To evaluate the performance of existing versus alternative cervical cancer screening protocols in Bhutan, cervical exfoliated cells were collected for cytology and high-risk human papillomavirus (HR-HPV) testing among 1,048 women aged 30-69 years. Conventional smears were prepared and read locally. HR-HPV was tested by GP5+/6+ polymerase chain reaction, followed by genotyping and human DNA methylation analysis among HR-HPV-positives, in Europe. Test positivity was 7.5% for ASCUS or worse (ASCUS+) cytology and 14.0% for HR-HPV. All women with ASCUS+ and/or HR-HPV positivity (n=192) were recalled for colposcopy, among whom a total of 29 cases of histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) were identified. An additional 7 CIN2+ cases were imputed among women without colposcopy. Corrected sensitivities for CIN2+ and CIN3+ were 61% and 74% for ASCUS+, 86% and 96% for HR-HPV, and 47% and 70% for ASCUS+ triage of HR-HPV. Specificity varied from 88% for HR-HPV up to 98% for ASCUS+ triage of HR-HPV, similarly for CIN2+ and CIN3+. Among HR-HPV-positive women with biopsies, methylation analysis offered similar discrimination of CIN2/3 and cervical cancer as ASCUS+, and better than HPV16/18 genotyping alone, but sample sizes were limited. In conclusion, the performance of cytology in Bhutan is in the mid-range of that reported in other screening settings. HR-HPV testing has the potential to improve detection of CIN2+, albeit with a higher referral rate for colposcopy. Cytological triage of HR-HPV-positives (performed in the absence of knowledge of HR-HPV status) reduced referral but missed more than one third of CIN2+. Cervical cancer represents the most common cancer among females in Bhutan , where aIndeed, during the last decade, cervical cancer screening has shifted towards the molecular detection of HR-HPV, the main cause of cervical cancer, allowing for increased automation of diagnostic procedures. Randomized trials in high-income countries among regularly screened women show that HR-HPV testing provides 60–70% greater protection against invasive cervical carcinomas over cytology, and allows extension of screening intervals  and prosWithin the framework of a collaboration between the Ministry of Health (MoH) of Bhutan and International Agency for Research on Cancer (IARC) , 24, we Of 1,048 women screened, mean age was 40 years [interquartile range=34-46 years], 66% had a previous Pap test, 86% reported one lifetime sexual partner, and 91% were currently married (data not shown [Table HR-HPV testing showed higher sensitivity (86% and 96% for CIN2+ and CIN3+ respectively) than cytology ASCUS+ (61% and 74%), equating to sensitivity ratios of 1.41 and 1.29, respectively. Negative predictive value was also higher for HR-HPV  than cytology ASCUS+ (98.6% and 99.4%). Specificity, however, was lower for HR-HPV (88% for both CIN2+ and CIN3+) than for cytology ASCUS+ (94% for both), as was positive predictive value. Of note, increasing the cytology threshold to LSIL+ was associated with sensitivity of only 42% and 48% for CIN2+ and CIN3+, respectively (data not shown).With respect to triage of primary cytology, triage of ASCUS by HR-HPV was associated with an improvement in specificity to 97% for CIN2+ and 96% for CIN3+, and improved positive predictive value (PPV), but with a decrease of sensitivity to 56% and 70%. Triage of primary HR-HPV by ASCUS+ was also associated with an improvement in specificity to 98% for both CIN2+ and CIN3+, and higher PPV, but also with a decrease of sensitivity to 47% for CIN2+ and 70% for CIN3+.To evaluate the potential utility of molecular markers for the triage of HR-HPV-positive women, we compared test positivity by histological diagnosis of HPV16/18 genotyping  and CADM1/MAL/miR124-2 methylation, with that of ASCUS+ cytology, among a subset of 101 HR-HPV positive women   and CIN3+ 74%) fell in the mid-range of estimates from similar studies that included colposcopy of HR-HPV-positive women, irrespective of whether they be in LMICs (26-87% for both CIN2+ and CIN3+) 4% fell i or HICs Use of HR-HPV as a primary screening test was associated with a higher detection of CIN2+ and CIN3+ than cytology (sensitivity ratio = 1.41 and 1.29 respectively), consistent with findings from previous studies , 26–28, Performance of HR-HPV testing for detection of CIN2+ and CIN3+ has been shown to be heterogeneous across studies in LMICs . In the HR-HPV testing offered a higher cross-sectional negative predictive value than ASCUS+ cytology in Bhutan. Indeed, a negative HR-HPV test has also been shown to offer greater reassurance against future CIN3+ –33 and cNevertheless, HR-HPV also resulted in a higher burden of referral to colposcopy and reduced specificity compared to cytology screening, consistent with results from previous cross-sectional studies . Whilst In Bhutan, primary HR-HPV testing followed by ASCUS+ triage of HR-HPV-positives was associated with high specificity and PPV, requiring referral of only 3.2% of screened women , offering an option for triage in Bhutan, where cytology is already established. Although the sensitivity of cytology triage might have been higher if cytotechnicians had known that they were triaging HR-HPV-positive women, cytology triage in Bhutan was nevertheless associated with a substantial loss of cross-sectional sensitivity, missing more than one third of CIN2+. So in a subset of HR-HPV-positive women, we compared the discriminating power of two molecular-based triage options, namely HPV16/18 genotyping and host DNA methylation, to that of cytology. Although sample sizes were limited, CADM1/MAL/miR124-2 methylation was strongly related to the severity of cervical disease and was always positive in cervical cancer, as shown previously . Indeed,Prior to becoming a recommended primary screening test, HR-HPV testing was recommended to triage ASCUS in primary cytology programs, being shown to have higher sensitivity and similar specificity than repeat cytology in this group of women . HoweverThe strengths of this study were the high proportion of screen-positive women who received colposcopy and biopsy, and the fact that histology was imputed among the few who did not . Furthermore, the population-based sample is expected to be broadly representative of women aged ≥30 years living in Bhutan, and the risk of HR-HPV-positivity and CIN3+ in this population was high, as reported previously . The majIn conclusion, despite the relatively good performance of the Bhutanese cytology program, a shift to primary HR-HPV screening has the potential to further improve detection of cervical pre-cancer, albeit with a higher referral rate for colposcopy and loss of specificity. Cytological triage of HR-HPV-positive women diminishes immediate referral to colposcopy but would have missed more than one third of CIN2+. Whilst methylation analysis was shown to be a promising and objective alternative to ASCUS+ cytology in the triage of HPV-positive women samples, our results remain preliminary.The study had the approval of both the Research Ethical Board of the Bhutan Ministry of Health and the IARC Ethics Committee.In 2012, during a population-based survey of HPV prevalence, 2,505 women aged 18-69 years were invited and underwent a gynecological examination in Jigme Dorji Wangchuck National Referral Hospital (JDWNRH) and Lungthenphu Hospital, Thimphu, Bhutan. Study procedures have been described in detail elsewhere . ExfoliaThe present study is restricted to the subset of these women among whom HR-HPV screening is recommended, namely 1,048 women aged ≥30 years. As Pap smear, and later HR-HPV, results became available, first all women with abnormal cytology (N=79), and subsequently, also HR-HPV-positive women with normal cytology (N=113), were referred for colposcopy, of whom 83% finally attended (159 of 192)  of 159 women who underwent colposcopy. Histology was performed at JDWNRH, Thimphu, and 29 cases were diagnosed as CIN2+  . Conventional screening indices of accuracy, including sensitivity, specificity, PPV, negative predictive value and their 95% confidence intervals were calculated for both CIN2+ and CIN3+. Firstly, crude indices using only CIN2+ and CIN3+ histologically confirmed among the 159 women attending colposcopy were calculated, assuming that all women without a biopsy were histologically negative. Secondly, corrected indices were calculated after imputation of missing data for the 33 HR-HPV-positive and/or cytologically abnormal women who did not attend colposcopy , 40. In


The prevalence of cervical Human Papillomavirus (HPV) infection in cervical cancer varies greatly worldwide, and HPV prevalence and genotypes in China are limited. The objective is to analyze the prevalence of HPV subtypes in cervical cancer patients in west China as well as the relationships between different histological types of cervical cancer and HPV subtypes.P < 0.05.2309 cases were included. 90.86% were infected with HPV, and the remaining 9.13% were negative. The most common subtypes were HPV16, HPV18, HPV58, HPV53, and HPV33. HPV16 and HPV18 appeared to be the most prevalent HPV subtypes across all age groups, while the prevalence of the other subtypes varied between age groups. A logistic regression analysis revealed that the occurrence of cervical squamous cell carcinoma and adenocarcinoma was most closely correlated to a persistent infection with HPV16 or HPV18, with The prevalence of 27 HPV subtypes in 2309 cervical cancer patients who received treatment at our hospital between June 2011 and January 2016 was analyzed based on the results of HPV testing using Liquid suspension chip technology (Luminex 200).HPV16 and HPV18 were the most prevalent HPV subtypes among the cervical cancer patients, followed by HPV58, HPV53, and HPV33. 