNA were expressed in the majority of the analysed HPV 16 positive tumors [a posteriori to clarify the possible causal association between tonsil cancers and HPV.Our results indicate that HPV has a high prevalence in tonsillar SCC. We emphasize the predominant role of HPV 16 since 89% HPV-positive tumors were linked with HPV 16; moreover 91% were associated with HPV 16 and/or 18. Unlike for cervical cancer, the association of HPV and oncogenic mechanisms remain to be clarified. These results are an indicator of the HPV distribution in tonsil cancers before HPV vaccine implementation among women. The HPV vaccination of young women will be able to contribute - Jean Lacau Saint Guily, MD: Member of the scientific EDiTH VI advisory board- Claire Okaïs, PharmD, is an employee of Sanofi Pasteur MSD- Anne-Carole Jacquard, PhD is an employee of Sanofi Pasteur MSD- Jean-Luc Prétet, PhD: occasional speaker for SPMSD- Agnès Beby-Defaux, MD, PhD: The author declares that he has no competing interest- Christine Clavel, PharMD, PhD, belongs to the steering committee of MERCK about vaccines- Gérard Agius: The author declares that he has no competing interest- Philippe Birembaut, MD: The author declares that he has no competing interest- Yann Leocmach, MD, is an employee of Sanofi Pasteur MSD- Benoît Soubeyrand, MD, is an employee of Sanofi Pasteur MSD- Didier Riethmuller, MD, PhD: expert for SPMSD- Christiane Mougin, MD: Travel support- François Denis, MD, PhD: The author declares that he has no competing interestJLSG participated to the study design, data analysis and interpretation and manuscript preparation and review. CC participated to the study design, data analysis and interpretation and manuscript preparation and review. CO participated to the data analysis and interpretation and manuscript preparation and review. JLP participated to the study design, data analysis and interpretation and manuscript preparation and review. ABD participated to the study design, data analysis and interpretation and manuscript preparation and review. GA participated to the study design, data analysis and interpretation and manuscript preparation and review. PB participated to the study design, data analysis and interpretation and manuscript preparation and review. ACJ participated to the study design, data analysis and interpretation. YL participated to the study design, data analysis and interpretation and manuscript preparation and review. BS participated to the study design, data analysis and interpretation and manuscript preparation and review. DR participated to the study design, data analysis and interpretation and manuscript preparation and review. FD participated to the study design, data analysis and interpretation and manuscript preparation and review. CM participated to the study design, data analysis and interpretation and manuscript preparation and review. All the authors have given approval of the version to be published.


Human Papillomavirus (HPV) is a very resistant, ubiquitous virus that can survive in the environment without a host. The decision to analyse HPV-related diseases in males was due to the broad dissemination of the virus, and, above all, by the need to stress the importance of primary and secondary prevention measures (currently available for women exclusively). The objective of the Consensus Conference was to make evidence-based recommendations that were designed to facilitate the adoption of a standard approach in clinical practice in Italy.The Sponsoring Panel put a series of questions to the members of the Scientific Committee who prepared a summary of the currently available information, relevant for each question, after the review and grading of the existing scientific literature. The summaries were presented to a Jury, also called multidisciplinary Consensus Panel, who drafted a series of recommendations.The prevalence of HPV in males ranges between 1.3–72.9%;. The prevalence curve in males is much higher than that in females and does not tend to decline with age. Women appear to have a higher probability of acquiring HPV genotypes associated with a high oncogenic risk, whereas in males the probability of acquiring low- or high-risk genotypes is similar. The HPV-related diseases that affect males are anogenital warts and cancers of the penis, anus and oropharynx. The quadrivalent vaccine against HPV has proved to be effective in preventing external genital lesions in males aged 16–26 years in 90.4%;  of cases. It has also proved to be effective in preventing precancerous anal lesions in 77.5%;  of cases in a per-protocol analysis and in 91.7%;  of cases in a post-hoc analysis. Early ecological studies demonstrate reduction of genital warts in vaccinated females and some herd immunity in males when vaccine coverage is high, although males who have sex with males gained no benefit at all. Males with an immunodeficiency disease are at greater risk of developing disease. Infertility seems to be caused by HPV in some cases. Studies demonstrate vaccination to both genders can be more efficacious and social equity matters are to be taken into consideration.The Jury made Recommendations based on the scientific evidence presented by the Scientific Committee. Accordingly, for prevention purposes and social fairness and equality, as both sexes are affected by the disease, the vaccination of 12-year-old males against HPV should be recommended in order to guaranty protection to everyone. Aspects related to healthcare policy and economic sustainability, are to