Pre-vaccination information on HPV type-specific prevalence in target populations is essential for designing and monitoring immunization strategies for cervical cancer (CC) prevention. Data on HPV prevalence in Italy are available for women over the age of 24 years, target of the population-based CC screening programmes; while data of HPV prevalence in younger ages are very limited. The present study enrolled Italian women aged 18–26 years in order to assess the prevalence and distribution of high-risk (HR) HPV types. Risk-factors correlated with HR-HPV positivity were also described.A sample of 2,289 women was randomly selected from the resident population lists of ten Local Health Units (LHUs) located in six Italian Regions scattered across the country; both rural and urban LHUs were involved. Women aged between 18 and 26 years and living in the selected LHUs were included in the study; pregnant women and women who did not speak Italian were excluded. A total of 1,102 women met the inclusion criteria and agreed to participate. Participants were offered pap test and Hybrid-Capture 2 (HC2) test for HR-HPV types and genotyping was performed on positive smears.adj = 4.15, 95%CI: 2.56-6.72) and ≥5 lifetime partners  and women who have used any contraceptive in the last six months  had a higher risk to be infected; women living with their partner had a lower risk  to acquire infection than women living with parents/friends/alone. Among HC2 positive women, HPV16 was the most prevalent type (30.9%), followed by 31 (19.6%), 66 (12.9%), 51 (11.3%), 18 (8.8%), 56 (8.8%). Co-infections of HR-HC2 targeted types were found in 20.4% of positive samples. The HR-HPV prevalence in women with abnormal cytology (52.4%) was significantly higher than in women with normal cytology (14.6%); however 33.0% of HR-HPV infected women had an abnormal cytology.Out of 1,094 valid samples, 205 (18.7%) were HR-HPV positive. Women with 2–4 (ORHR-HPV prevalence in Italian women aged 18–26 years was 19%, higher than what detected for older women, by other studies using the same molecular method and laboratory network; this result supports the choice of electing girls before the sexual debut as the primary target of HPV vaccination. The HPV type distribution found in this study may represent a baseline picture; an accurate post-vaccine surveillance is necessary to early detect a possible genotype replacement. The high prevalence of viral types other than vaccine-HPV types supports the necessity to guarantee the progression of CC screening programmes in vaccinated women. In the last decade many studies have shown that infection with high-risk (HR) types of human papillomavirus (HPV) is a necessary condition for the development of invasive cervical cancer (CC) . GenitalAs of July 2010, 18 European countries had integrated HPV vaccination in their national immunization programmes, most of them targeting pre-adolescent girls; nine countries had also planned catch up programmes for older girls . In ItalPre-vaccination distribution of HPV genotypes in target populations is essential for designing, monitoring and evaluating immunization strategies. Baseline prevalence is relevant to estimate vaccine effectiveness against the HPV-vaccine types, to evaluate the cross-protection of the vaccines, and to monitor over time the relative frequency of genotypes under the selective pressure of the vaccines.Data on HPV prevalence in Italy are available for women over the age of 24 years -10, targThis cross-sectional study was carried out in the period 2007–2009, coordinated by the Istituto Superiore di Sanità  and partially sponsored by the Ministry of Health. It was part of a national project (PreGio), which also included a KAP (knowledge-attitudes-practices) survey on HPV infection and CC prevention  and a stA sample of 2,289 women was randomly selected from the resident population lists of ten Local Health Units (LHU) located in six Italian Regions scattered across the country: Abruzzo and Campania in southern Italy , Lazio and Tuscany in central Italy (388) and Emilia-Romagna and Piedmont in northern Italy (875). Both rural and urban LHUs were involved; all 10 LHUs have population-based CC screening programmes.The inclusion criteria for the study were: women aged between 18 and 26 years and living in the selected LHUs. Pregnant women and women who did not speak Italian were excluded. The women were stratified into two age groups (18–24 and 25–26 years), partially overlapping (25–26 years) with the target age of population-based CC screening. Procedures for enrolling participants as well as study methodology have already been described ,14.All women were offered Pap-test and HPV test; a written informed consent was obtained by each participant. Selected midwives, especially trained for this study, were in charge of contacting sampled women, collecting socio-demographic data and information regarding sexual and reproductive history, getting informed consent and performing cervical smears. The study was approved by the national ethics committee of the ISS.Cervical smears were taken according to the consolidate CC-screening procedures at the LHU. In most Regions  two cervical samples were taken: one for Pap-test and one for the HPV test in Specimen Transport Medium ; for STM sample, cytobrush was used to obtain samples. In Abruzzo Region the cervical cell specimens were put in PreservCyt solution  and used both for HPV testing and for cytological examination; for Preservcyt sample, Ayre spatula and cytobrush were used. The cytology was interpreted locally according to Bethesda 2001 system . InformaWomen aged 25–26 years followed the routine protocol of treatment or follow-up according to cytological results. A specific protocol for 18–24 year-old women was adopted: colposcopy was offered to women with HSIL  and ASCH , while women with LSIL  or less severe lesions were invited to repeat pap-test two years later and colposcopy was offered only if, after two years, the diagnosis was still LSIL or it progressed to a more severe lesion.The HPV test was performed using Hybrid Capture 2  according to the manufacturer’s instructions. It is a hybridization assay which detects the presence of HPV-DNA using cocktails of RNA probes and an amplified, chemiluminescent signal. The high-risk group of probes B, designed to detect HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, was used. The assay is calibrated on a positive cut off (pc) of 1 pg/ml of HPV-DNA. Samples were considered positive when the ratio between the Relative Light Units (RLU) of specimen and the pc attained or exceeded the value of 1.0.A regional laboratory was identified in Tuscany, Piedmont, Abruzzo and Emilia-Romagna Regions for performing HPV testing; the Analytical and Biomolecular Cytology Unit of the Cancer Prevention and Research Institute (ISPO) in Tuscany also analysed samples from Campania and Lazio Regions.Genotyping was performed on HR-HC2 positive samples. DNA was extracted using the QIAamp DNAMini kit (Qiagen) according to the manufacturer’s instructions. For PreservCyt™ samples the elution volume varied from 80 to 100 μl depending on the size of the pellet, while for STM samples 100 μl of elution buffer (Buffer AE) were used. To facilitate a higher recovery of DNA a double elution was done for all the samples. HR-HC2 positive samples were typed using the “Consensus High Risk HPV genotyping” kit assay . The test is based on the reverse hybridization principle. Denaturated biotynilated amplicons, resulting from the amplification of a part of the L1 region with GP5+/GP6+ primers, were hybridized with specific oligonucleotide probes, which were immobilized as parallel lines on membrane strips (Reverse Line Blot Hybridization). The hybrids were detected with alkaline phosphatase–streptavidin conjugate and substrate (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium), resulting in a purple precipitate at positive probe lines. The kit allows the identification of 18 HPV types . One HPV-positive control and two negative PCR controls (a purified DNA sample negative for HPV and a DNA free sample) were included in each PCR run and subsequent Reverse Line Blot (RLB). GP5+/GP6+ PCR-negative and RLB-negative samples were amplified for the β-globin gene sequence using GH20-PC04 primers (268-bp amplicon length) to assess DNA integrity (Bauer).The molecular Laboratory of ISPO performed genotyping of samples collected in all Italian regions involved in the study except samples collected in Piedmont Region, which were genotyped at the Center for Experimental Research and Medical Science of the Turin University. ISPO also coordinated the molecular activities of the whole project, standardizing the procedures for storing and analysing the samples according to a protocol defined to guarantee the quality of molecular methods .t-test or Mann–Whitney nonparametric test was used to compare continuous variables. Crude and adjusted odds ratios (OR) and 95% confidence intervals (95%CI) were calculated to evaluate the association between the HR-HPV positivity and socio-behavioural characteristics.Categorical variables were summarized by absolute frequencies and percentages, and continuous variables by means and Standard Error (SE). The Chi-square test and Fisher’s exact test were used to compare proportions; the Confounding was assessed by a multivariate logistic regression approach; all variables with a p-value ≤0.15 in the univariate analysis were included in the multivariate model and retained in the final model according to a log-likelihood-ratio test for goodness-of-fit. Statistical analysis was performed using STATA 11.2 .Recruitment was performed in the period ranging from February 2008 to April 2009. The initial sample included 2,289 women; 239 did not meet the inclusion criteria  and were replaced, and 290 could not be contacted. Thus 1,999 women were offered Pap test and HPV test. Among them, 897 (44.9%) declined participation for several issues   were enrolled in the study. In order to calculate the participation rate to the prevalence study, we considered more appropriate to exclude from the denominator  the 154 women (among the 897 women who declined participation) who spontaneously declared not to be sexually active: therefore the participation rate was 51.6% , with a range among LHUs of 23.9-80.7.The mean age of participants was 23.8 ± 0.07 years; 40.7% lived in northern Italy, 37.8% in southern Italy, and 21.5% in central Italy. Most women were Italian (93.7%), unmarried (89.3%) and