9.13% of the cases appeared to not be associated with HPV. This suggests that HPV testing without the use of cytology may overlook some special types of cervical cancer that account for approximately 10% of all cervical cancer cases. In 2015, China reported 98,900 new cases of cervical cancer and 30,500 cervical cancer deaths . CurrentStudies have found that the type-specific prevalence of HPV varies obviously by age and region. For example, in the southern part of China, HPV infections primarily occur among women under 30 years old. The HPV prevalence was estimated to be 26%, with 21.12% infected with high-risk HPV, and HPV52, HPV16, and HPV58 were the most prevalent subtypes .The prevalence and HPV type also vary according to the grades of cervical cancer and cervical precancerous lesions. A study found that the prevalence of high-risk HPV was responsible for up to 89.8% of the invasive cervical cancer patients in Ghana. HPV18 appeared to be the most prevalent HPV subtype, and HPV59, HPV45, and HPV16 were the second most prevalent ones [An analysis of the HPV subtypes of different ethnic groups in a country or a region is highly significant to prognosis evaluation, creating reexamination plans and clinical treatment plans, and developing preventive and therapeutic HPV vaccines. However, the HPV vaccine has not been widely administered in China. The HPV vaccine was not marketed in China until after 2016, and as a result, it has not been widely used. While HVP infections in healthy women and patients with cervical precancerous lesions have been reported in different provinces of China, there have been relatively few research reports on HPV infections among cervical cancer patients in China. Therefore, our research group analyzed HPV infections among cervical cancer patients that received treatment between June 2011 and January 2016 at the largest gynecological cancer prevention and treatment center in West China. Our aim was to provide a basis for cervical cancer prevention and treatment.Figure Table The age-specific prevalence of HPV among the cases was as follows: under 30: 91.40%; 30 to 40: 88.63%; 40 to 50: 92.83%; 50 to 65: 89%; and over 65: 94%. The three most prevalent subtypes were HPV16, HPV18, and HPV58, while the prevalence of the other subtypes differed between age groups. See Table As can be seen from Table P < 0.001. Adenosquamous carcinoma of the cervix may also be correlated with persistent HPV45 infection, with P = 0.007. Figure The logistic regression analysis reveals that squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma of the cervix were most closely correlated to persistent infection with HPV16 or HPV18, with A systematic survey and analysis of HPV infections among women in different regions can help effectively identify populations that are at a high risk of cervical cancer. Such information will ultimately provide an important basis for the prevention and treatment of cervical cancer as well as the development of HPV vaccines. HPV infection in cervical patients is different in different countries and in different regions , 13. An In contrast, we obtained different results. This study analyzed HPV infections among 2309 cervical cancer patients.90.87% of the cases in this study were currently infected with HPV, with 90.82% infected with high-risk HPV types. The most common subtypes among these cases were HPV16, HPV18, HPV58, HPV53, and HPV33.et al. [High-risk HPVs are known to be the primary cause of cervical cancer. HPV prevalence varies with the grades of cervical cancer and cervical precancerous lesions as well as differs between regions and ages. Xiao Meizhu et al.  analyzedet al.  estimateP < 0.001. Adenosquamous carcinoma of the cervix may also be correlated with a persistent HPV45 infection, with P = 0.007. The results suggest that some special histological types of cervical cancer may be associated with infection by certain HPV subtypes. However, due to the small sample size, further research based on a large sample is needed in order to validate these findings. These findings may provide a new approach for the prevention, screening, and treatment of cervical cancer.A few studies have suggested that persistent infections with different HPV subtypes may be responsible for different histological types of cervical cancer, and the corresponding prognosis tends to vary. In this study, a logistic regression analysis was conducted to determine whether there is a link between the occurrence of different histological cervical cancer types and specific HPV subtypes. The results show that squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma of the cervix areclosely correlated with a persistent infection with HPV16 or HPV18, with HPV has been proven to be the primary cause of cervical lesions, however, it is not the only cause. The occurrence and development of cervical lesions result from the combined effect of multiple factors. Furthermore, 9.13% of the cervical cancer cases included in this study tested negative for HPV, with squamous cell carcinomas, adenocarcinomas, and adenosquamous carcinomas accounting for the majority of these cases. Research has shown that an estimated 80% of women get infected with HPV at some point in their lifetime; however, most of these cases are transient infection. Most HPV infections only last for 8 to 24 months before the immune system clears them. Nonetheless, in very few cases, the infections tend to persist and progress into lesions . These cet al. found that the percentage of HPV-negative cases in cervical adenocarcinoma patients was 15.6% as compared to 2.9% of squamous cell carcinomas cases [et al., HPV-DNA was detected in 91% of mucinous adenocarcinomas  and in all adenosquamous carcinoma cases. In contrast, no HPV-DNA was detected in the non-mucinous adenocarcinoma cases . The above data demonstrate that some common types and rare subtypes of cervical adenocarcinomas are uncorrelated with HPV infection [Similar to the previous study, HPV-negative cases made up a relatively high percentage of the women with special histological types of cervical cancer, and the percentage was higher among the cervical adenocarcinoma cases than among the squamous cell carcinoma cases. In an observational study of 136 cervical cancer patients, Rodriquez-Carunchio L. as cases . The ceras cases . In a stnfection . PerhapsThe study has limitations. Although the sample size is large, the study was only a single-center retrospective study. Some special types of cervical cancer are relatively small, which can affect the accuracy of the results of the logistic regression analysis. A logistic regression analysis was used to determine whether there is a link between the occurrence of different histological cervical cancer types and specific HPV subtypes, and it was found that there is an association between cervical cancer and persistent infection with HPV16 or HPV18. Nonetheless, it is very difficult to define the case with persistent infection. A large-scale multicenter prospective study is to be needed to confirm the results.The results of this study show that HPV16 and HPV18 were the most prevalent HPV subtypes in cervical cancer patients across all age groups in West China, followed by HPV58, HPV53, and HPV33. The prevalence of the HPV subtypes, excluding HPV16 and HPV18, varied significantly between the age groups. Additionally, 9.13% of the cases included in this study were not associated with HPV. Most of these cases were diagnosed with special types of cervical cancer. This suggests that HPV testing without the use of cytology may overlook some special types of cervical cancer that account for approximately 10% of all cervical cancer cases. Therefore, clinical attention should be paid to this finding.This is a retrospective study. The West China Second University Hospital of Sichuan University is known as the largest gynecological cancer prevention and treatment center in West China. This study focused on women with pathologically confirmed invasive cervical cancer who received treatment between June 2011 and January 2016 at this hospital. Two pathologists reexamined pathological sections of paraffin-embedded tissues from the cervical cancers. If the two pathologists had inconsistent views, then a third pathologist was consulted to resolve the issue. Patients with uterine sarcoma, metastatic cervical cancer, or other malignant tumors associated with the cervix were excluded from this study. Because the study is a retrospective study, there is no ethical committee approval. This is in accordance with institutional or national policies concerning research approvals.Five 10 μm thick sections were cut from each paraffin-embedded tissue specimen, and these sections were then placed into 1.5 mL centrifuge tubes. Next, 1 mL of xylene was added to each centrifuge tube and blended with the tissues by oscillation at room temperature. After centrifugation, the supernatant was removed, and 1 mL of absolute ethanol was added to the tubes to eliminate the xylene. Subsequently, a kit (Qiagen RNA/DNA Mini kit) for purifying DNA from tissue produced by Qiagen was used to extract DNA from the specimens according to the kit handbook. Afterwards, the extracted DNA was dissolved in 50 μL of elution buffer and stored at −20°C for use.The DNA purified from the tissues was placed in an HPV genotyping kit (Human papillomavirus nucleic acid typing detection kit (flow fluorescence hybridization) produced by Shanghai Tellgen Corporation). There were 27 HPV subtypes of interest, including 17 high-risk subtypes  and 10 low-risk subtypes . The hybridization products were tested using a Luminex 200, which is a multifunctional suspension array system.t-test. The enumeration data, expressed in %, were tested using the χ2 test. A logistic regression analysis was performed to examine the relationships between the different histological types of cervical cancer and the HPV subtypes. P-values were calculated and used to determine whether a certain HPV subtype had an influence on a particular histological type of cervical cancer. In this study, α = 0.05 was taken as the standard level of significance; thus, the difference between data is considered statistically significant when P < 0.05.The data from the test was analyzed using the statistics package SPSS 22.0. The measurement data, expressed as x ± s, were tested through a


Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is frequently impervious to curative treatment efforts. Similar to other cancers associated with prolonged exposure to carcinogens, HNSCCs often have a high burden of mutations, contributing to substantial inter- and intra-tumor heterogeneity. The heterogeneity of this malignancy is further increased by the rising rate of human papillomavirus (HPV)-associated (HPV+) HNSCC, which defines an etiological subtype significantly different from the more common tobacco and alcohol associated HPV-negative (HPV-) HNSCC. Since 2011, application of large scale genome sequencing projects by The Cancer Genome Atlas (TCGA) network and other groups have established extensive datasets to characterize HPV- and HPV+ HNSCC, providing a foundation for advanced molecular diagnoses, identification of potential biomarkers, and therapeutic insights. Some genomic lesions are now appreciated as widely dispersed. For example, HPV- HNSCC characteristically inactivates the cell cycle suppressors TP53 (p53) and CDKN2A (p16), and often amplifies CCND1 (cyclin D), which phosphorylates RB1 to promote cell cycle progression from G1 to S. By contrast, HPV+ HNSCC expresses viral oncogenes E6 and E7, which inhibit TP53 and RB1, and activates the cell cycle regulator E2F1. Frequent activating mutations in PIK3CA and inactivating mutations in NOTCH1 are seen in both subtypes of HNSCC, emphasizing the importance of these pathways. Studies of large patient cohorts have also begun to identify less common genetic alterations, predominantly found in HPV- tumors, which suggest new mechanisms relevant to disease pathogenesis. Targets of these alterations including AJUBA and