be discussed by respective public system representatives. More campaigns to raise awareness through all institutional channels are needed, not only regarding anogenital warts, but for HPV-related diseases in general in males in accordance to new scientific evidences. The Human Papillomavirus (HPV) is a very resistant, ubiquitous virus that can survive in the environment without a host . It may The dynamics of heterosexual transmission of HPV (the major route of infection) are still being investigated and their relative importance has not been established yet. The ability of HPV to survive for a long time on various surfaces, objects, and parts of the body that have had contact with infected mucous membranes makes potential transmission to the genitals via the hands, the perigenital skin (the scrotum or pubis) or even inanimate objects possible .HPV has been found on the skin and the mucous membranes of individual exposed to the virus, and causes both anogenital and oropharyngeal infections. In women, the prevalence of the different genotypes of HPV has been determined and analyzed within the setting of cytologic examinations with the purpose of screening for and diagnosing precancerous and cancerous lesions. In men, the first studies focused on HPV infection and the risk of cancer in the homosexual population. More recently, studies on the importance and clinical consequences of HPV infection have been extended to the heterosexual male population, and have also examined the role of males in the transmission of HPV to women.The frequency of HPV infection in various groups of male subjects was analyzed in a systematic review of the literature, which included 40 papers published from 1999 to 2006 . OverallOne finding is difficult to explain: the prevalence of HPV infection in men does not vary among different age groups, although its incidence is similar in both genders and its clearance is fairly rapid. A possible explanation is that repeated infections occur during sexual activity and persist for variable periods of time. However, a longitudinal assessment of a cohort of males who are followed up in the long term is needed to provide more information on this phenomenon. The following points have become apparent with respect to HPV-related diseases in males:– - Several issues of the diseases are controversial and should be addressed by adopting a multidisciplinary and multiprofessional approach;– - Such diseases are a public health matter and have aroused the interest of healthcare professionals and users (patients/citizens), with potential impacts on clinical practice;– - The scientific data on these diseases are incomplete, but nevertheless enable the drafting of a reliable consensus document that is based on expert opinions;– - Exhaustive information on HPV-related diseases in males is not available, so it is important to make recommendations for further research;– - A document that lays down recommendations for the prevention and management of HPV-related diseases in males, may reduce variability in healthcare and, possibly, related costs.The decision to analyze HPV-related diseases in males by establishing a Consensus Conference (CC) was due to the broad dissemination of the virus, and, above all, by the need to stress the importance of measures for primary prevention and organized screening programs, which, at present, are the only tools we have to reduce the incidence of HPV-related diseases. It is worth noting that in clinical practice there are neither validated tests nor screening programs for HPV in males at the moment.The objective of the CC was to make evidence-based recommendations, starting from a review of existing literature that was designed to assess the best scientific evidence available currently.A CC is one of the tools that enable agreement to be reached, through a formal process, on healthcare issues that are particularly complex and controversial. The outcome facilitates a uniform approach to such issues in clinical practice.The consensus conference on Human Papillomavirus Diseases in Males was called by a Sponsor Panel (SP), which included representatives of three Italian scientific societies: SIA – , SIAMS , SIU . They appointed ten acknowledged experts in the field to set up the Scientific Committee (SC) of experts.In January 2010 the SP gathered the Scientific Committee to submit four specific predefined questions of importance to better understand HPV in males about which the SC was asked to draw a synthesis of the available knowledge starting from a revision of the scientific literature on the topics.The expert panel revised the scientific literature, classifying it into the following areas:– - HPV epidemiology– - Natural history of HPV infection– - HPV burden of disease– - HPV prevention strategies– - HPV vaccines– - Social impact of HPV diseasesInclusion criteria and paper selection process were established in accordance with the Sponsor Panel and the Panel of experts. To be included in the review, each paper had to: a) contain data from males or separately presented from females; and b) contain country-specific/racial/sexual-habits data for male subjects or c) report on HPV prevalent in males or d) report on incidence of HPV–related events in males or f) report on relationship between HPV and fertility or g) report results on diagnostic testing methods on males or separately presented from females.Studies that did not meet criteria a) and b) and at least one of criteria c), d), f), g) were ineligible. Literature collection