had a high educational level, defined as > 8 years of education (80.2%). Detailed socio-behavioural characteristics are shown in Table The Pap-test was performed on 1,099 cervical smears because three samples were missed  and the HR-HPV testing was performed on 1,094 out of 1,999 samples . The proportion of HR-HC2 positive samples was 18.7%  and did not differ by geographic area and age group  and 194 (96%) showed positivity at least for one of the 18 types identifiable by the kit assay used for genotyping. In contrast, eight samples (4%) did not result positive at any of these 18 types: this proportion is in line with data reported in literature ,18 and iAmong Hybrid Capture positive women HPV16 was the most prevalent type 30.9%), followed by 31 (19.6%), 66 (12.9%), 51 (11.3%), 18 (8.8%), 56 (8.8%) and 58 (8.3%); all the other types were detected at frequencies lower than 5.2%  had a multiple infection (36 infected by two types and one by three types). No difference in socio-demographic characteristics and sexual and reproductive history was found between women presenting a multiple and single infection. HPV16 was the most common genotype detected in multiple infections, followed by HPV31 and 18. Table % had a mThe univariate analysis of socio-behavioural characteristics and HR-HPV infection is reported in Table The following variables were included in the multivariate model: marital status, “living with”, age at first sexual intercourse, age at first delivery, parity, number of lifetime partners, number of partners in the last six months, use of contraceptives in the last six months.adj = 4.15, 95%CI: 2.56-6.72 if 2–4 partners and ORadj = 10.63, 95%CI: 6.16-18.36 if ≥5 partners). Also, living with the sexual partner and having used any contraceptive in the last six months remained significantly associated with the detection of HR-HPV infection .In the multivariate logistic regression model Table , the effThe pap-test result was available for 1,093/1,094 cervical smears tested for HR-HC2; 5.7%  of samples were inadequate, cytology was normal in 83.0% samples  and abnormal in 11.3%  samples: 62 ASCUS/AGUS , 52 LSIL, 6 ASCH and 4 HSIL. The proportion of HR-HPV positivity was significantly higher in women with abnormal cytology  than in women with normal cytology  . Among the 197 HPV-infected women with adequate cytology, 65 (33.0%) had an abnormal finding.HPV16 was the most frequent virus strain in normal cytology, ASCUS/AGUS, LSIL and ASCH, detected in 29.3, 47.8, 24.2 and 66.7% of the samples respectively. All the four HSIL were HR-HPV positive and four different types were detected respectively .We found a prevalence of HR-HPV infections of 19% in a sample of Italian women aged 18–26 years. This percentage is in agreement with an Italian study which used a similar methodology to measure the HR-HPV prevalence in 18–24 year-old women from Tuscany Region . This vaCervical cancer incidence is lower in the South than in the North of Italy, therefore a lower HR-HPV prevalence in the general population was expected in the South. Instead, we did not find any difference in HR-HPV prevalence rates between northern, central and southern regions of Italy, confirming what reported by Agarossi and coll. . This fiGenotype HPV16 was detected in 31% of HR-HC2 positive samples, followed by 31, 66, 51 and 18. This is well consistent with the results of a comprehensive meta-analysis on HPV positive women with normal cytology conducted by de Sanjosé and coll. , which rAccording to the International Agency for Research on Cancer (IARC) classification, HPV16, 31, 51 and 18 are classified as “carcinogenic to humans”, while HPV 66 is classified as “possibly carcinogenic” . It shouAs expected, we found that women with cervical cytological abnormalities were at significantly increased risk for being infected by HR-HPV types than women with normal cytology (52 vs. 15%). Current Italian studies involving women with cytological abnormalities (different ages and enrolment criteria) reported HR-HPV prevalence of 34-68%, increasing with cytology severity: 24-56% in case of diagnosis of ASCUS/AGUS, 42-72% in LSIL and 73-95% in HSIL ,27,30-32Co-infections of HR-HC2 targeted types represented the 20% of HPV positive samples. This value is included in the range (15-50%) reported in literature ; similarThe multivariate analysis evidenced the role of the number of lifetime sexual partners as determinant of HPV infection, consistently with other studies ,12,21,37The major strengths of this study are that the sample was large and it targeted an age group that has not been investigated extensively.Among the limitations, it should be mentioned the fact that our sample may not be entirely representative of Italy’s general population of females aged 18–26 years because only ten LHUs in six Regions participated, though local probabilistic samples were population-based and both urban and rural LHUs in northern, central and southern Italy were involved.In addition the participation rate was 52%, therefore our findings could not be representative of the entire study population; however differences in socio-demographic characteristics between participants and people who declined participation were minimal.The participation rate could be underestimated because we have excluded from the initial sample the 154 women who spontaneously declared not to be sexually active, but we do not know if other women, who declined participation, were virgin too (we did not collect this information). This age group represents a difficult target for prevention measures, because in Italy young adult women are not accustomed to being targeted by preventive programmes; moreover, enrolled women were offered the participation to a package of activities within the PreGio project, which is more difficult to be accepted than a single cervical smear. On the other hand, it should be noted that the study personnel had received special training and that a high number of attempts were made to contact non-respondents. Two other studies ,12, whicAs already mentioned, another limitation is that the prevalence of HPV 26, 53, 66, 73 and 82 could be heavily underestimated because they are not targeted by HR-HC2 test. In fact HPV 73 and 82 were observed only in co-infection with other HR-HPV types, suggesting that they could have been found only because coexisted with HPV types detected by HR-HC2 probes B .We measured the genital HR-HPV prevalence in Italian women aged 18–26 years before the introduction of HPV vaccination across all the areas of Italy; we found a prevalence of 19%, higher than what detected for older women . The useThe HPV type distribution found in this study may represent a baseline picture; an accurate post-vaccine surveillance is, in fact, necessary to early detect a possible genotype replacement. The high prevalence of viral types other than vaccine-HPV types supports the necessity to guarantee the progression of CC screening programmes in vaccinated women. Studies linking screening programmes with vaccine immunization registries should be performed to evaluate the HPV vaccine effectiveness on vaccine-related and not-vaccine related types.The authors declare that they have no competing interests.CG coordinated and monitored the project activities, analysed the data, interpreted the results, drafted and edited the manuscript. SDo designed the study, coordinated and monitored the project activities, interpreted the results and revised the manuscript. FC and GR participated in the study design, carried out the immunoassays, coordinated the activities of laboratories and revised the manuscript. SS designed the study, interpreted the results and revised the manuscript. SDe coordinated and monitored the project activities and revised the manuscript. MLCdA designed the study, coordinated and monitored the project activities in the first phase of the project and revised the manuscript. AB analysed the data and revised the manuscript. MPA, SB, NC, DF, AL, MCM, RN, EB participated in the study design and coordinated the activities in their local health units. EB, AG, VM, PP carried out the immunoassays. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/74/prepub


Background. Infection with and persistence of high-risk human papillomavirus (HR-HPV) are the strongest risk factors for cervical cancer. In addition, other genital microorganisms may also be involved in the progression of HPV-associated lesions. Objetive. To evaluate the association of the vaginal microbiota  with HR-HPV infection in Spanish female sex workers (FSWs). Methods. This cross-sectional study involved 208  who visited a sexually transmitted infection (STI) information and prevention center (SERGAS) between January 2010 and December 2011. Face-to-face interviews were carried out. Cervical and vaginal samples were examined for human papillomavirus (HPV), Trichomonas vaginalis, Candida spp., and microorganisms related to bacterial vaginosis (BV). Results. HR-HPV was found to be significantly associated with BV in FSWs with positive results for HPV16-related types . T. vaginalis was isolated in FSWs with the following HR-HPVs: 18, 45, 66, and 68. Candida spp. were isolated only in FSWs with HPV 18-positive infection. Conclusion. We demonstrate a significant prevalence of HR-HPVs in FSWs with disturbances in the vaginal microbiota. High-risk human papillomavirus (HR-HPV) infection is the most common sexually transmitted infection (STI) among young adult women and plays a critical role in the development of genital cancer. Recently, 15 HR oncological viral strains have been identified, including the HPV 16 group  of the alpha-papillomavirus genus  and the HPV 18 group  [Candida albicans is the most prevalent species in the majority of cases of asymptomatic colonization and vulvovaginal candidiasis. However, certain species of Candida are more pathogenic than others, induce hyphal and pseudohyphal formation, and enhance proteolytic activity and antigen modulation. These properties enable Candida to penetrate the mucosal surface and induce mucosal swelling, erythema, and exfoliation of cells [Candida spp. are more prone to acquiring cervical cytologic abnormalities over time than women who have a known, disturbed, bacterial vaginal microbiota [Earlier reports have suggested a link between genital tract inflammation and cervical cancer, only a few studies have controlled for HR-HPV genotypes, and no study has examined this infection in female sex workers (FSWs). The association between self-reported abnormal vaginal discharge and cervical intraepithelial neoplasia (CIN) in HR-HPV-infected women further suggests a link between genital tract anomalies and cervical cancer , 5. Bactof cells . In addicrobiota , 10.Trichomonas vaginalis, Candida spp., or BV in a group of FSWs with HR-HPV infection.Therefore, the purpose of the current study was to determine the prevalence of vulvovaginitis caused by Approval for the study was obtained from the Ethics Committee of Galician (Spain) Human Research. HIV-negative FSWs  who visited an STI information and prevention center in the north-eastern region of Spain between January 2010 and December 2011 were included in the study. The sample size was calculated to allow for a prevalence of 50% or higher for HR-HPV, with a confidence interval of 95% and an error of 6.5%.