FAT1, both involved in the regulation of NOTCH/CTNNB1 signaling. Genes involved in oxidative stress, particularly CUL3, KEAP1 and NFE2L2, strongly associated with smoking, have also been identified, and are less well understood mechanistically. Application of sophisticated data-mining approaches, integrating genomic information with profiles of tumor methylation and gene expression, have helped to further yield insights, and in some cases suggest additional approaches to stratify patients for clinical treatment. We here discuss some recent insights built on TCGA and other genomic foundations. This o= 0.037; . Further= 0.037; , 22, 56)= 0.037; , 56, butin vivo results in which dual kinase inhibition of EGFR and HER2 enhances response to cetuximab [In the TCGA cohort, EGFR was amplified in 12 % of HPV- cases , the sametuximab . In addietuximab , 95, 96.etuximab –99).For two additional genes, AJUBA and FAT1, almost all detected alterations were found in HPV- tumors Fig.  and d. BThe TCGA analysis also identified a set of less well studied alterations associated with oxidative stress, specifically involving CUL3, KEAP1 and NFE2L2 . KEAP1 aLastly, the TCGA network detected co-amplification of chromosome regions 11q13 and 11q22. Found within region 11q22, an amplicon previously described in lung, esophageal and cervical cancer –111, areAt the molecular level, HPV+ carcinomas significantly differ from HPV- cases, highlighted in great detail by the TCGA network and others , 27, 92.HPV+ HNSCC is defined by infection of tumor cells with HPV. HPV DNA can exist either integrated into the human genome or in a nonintegrated form , 119. UpHPV oncoproteins include E6 and E7 Fig.  and b.Also associated with HPV+ disease is the RTK FGFR3, which is activated in 11 % of cases through either mutation or a gene fusion event, and the aforementioned TNF receptor associated factor TRAF3, inactivated in 22 % of cases (versus 1 % in HPV- disease). The FGFR3 fusion partner is TACC3, a protein critical for nucleation of microtubules at the centrosome , aberranHPV- and HPV+ HNSCC share one particularly challenging feature: tumor heterogeneity . This asProvocatively, in a ten-variable multivariate analysis of TCGA HNSCC data incorporating MATH scores, no prognostic significance of HPV status, N classification or TP53 mutational status was determined . While tAdditional innovative and detailed analysis by McGranahan et al. utilized TCGA datasets for nine tumor types, including HNSCC, to highlight important aspects of cancer evolution and clonality . In ordeAs an emerging concept for this disease, consideration of germline variants may be relevant to fully appreciate tumor heterogeneity. Recent analysis of TCGA data suggested that 15 %  of HNSCThe majority of patients with HNSCC are treated with surgery and/or radiation and in some cases adjuvant chemotherapy , 11, 27.Figure The perhaps most exciting recent development in the treatment of cancer is immunotherapy . ImmunotFuture endeavors are likely to include more extensive elucidation of the role of DNA methylation in HNSCC, in part to substantiate publications based on the TCGA dataset. DNA methylation is important in the regulation of gene expression, and aberrant methylation has been described for essentially all cancer types and as a critical aspect of cancer genomics , 182. PrInterestingly, prominent differential methylation of three members of the zinc finger gene family, ZNF14, ZNF160 and ZNF420, has been identified as suitable to detect HNSCC with 100 % specificity in primary tissue and saliva samples; subsequently, the three ZNF methylation signature was validated using the 273 TCGA cohort . For mosDetailed profiling of HNSCC by the TCGA network and other research groups has greatly enhanced our understanding of this malignancy. First and foremost, the composite results have highlighted the tremendous inter- and intra-tumor heterogeneity, complicated by the increasing incidence of HPV-associated tumors. Efforts have started to focus on classifying tumors based on molecular profiles –193; howThe greatest potential therapeutic advantage to come from the detailed parsing of HNSCC heterogeneity is advanced and eventually precise treatment with immunotherapy. For example, consistent identification of tumors with highly immunogenic alterations would significantly help guide therapeutic decision-making . Immunot


Estimate the prevalence of cervical HPV infection among women assisted by the Family Health Strategy and identify the factors related to the infection. A cross-sectional study involving 2,076 women aged 20–59 years old residing in Juiz de Fora, State of Minas Gerais, who were asked to participate in an organized screening carried out in units were the Family Health Strategy had been implemented. Participants answered the standardized questionnaire and underwent a conventional cervical cytology test and HPV test for high oncogenic risk. Estimates of HPV infection prevalence were calculated according to selected characteristics referenced in the literature and related to socioeconomic status, reproductive health and lifestyle. The overall prevalence of HPV infection was 12.6% (95%CI 11.16–14.05). The prevalence for the pooled primer contained 12 oncogenic HPV types  was 8.6% (95%CI 7.3–9.77). In the multivariate analysis, it was observed that the following variables were significantly associated with a higher prevalence of HPV infection: marital status , alcohol consumption  and number of lifetime sexual partners . The prevalence of HPV infection in the study population ranges from average to particularly high among young women. The prevalence of HPV16 and HPV18 infection is similar to the worldwide prevalence. Homogeneous distribution among the pooled primer types would precede the isolated infection by HPV18 in magnitude, which may be a difference greater than the one observed. The identification of high-risk oncogenic HPV prevalence may help identify women at higher risk of developing preneoplastic lesions. Human papillomavirus (HPV) infection is one of the most common genital infections in the world and is a necessary cause for cervical cancerThe global strategy is the screening of pre-invasive lesions with a cervical cytological examination by smear exam. In developing countries, the impact of introducing and scaling up the screening as a health policy was lower than in developed countries, due to poor organization, poor coverage, and lack of quality assurance,,These failures motivated the search for alternative techniques (associated or substitute) that would contribute to the reduction of losses and more accurate identification of women with HPV types with higher oncogenic risk. Molecular biology techniques have contributed to understanding genital HPV infection in different settings, and are used in scientific research since the beginning of the 1980s, but only recently have been incorporated into the health servicesIn Brazil, the identification of HPV-infected women is based on heterogeneous studies and, therefore, is of difficult comparability and limited reproducibility since estimates made from studies that consider referenced women may result in overestimated measuresThus, the objective of the present study was to estimate the prevalence and risk factors for HPV infection among women residing in the coverage area of the Family Health Strategy (FHS).A cross-sectional study was carried out with a population of residents of an area attached to the two units of the Family Health Strategy located on the outskirts of the city of Juiz de Fora, state of Minas Gerais. All women residing in the FHS coverage area between 20 and 59 years old, asymptomatic, were considered eligible. Pregnant women, those who were immunocompromised or had been previously submitted to uterus excision procedures  were excluded. Approximately 3,500 women were recruited in their homes by community health agents to undergo the preventive examination in the proposed area as a programmatic action developed by the family health units, with an emphasis on the organized screening of absences through the FHS register.Data were collected from September 2010 to August 2012. Study participants were interviewed by trained health professionals, using a standardized questionnaire adapted from the National Health Survey, applied in the Federal District in 2010 and improved from 2013 onwards through the partnership between the Oswaldo Cruz Foundation and the Brazilian Institute of Geography and Statistic. The final instrument encompassed question groups distributed in nine modules: identification, sociodemographic characteristics, social support, self-assessment of health status, lifestyle, morbidity, women’s health, sexual behavior, and sexually transmitted infections. These data were measured. The instrument, its detailing, and the definition of each category used are available for consultation. Anthropometric data and morbidity data (blood pressure) were measured, and the results of exams refer to reports presented by participantsThe women were then submitted to conventional cervical cytology examination at the primary health units, while simultaneously being tested for HPV infection, and the samples were collected by the nurses and trained physicians working in the units.® automated platform , which uses primers to amplify the DNA of 14 high-risk HPV types: HPV16, HPV18 and a pooled primer with 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, e 68 HR-HPV. Negative results were those specimens in which there was no amplification of viral DNA and positive ones were those in which there was an amplification of the viral DNA corresponding to the test primers. The procedures for analyzing the sample quality occurred according to pre-established protocols adopted by the executing Laboratory.HPV testing was performed at the Laboratory of Virology of the Institute of Tropical Medicine of the Universidade de São Paulo. The uterine cervix samples were preserved in a PreservCyt solution and analyzed by the Polymerase Chain Reaction (PCR) method using the HPV-HR test + GT 16/18 test on the cobas 4800 HPV TestIn the study period, 2,076 eligible women, approximately 60% of the population of women residing in the areas assigned to the FHS, answered to the recruitment, attended the FHS units and were included in the study. Due to reading problems, it was not possible to obtain HPV test results for 54 women (2.6%), because 40 samples had invalid results and 14 had inconclusive results. Thus, the study population had 2,022 women. In one of the health units, the number of women studied was almost twice the one obtained in the other unit, maintaining the proportionality of the size of the populations assigned to these two units.We performed a univariate analysis of the data, and the absolute and relative frequencies were calculated for the nominal categorical variables, according to the family health unit. Then, the