started in January 2010; four coordinators (one for each question of the Consensus Conference) were encharged of retrieving all the relevant documents through November (2010), following the up-reported selection criteria. Longitudinal, retrospective and cross-sectional studies, as well as randomized clinical trials were considered, upon availability. The search process used the following database to identify articles published until 2010 included:Medline, Embase, Cochrane Controlled Trials Register, PubMed , and the Cochrane Library were searched to identify articles published and the following keywords: HPV or Papillomavirus” OR “HPV Vaccination” OR “HPV infection/epidemiology” OR “Papillomavirus natural history” And  OR  And Male.See flow diagram in Figure All the eligible papers were used by the four coordinators to argue on their specific question and were included in the references section. Among the eligible papers, those supporting the question conclusions were reviewed by an independent methodological expert and assessed with the quality rating reports in Table During year 2010 the SP called 3 meetings with the SC in order to examine the various revisions of the document drawn together from scientific evidence for the presentation of the results.The final document with the results was presented on 22nd November 2010, by the SP and the SC to a Jury called also a multidisciplinary Consensus panel of experts composed of the following: 1 representative of the ethic committee, 1 oncological gynecology, 1 pediatrician, 2 specialist of public health and epidemiology, 1 representative of the Italian Medicines Agency (AIFA), 1 representative of National Institute of Health (ISS), 1 representative of the Ministry of Equal Opportunities, 1 Member of “Cittadinanzattiva” .Each question posed to the SC was discussed during the day of the consensus.On the basis of the document which was presented and the lectures held by the SC during the consensus meeting, the Jury reached a general agreement about the information provided and how to answer to the key questions, drawing from each questions recommendations based on their observations.The document with the results of the work and there commendations of the Jury was printed after the Consensus Conference.Various methods have been designed to assess the robustness of clinical recommendations. At present, an international method is being developed, although it will be difficult to reconcile different approaches and techniques and include them all in a single assessment system In the present case, the outline published in the British Medical Journal in 2001 was followed  Prevalence of the infection  – GradinIn a systematic review by Dunne et al. on prevalence of HPV in men  it was fB) The prevalence of infection by age  – GradinIn males, the prevalence of HPV does not differ significantly among age group as it does in females . Indeed,C) Incidence and clearance of the infection  – GradinThe natural course of the disease has not been investigated properly in males from an epidemiological point of view, namely, by establishing rates of acquisition and time to clearance of HPV infection. A few prospective studies have estimated the probability of acquiring the infection for heterosexual males. In a population of 290 male participants aged between 18 and 44 years , who were followed up for a mean period of 15.5 months , the oveThe study by Partridge , which wD) HPV-related diseases in malesThe HPV-related diseases that occur in males are genital warts and cancers of the penis, anus, oral cavity, and oropharynx (carcinomas of the head and neck).➣ Anogenital warts ,28,29 , anogenital warts are caused by HPV genotypes 6 and 11 . In ItalHaving ≥ 2 sexual partners in the last 6 months  and the presence of other concomitant STDs  were risk factors . In a st➣ Cancer of the penis -32 . In the USA, the incidence of cancer of the anus increased considerably between 1970 and 2000. Its incidence is particularly high in homosexual males and the risk is increased even further in the presence of human immunodeficiency virus (HIV) infection. It has been estimated that, in Europe, something like 2,000 cases of cancer of the anus occur in males every year ,34.➣ Carcinomas of the head and neck  . Furthermore, a significant association has been found between HPV in the oral cavity and sexual habits [E) HPV and fertility -38 – GraPreliminary studies show not only that HPV is present in semen, but that it might reduce sperm motility probably causing reduced fertility in males. Moreover, the virus might interfere with the development of the embryo in animal models according to some studies -38. HPV F) Diagnosis of HPV in males -41 – GraAt the moment, there is no generally accepted and validated test for screening HPV in males in the clinical practice. However, there is a general consensus on when diagnostic testing should be performed, which may be summarized as follows -41:1. When the patient has a partner who is HPV positive or has an HPV-related disease;2. When HPV-related clinical manifestations are present;3. When the patient has sex with men.At present, molecular methods are used most commonly, both to detect infection and to identify the virus genotype. The tests are based on amplification methods (PCR) with hybridization . Cell saFor males, a test that identifies not only HR-HPV but also LR-HPV and genotypes associated with intermediate risk or that have not been classified yet would be useful, because it may enable differential diagnoses