χ2 test was used for statistical analysis, while the odds ratio (OR) was used to measure the strength of the association between vaginal infections and high-risk human papillomavirus types. Statistical tests were considered significant if the P value was ≤0.05. Logistic regression analysis was used to assess the simultaneous effect of more than 1 variable on the risk of HPV infection and to identify possible confounding factors. We determined the OR by using nonconditional logistic regression but using the correct definition; we could not estimate the prevalence ratio (PR) because we did not have any patients who were not exposed to the infection. The software used for the statistical analysis was the Statistical Package for the Social Sciences (SPSS), version 18.0.1.The Cervical samples were collected with a cervical brush, and all cervical swabs were placed a 1 mL a tube containing of the Digene specimen transport medium  and were stored at −20°C until testing. The presence of HR-HPV infection was determined through the Digene HR-HPV test—Hybrid Capture II (HC2)—by using the HR-HPV probe B cocktail, which identified types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, in accordance with the manufacturer's instructions. To minimize the cost of testing, an HC2 HPV DNA test was performed for all samples with only the high-risk HPV probe mixture. Positive specimens were detected by binding of hybridization complexes onto the surface of the microplate wells coated with monoclonal antibodies specific to RNA-DNA hybrids. Immobilized hybrids were detected by the addition of an alkaline phosphatase-conjugated antibody to RNA-DNA hybrids, followed by the addition of a chemiluminescent substrate.The presence of BV was evaluated microscopically in samples collected from the posterior vaginal fornix. BV was diagnosed on the basis of the presence of 3 of the following clinical and microscopic findings: vaginal pH greater than 4.5, presence of clue cells, grey homogenous vaginal discharge, and positive whiff test, in which a fishy odor is released after the addition of 10% potassium hydroxide solution to the vaginal fluid.T. vaginalis within 15 min. Sample collection was performed using calcium alginate swabs, which were transported in the Stuart Amies transport medium (Copan Innovation), kept at room temperature, and sent to the laboratory within 6 h of collection for culture on Roiron medium (MAIM S.L.). T. vaginalis was first detected by direct microscopic visualization of the fresh sample, followed by culture of the vaginal discharge on the Roiron medium and aerobic incubation at 37°C. The broth was examined microscopically for motile trichomonads on days 1, 3, and 5 of culture.The vaginal swab was placed in 0.2 mL of sterile physiologic saline for wet mount evaluation and examined microscopically (400x) for motile Candida ID2 (CAN2); bioMérieux) in the case of women for whom a positive diagnosis was suspected but for whom a negative result was obtained by direct visualization. For samples that did not test positive following microbiological culture, complementary diagnosis analysis using the Vitek ID-YST system (bioMérieux) was also performed.Samples were collected using calcium alginate swabs and transported in the Stuart Amies transport medium. The samples were kept in a T° environment and then sent to the laboratory within 6 h, as recommended by the manufacturer. Confirmatory microbiological culture of vaginal discharge was carried out in Sabouraud chromogenic medium . HR-HPV infection was present in 7.5–35.5% of these women, and the peak prevalence of this infection occurred 20 years earlier than that for T. vaginalis.Finally, 208 HIV-negative FSWs aged 18–49 years were involved in this study. The mean age of the study population was 27 years, whilst 60% of participants belonged to the target age group of 18–25 years. HR-HPV prevalence peaked in younger women aged 18–25 years. As expected, the prevalence of Data for HR-HPV-positive samples were grouped as belonging to HPV16-related genotypes , HPV18-related genotypes , or other genotypes, including HPV51 and HPV66. In the study group of 208 FSWs, 81 women had positive results for HPV16 infection and 48 for HPV18 infection. The distribution of frequencies for different genotypes of HPV is shown in T. vaginalis. We found no relationship between Candida spp. and HR-HPV, with the exception of HPV18 . Moreover, among women with BV, vaginal Candida spp.colonization was associated with HPV18 infections . When both vaginal Candida spp. and T. vaginalis were removed from the model, BV was still significantly associated with HR-HPV in all the FSWs. In contrast, when BV was removed from the model, vaginal Candida spp.colonization was not associated with HR-HPV, except for HPV18 . The presence of vaginal Candida spp.colonization alone was not significantly associated with all the HR-HPV genotypes, except for HPV18 . However, when Candida spp. colonization was present concurrently with BV or trichomoniasis, it negatively affected the outcomes for certain HPV types.Among women with vaginal T. vaginalis was statistically significant in women with HPV18, HPV45, HPV66, or HPV68; the presence of Candida spp. was found to be statistically significant only when HPV18 was detected. This result was further associated with both Candida spp. and T. vaginalis . Thus, we expect that, among the 16 HR-HPV-negative participants, 88% did not have BV. This equates to 66% of HPV31-negative, 82% of HPV33-negative, 75% of HPV35-negative, and 72% of HPV52-negative participants. In contrast, the presence of aginalis .Gardnerella vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum) [According to Cheng et al. and Muñoz et al., HPV types may be organized according to 1) HPV16-related types  or HPV types belonging to the alpha-papillomavirus-9 species, (2) HPV18-related types  or HPV types belonging to the alpha-papillomavirus-7 species, or (3) others, including HPV51 and HPV66. We have used this classification system in our study  HPV16-re–16. BV ilyticum) . In womelyticum) , 20. BV-lyticum) . Such enlyticum) –25. Howelyticum) .Candida spp. infection is the most prevalent mycosis in the majority of cases of asymptomatic colonization and vulvovaginal candidiasis. However, certain species of Candida are more pathogenic than the other species and induce hyphal and pseudohyphal formation and enhance proteolytic activity and antigen modulation [Candida spp. to penetrate the mucosal surface and induce mucosal swelling, erythema, and cell exfoliation. The role of T. vaginalis and Candida spp. in HR-HPV pathogenesis has been evaluated by some authors, including Engberts et al. [Candida spp. is not associated with an increased risk of acquiring HR-HPV.dulation . These fs et al.  who estaG. vaginalis was the most frequent bacterial agent found in women with HR-HPV infection , while in the control group the most frequent agent was Candida spp.  [Candida spp. and cytologic changes in the vagina. Candida spp. is not a cofactor in the presence of HR-HPV. However, it has been found that Candida spp. is more frequently found in women without cervical intraepithelial neoplasia (CIN) [T. vaginalis and vaginal Candida spp. colonizations were not associated with HR-HPV infection in HIV-negative women.In a study involving cytological analysis of samples obtained from 17,391 outpatients during January 1997–August 1998 in Brazil, 390 samples (2.24%) had alterations that were consistent with HPV infection and were sometimes associated with CIN I. The results showed that < 0.001) . This re< 0.001)  and Chak< 0.001) , who alsia (CIN) . As repoia (CIN) , T. vagiT. vaginalis, it appears that the presence of Candida spp. in the vagina is not a cofactor in the development of cervical cancer. This is consistent with the hypothesis that the local cervicovaginal milieu plays a role in susceptibility to HR-HPV infection since women who carry Candida spp. are likely to possess a healthy Lactobacillus-predominated vaginal microbiota, in contrast to women with dysbacteriosis [T. vaginalis and CIN or an association between T. vaginalis and HR-HPV infection [T. vaginalis. One hypothesis is that the parasite can act as a potential catalyst in the development of secondary infections, including HR-HPV, by producing a wide array of enzymes such as cysteine proteinase enzymes that are linked with cytotoxicity and the degradation of basement membrane components. Furthermore, some studies have shown that the double-stranded RNA of T. vaginalis is also associated with differential expression of enzymes and may therefore affect virulence [T. vaginalis could alter the natural history of various sexually transmitted diseases and, in particular, HR-HPV, by increasing virulence. This study clearly shows that women with positive cytological results for T. vaginalis are at significant risk of acquiring HR-HPV infection [In contrast to the finding for BV and teriosis . Only a nfection . These fnfection . Severalirulence –39. Thernfection , 41.Neisseria gonorrhoeae, Chlamydia trachomatis, and T. vaginalis, HR-HPV infection has been found to be common (36%), and oncogenic genotypes have been found in 44% of HR-HPV-positive patients [In addition to the conventional STIs caused by patients .T. vaginalis was found in 80 of the patients (0.2%) [T. vaginalis had a significantly higher prevalence of HR-HPV than women with normal results for cervical smear tests, irrespective of age (P < 0.01). Noël stated that these data suggest a potential association between these 2 infectious agents because of sexual intercourse and probably because of biochemical or immunological reasons [T. vaginalis in Flanders is low, with only 3.7/1,000 women infected. HR-HPV