bivariate analysis was performed parallel to the comparison of the basic characteristics between the groups, using the chi-square test for proportions, which were considered significant if p ≤ 0.20. We measured the HPV infection prevalence, general and stratified according to selected variables with respective 95% confidence intervals (95%CI) and p value.per capita family income, health evaluation, lifestyle , reproductive history  and sexual behavior . The variable categories were defined according to classic literature references, selected in the development of the questionnaire. The variable per capita income was categorized based on the minimum wage values at the time of the study . This way, low income corresponded to values below 50% of the minimum wage, average income corresponded to values between 50% and 100% minimum wage and high income was more than one minimum wage. The total income of the family, including all residents of the household, was considered.To evaluate the factors related to the infection, identified as the positive result for the HPV test, variables were selected according to epidemiological criteria and biological plausibility. Included in the study were characteristics related to: age, schooling, skin color, Gross and adjusted prevalence ratios were calculated using Poisson regression with robust variance and respective 95% confidence intervals. The variables that in the bivariate analysis had p ≤ 0.20 were selected for the final model, which, in addition, included the age group and health unit of the family in which the woman resided and was assisted. All analyses were performed using the statistical software Stata, version 12 .The project was approved by the Ethics Committee of the Institute of Social Medicine of the Universidade do Estado do Rio de Janeiro (Position 0026.1.259.180-09), following all the proposed recommendations. All the women included in the study did so by reading and signing the informed consent form, ensuring the confidentiality of the data and the privacy of the women participating in all the stages of the study.The sociodemographic characteristics, as well as those related to the self-assessment of health, lifestyle, reproductive health and sexual behavior of the women studied, are described in The overall prevalence of infection by at least one high-risk type of HPV among women of both FHS units was 12.6% (95%CI 11.16–14.05). The prevalence of infection by at least one type of HPV-AR, except HPV16 and HPV18 (pooled primer with 12 types) was 8.6% (95%CI 7.30–9.77), while the prevalence of HPV infection by HPV 16 and 18 was 1.8% (95%CI 1.20–2.35) and 0.5% (95%CI 0.22–0.86), respectively. The prevalence of coinfection by HPV16 or HPV18 and at least one of the 12 types of pooled primer  was equal to 1.7% (95%CI 1.16–2.29) .The prevalence of HPV infection according to selected independent variables is shown in In the multivariate analysis, the following variables remained significantly related to HPV infection: single , alcohol consumption at any frequency  and had three or more sexual partners throughout life  .,The prevalence of cervical HPV infection in women assisted by the Family Health Strategy was 12.6%. This measure is close to the prevalence (12.8%) observed by Girianelli et al. (2010) in a study with the household recruitment of low-income women living in the municipalities of Baixada Fluminense, Rio de Janeiro, which used the hybrid capture technique to detect HPV. Although the PCR technique will present higher prevalence estimates than those obtained using the hybrid capture technique, the measures obtained in the study by Girianelli et al. (2010) resulted from the inclusion of women who had not received preventive examination for more than three years, while the present study considered data from all the women in the target population to be screenedThe target population women’s attendance to the cervical cancer screening in FHS units participating in the present study was lower than expected (60%). It was expected that, as an action started with household recruitment and appointment scheduling at the health unit, women’s demand would be higher. Reduced adherence to screening reflects the behavior observed in other Brazilian studies and may have influenced the estimates observed for the group. This shows that, even in organized screening, there seem to be factors that influence the response to recruitment, resulting in partial adherenceRegarding losses in HPV testing, despite periodic training and supervision, there were failures in capturing HPV typing and detection and cytology results, with no significant impact on the analysis of results. Such losses were somewhat predicted, considering that the study was operationalized through the health service.The results concerning the circulating types of HPV in the present study were discordant of some studies that evaluated the prevalence of HPV among Brazilian women,The prevalence of cervical infection by HPV varies from medium to high, especially among young women, who initiate sexual activity with a risk of exposure to oncogenic types. The incorporation of HPV testing in the screened women would have the advantage of increasing the follow-up interval, with HPV-positive women having a cytological examination, whereas HPV-negative women would only need five-year intervals, two years longer than recommended for follow-up with a cytological examination after two years with normal resultsThe prevalence of HPV infection in Brazilian studies performed with women recruited from health units appears to be considerably higher than the prevalence observed in population-based studies. In the latter, the possibilities of selection bias and consequent overestimation of prevalence estimates are practically eliminated, since the studied population does not only include women who were assisted by, or referenced to, health services for being symptomatic.,,,,,,-,Regarding the factors related to cervical HPV infection observed, marital status (single), alcohol consumption (any frequency), and the number of lifetime sexual partners (three or more) remained as independent predictors of HPV infection, even after adjustment for other variables. These findings confirm the results of other studies that also related the risk of HPV infection to marital status, abusive alcohol consumption and the sexual history of the women,Self-assessment of health status, smoking, contraceptive use, parity and age at sexarche did not remain as independent predictors of HPV infection in the final model. Vaccarella et al. (2006) also found no statistically significant association between HPV infection and parity, prolonged use of oral contraceptives or use of condoms by the sexual partner and age at sexarche,,It should be noted that the prevalence of infection in the different age groups is close to that estimated in similar studies, with the highest proportion of infected women found between 25 and 34 years of age, which is also the age group with the highest incidence of in situ carcinomaThus, the prevalence of infection among the studied women is similar to that found in other Brazilian studies, being higher among younger women. Independent predictors of HPV infection – marital status, alcohol consumption, and the number of lifetime sexual partners – are highly influenced by economic, cultural, and social issues. Further studies are needed to understand if the relationship between the factors associated with increased prevalence of HPV infection changes when analyzed against the incidence of pre-invasive and invasive cervical cancer lesions among these women to verify whether predictive factors would be the same in a population of women with carcinoma (in situ or invasive).Organized screening, an alternative to opportunistic screening, provides for the capture of women in the target age groups, while at the same time not unnecessarily repeating the pap smear. However, factors related to non-adherence to the exam within the recommended periodicity need to be recognized and become the target of intervention, since the simple change in the recruitment strategy does not guarantee that the woman will attend the health unit for examination. Testing for HPV, as well as screening, would perform better on an organized, non-opportunistic screening system.Testing for HPV should be evaluated economically for its implementation to be recommended. As a technology to be incorporated into the screening, it requires team training and strict logistic supervision to avoid contamination of negative samples or cross-contamination by types between samples. It is necessary to adopt methods based on polymerase chain reaction that recognize each type of HPV in the prevalence of infection among the women of the different regions of the country. The higher sensitivity and lower specificity of the HPV-DNA test compared to screening with conventional cytology could overload secondary care referrals. The impact of this increase in positivity on services needs to be evaluated, including its cost-effectiveness ratio. On the other hand, a change in the age range of women screened and the use of the examination in regions of restricted access to health units would benefit women living with unequal access to cervical cancer prevention, identifying women at higher risk for acquisition and persistence of the infection and, consequently, for the evolution of preneoplastic lesions. The ethical implications of HPV testing need to be considered, and the psychological impact judged carefully, since women who have HPV infection are not ill, and it may be hard to understand this condition. Screening provides an overview of the HPV infection prevalence at a time when circulatory type surveillance is needed, including ecological niche filling, subsequent to vaccine implantation against types 16 and 18, and this action can be viewed as an effort within priorities of policies aimed at the control of cervical cancer and, therefore, at women’s health.In general, actions that focus on the social determinants of health, course approach, quality of life, and lifestyle promote improvements that are essential to modifying exposure to risk factors related to HPV infection, as well as to many other exposures, diseases, and aggravations. Gender-related issues and their vulnerabilities should guide the planning of educational actions among women, permeating prevention and control of injuries even before sexual activity begins. The health sector must work in an integrated manner with other sectors to ensure that inequalities in access to skilled services and information on women’s health are overcome and to identify opportunities for integration. The search for a solution to these problems will not be complete without dialogue between epidemiological research and health service, in a mutually beneficial process, where the most favored will certainly be the female population.