to be made between benign and malignant lesions and those related or not related to HPV (e.g. molluscum contagiosum) -46.G) Follow-up of male patients with HPV-related diseases ,48 – GraThe following investigations are suggested for the follow-up of HPV-related diseases:➣ According to the site involved, benign lesions should be checked clinically with genitoscopy and/or anoscopy every 4 months for the first year, as appropriate. Molecular analysis to screen for HPV should be carried out with the same frequency;➣ In subjects at risk (those with neoplastic lesions or with a history of HPV-related neoplasias), testing for HPV DNA should be carried out every 4 to 6 months;➣ In subjects with a greater risk of infection and disease development , testing for HPV should be carried out every 8 to 12 months;➣ In asymptomatic HPV-positive subjects, laboratory follow-up is recommended after 8 to 12 months to assess the persistence or clearance of the infection and to check whether any lesions have developed.According to the answers given by the SC, Impact of HPV-related diseases seem to be high in males. Not the same burden as in women for high risk HPV types related lesions but more when taking into account only low grade HPV related lesions such as genital warts. Prevalence seems to be higher than in women irrespective of age and no standardized HPV test guidelines are recommended as in women. Preliminary fertility studies in men seem to show that HPV might have an important role, more studies and data need to be done in order to prove HPV relation with fertility.➣ More prevalence studies in males should be conducted. In Italy, notification of HPV related diseases such as genital warts are not mandatory, more epidemiological data is needed in order to evaluate disease impact.➣ More scientific studies should be made about data on the standardized collection of biological samples and method of testing used for HPV detection in males in order to implement in the future standard recognized sampling and diagnostic tests in eventual prevention and screening programs.➣ Further research on the issue of HPV and fertility worldwide is required in order to fully understand if it is related and affected by HPV and in what measures HPV vaccination might reduce this.A) Transmission potential of the virus ,2,27,49 HPV is a highly contagious virus that is widespread in the environment. It is transmitted mainly through sexual contact. However, indirect secondary modes of transmission also exist  ,2,27,49.B) Transmission dynamics ,52-54 – Transmission of the virus from one partner to another varies also according to sexual practices. Many male partners may be classified as «healthy carriers» who unknowingly serve as an asymptomatic reservoir and could contribute to the development of HPV-related diseases in women -54.C) Risk factors and monitoring ,55-58 – In males, age does not appear to be related clearly to the acquisition of the infection. In general, sexual habits  correlate with the presence of infection, in both males and females. Individual factors  or factors related to the female partner  are not related consistently and significantly to the acquisition of infection and its presence in males ,4,27. InBased on the previous statements and data, HPV DNA sero status seem to play a fundamental role in HPV transmission and diseases development between partners. In males, there are several risk factors that play a fundamental role in HPV infection and development of the disease such as immunodeficiency disease.➣ Awareness campaigns should be implemented not only for anogenital warts but for HPV-related diseases in general, through all institutional channels, in order to increase HPV knowledge and reduce, when possible, HPV transmissibility at a population level.➣ Subjects with an immunodeficiency status, such as HIV, have a greater risk of HPV infection; prevention strategies should also target high risk populations.A) Primary Prevention -68 – GraPrimary prevention through vaccination has proved to be very effective in preventing precancerous lesions and cancers of the cervix, vulva, and vagina, as well as anogenital warts, in women up to the age of 45 years -66. The Clinical trials have shown that the quadrivalent vaccine is effective in preventing external genital lesions  in males aged between 16 and 26 years in 90.4%;  of cases . On the At present, efficacy data on the quadrivalent vaccine with respect to the prevention of external genital and anal precancerous lesions are available Table . In a suNotes of importance after the consensus:In accordance to the importance and recommendations already stated by the CC document about HPV in males, in late 2010 and 2011, on the basis of vaccine efficacy data against HPV-related anal precancerous lesions, in December 2010, the FDA approved the extension of the indications for the quadrivalent vaccine to the prevention of anal cancer and anal intraepithelial lesions in males and females aged between 9 and 26 years . In addiB) Condoms ,75 – GraThe use of a condom reduces, albeit not completely, the risk of transmission of HPV infection between heterosexual partners. Moreover, correct and consistent use of a condom reduces the risk of transmission by approximately 50%; and appears to promote clearance of the infection as seen on a randomized trial conducted in women diagnosed with CIN lesions and their partners, in the group that used condom, correct usage seemed to promote regression of CIN lesions and clearance of HPV infection on both . Condom Vaccination on males, according to recent data, seems to be effective with the quadrivalent vaccine as it is in women. Vaccination is the most effective measure to reduce HPV related diseases impact in males with efficacy rates around 90%;. Condom usage seems to be also effective in reducing at least in 50%; the risk of transmission when used correctly.