is present in 15% of these women and the peak of its prevalence occurs 20 years earlier than that of detection of T. vaginalis. The present study design does not allow for a definitive answer for this paradoxical increase in T. vaginalis prevalence in the absence of HR-HPV in the age group of 40–50 years [ In another study, s (0.2%) . Of thes reasons . Depuydt50 years .T. vaginalis infection was common, with oncogenic genotypes (HPV18 and HPV18-related types 59 and 68) being present in 61%, 45.5%, and 69% of positive samples. Moreover, T. vaginalis was present in 86% of HPV66-positive samples. These findings emphasize the importance of a cervical cancer prevention strategy, such as a cervical cytology screening program for HR-HPV, which is to be introduced soon.In our study, BV and T. vaginalis was statistically significant, in women with HPV 18, 45, 66, and 68. Candida spp. were not significantly associated with HR-HPV; they were more frequently among HR-HPV-negative women. These data confirm that screening for genital infections may reveal the simultaneous presence of different sexually transmitted microorganisms. These results suggest and emphasize the value of screening for genital infections in HR-HPV-positive patients in order to decrease the presence of other microorganisms and to reduce the probable synergistic effects of the vaginal microbiota. Prevention is important not only to avoid other STIs and their sequelae but also to reduce the influence of concomitant infection with other microorganisms on HR-HPV infection.This study suggests a positive association between BV and HR-HPVs . Presence of


Delivered via partnerships between Veterans Affairs medical centers and Alzheimer’s Association chapters, PDC targeted both patients and caregivers, distinguishing it from many non-pharmacological interventions. Hypotheses posited PDC would improve five veteran self-reported outcomes: 1) unmet need, 2) embarrassment about memory problems, 3) isolation, 4) relationship strain and 5) depression. Greater impact was expected for more impaired veterans. A unique feature was self-reported research data collected from veterans with dementia.Five matched communities were study sites. Two randomly selected sites received PDC for 12 months; comparison sites received usual care. Three structured telephone interviews were completed every 6 months with veterans who could participate.Of 508 consenting veterans, 333 (65.6%) completed baseline interviews. Among those who completed baseline interviews, 263 (79.0%) completed 6-month follow-ups and 194 (58.3%) completed 12-month follow-ups. Regression analyses showed PDC veterans had significantly less adverse outcomes than those receiving usual care, particularly for more impaired veterans after 6 months, including reduced relationship strain , depression , and unmet need . PDC veterans also had less embarrassment about memory problems . At 12 months, more impaired veterans had further reductions in unmet need  and embarrassment . Limitations included use of matched comparison sites rather than within-site randomization and lack of consideration for variation within the PDC group in amounts and types of assistance provided.Partnerships between community and health organizations have the potential to meet the dementia-related needs and improve the psychosocial functioning of persons with dementia.NCT00291161 More than 300,000 veterans with dementia receive care from the Department of Veterans Affairs (VA), the largest healthcare system in the United States . To addrPDC is a version of the evidence-based program, ‘BRI Care Consultation,’ which was developed by a research team led by the Benjamin Rose Institute on Aging to assist older adults and caregivers dealing with chronic health conditions -8. The BA key feature of PDC is its basis in a formal partnership between a healthcare organization  and a community service organization . Bringing healthcare and community providers together in this partnership was designed to facilitate more holistic care that: 1) improved fragmentation and lack of coordination between medical care and community services ,10; 2) rPDC gives equal attention to veterans with dementia and their primary informal caregivers . Veterans are active participants in PDC whenever possible, despite their dementia. Involving persons with dementia, as well as their caregivers, distinguishes PDC from many of the more than 40 evidence-based, non-pharmacological interventions that focus exclusively on caregivers . MoreoveThis research was a controlled trial that tested the effectiveness of PDC by examining the impact on self-reported outcomes by veterans with dementia. The study was approved by the institutional review boards of the Providence VA Medical Center, VA Boston Healthcare System, University of Oklahoma Health Sciences Center and Baylor College of Medicine.The Stress Process Model, which has been widely used in research on stress and coping for more than 30 years and recently was adapted for individuals with dementia (9), guided the two study hypotheses -23. Key This study’s two hypotheses are based on conceptualizing PDC as an external support resource (domain 3) that may directly or indirectly reduce negative consequences of primary stressors, which are veterans’ impairments. The first hypothesis focuses on direct benefits and posits that veterans who receive PDC will have decreases in five adverse veteran-reported outcomes that represent four types of role and intra-psychic strains (domain 2): 1) unmet need for help or information, 2) embarrassment about memory problems, 3) isolation from others and 4) relationship strain between veterans and their primary informal caregivers), and 5) one component of well-being (domain 5) (symptoms of depression). The second hypothesis focuses on indirect effects of PDC on the same five outcomes, with the potential benefits being conditional or dependent on severity of, or difficulty with, veterans’ impairments. The conditional-effects hypothesis posits that PDC will have greater benefits for veterans with more severe cognitive impairment or more dependencies in personal care. This hypothesis is based on the Stress Process Model’s assumption that negative effects of a chronic stressor, such as dementia, are not uniform across all individuals but depend upon the severity of symptoms and perceptions of difficulties associated with the stressor ,25.There were five study sites: Boston, MA; Houston, TX; Providence, RI; Oklahoma City, OK; and Beaumont, TX, with all sites located in one of two selected Veterans Integrated Service Networks (VISNs). VISNs provide a unifying administrative structure for all VA services within a given geographic region. Study sites were matched by VISN to assure uniformity in this overarching administrative structure. One of the two selected VISNs  was chosen because it was the location of the study’s VA principal investigator. The other selected VISN  had a similar array of VA services as VISN 16.Within each of the two selected VISNs, VA study sites were matched to be similar in: size, services offered (both inpatient and outpatient), academic affiliations, research missions and medical-residency training programs. Alzheimer’s Associations chapters within selected VISNs were similar in size, with comparable core programs and services. After selecting matched sites within each VISN, one was randomly selected to deliver PDC and the other was deemed the comparison site that would deliver usual care (UC). Specifically, in one VISN, Boston was randomly selected to deliver PDC and Providence was selected as its matched UC site. In the other VISN, Houston was randomly selected to deliver PDC and Oklahoma City was selected as its matched UC site. Beaumont was paired with Oklahoma City as part of the same UC site to assure a sufficiently sized comparison sample. Matched PDC and UC sites, rather than within-site randomization, were used to allow PDC implementation throughout partnering organizations, without concerns about diffusion of PDC to the UC group.Veterans and caregivers at PDC sites received the care-coordination program and an initial, basic packet of educational materials on dementia; UC-site veterans and caregivers received the same basic educational materials and usual care from the VA and chapters. Aside from PDC care coordination, there were no restrictions on services or care that could be obtained by the PDC or UC groups from the VA, chapters or other organizations.Eligibility requirements for veterans included receiving primary healthcare from the VA, residing outside a residential care facility at the time of enrollment, living within a partnering chapter’s service area, being 60+ years of age and having at least one of the following dementia diagnostic codes from the International Classification of Diseases, Ninth Revision recorded in the VA medical record: 290.41–43, 291.2, 292.82, 294.1, 294.8 and 331.0. There was no enrollment restriction based on type or severity of veterans’ impairments, or availability of a family caregiver. VA primary care physicians confirmed veterans’ diagnoses and eligibility prior to sample selection.The sample was recruited from 18 January 2007 to 22 July 22 2009. Research interviews occurred from 21 March 2007 to 15 September 2010. Veterans’ ability to complete research interviews that provided self-reported outcomes was determined by a telephone screening, using the short Blessed Orientation-Memory-Concentration Test . The BleSample-size estimates were based on a one-tailed significance test, a modest effect size (.12), .80 power, an alpha of .05, 10 independent variables in a regression equation and an estimated squared multiple correlation of .55. A minimum sample size of 247 was determined based on these assumptions.Two half-time care coordinators, with part-time administrative assistant support, delivered PDC at each intervention site. One care coordinator worked in the local VA medical center  and the other worked in the partnering Alzheimer’s Association chapter (community service organization). Although from different organizations, the two care coordinators worked as a team, with one shared electronic Care Coordination Information System (CCIS) and regularly scheduled planning and case-conference meetings. Care coordinators had bachelor’s or master’s degrees in social work, nursing or other helping professions.The care coordinator from the VA medical centers had primary responsibility for assisting veterans with medical-related concerns  