TTN gene truncating variants are common in dilated cardiomyopathy (DCM), although data on their clinical significance is still limited. We sought to examine the frequency of truncating variants in TTN in patients with DCM, including familial DCM (FDCM), and to look for genotype-phenotype correlations. Clinical cardiovascular data, family histories and blood samples were collected from 72 DCM probands, mean age of 34 years, 45.8% FDCM. DNA samples were examined by next generation sequencing (NGS) with a focus on the TTN gene. Truncating mutations were followed up by segregation study among family members. We identified 16 TTN truncating variants (TTN trunc) in 17 probands . During mean 63 months from diagnosis, there was no difference in adverse cardiac events between probands with and without TTN truncating mutations. Among relatives 29 mutation carriers were identified, nine were definitely affected (31%), eight probably affected (27.6%) one possibly affected (3.4%) and eleven were not affected (37.9%). When relatives with all affected statuses were combined, disease penetrance was still incomplete (62.1%) even after exclusion of unaffected relatives under 40 (82%) and was higher in males versus females. In all mutation carriers, during follow-up, 17.4% had major adverse cardiac events, and prognosis was significantly worse in men than in women. In conclusion, TTN truncating variants were observed in nearly one fourth of young DCM patient population, in vast majority without conduction system disease. Incomplete penetrance suggests possible influence of other genetic and/or environmental factors on the course of cardiotitinopathy. Counseling should take into account sex and incomplete penetrance. TTN) emerged as a major DCM locus with truncating mutations found in one fifth to one fourth of patients, especially those with familial forms of the disease [TTN truncating variants was raised by their presence in 1% of hypertrophic cardiomyopathy (HCM) patients and in 3% of controls [TTN truncated transcripts expression in the heart muscle was published showing that unlike in controls, TTN truncating mutations in DCM patients were predominantly located in the sarcomeric A-band region of the protein, occurred closer to the protein’s carboxyl terminus and were present in exons that were abundantly transcribed in the heart muscle [Whereas mutations in numerous loci have been known to predispose to dilated cardiomyopathy (DCM) , recentl disease –4. Howevcontrols , and by controls . Recentlt muscle .TTN truncating mutations, and (2): to examine genotype-phenotype correlations.Our aims were: (1) to identify genetic background in a cohort of DCM patients referred for clinical genetic diagnosis with a focus on All patients and relatives signed written informed consent in accordance with the Declaration of Helsinki. The study was approved by local Bioethics Committee of Institute of Cardiology with approval number 1276.TTN truncating mutation positive probands. DCM was defined according to ESC (European Society of Cardiology) criteria, with left ventricular ejection fraction below 45% and left ventricular end-diastolic diameter >117% of the predicted value corrected for age and body surface area [The study cohort was drawn from all index patients referred for clinical genetic testing to Unit for Screening Studies in Inherited Cardiovascular Diseases with the diagnosis of DCM from 2012 to 2014. The cohort comprised of 72 unrelated DCM probands, all were Caucasian, 48 were male (66.7%). In addition, we performed segregation study among available 44 relatives of ace area  in relatMean age of probands at diagnosis was 33.8±13.6 years, and mean left ventricular ejection fraction at diagnosis was 24%. Of 72 probands, 33 (45.8%) were with familial DCM (FDCM), and 39 (54.2%) with sporadic DCM.ABCC9, ACTC1, ACTN2, ANKRD1, BAG3, CRYAB, CSRP3, DES, EMD, ILK, LAMA4, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYPN, PDLIM3, PLN, PSEN1, PSEN2, RBM20, SCN5A, SGCD, TAZ, TCAP, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TTN, VCL) were performed. The detailed NGS approach for each proband with TTN truncating variant found is given in TTN defined as TTN N2BA transcript NM_001256850.1. Mean ge10 was 97.5±1.99 for N2BA TTN transcript. The mean coverage was 61.5±43.1 for NGS target regions and 66.6±41.0 for N2BA transcript NM_001256850.1. The NGS parameters are given for N2BA transcript as we considered only variants in cardiac TTN transcript NM_001256850.1.DNA was extracted from the peripheral blood by phenol extraction. Genetic testing in probands was performed using next generation sequencing (NGS). 44 patients were analyzed by whole exome sequencing (WES), in 22 TruSight One (TSO) sequencing panel and in 6 sequencing of a custom panel of 35 genes involved in cardiomyopathies  and BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) according to the manufacturer’s instructions. The results were analyzed with Variant Reporter 1.1 Software (Life Technologies). List of primers specific to each TTN truncating variant is available in http://www.1000genomes.org/), NHLBI GO Exome Sequencing Project (ESP) 6500  and Version 0.3 of ExAC (http://exac.broadinstitute.org/).All results for categorical variables were presented as numbers and percentages and for continuous variables as mean and standard deviation (SD) or median. The Fisher’s exact test was used for comparison of categorical variables. All tests were two-sided with the significance level of p<0.05. Kaplan-Meier curves were compared with log-rank test. Statistical analyses were performed with statistical package STATISTICA v6.TTN truncating variants  in 17 (23.6%) of 72 subjects (TTN truncating variant (TTN trunc) compared to seven of 39 sporadic DCM cases (17.9%). There were ten nonsense and six frameshift mutations but no splice variants. Of the 16 variants, 3 were described before—two were found in large population study by Roberts et al. [TTN mutations were located in symmetric exons. The majority of probands  had mutations localized in the A-band. The only variant found outside A-band and adjacent region of I-band was found in a family where cosegregation was nearly full with only one of 6 family members, 18 years old woman, being unaffected  variants in coding sequence excluding sex chromosomes. After such filtering among 376 variants in proband from DCM082 family and 223 variants in proband from DCM102 family the only one shared variant between these patients was TTN truncating variant which suggest no relatedness. This conclusion is further supported by internal comparisons between samples from related and unrelated subjects analyzed by WES in our lab for other purposes .To verify the possibility of relatedness of two probands sharing the same TTN trunc carriers (58.8% of carriers), 30 in non-carriers (54.5% of non-carriers) and 1 shared between two probands one with and the other without TTN truncating variant. Full list of missense TTN variants in our cohort is shown in TTN truncating variants similar prevalence among carriers vs. non-carriers argues against significant effect among the latter.In our group of 72 probands we also found 41 missense variants in cardiac transcript N2BA– 10 in 17 TTN trunc positive (n = 17) versus TTN trunc negative DCM patients (n = 55). Conduction disease was defined as the presence of left bundle branch block (LBBB) and/or atrioventricular block (AVB). One TTN trunc positive proband had LBBB (5.9%) whereas among 55 TTN trunc negative probands 21 (38.2%) had conduction disease . However, the difference in prevalence of conduction disease between TTN trunc carriers and noncarriers was not significant after correction for number of comparisons (n = 21) ((n = 21) . There wTTN trunc carriers versus noncarriers  major adverse cardiac events in the whole group (n = 72). Kaplan-Meier survival curve did not show difference in outcome in TTN mutations 9 relatives (31%) had definitely affected status, 8 (27.6%) had probably affected status and one (3.4%) had possibly affected status  and comparison of TTN trunc positive probands (n = 17) with TTN trunc positive DCM relatives (n = 9). There was no difference in the severity of the disease at diagnosis  and outcome between these two DCM patients’ subgroups, namely major adverse cardiac events were present in 5/17 probands and in 3/9 affected DCM relatives, p = NS.TTN truncating mutations was 62.1% and did not clearly increase with age being 60% by age 50 (n = 20), 56% by age 60 (n = 25) and 62% by age 70 (n = 29). After excluding all unaffected TTN trunc relatives younger than 40 years the penetrance was 82%. However, we noted that in TTN trunc relatives older than 59 years  the penetrance was 100%.Overall disease penetrance among relatives carrying TTN trunc carriers stratified by sex. Median age of onset in males was estimated as 28 years (range from 26 to 30) and in females as 56 years (range from 33 to 79) . Though we believe that there is no reason to exclude probands, as the criterion used in this analysis is age of onset, which is independent from patient’s proband/relative status, when the analysis was restricted to TTN trunc relatives the effect was still statistically significant . Furthermore, according to Kaplan-Meier analysis based on all TTN trunc individuals the estimated cumulative risk of DCM was 60.8% at the age of 33 and 95.1% at 61 years in males, and 17.6% and 53.9% in females, respectively.We observed that the penetrance was sex dependent. TTN mutations was also sex dependent. In all mutation carriers (n = 46), during 45.3±56.3 months of follow-up, 8 (17.4%) had major adverse cardiac events. TTN truncating variants in comparison to female carriers. The difference between these two groups was statistically significant .The incidence of major adverse cardiac events among those with TTN trunc positive probands additional rare  variants in known cardiomyopathies’ genes were found, namely ACTN2, DSP, LDB3, MYH6, MYH7, PKP2, SCN5A, TNNI3. All of them were missense variants with variable bioinformatics predictions of pathogenicity and clinical significance  of ificance . Two of myopathy  and MYH7myopathy . The segTTN trunc raised a question about their possible influence on outcome. Among 9 affected relatives of these probands, 7 (78%) had also additional variants, there were only 2 affected TTN trunc positive relatives in whom additional variant was not present whereas none of the affected relatives carried only the additional variant. Taken together these data are consistent with the hypothesis that in these families DCM is caused by TTN trunc but the numbers are too small for definite conclusions.Identification of additional variants in nearly half of probands carrying the TTN truncating variants, 13 of them novel. An important feature of our study is the examination of young DCM patient population with a mean age of 34 years.While studying 72 DCM patients we identified 16 different TTN truncating variants among our unselected young DCM patient population (23.6%) is similar as in previously published cohorts, i.e. between 11 and 22% [TTN trunc variants in familial DCM again similar to previous reports (between 18 and 27%). Our young DCM cohort was characterized by advanced heart failure with nearly half of the patients with documented familial disease. This is in agreement with current guidelines recommending genetic testing mainly in patients with clear-cut phenotype. As in the study by Herman et al. [TTN truncating variants, had generally similar clinical manifestations including the risk of major cardiac events. Similarly, as in the study by Herman et al., we found that in our patients DCM caused by TTN trunc is usually unaccompanied by conduction disease (in particular none of the TTN trunc carriers had AVB).The observed frequency of pulation 3.6% is s and 22% , 15. We TTN truncating mutations were located in the symmetric exons of an A-band region. The A-band region, critical for biomechanical sensing and signaling, is organized in repeats containing numerous FN-III domains interspersed with Ig domains providing repetitive binding sites for myosin and thick filament associated proteins [TTN truncating mutations occur predominantly in the A-band [In our group 82.2% proteins . Our fine A-band .TTN truncating variant in the Z-disc region which confirms that this region is rarely mutated in patients with DCM. To our best knowledge only a few TTN variants located in Z-disc have been published so far in patients with DCM. These include three missense mutations [Only one proband had utations , 17 and utations . As peneTTN truncating mutations are more likely to be disease-causing when they occur in exons which are abundantly transcribed and located closer to the carboxyl end of the protein [TTN truncating variants, even when they meet the criteria mentioned above, are characterized by incomplete and sex-related penetrance. We found incomplete penetrance in TTN trunc relatives carriers that was 62.1% when all affected statuses were combined and 82% after exclusion of unaffected relatives under 40. In the study by Akinrinade et al. disease penetrance increased from 53.8% at age 50 to 84.6% at age 60 and 100% at age 70 [TTN relatives was 29% at the age of 50 (n = 49), had increased to 58% at the age of 60 (n = 43), and further to 83% at the age of 70 (n = 36) [MYBPC3 mutations [LMNA [TTN truncating mutations which is similar as in LMNA mutation carriers [ protein . Our res(n = 36) . The var(n = 36) . Furtherutations  or Emeryns [LMNA . Our datcarriers , and concarriers .Of note, severity of the disease and rates of major adverse cardiac events defined as cardiac transplantation, implantation of a left ventricular assist device and death from cardiac causes, were not different when comparing DCM probands and relatives affected with DCM.Our population drawn from patients referred for clinical genetic testing introduces some bias towards more severely ill, with prominent family history. Also our Institute is one of leading cardiac transplant centers in Poland, patients with more advanced diseases are referred here, and patients with familial background are more willing to have their disease thoroughly clarified. Furthermore, we acknowledge that our findings should be interpreted with caution due to small patient numbers.During a long period of time when probands were tested NGS procedures were improved and refined which led to wide ranges of NGS parameters like ge10 or sequencing depth. The minimum coverage of sequencing was showed as mean ge10 instead of widely used mean ge20 because this parameter was more stable than ge20.TTN truncating variants were observed in nearly one fourth of young DCM probands with sex-related differences in disease penetrance and severity which should be taken into account in genetic counselling.S1 FigTTN mutation is indicated by a + or − symbol respectively. The presence or absence of additional variants are noted in parenthesis (+) or (-) respectively. DCM008: TTN p.Arg31056*, DCM019: TTN p.Arg21009*, DCM023: TTN p.Gly18918Valfs*17 (and TNNI3 p.His34Gln), DCM029: TTN p.Gln27004*, DCM033: TTN p.Ile26829Metfs*15, DCM036: TTN p.Lys27131*, DCM075: TTN p.Arg22817*, DCM078: TTN p.Lys14528* (and LDB3 p.Gly19Ala and SCN5A p.Ala572Asp), DCM081: TTN p. Ser28693Ilefs*2 (and MYH6 p.Arg204His).Squares represent males and circles represent females. An arrowhead denotes the proband. A diagonal line marks deceased individuals. Solid symbols denote affected status, half blackened symbols denote probably affected, quarter blackened symbols denote possibly affected status, open symbols with “N” denote not-affected individuals. Grey symbols denote individuals treated for heart failure but not tested at the reference center. The presence or absence of (TIF)Click here for additional data file.S2 FigTTN p.Ala29119Leufs*17, DCM092: TTN p.Ser29255Alafs*18, DCM097: TTN p.Ser493* (and DSP p.Ala566Thr), DCM102: TTN p.Ala29119Leufs*17, DCM109: TTN p.Asn30734Glnfs*17 (and PKP2 p.Pro7Ser), DCM113: TTN p.Arg17736* (and ACTN2 p.Arg298His), DCM132: TTN p.Glu23514*, DCM134: TTN p.Gln26147* (and MYH7 p.Arg237Trp).For Fig legend see (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 TableVariants annotated to NM_001267550.2 transcript.(DOCX)Click here for additional data file.S5 TableFor Table Legend see (DOC)Click here for additional data file.S6 TableLegend: PolyPhen2: D-probably damaging, P-possibly damaging, B-benign; MutationTaster: D-disease casing, N-polymorphism.(DOCX)Click here for additional data file.