➣ An awareness campaign should be implemented about available vaccines, the importance of vaccination and its high efficacy in males. Also about other prevention measures such as condom usage, through all institutional channels.➣ The quadrivalent HPV vaccine has proven to be effective in protecting males against genital warts and HPV related precancerous anal lesions. Based on vaccine efficacy results, male vaccination should be recommended as in females as they can benefit too from vaccination.A) Vaccination  – GradinLittle data is still available about vaccination effect on a population level. Only on the years to come data will become more available and valid. Only one ecological study, obtained from vaccination programs carried out in women in Australia, were vaccination coverage is high on women, although still early have shown that vaccination induces also some herd immunity in men i.e. there is a partial reduction in the occurrence of anogenital warts in heterosexual males compared to vaccinated women. In Australia, the quadrivalent vaccine has been administered to schoolgirls aged between 12 and 18 years and to women younger than 26 years since 2007. The coverage rate in the area where the study was conducted ranged from 65%; to 75%;. One year after the implementation of the mass vaccination program, comparison of the rates of diagnosis of anogenital warts before and after the introduction of vaccination showed a 48%; reduction in the diagnosis of anogenital warts in women younger than 28 years, but no corresponding reduction was obtained in women older than 28 years and in the males who have sex with males population .Over the same period, a 17%; reduction in the diagnosis of genital warts among heterosexual males was recorded. This reduction was not observed in homosexual males, a finding that suggests some herd immunity effect. In other words, use of the vaccine confers indirect protection on the unvaccinated population as a result of the reduction in the total number of infected subjects, which in turn decreases the number of potentially infectious contacts.B) Reduction in HPV-related events ,78 – GraIn view that natural history in males, although explained, still lacks further research as today we know that although similar, it does not behave as in females and because of the paucity of established epidemiological data, it is impossible (at the moment) to provide details on the impact of a primary prevention strategy on the reduction of HPV-related disease in partnersThe complexity of the scenario produced by the implementation of primary prevention in males is due to a number of general considerations: 1) there is limited information on the natural history of HPV infection in males, that behaves differently than females , information is still scarce and more studies are still needed; 2) the inconsistencies of epidemiological data and rates of concordance of genotypes among couples ; 3) The lack of a reliable and validated monitoring and diagnosis system (in the long and short term) for males.Given that HPV infection is a sexually transmitted disease, interventions for primary prevention in one partner will evidently have a positive effect in terms of reducing HPV-related disease in the other partner. Mathematical models indicate that the cost–effectiveness ratio for the vaccination of males becomes favorable in the presence of a low coverage rate for females, because it can contribute towards the containment of infection in the community ,78, someAlthough herd immunity exist, primary vaccination exclusively of males or females doesn’t seem to be effective when coverage rates are less than high in reducing HPV related disease on partners in a significant way. In Italy coverage rates are not high, only 53%; of 12 years old females have been vaccinated with 3 doses. Social equity right to get the vaccine in males and higher risk groups, as well as social burden  should be taken into consideration as they seem relevant.➣ When vaccination campaigns targets only one gender, vaccine coverage should be very high in order to expect significant effects in the other gender morbidity.➣ Were possible, vaccine programs to both genders should be implemented as they seem to be more effective.➣ More initiatives should be directed towards the population raising awareness of primary prevention, in order to achieve high vaccination coverage in the already implemented vaccination campaign in females to guaranty at least some herd immunity in males.➣ Specific efforts should be made to vaccinate higher risk groups such as immunosuppressed subjects.On the evidence presented by the 4 main questions made to the SC by the SP, the Jury composed by the multidisciplinary consensus panel agrees that HPV (human papillomavirus) impact is important in males. Based on the specific scientific data analyzed and the conclusive considerations the multidisciplinary consensus panel made, they conclude the following recommendations:➣ For prevention purposes and social fairness, 12-year-old males should be vaccinated. The jury did not deal with aspects related to healthcare policy and economic sustainability, which are to be discussed elsewhere by others.