while the care coordinator from the Alzheimer’s Association chapter had primary responsibility for assisting caregivers with nonmedical concerns . The VA care coordinator also focused on helping families access VA services and benefits, whereas the Alzheimer’s Association care coordinator focused on helping families use community services, including those offered by the Alzheimer’s Association. This division of labor between care coordinators capitalized on the complementary strengths of each partner organization and represented a bridge between health care and community services.Training for care coordinators consisted of a 1.5-day initial session on the PDC philosophy, service-delivery protocol and the CCIS that guides service delivery. Additionally, one- to two-hour biweekly refresher trainings were completed throughout the study period. These sessions focused on case reviews to monitor fidelity to the intervention protocol, strategies for working with a partner organization, using the CCIS and handling difficult cases. Continuing education also was provided on special topics, such as differences among illnesses that cause dementia, helping families respond to emergencies and respite for caregivers.PDC is a coaching model driven by consumer choice, with care coordinators helping find solutions to concerns that are the priorities of veterans and caregivers. PDC followed a set, standardized protocol that required a minimum of at least one contact between care coordinators and consumers per month; more-frequent contacts occurred as needed. The protocol required care coordinators to discuss with veterans and/or caregivers a broad range of medical and nonmedical concerns, although the specific content was customized to consumers’ preferences and needs.PDC is a low-cost service delivered by telephone, mail and e-mail, with in-person contacts rarely needed. The two half-time care coordinators from the partnering organizations ) maintained caseloads of 75 to 125 families. All expenses to deliver PDC  can be recovered by charging a fee of $60 to $80 per month per family.PDC gives equal attention to preferences and needs of veterans and caregivers. Veterans with dementia are engaged in the program whenever possible, despite their impairments. Veterans without caregivers are able to use PDC, so long as they can communicate by telephone. If veterans are too impaired to communicate by telephone, their caregivers can be the sole participant in the program.PDC has three main components: 1) initial assessment, 2) action plan, and 3) ongoing monitoring and reassessment.The initial assessment is completed gradually during the first four weeks of enrollment. It is designed to be brief, with the action plan to address assessed concerns implemented simultaneously with or prior to completion of the entire initial assessment. The initial assessment covers a broad range of domains or potential problem areas: 23 for veterans  and 16 for caregivers . The required initial assessment consists of a single-item trigger question for each domain; trigger questions can be formally asked or covered informally during conversations. More extensive detailed assessment questions are available for each domain as optional tools, if additional probing is necessary to clarify a problem.The action plan is the core of PDC. It comprises simple behavioral tasks called action steps that, if accomplished, move veterans and caregivers toward solutions to concerns they identified as important. Action steps should be easy to complete and include, for example, calling an organization to inquire about the availability of a service, reading an educational resource on a topic of concern or contacting another family member to ask whether he or she is willing to help with a caregiving task. With coaching and guidance from care coordinators, veterans and caregivers determine the content of action steps, who will complete the action steps and the projected dates of completion. New action steps are continuously added and build upon prior action steps. Multiple action steps, spread over a period of weeks or months, often are needed to find solutions to specific problems. As action steps are completed, veterans and caregivers move toward solutions and gain confidence in their self-management abilities. Copies of action plans are mailed to veterans and caregivers and summarized in the larger medical record.On average, each veteran and his or her caregiver had more than seven action steps. The most common pertained to accessing and coordinating services and benefits available from the VA, Alzheimer’s Association or other community agencies. Specifically, 78% of veterans and caregivers had action steps related to accessing VA services or benefits, 59% to accessing Alzheimer’s Association services and 76% to accessing other community organizations. Other common action steps focused on improving care from the informal network (57%), managing symptoms (40%), improving communication with healthcare providers (33%) and home safety (29%).The hallmark of PDC is a long-term relationship to provide continuous support to veterans and caregivers. Ideally, care coordinators become knowledgeable and familiar experts who are trusted by families. On average, families had over 14 contacts with coordinators during the twelve-month study period, which focused on completing the required initial assessment and reassessments, adding new action steps and checking the disposition of pending action steps, and completing required routine checking.Reassessments involved readministering trigger questions used in the initial assessment. They were required at least every six months. More frequent reassessments for selected domains are recommended for persistent or ongoing problems. Reassessment helps care coordinators and consumers gauge progress in finding solutions to problems.et al. [Consistent with the design of PDC, the most contacts between care coordinators and veterans or caregivers were by telephone (80%) and regular mail and e-mail (16%), with a small number in person (4%). The number of contacts was evenly split between care coordinators from the VA and the Alzheimer’s Association, which reflected PDC’s team-based delivery model. Care coordinators initiated approximately 90% of contacts; veterans or caregivers initiated 10%. .The study period was twelve months, with three research telephone interviews attempted with participating veterans who were able to pass the baseline telephone screening with the adapted Blessed Test and three research interviews with veterans’ caregivers. Research interviews were administered by trained interviewers. Baseline interviews were conducted as soon as possible after written consents were received. To assess the impact of PDC on study outcomes, second and third research interviews were conducted at six and twelve months postbaseline.Five self-reported outcomes from veterans with dementia  unmet needs, 2) embarrassment about memory problems, 3) isolation, 4) relationship strain and 5) depression) were examined after six and twelve months. As described below, psychometric properties of these outcomes were tested with the current sample of veterans to reaffirm their reliability and structural validity, which had been established in previous studies for all but one outcome (citations provided below). Overall, psychometric analyses showed individual items comprising these outcomes had good internal reliability, based on Cronbach’s alpha, and good structural validity, based on high factor loadings on a single factor and low cross-loadings on factors representing other outcomes. Thus, psychometric analyses reinforced the feasibility of collecting information from persons with dementia, despite mild-to-moderate levels of cognitive impairment. However, a limitation of the outcomes was the use of simple ‘yes/no’ response choices for most questions comprising these measures to facilitate administration with cognitively impaired respondents .Developed for this study, this outcome was based on 24 dichotomous questions that were summed to measure veterans’ perceptions of unmet need across eight domains: 1) understanding dementia, 2) daily living tasks, 3) accessing VA and other services, 4) legal and financial issues, 5) organizing family care, 6) alternative living arrangements, 7) emotional support and 8) medications. This measure had good structural validity, with factor loadings from .83 to .62 on a single factor and good internal reliability .This previously published outcome  was the This previously published outcome  includedThis outcome was adapted from a published family caregiving measure  and was Veteran depression was measured by the 11-item Center for Epidemiologic Studies Depression Scale, which had good reliability at all three data-collection waves  .Two multi-item scales representing impairment were used to test the conditional-effects hypothesis: 1) cognitive impairment was measured by the Blessed Test ; and 2) A wide range of baseline measures was used to test for initial differences between PDC and UC groups that may have resulted from using matched sites rather than randomization.Multiple regression equations tested whether veterans in the PDC and UC groups differed across the five outcomes: 1) unmet need, 2) embarrassment, 3) isolation, 4) relationship strain, and 5) depression. Two equations were estimated for each: one representing change from baseline to six months and one representing change from six to twelve months. In each equation, the effect of PDC on an outcome was represented by the regression coefficient for a dichotomous variable that distinguished between veterans in the PDC and UC groups. In addition, two product or interaction terms tested the conditional-effects hypothesis that posited the benefits of PDC would be greater for more impaired veterans . Product terms were constructed by multiplying the dichotomous intervention variable by the two variables representing impairments. To facilitate interpretation, impairment measures were centered to have a mean of zero .Regression coefficients for the PDC variable and product terms were of primary interest to this study. When there were no significant product terms and the regression coefficient for the PDC variable was significant, it meant the difference between the PDC and UC groups pertained to all veterans, regardless of level of impairment . When there was a significant product term, the regression coefficient for the PDC variable represented the difference between the PDC