This study is the first to examine associations between several area-based socioeconomic factors and human papillomavirus (HPV) vaccine uptake among boys in the United States (U.S.).N = 19,518) with several individual-level and ZIP Code Tabulation Area (ZCTA) census measures. Multivariable logistic regression was used to estimate the odds of HPV vaccination initiation and series completion separately.Data from the 2012-2013 National Immunization Survey-Teen restricted-use data were analyzed to examine associations of HPV vaccination initiation (receipt of ≥1 dose) and series completion (receipt of three doses) among boys aged 13-17 years (In 2012-2013 approximately 27.9% (95% CI 26.6%-29.2%) of boys initiated and 10.38% (95% CI 9.48%-11.29%) completed the HPV vaccine series. Area-based poverty was not statistically significantly associated with HPV vaccination initiation. It was, however, associated with series completion, with boys living in high-poverty areas (≥20% of residents living below poverty) having higher odds of completing the series  than boys in low-poverty areas (0-4.99%). Interactions between race/ethnicity and ZIP code-level poverty indicated that Hispanic boys living in high-poverty areas had a statistically significantly higher odds of  HPV vaccine initiation  and series completion   than Hispanic boys in  low-poverty areas. Non-Hispanic Black boys in high poverty areas had higher odds of initiation  and completion  than non-Hispanic Black boys in low-poverty areas. Rural/urban residence and population density were also significant factors, with boys from urban or densely populated areas having higher odds of initiation and completion compared to boys living in non-urban, less densely populated areas.Higher HPV vaccination coverage in urban areas and among racial/ethnic minorities in areas with high poverty may be attributable to factors such as vaccine acceptance, health-care practices, and their access to HPV vaccines through the Vaccines for Children Program, which provides free vaccines to uninsured and under-insured children. Given the low HPV vaccination rates among boys in the U.S., these results provide important evidence to inform public health interventions to increase HPV vaccination. In the United States (U.S.), approximately 39,000 HPV-associated cancers among women and men are diagnosed each year, many of which are preventable with the current HPV vaccine. Fifty-nine percent of these cancers occur in women . HPV is Racial/ethnic and socioeconomic disparities in HPV-associated cancers have been documented in the U.S. Recent data indicate Hispanic men have higher rates of penile cancer compared to non-Hispanic (NH) men. White and Black men have higher rates of oropharyngeal and anal cancers compared to men of other races , 5. FurtIn 2006, the Centers for Disease Control and Prevention (CDC) Advisory Committee on Immunization Practices (ACIP) recommended that females aged 11 and 12 years receive a routine three-dose HPV vaccine . ACIP exDespite a safe and effective vaccine to reduce the risk of HPV-related cancers, HPV vaccination coverage in the U.S. remain low. The Healthy People’s 2020 goal for teens completing the recommended HPV vaccine three-dose series is 80% , yet in The low U.S. HPV vaccination rates have led to numerous studies on HPV vaccination among teens to better understand the reasons for the low coverage. These studies suggest that the low number of HPV-immunized persons are in part due to lack of parental knowledge about HPV and the HPV vaccine, lack of health-care provider recommendations for the vaccine, missed opportunities for vaccination, religious and cultural factors, and beliefs that vaccinating adolescents against a sexually transmitted infection early is unnecessary and/or may promote sexual activity , 15.The majority of research examining factors associated with HPV vaccination in the U.S. has focused on teen girls , 14, 15.Given that uptake of cancer prevention and screening activities are generally higher among high-income, more educated populations –34, convIn this study, we analyzed data from the National Immunization Survey-Teen (NIS-Teen) to examine associations between both individual-level and area-based factors and HPV vaccine initiation and completion among boys. This study is the first to explore area-based factors that may be associated with HPV vaccine uptake among boys in the U.S.We conducted a secondary data analysis of restricted-use data from the 2012 and 2013 NIS-Teen, an annual survey conducted by the CDC to monitor vaccination uptake in the U.S. The NIS-Teen includes a nationally representative stratified sample of girls and boys aged 13 to 17 years in all 50 states and the District of Columbia. The survey is based on random-digit dialing of both landline and cellular telephone numbers to identify eligible households. It includes two parts: 1) a survey of parents or guardians of 13- to 17-year-olds to collect information about demographic and socioeconomic characteristics and 2) a survey mailed to all vaccination providers whom the parents identified and consented to verification of their teen’s immunization histories. The survey sampling procedures have been described elsewhere . The NatThe present study was limited to only provider-verified vaccination records. An additional 3.3% of these cases were excluded from the statistical models because of missing ZIP codes  and 0.9% were excluded. There were no differences between participants in the final sample and those excluded with regard to any of the individual-level or area-based socioeconomic measures. The final dataset included 19,518 boys.We examined two primary HPV vaccination outcomes that were based on provider-verified vaccination records: 1) initiation: receipt of at least one dose of the three-dose HPV vaccine series; and 2) completion: receipt of three doses of HPV vaccine. We included several individual-level variables previously shown to be associated with HPV vaccine uptake , 37–41. Several area-based socioeconomic measures created using U.S. Census ZIP Code Tabulation Areas (ZCTAs) data were merged with the ZIP codes of participants’ current residences at the CDC Research Data Center (RDC). ZIP codes are restricted variables; therefore, these data were accessed through the RDC. The ZCTAs are generalized areal representations of U.S. Postal Service areas, and in most instances the ZCTA is the same as the ZIP code . ZCTAs wWe included several area-based measures previously incorporated in health disparities research , 45 and A measure of racial/ethnic composition or density of a ZCTA was also created. Racial/ethnic composition refers to proportions of people of the same race/ethnicity in a defined area and has been used as a proxy measure of segregation , 50. SegWe also included a ZCTA measure of population density , which has been used in previous research as an indicator of the built environment, crowding  and a prNIS-Teen data for years 2012 and 2013 were combined and the sampling weights for the provider verified data (PROVWT) was recalculated using suggested methods in the NIS-Teen Users Guide  to ensurp < 0.05) into a multivariable logistic regression model. The multivariate models produced adjusted odds ratios (AORs) and 95% confidence intervals (CIs). One multivariable model included only individual and provider factors, and two separate multivariable models were run to examine the independent associations of ZCTA poverty and racial/ethnic composition with vaccination outcomes. In the two separate multivariable models that included area-based measures we also included population density and in an additional analysis replaced population density with rural/urban residence. Joint contributions of area poverty, racial composition, and race/ethnicity were assessed with interaction terms between individual-level race/ethnicity and ZCTA poverty and individual-level race/ethnicity and ZCTA racial composition. Population density was also included in the multivariable models that were run to examine the associations of ZCTA poverty and racial/ethnic composition with vaccination outcomes. The surveyed state was included as a random effect to account for dependency by state residence .We performed bivariate association tests between all the variables and our two primary HPV vaccination outcomes (initiation and completion) with Wald chi-square tests. We also used logistic regression to identify variables associated with our two primary HPV vaccination outcomes. For each outcome separately, we entered all variables associated with the outcome in bivariate models  . LogistiSociodemographic and geographic characteristics of the survey participants are presented in Table Overall, for the years 2012-2013, 27.9% of boys received at least one dose of HPV vaccine , but later completed the three-dose series within the recommended duration. Second, the parents’ role in the survey is subject to recall bias because some parents may have incorrectly recalled whether they had ever received a provider recommendation for the vaccine. Finally, ZCTA was the smallest unit available for the NIS-Teen survey, which has been shown to be more heterogeneous than smaller units such as census tracts . Using dThis study found that HPV vaccination coverage for boys varied by their race/ethnicity depending on the level of poverty in their ZIP Code Tabulation Area. Racial/ethnic minorities from areas with high levels of poverty had higher odds of both HPV vaccination initiation and completion compared to those from low-poverty areas. We also found that boys from areas where the majority race/ethnicity group was Hispanic had greater odds of vaccine initiation compared to boys from majority NHW or NHB areas. Higher HPV vaccination coverage in areas with high poverty may be attributable to targeted interventions by the Vaccines for Children program, which provides free recommended vaccines to uninsured and under-insured children. Given the low HPV vaccination rates in the U.S., these results provide important evidence to inform public health interventions to increase HPV vaccination.