➣ The Regions of Italy should introduce an HPV vaccination program with facilitated access (reduced price) for males, as they have done already for females.➣ More initiatives should be directed towards the population raising awareness of primary prevention and disease. As a result, vaccination coverage rates may improve in the ongoing vaccination campaign in females to indirectly reduce some of male’s morbidity.➣ Specific efforts should be made to vaccinate groups at higher risk of infection and HPV disease development such as immunosuppressed subjects, especially HIV + .➣ Awareness campaigns of new scientific evidence should be implemented not only for anogenital warts but for HPV-related diseases in general, through all institutional channels.➣ Further research on the issue of HPV and fertility worldwide is required in order to fully understand if it is really caused by HPV and in what measures infertility rates can be reduced by HPV prevention measures such as vaccination.➣ Prevalence and incidence studies in males should be further conducted. In Italy, notification of HPV related diseases such as genital warts are not mandatory, more epidemiological data is needed in order to evaluate disease burden.➣ Scientific studies should be conducted about the standardized collection of biological samples and testing methods used for HPV detection in males in order to implement in the future standardized guidelines on sampling and diagnostic tests in males.The study was supported by an unrestricted funding from Sanofi Pasteur MSD, Italy.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/117/prepub


The knowledge on type specificity and factors that increase or decrease the risk of incident HPV-infections is important to better understand the dynamics of HPV-infections.rd trimester of pregnancy and followed-up for 6 years, during which 203 baseline HPV-negative women acquired incident HPV infection. Incidence times and incidence rates (IR) were calculated for 24 low-and high-risk HPV-types detected by Multiplex-HPV-genotyping at each visit. Poison regression was used to estimate predictors of incident HPV infections of species 7 and 9 HPV-genotypes.A series of 329 pregnant women were enrolled in Finnish Family HPV Study at 3HPV16 was the most frequent (47.8%) incident genotype followed by multiple-type infections (25.1%), and single infection with HPV18, 70, 6 and 45. Actuarial mean times to incident event were longest for HPV31 (34.5 months) and HPV45 (32.8 months), while crude mean times were longest for HPV56 (42.4 months) and HPV16 (23.1 months). Actuarial IR was highest for HPV16 and multiple-type infections. Independent protective factors against incident infections were 1) > 2 life-time sexual partners (p = 0.014), 2) later initiation of oral contraceptives (age > 20 years) (p = 0.017) and 3) pregnancy at FU visit (p = 0.0001).Among newly delivered mothers, higher number of life-time sexual partners, initiation of OC use after age 20 and becoming pregnant during FU decreased the risk for incident species 7/9 HPV infections. Human papillomavirus (HPV)-types that infect the female genital tract belong to the alpha-papillomavirus-genus which includes 15 species and 58 HPV-genotypes. According to their clinical behavior, 15 HPV-types are high-risk (HR)-types, 12 are low-risks (LR)-HPV-types, and three probable HR-types . The stuAmong young women, incident infections with HR-HPV-types especially with HPV16 -11,14-16The aim of this study was to assess the frequency of type-specific incident HPV-infections in addition to actuarial and crude incidence-times and -rates of the most common LR- and HR-HPV- genotypes in newly delivered mothers, prospectively followed-up for 6 years in the Finnish Family HPV Study. Risk-factors of incident HPV-infection were analyzed with univariate and multivariate Poisson regression for panel data.The Finnish Family HPV Study is a prospective cohort study conducted at the Department of Obstetrics and Gynecology, Turku University Hospital and the Institute of Dentistry, Faculty of Medicine, University of Turku. The study was designed to evaluate the dynamics of HPV infections in mothers, fathers and their newborn infants. A total of 329 women, 131 men and 331 children were recruited to this study between 1998 and 2002. An extended 6 years follow-up was performed between 2006 and 2008, and invitation to this follow-up visit was send to all women and men in this cohort; a total of 171 women and 44 men was finally reached and examined.Subjects in this cohort comprise mothers-to-be who were recruited at the minimum of 36 weeks of pregnancy  and follThe scrapings for HPV-testing were taken at the baseline and at 2-,12-,24-,36-month and 6-year FU-visits. Sampling was done with a cytobrush from the uterine cervix  using sampling media of 0.05 M phosphate-buffered saline with 100 μg gentamycin. The samples were immediately frozen at -20°C and stored at -70°C. Scrapings from the oral mucosa were also collected at each visit , but onlA routine pap smear was taken from all women at baseline, and 2-,12-,24- and 36-month, by using conventional three-sample technique with wooden spatula and cytobrush  as described earlier .The HPV genotyping was done by multiplex-HPV-genotyping kit  as outlined in our recent study . The kitHPV genotyping was done using the earlier PCR product which was now re-amplified with GP05+ and bio-GP06+-primers . OriginaAs HPV16 is the most prevalent genotype, we retested all HPV16-positive