and UC groups among veterans with mean levels of cognitive impairment or personal-care dependencies. The regression coefficient for a significant product term represented the average difference in an outcome between PDC and UC groups among veterans with higher- and lower-than-average levels of impairment. , results consistent with hypotheses were indicated by significant regression coefficients with negative values. Analyses were run using IBM SPSS Statistics, Version 21 .A total of 1,775 veterans were referred to the project by VA primary care physicians and mailed IRB-approved study invitations and consent forms. Signed consent forms were received for 508 veterans (28.6%). All but 22 consenting veterans had a family or friend caregiver who also participated in the study. Among nonparticipating veterans, 207 (11.7%) were ineligible , 305 (17.2%) were not able to be reached by telephone after mailing the study invitations and 755 (42.5%) actively or passively declined. Figure Nearly all veterans were men (97.5%) and nearly all caregivers were women (94.9%). The majority of veterans (76.4%) completed at least high school, with 24.8% having a college degree; and 19.0% identified themselves as a member of a minority group. On average, veterans received their dementia diagnosis 2.03 years (SD = 1.97) prior to the study.Of the 508 consenting veterans, 333 (65.6%) passed the telephone screening using the adapted Blessed Test and completed the baseline research interview. The second research interview, conducted six months post-baseline, measured outcomes after a shorter period of study participation and was completed by 263 of the 333 veterans (79.0%). The third research interview, conducted 12 months post-baseline, measured outcomes after a longer period of study participation and was completed by 194 of the 333 veterans (58.3%). Attrition analysis comparing veterans who completed baseline interviews but who did not complete six- and/or twelve-month follow-ups indicated that those who stopped participating were significantly more likely to be members of a minority group, more impaired in personal care at baseline, and more isolated from others at baseline. These differences suggest study results may not fully represent the experiences of veterans who are more vulnerable or disadvantaged in terms of health, social support and socioeconomic status.Although this study demonstrates the feasibility of collecting data directly from persons with dementia, it is important to note that 175 (34.4%) veterans participating in PDC were too impaired to be interviewed at baseline and were not represented. Additionally, as described in Figure Table Table Table P = .08). Among those more cognitively impaired, six-month unmet need also was less for veterans receiving PDC than for those receiving UC . For six-month relationship strain, the significant conditional effect was for those with more personal-care dependencies ; veterans receiving PDC had lower levels of this adverse outcome than those receiving UC. For six-month depression, the significant conditional effect was for the more cognitively impaired, with veterans receiving PDC having fewer symptoms of depression than those receiving UC . The other significant six-month effect of PDC was a main effect (hypothesis 1) for embarrassment about memory problems. Veterans receiving PDC reported lower levels of six-month embarrassment than veterans receiving UC .Regression equations for six-month outcomes in Table P < .01).The second part of Table P = .02). The other was a conditional effect that was consistent with hypothesis 2 . Interpreted together, these two significant effects suggest that, among less cognitively impaired veterans , the PDC group had significantly higher scores than the UC group for twelve-month embarrassment. However, among veterans who were more cognitively impaired (above average levels in this sample), scores for twelve-month embarrassment were similar between the PDC and UC groups. For example, a separate regression analysis of only veterans with higher-than-average cognitive impairment showed no significant group difference between PDC and UC groups for twelve-month embarrassment.The other equation with significant differences was for twelve-month embarrassment about memory problems. There were two significant regression coefficients; one was a main effect that was opposite hypothesis 1  by: 1) using a larger, more diverse sample than prior studies; and 2) delivering the program in a manner similar to the way it would be implemented if it were part of usual care.PDC was associated with significantly less adverse outcomes, particularly for more impaired veterans. Improvements in all but one outcome were conditional effects (hypothesis 2) pertaining to veterans who were more cognitively impaired or had more difficulties with personal care. There was one outcome that significantly improved for all veterans receiving PDC (hypothesis 1). Additionally, most beneficial effects of PDC were evident from baseline to six months, with fewer outcomes improving from months six to twelve. However, beneficial effects at six months were maintained throughout the twelve-month study period for all but one outcome .Beneficial program effects after six months were evident in reduced relationship strain, depression and unmet need for more impaired veterans, and reduced embarrassment about memory problems for all veterans. Between months six and twelve, there were further reductions in unmet need for more impaired veterans. The one unexpected finding contrary to hypotheses was that embarrassment about memory problems increased for the PDC group between months six and twelve among less cognitively impaired veterans. Embarrassment did not differ between the groups from six to twelve months among more cognitively impaired veterans.Further research is needed to understand the unexpected findings for embarrassment at twelve months. One possibility is that embarrassment increased for those with mild cognitive impairment because PDC brought attention to symptoms previously normalized, denied or downplayed. PDC initially may have reduced feelings of embarrassment by improving understandings of the diagnosis and symptoms. But, in the longer-term, it may have increased the focus on dementia, which, in turn, may have negatively affected veterans’ feelings about themselves.This investigation tested the effectiveness of PDC by examining improvements in psychosocial outcomes representing veterans’ subjective perceptions of the consequences of their dementia. Outcomes based on information provided directly by persons with dementia contrast with the more common approach of relying exclusively on proxy reports to estimate their perceptions and experiences ,20,36. TBecause PDC targeted both persons with dementia and their caregivers, assessing outcomes from both was a logical extension of the program. Asking veterans, along with caregivers, for their opinions also honored the autonomy and dignity of the former and recognized them as active participants in their own care and care-related decisions. At the same time, only a subsample of all enrolled veterans with dementia was able to communicate their perceptions using a structured research data-collection tool, with more severely impaired veterans not represented by the findings.There were two notable limitations to the study. First, use of matched comparison sites, rather than within-site randomization, made it less certain that the intervention and comparison groups were equivalent at baseline, although observed baseline differences were statistically controlled. Matched sites were used because it facilitated sustainable implementation procedures at PDC sites for subject recruitment and integration of PDC with other services offered by partnering organizations. Second, this investigation did not test whether outcomes differed among veterans within the PDC group, depending on the amounts and types of assistance provided by PDC. Although PDC has a standardized protocol specifying a required minimum exposure to the intervention, the content and number of action steps beyond the minimum varied by individuals and were tailored to veterans’ preferences and needs.Positive results from this research suggest that PDC is a promising new model of care coordination. It is being considered for broader implementation by the VA, Alzheimer’s Association, other health systems and community agencies. PDC is consistent with the goals and priorities of a number of national policy and research initiatives charged with developing improved ways of linking healthcare and community services. For example, the National Plan to Address Alzheimer’s Disease  has the Large-scale implementation of PDC outside of a research study will require more information on the cost, financing, reimbursement, marketing strategies and ability to integrate it with existing services and information systems . Many ofBRI: Benjamin Rose Institute; CCIS: Care Coordination Information System; PDC: Partners in Dementia Care; UC: usual care; VA: Veterans Administration; VISN: Veterans Integrated Service Networks.Authors currently and formerly employed by the non-profit Benjamin Rose Institute on Aging  led the research team that developed the BRI Consultation Program, which provided a foundation for Partners in Dementia Care. BRI holds the copyright to and currently licenses and trains organizations to deliver BRI Care Consultation.There are no additional competing interests.DMB and MEK: conception and design; acquisition, analysis and interpretation of data; drafting and revising the manuscript; final approval of the version to be published; and agree to be accountable for all aspects of the work; KSJ, ALS, NLW, and CAM: conception and design, acquisition, analysis and interpretation of data; drafting and revising the manuscript; and final approval of the version to be published; ROM: conception and design; analysis and interpretation of data, drafting and revising the manuscript and final approval of the version to be published; KM: analysis and interpretation of data, drafting and revising the manuscript and final approval of the version to be published; RR, JAM, GLO, EA, RE, PP, and TAT: acquisition of data, drafting and revising the manuscript and final approval of the version to be published; WJL: acquisition, analysis and interpretation of data; drafting and revising the manuscript and final approval of the version to be published. All authors read and approved the final manuscript.


Renal allograft recipients are reported to have a higher incidence of malignancy than the general population. This single hospital-based study examined the incidence and clinicopathologic behavior of uterine cervical carcinoma in renal transplant recipients.Among 453 women receiving renal transplantation from January 1990 to December 2008, 5 patients were diagnosed with cervical carcinoma. Medical records of these 5 patients were retrospectively reviewed, and clinicopathologic data were collected and analyzed.The incidence of cervical carcinoma in renal transplant recipients was 58.1 out of 100,000 per year, which is 3.5 times higher than in the general Korean population. The mean interval between the time of renal transplantation and the time of cervical carcinoma diagnosis was 80.7 months. After a median follow-up of 96.2 months, there was no recurrence of the disease or death. In 4 patients who were positive from human papillomavirus in situ hybridization (HPV ISH), high or probably high risk HPV DNA was detected in all. Punctate staining of HPV ISH was detected in 3 out of 4 patients.Higher incidence of cervical carcinoma is expected in renal transplant recipients, so appropriate surveillance is needed to ensure early detection and treatment of cervical carcinoma. Recent advancements in immunosuppressive therapy have reduced cases of acute rejection and improved the long-term survival rate of grafts in organ transplant patients. Long-term use of immunosuppressants has likewise increased the incidence of infectious diseases, autoimmune diseases, and malignancy in organ transplant patients.Particularly, the incidence of malignancy was reported to be 2-31% in patients who had received renal transplants , and wasHPV is known as the major cause of cervical carcinoma and cervical intraepithelial neoplasia (CIN),11. MoreIn the early stage of the developmental course of CIN to invasive carcinoma, the phase at which the HPV DNA integrates into the host's DNA has been known to play an important role, although such developmental course has yet to be clearly identified. It was reported that the E2 gene loses its regulating function through this process, and that subsequent over-expression of the E6 and E7 genes causes loss of control over the cell cycle. Taking these into account, the accumulation of variations of multiple genes causes invasive cervical carcinoma .Recent studies reported the usefulness of In Situ Hybridization (ISH) for HPV, a method of testing whether or not the HPV DNA, which has an important role in early oncogenesis, is integrated into the host's DNA. It was also reported that when the HPV DNA integrated into the host's DNA, a punctate pattern occurred in which obvious round spots were observable in the nucleus. When unitegrated, the episomal HPV DNA existed, and a diffuse pattern was observable in which the nucleus was uniformly stained .As described, immunosuppresants used after a renal transplant to reduce the rejection reaction to graft may increase the chances of opportunistic infection with HPV, and the immunosuppressive condition itself may also increase the risk of cervical carcinoma through immunologic tolerance to oncocytes with accumulated genetic variations ,9.In South Korea, statistical data on a national level on the incidence of malignancy in patients who had received a renal transplant are not available, and no proper screening method for these patients has been established. As such, this study was performed to investigate the incidence of cervical carcinoma and other malignant carcinomas in female patients who had received a renal transplant at a single hospital, to analyze the clinicopathological features of patients with cervical cancer and to analyze whether or not the HPV DNA integrated into the host's DNA with the use of ISH.This study was conducted according to the Declaration of Helsinki, and approval was obtained from the Internal Review Board for the clinical trial. A total of 453 female patients who had received a renal transplant from January 1990 to December 2008 at Kangnam St. Mary's Hospital of Catholic University were included in the study. According to a retrospective review of the medical charts of these patients, 36 of them had malignancy. The incidence of carcinoma was analyzed according to the type of carcinoma. The clinical factors related to the renal transplant and the clinicopathological factors related to cervical carcinoma were analyzed based on the medical records of the five patients who were diagnosed with cervical carcinoma. In addition, for the HPV ISH and the analysis of the HPV type, a TMA core with a size of 5 mm, which is sufficient for observation, was prepared using the specimen from the uterine cervix that was removed through hysterectomy. Using the TMA of the uterine cervix, a 4 mm-thick slice was prepared and attached to the coated slide, and then used for the ISH .The Inform HPV III  probe that can identify 12 types of high-risk HPV was used. The HPV ISH was performed using the BenchMark Automated Slide Staining System  according to the manufacturer's instructions. The result of the nuclear staining was interpreted as positive, and the pattern of the nuclear staining was classified as diffuse, punctated, and mixed . "DiffusThe paraffin block, in which the patient's tissue was stored, was deparaffined, and the DNA was extracted . SubsequIn brief, this genotyping was for a microarray system that included probes of 40 types of HPV . The detA total of 453 female patients had received a renal transplant from January 1990 to December 2008 at Kangnam St. Mary's Hospital. Of these, 36 patients (7.9%) were found to have had malignancy during the follow-up. The most common type of malignancy was thyroid carcinoma followed by cervical carcinoma and bladder carcinoma. . Three of the five patients used Cyclosporin as an immunosuppressant, and two switched to Tacrolimus due to their acute rejection reaction. The grafts were well-maintained in four of the five patients, whereas in one patient, the graft lost its function, which led to hemodialysis.The mean period of the dialysis before the renal transplant was 13.4 months (1-51 months) Table . The meaAccording to the HPV ISH of tissues from five patients, four patients (80%) tested positive for HPV. Punctate staining pattern was observed in three (75%) of these four patients, and one manifested the diffuse staining pattern. According to the HPV typing, high-risk HPV (types 16 or 58) was detected in three patients, and probably high-risk HPV (type 66) was detected in one patient Table .Statistics from many developed countries show that the overall incidence of malignancy in patients who had received an organ transplant is about 2-4 times higher than in the general population, and that skin cancer and malignant lymphoma are the most common types of carcinoma in such patients. Moreover, the incidence of malignancy such as cervical carcinoma, malignant lymphoma, and Kaposi's sarcoma, which are attributable to a carcinogenic virus, are also higher than in the general population -18. HoweFactors that increased the incidence of malignancy after the organ transplant were reported to be related to the age at the time of the organ transplant, the sex, the race, the type of immunosuppressant, a history of renal disease that required a transplant, and the period during which dialysis was performed prior to the renal transplant. Such factors, however, varied by study ,7,23-25.Based on some studies, malignancy that developed in patients who received an organ transplant had a worse prognosis than that which developed in the general population ,26,27, wThe incidence of HPV infection after a renal transplant was reported to be 15-45%,30-33. I13 Eighty percent of the cervical lesions tested positive for HPV by the HPV ISH. Of these HPV-positive tissues, 75% had a punctate staining pattern, and 25%, a diffuse staining pattern  in three of four patients, and probably high-risk HPV (type 66) in one patient Table . This suIn this study, the incidence of cervical carcinoma in patients who had received a renal transplant at the authors' hospital increased by 3.5 times compared to the general population. Based on this risk, it is suggested that appropriate screening tools be required for women who had received a renal transplant. Analysis of the clinical factors related to cervical carcinoma that develops after a renal transplant and analysis of the prognosis of such carcinoma are required, since there were only a few cases in this study. It is considered that collection and management of statistical data on patients who had received a renal transplant should be performed at the national level for more efficient analysis.The authors declare that they have no competing interests.STP contributed mainly in the design, literature review and writing of this work, while corresponding author CWL provided the idea, planned, edited and approved the written work. Both SYH and JSP gave valuable advices and edited the discussion. MJS worked on the clinical presentation. All authors read and approved the manuscript.


Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types. Human papillomaviruses (HPVs) have been demonstrated to cause a diverse range of epithelial lesions. To date, 120 HPV’s have been identified and each of these types are associated with infection at particular anatomical sites Although the majority of HPV related non-malignant diseases are attributable to HPV 6 and 11 Anal cancer is uncommon in the general population, although incidence in the general Australian population is increasing Cervical cancer and its precursors namely high grade cervical intraepithelial neoplasia (CIN2/3) and adenocarcinoma insitu (AIS) are frequently associated with persistent HPV infection of types HPV16 and HPV18 Several key gene regions and their role in malignant transformation have been extensively studied in high-risk HPV types 16 and 18. For example, viral E6 and E7 oncogenes are key mediators in cell transformation through the disruption of tumour suppressor Rb/E2F and subsequent p53 pathways respectively, which are critical in regulating the initiation of DNA replication. Loss of function or disruption of these pathways due to integration of HPV DNA, leading to increased expression of E6 and E7 proteins, has been demonstrated to result in the development of cervical carcinoma Previous studies investigating genetic variation of these gene regions have used standard reference genomes for comparative purposes. These standard reference genomes for HPV6 are denoted prototypic HPV6b, and non-prototypic HPV6a and HPV6vc, and one standard reference genome for HPV11. A recent study based on phylogenetic analysis of complete genomes derived from published HPV6 and HPV11 variants has proposed a new standard nomenclature for HPV6 and HPV11 This study examines the genetic variability of the LCR, E6 and E7 gene regions in both HPV6 and HPV11 across different lesion types to determine whether different disease states resulting from single HPV6 or HPV11 infection cluster into distinct variant groups. In addition, current data on the genetic diversity of HPV6 and HPV11 variants present within the Australian population are very limited. Only one study has been conducted within the Australian