samples as described by Schmidt and co-workers  and discBlood samples were taken at baseline and at 2-,12-,24-and 36-month and 6 years of FU. Samples were analysed for the antibodies to the major capsid protein L1 of HPV types 6,11,16,18 and 45, and were analysed by multiplex HPV serology based on glutathione S-transferase fusion-protein capture on fluorescent beads. These results were interpreted using the technique and cut-off values as recently described . The Sel®  and STATA  software packages . Frequency tables were analyzed using the χ2-test, with the likelihood ratio or Fisher's exact test for categorical variables. Differences in the means of continuous variables were analyzed using non-parametric  tests for two- and multiple independent samples, respectively.All statistical analyses were run using SPSSThe genotype-specific outcomes in each woman were assessed by comparing the viral events at each FU visit to the baseline HPV status and are presented in Figure Times in months to incident infections were calculated from baseline visit to the first incident event separately for actuarial and crude times. To calculate the former, all baseline HPV-negative women (n = 255) were included in Kaplan-Meier analysis, cases being censored at the first incident event (those who had) or at the end of FU (those who did not develop incident event). Only those women who had completed at least two FU visits were included to this study. To calculate the crude incidence times, only those women with an incident infection with an individual genotype (or species) were included. Both actuarial and crude incidence rates (IR) were calculated, and expressed per 1000 women months at risk (wmr). To get genotype-specific actuarial IRs, the number of incident events for each individual genotypes (and species) was divided by the total wmr , including also the women with no incident event. To calculate crude IRs, only the women with incident events were included and the number of incident events for each individual genotype (or species) was divided by the wmr of those women only. To compare the individual IRs, the rate ratio statistics (STATA) was used, with test-specific 95% confidence intervals (95%CI).To analyze the predictors of incident HPV-infections, we used the species-specific incident infection approach to avoid stratifying the material into individual genotypes with single or few cases only. Furthermore, we were only interested in incident HPV-infections by the key HR-HPV-types, i.e., those of Species 7 and Species 9. Women that had an incident infection recorded by a type or types from species 7 and/or 9 were included to these analyses (n = 133).Poisson regression analysis was used for panel data, clustered by mother-ID and executed (in univariate and multivariate-mode) using population-averaged (PA) model ,27. FU vIn univariate Poisson, we first tested all covariates recorded at baseline questionnaire as well as some selected variables from the FU questionnaire (e.g. a new partner), previously implicated as potential risk factors of HPV in this cohort ,28. In tOf the 255 baseline HPV-negative mothers enrolled in the cohort, 203 experienced an incident event during the FU. The mean follow-up time of these 203 mothers was 58.6 ± 25.2 (SD) months . HPV16 caused most frequently incident infection , followed by HPV18 , HPV70 , HPV6 and HPV45 . Multiple-type infections were the second most common incident events after HPV16, with 25.1% (n = 51) frequency. HPV16 and HPV18 were detected in 70.6% (n = 36) and 27.2% (n = 14) of the multiple-type infections, respectively.The mean times to the first incident event are shown in Table Apart from the single case of HPV33, HPV56 showed the second longest crude mean time of 42.4 months, followed by HPV16, 18 and 31, with 23.1, 20.6 and 20.1 months, respectively. Remaining genotypes had crude mean times between 6.3 and 17 months.Species 9 was the most dominant species covering 54.7% (n = 111) of all incident infections and had actuarial mean time of 27.5 months (95%CI23.3-31.7) and crude mean time of 22.6 months (95%CI19.7-25.4). The second and third most common species were species 7 and species 10, with 10.8% (n = 22) and 3.9% (n = 8) prevalence, respectively. Actuarial mean time for species 7, 9 and 10 was similar, 27.5, 27.5 and 27.0 months, respectively. Species 6 and 9 had the longest crude mean incidence times, 25.0 and 22.6 months, respectively.The cumulative incidence of species 7 and 9 was compared with species 10 in univariate survival (Kaplan-Meier) analysis Figure . DespiteOf single genotypes, HPV16 had by far the highest actuarial IR, 15.6/1000 wmr 95%CI12.5-18.6), followed by multiple-type infections, with 8.2/1000 wmr (95%CI5.96-10.45). Actuarial IR for HPV18 was markedly lower, 1.3/1000 wmr (95%CI0.39-2.17). Due to the dominant role of HPV16, species 9 showed the highest IR of 17.9/1000 wmr (95%CI14.6-21.2), far exceeding that (3.4/1000 wmr) of species 7  is excluded  > 2 life-time sexual partners (p = 0.011), 2) late (> 20 years) initiation of OCs (p = 0.017), and 3) pregnancy at FU-visit , with the details of estimates in sub-categories shown in Table This is one of the first studies to assess type-specific incident HPV-infection -16, in cWe previously analysed crude incidence times collectively for 13 HR-HPV-types detected by Hybrid capture 2-assay . It tookAs evident in Figure Also the IR can be calculated as actuarial and crude, as done here. These two IRs are markedly different, because of the significantly different person months at risk. This also makes comparison between individual studies difficult, particularly when the FU-times and cohort size are substantially different. If one compares a setting where 1000 women are followed-up for 10 months (10.000 wmr), and another one with 100 women followed-up for 100 months (10.000 wmr), certainly the IRs in the latter are higher. This is because the median incidence time is around 12-13 months and only a fraction of events can be detected with a short FU-time. In addition, it is essential to know the age-profile of the cohort, because IR for HPV infection is critically age-dependent .In our cohort, actuarial IR was by far the highest for HPV16, 15.6/1000 wmr. This is consistent with other studies, where HPV16 had the highest actuarial IR, albeit the absolute IRs of these cohorts was much lower. A young women's health study-cohort from USA reported an actuarial IR of 5.9/1000 wmr for HPV16 , which iThis is one of the first studies on crude IRs, which reflect the differences in the rates at which individual genotypes accumulate incident events among women who have these events. Although firm conclusions on single genotypes are difficult because of the rarity of many individual genotypes, it is interesting to note that the crude IR of the key LR-HPV-types , is almost twice as high as that of the two main HR-types . This indicates that the former develop incident infections at much higher rate, which is also shown by their significantly shorter times to incident events Table . Using rAs to the risk-factors for incident species 7/9 infections, seven variables were significant predictors in univariate analysis. First of these was linked with HPV-serology ; mothersInitiation of smoking beyond 13 years of age was associated with an increased risk of incident HPV-infection, but lost its significance in multivariate model. Some studies have found no association between smoking and incident infection ,16, wherWomen who started their sexual activity before 13 years of age were found to have a lower risk for incident infections during the FU. Also this association is in line with our recent analysis disclosing early age of sexual debut as a risk factor for HR-HPV persistence . It is lThe same analogy applies to life-time number of sexual partners , shown in this study to be inversely related to the risk of incident infections, i.e., lower partner number was associated with an increased risk. It seems likely that those with more partners have already experienced such an event before enrolled in the study, i.e., are baseline HPV+ and by definition, no longer at risk for incident infection by the same HPV genotype.The initiation of OC usage before the age of 20 increased the risk for incident infection and was a significant predictor also in the multivariate model. Previous studies report conflicting results; current use of OCs has been shown to increase  or decreAnother significant protective predictor was the second pregnancy during the FU Table . No prevThe main limitations of this current study are firstly the small cohort size of the women; the total number of incidence infections was 203. To gain more information on genotype specific incident HPV-infections, times and rates, and to increase the statistical power, a much larger cohort is needed. Secondly the women were all pregnant at the time of enrolling to this study, as it is the special strength of this study it also may be considered as a limitation because the detailed mechanism how pregnancy and HPV infections interacts are not that well understood.Data obtained from this study indicate that the probability of mothers, who test HPV-negative just before delivery, to contract an incident infection by species 7/9-genotypes during the subsequent follow-up is less likely among those who report more than 2 life-time sexual partners at baseline, start using of OCs after age 20, and become pregnant for the second time during the follow-up. To the best of our knowledge, this is the first study where actuarial and crude times to the incident events as well as actuarial and crude IRs are evaluated at the genotype- and HPV-species level. Obtaining genotype-specific data during long-term follow-up is needed for better understanding of the natural history of genotype specific HPV-infections, e.g. the times and rates at which each genotype accumulates incident events. It sounds feasible to reason that in the future, those genotypes with shortest times to incident infections , could be the most appropriate targets for early prevention, e.g. by genotype-specific multivalent HPV vaccines.The authors declare that they have no competing interests.KL is the first author in this study, participated in execution of the work at all phases, this study is part of her PhD project. KS is a pathologist and senior author in the project, with main responsibility in maintaining the databank as well as designing and running the statistical analyses. SG is the clinical principal investigator and coordinator at the Department of Obstetrics and Gynecology, and has participated in the design of the study and analysis and writing of the results. MR has been responsible for enrolment and examination of all mothers during the follow-up and writing of the results. SS is the coordinator and principal investigator of the Finnish Family HPV study and the head of the HPV research laboratory. All authors have read and approved the final version of this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/179/prepub