population, sequencing only a portion of the LCR for 47 HPV6 or HPV11 RRP isolates All patients provided written informed consent at time of treatment whereby ethics approval was received from: the human research ethics committee at St Vincent’s Hospital, Sydney, Australia for anal cancer biopsies, the health ethics research committee at the Royal Perth Hospital, Perth, Australia for genital wart biopsies, and the human research ethics committee at the Royal Women’s Hospital, Melbourne, Australia for the collection of cervical cells. In addition, ethics approval was obtained from the ethics in human research committee of the Royal Children’s Hospital, Melbourne, Australia for juvenile RRP biopsies whereby written informed consent was provided by either the parent or guardian at time of treatment.Based on single HPV infection status a total of 71 HPV 6 and HPV11 isolates from four different disease states being: RRP, genital warts, anal cancer, and cervical neoplasia cells were included in this study. Formalin fixed, paraffin embedded (FFPE) biopsies collected at the Royal Children’s Hospital, Melbourne between 1964 and 1994 from 17 juvenile RRP patients described previously For FFPE archival tissue, 5 – 10 µm sections were deparaffinised with 800 µl histolene, then mixed with 400 µl absolute ethanol. The tissue was centrifuged at 14,000 × g for 2 minutes. The resultant pellet was washed with 1 ml of absolute ethanol, centrifuged for 2 minutes and the supernatant was discarded. The pellet was air dried and subsequently incubated for 4 hrs at 55°C with 160 µl tissue lysis buffer  and proteinase K at a final concentration of 1 mg/ml. DNA was then isolated from digested tissue using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics) on the automated MagNA Pure LC extraction system, according to the manufacture’s protocol, with the addition of 33.3 µg/ml of poly(A) RNA carrier to the lysis buffer which has been demonstrated to improve extraction efficiency. Nucleic acid was eluted into a final volume of 100 µl.Fresh 1–2 mm biopsies were homogenised using MagNA Lyser (Roche Diagnostic) with 250 µl of Tissue Lysis Buffer (Roche Diagnostics), by two repetitions of 30 seconds at a speed of 7000 rpm in the MagNA Lyser Instrument. Proteinase K was added to a final concentration of 1 mg/ml, and samples were incubated at 55°C until fully digested. DNA was then isolated from the digested tissue as described above. DNA was isolated from the PreservCyt samples of cervical cells using the MagNA Pure LC DNA Isolation Kit I on the automated MagNA Pure LC extraction system, according to a modified protocol HPV genotyping of all archival FFPE tissues were confirmed by using the INNO-LiPA HPV genotyping test  which has demonstrated increased sensitivity and accuracy for archival tissue DNA positive for HPV6 or HPV11 samples were subject to type and gene specific PCRs. All primers used for genetic variation analysis of HPV6 LCR, E6 and E7 , were de2, 1 U AmpliTaq® Gold DNA Polymerase and 1X reaction buffer , and 2.5 to 5 µl of DNA template. Following a denaturation step of 94°C for 9 min, PCR steps at 94°C for 30 sec, 55°C for 20 sec and 72°C for 30 sec for 45 cycles, followed by an extension step at 72°C for 10 min, on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). Amplicons were run on a 2% agarose gel for visualization, using 1000 base pair HyperLadder IV  for sizing.All PCR reactions were performed in a 50 µl reaction volume containing 0.5 µM of each primer, 2 mM of each deoxynucleoside triphosphate, 2.5 mM MgClAmplicons were sent to the Australian Genome Research Facility for purification and Sanger DNA sequencing using a AB3730xl capillary sequencer (Applied Biosystems). To minimise PCR and sequencing errors, sequencing was performed on both forward and reverse stands with predicted sequencing error of 0.01% based on sequencing of control DNA. For regions which did not meet our quality control criteria  confirmation of HPV sequence was carried out by repeating both the forward and reverse strands in duplicate. Sequences were assembled using SeqMan version 8  and aligned against reference genomes using SeaView version 4 The HPV6 nucleotide sequences for the LCR, E6, E7 gene regions are deposited in the GenBank database under the accession numbers: LCR sequences (KC300093-KC300140), E6 sequences , E7 sequences (KC300189-KC300237).The HPV11 nucleotide sequences for the LCR, E6, E7 gene regions are deposited in the GenBank database under the accession numbers: LCR sequences (KC329850-KC329871), E6 sequences (KC329872-KC329893), E7 sequences (KC329894-KC329915).Data in categories were evaluated using two-tailed Fisher’s exact test.This study used samples with either HPV6 or HPV11 infection. Of the entire sample set for each lesion type a total of 49 specimens were identified as HPV6 isolates, that is 8 from RRP lesions, 33 genital warts, 4 anal cancer biopsy specimens and 4 cervical cell samples. In addition, a total of 22 specimens were identified as HPV11 isolates, that is 9 RRP lesions, 10 genital warts, 2 anal cancer biopsy specimens and 1 cervical cell sample . For theAll 49 HPV6 isolates were successfully amplified and sequenced across the E6 (nt 102–554) and E7 (nt 530–826) ORFs. For HPV6 E6/E7, a total of 18 single nucleotide variations (13 in the E6 and 5 in the E7 ORF) from the lineage A reference genome were identified . No isolA total of 48 HPV6 isolates were successfully amplified and sequenced across the LCR . Single nucleotide variations from the lineage A reference genome were identified at 45 sites across the LCR. In addition, 5 insertions and 4 deletions were identified . Three vA total of 105 HPV6 LCR sequences were used to perform a comparative phylogenetic analysis, which included 48 Australian isolates from this study, 45 Slovenian All 22 HPV11 isolates were successfully amplified and sequenced across the E6 (nt 102–554) and E7 (nt 530–826) ORFs. Three genomic variants were identified across the E6/E7 ORFs for HPV11 . None ofThe LCR from 22 HPV 11 isolates was successfully amplified and sequenced . Comparative analysis with the HPV11 sublineage A1 reference sequence revealed 13 single nucleotide variations, 1 insertion and 3 deletions in the LCR . A totalComparative phylogenetic nucleotide sequence analysis was carried out on a total of 94 HPV11 LCR sequences. These publicly available sequences included 22 Australian isolates from this study, 63 Slovenian We present one of the largest studies of Australian isolates to date which investigates genetic variation for HPV6 or HPV11 in some of the key HPV gene regions thought to be associated with development of lesions and disease progression in epithelial cells. This study compares genetic variation within various lesion types sampled from the Australian population for the two most prevalent types of low risk HPV associated with these diseases. Analysis of genetic variance revealed slight association with HPV6 and anogenital lesion and HPV11 and RRP with genetic variants HPV 6 B1 lineage being more strongly associated with anogenital lesion.The majority of HPV11 isolates from this study clustered to the variant A2–1 for E6 and E7 . This vaThis study identified a series of variations in the E6 and E7 ORFs of the HPV6 genome within the Australian lesions sampled. A total of three amino acid alterations in each of the E6 and E7 ORFs of different HPV6 isolates were found, some of which were unique to a single isolate, while others were found in several or multiple cases. Of these, several unique amino acid alterations were identified: E113D in E6 and E73K in E7. Conversely, amino acid alterations E19K and H50Q in the E6 ORF and F52Y and N88D in the E7 ORF, present in numerous isolates, have been described previously When the prevalence of HPV-type in genital warts and RRP (lesions for which n>10) was considered, a type specific distribution was indicated for genital warts but not RRP. The prevalence of HPV 6 was found to be higher for genital warts (77% HPV6 vs 23% HPV11) in this sample set, as described previously by Variant analysis of the LCR within Australian isolates was completed for both HPV6 and HPV11. In recent years, a growing number of studies have described HPV6 LCR variations in isolates from the nucleotide positions of the lineage A reference genome Genomic regions such as the LCR have been used by previous studies to identify genetic variants as it provides enough genetic variation to differentiate between variants in both HPV6 and HPV11. For this reason, the LCR was selected for comparative phylogenetic sequence analysis between this and previous studies to determine whether genetic lineages previously described This current study also provides unique pre-vaccination data on the genetic variation present in RRP, anal cancer, cervical cells samples for HPV6 and HPV11 within the Australian population. For genital warts, based on age  and sex , at least 88% of these samples would have come from women and men ineligible for publically funded prophylactic HPV vaccination. The remaining 12% of genital warts were derived from women <26 years, whose vaccination status was unknown. The high proportion of genital wart isolates positive for HPV6 or HPV11 suggests that lesions developed either in unvaccinated patients, or in women who were vaccinated with prevalent infection. Australia was the first country to introduce a government-funded, population-based mass vaccination program in 2007 of the female population (12 to 26 years), using the quadrivalent vaccine Garadasil®  targeting HPV types 16, 18, 6 and 11. It has already seen a reduction in the number of cases of genital warts due to a high coverage of vaccine In summary, we found slight association between HPV6 with anogenital lesions with sublineage B1 being predominate. This is consistent with the findings of other studies and suggests that viral and/or host epigenetic and other host factors (such as those associated with immune response) may play significant roles in disease susceptibility and progression. In addition, larger studies investigating whole genome sequences may shed further light on possible associations with pathogenesis. However, this study makes a significant contribution toward the slowly expanding knowledge about HPV6, and in particular HPV11 genomic diversity and pre-vaccination circulating HPV6/11 variants within the Australian population.Table S1Summary of HPV6 and HPV11 lesion types.(DOCX)Click here for additional data file.Table S2Summary of genetic variation identified across all lesion types for HPV6 and HPV11.(DOCX)Click here for additional data file.Table S3Primers used for amplifying and sequencing the LCR, E6 and E7 ORFs.(DOCX)Click here for additional data file.Table S4HPV6 nucleotide and amino acid sequence variation in the ORF of E6 and E7 from 49 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S5HPV6 nucleotide sequence variation in the LCR from 48 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S6HPV11 nucleotide and amino acid sequence variation in the ORF of E6 and E7 from 22 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.Table S7HPV11 nucleotide sequence variation in the LCR from 22 clinical isolates representing four different lesion types.(DOCX)Click here for additional data file.