Despite the existence of human papilloma virus (HPV) vaccines with demonstrated safety and effectiveness and funded HPV vaccination programs, coverage rates are persistently lower and cervical cancer burden higher among Canadian Indigenous peoples. Barriers and supports to HPV vaccination in Indigenous peoples have not been systematically documented, nor have interventions to increase uptake in this population. This protocol aims to appraise the literature in Canadian and global Indigenous peoples, relating to documented barriers and supports to vaccination and interventions to increase acceptability/uptake or reduce hesitancy of vaccination. Although HPV vaccination is the primary focus, we anticipate only a small number of relevant studies to emerge from the search and will, therefore, employ a broad search strategy to capture literature related to both HPV vaccination and vaccination in general in global Indigenous peoples.Eligible studies will include global Indigenous peoples and discuss barriers or supports and/or interventions to improve uptake or to reduce hesitancy, for the HPV vaccine and/or other vaccines. Primary outcomes are documented barriers or supports or interventions. All study designs meeting inclusion criteria will be considered, without restricting by language, location, or data type. We will use an a priori search strategy, comprised of key words and controlled vocabulary terms, developed in consultation with an academic librarian, and reviewed by a second academic librarian using the PRESS checklist. We will search several electronic databases from date of inception, without restrictions. A pre-defined group of global Indigenous websites will be reviewed for relevant gray literature. Bibliographic searches will be conducted for all included studies to identify relevant reviews. Data analysis will include an inductive, qualitative, thematic synthesis and a quantitative analysis of measured barriers and supports, as well as a descriptive synthesis and quantitative summary of measures for interventions.To our knowledge, this study will contribute the first systematic review of documented barriers, supports, and interventions for vaccination in general and for HPV vaccination. The results of this study are expected to inform future research, policies, programs, and community-driven initiatives to enhance acceptability and uptake of HPV vaccination among Indigenous peoples.PROSPERO Registration Number: CRD42017048844The online version of this article (10.1186/s13643-018-0692-y) contains supplementary material, which is available to authorized users. Persistent infection with human papillomavirus (HPV) strains 16 and 18 can cause cervical and other cancers , 3. TherEnhancing HPV vaccination is a potentially high-impact, low-cost opportunity for cancer risk reduction among Indigenous peoples. As part of a tri-phase study in First Nations people to (1) establish baseline HPV vaccination rates, (2) systematically identify and validate the barriers and supports to HPV vaccination, and (3) comprehensively map documented and reported supports and barriers to HPV vaccination to develop a theory-informed, context-appropriate knowledge translation intervention, this synthesis aims to identify documented barriers, supports, and intervention strategies for HPV vaccination in Canadian and global Indigenous populations.Despite its success as a public health intervention –34, vaccGeneral barriers and supports to vaccine acceptability and uptake of childhood vaccines have been documented , 43, 44.In Canada, the HPV vaccine was introduced into national and regional vaccination programs targeting pre-adolescents in school settings , to prevA recent systematic review  summarizAlthough a recent systematic review reported a lack of consistency and detailed reporting of interventions to increase HPV vaccine uptake , it founHPV vaccine coverage levels in the general population of pre-adolescents may hide sub-populations with lower coverage, including Indigenous peoples who are known to have higher HPV infection rates , 11, 66,The evidence compiled thus far on the barriers, supports, and interventions for HPV vaccination reveals considerable gaps in data for Indigenous peoples. Even so, any evidence on vaccination in this population may provide important guidance for future work. To our knowledge, this will be the first synthesis of the barriers, supports, and interventions for improving vaccination and HPV vaccination in Indigenous peoples. Synthesis-level knowledge in this area is expected to support Indigenous peoples, researchers, clinicians, policy makers, and communities in their efforts to ameliorate well-documented and disproportionate burdens of disease that persist in Indigenous populations worldwide.Because we anticipate only a small number of relevant studies on HPV vaccination to emerge from the search, we will employ a broad search strategy to capture literature related to both HPV vaccination and vaccination in general among Indigenous peoples in Canada and globally. This systematic review will locate, gather, and critically appraise the global literature in Indigenous peoples , relatinThe design of this mixed methods –84 systeWhat are the documented barriers and supports to V/HPV-V, in Canadian Indigenous and global Indigenous peoples?What are the documented interventions to increase acceptability and uptake or reduce hesitancy of V/HPV-V, in Canadian Indigenous and global Indigenous peoples?Primary questions of interest are:3.What are the documented barriers and supports to V/HPV-V, in youth (< 18 years) within Canadian Indigenous and global Indigenous peoples?4.What are the documented interventions to increase acceptability and uptake or reduce hesitancy of V/HPV-V, in youth (< 18 years) within Canadian Indigenous and global Indigenous peoples?Secondary questions of interest are:and (b) discuss barriers or supports and/or interventions to improve uptake/acceptability or to reduce vaccine hesitancy for V/HPV-V. Studies will be excluded if they do not (a) include global Indigenous peoples or include subpopulation(s) of one or more global Indigenous peoples comprising more than 50% of study participants, or for which separate analyses for global Indigenous peoples or subpopulation(s) are not presented; (b) discuss barriers, supports or interventions for enhancing acceptability or uptake or reducing hesitancy for V/HPV-V; (c) report extractable data; or (d) report original research.We will include studies that involve global Indigenous peoples, including First Nations, Inuit, and Métis (Canada), American Indians and Alaskan Natives (USA), and Indigenous peoples in other countries including, but not limited to, China, South Asia, former Soviet Union, Southeast Asia, South America, Africa, Central America/Mexico, Arabia, Japan and the Pacific Islands, Australia, New Zealand, Greenland, and Scandinavia , 94, 95.or interventions that increase or enhance acceptability/uptake or interventions to reduce hesitancy for V/HPV-V. Secondary outcomes will investigate these same factors, specific to youth (< 18 years) in Canadian and global Indigenous peoples. For this review, we define barriers as single or multilevel factors that are negatively associated with, or hinder the acceptability or uptake of V/HPV-V or increase hesitancy. Supports include single or multilevel factors that are positively associated with, or enhance the acceptability and uptake of V/HPV-V or reduce hesitancy. Vaccination acceptability is defined as individuals’ willingness to accept vaccination  and vaccine uptake is defined as the behavior of getting vaccinated  and discrepancies resolved by a third investigator.The anticipated outcomes of interest are barriers and supports to V/HPV-V  and interventions to enhance V/HPV-V acceptability A draft data abstraction tool will be generated in MS Excel , based oMethodological quality will be assessed using a universal appraisal tool  developed for the assessment of quality in diverse study designs . The 4-pA secondary quality analysis of the included effective intervention studies will be undertaken using the domains of the Consolidated Framework for Implementation Research (CFIR) . The maiThe overall study flow will be reported using a PRISMA diagram , providiWe anticipate the ability to provide both a descriptive synthesis and quantitative summary of relevant measures for interventions; however, recent reviews examining barriers and supports in the area of HPV vaccination revealed a level of heterogeneity among studies precluding meta-analysis . All incWe plan to report findings pertaining to Canadian Indigenous and global Indigenous peoples separately, contingent on the volume of eligible literature identified in each group. We will report results for HPV vaccines separately from results arising from studies of other vaccines, given the characteristics and contextual factors associated with HPV compared with other vaccines and target populations. A draft analysis plan and anticipated products of the synthesis are outlined in Table To date, we have not located a systematic review describing the barriers and supports to HPV vaccination or one that gathered and assessed effective interventions to increase acceptability and uptake of HPV vaccines in Indigenous peoples. Previous systematic reviews have been limited to vaccine efficacy in Indigenous peoples –121. In While the importance of multiple , 80, 122We have chosen to include global Indigenous peoples , 134 andThe results of this study are expected to inform future research, policies, programs, and community-driven initiatives to enhance acceptability and uptake of vaccination among Indigenous peoples. Specifically, this review will identify and describe the documented barriers, supports, and interventions for V/HPV-V. These will provide a foundation to inform the development of new vaccination strategies among Indigenous peoples. We anticipate the review will identify the nature of evidence gaps and highlight areas requiring further study.To our knowledge, this study will contribute the first systematic review of the global literature in this area. We will take an integrated knowledge translation , 136 appAdditional file 1:PRISMA-P Checklist. (DOCX 39 kb)


Papillomaviridae family. The prevalence of HPV genotypes may vary according to the region and the population studied. Quilombo communities are ethnic and racial groups with difficult access to health services compared to the general population in Brazil. The aim of this study was to identify specific HPV types correlating with sociodemographic/behavioral characteristics and cervical smear cytological abnormalities in Quilombola women.Human papillomavirus (HPV) is a member of the Linear Array HPV Genotyping Test kit by Roche Molecular System®. The association between HPV types and cytological diagnosis was investigated according to the different age groups.This cross-sectional study included 395 Quilombola women users of the Unified Health System of the Municipalities of Maranhão for the screening of cervical cancer. The samples were analyzed for the presence of cytological abnormalities by conventional methods and tested for 37 HPV genotypes using polymerase chain reaction with primers PGMY09/11 followed by reverse line blot hybridization performed with the HPV infection was detected in 12.6% (50/395) of the women. Infections by high-risk HPV types were more frequent. Genotypes 68 (26.0%); 58 and 52 (20.0%); 31 (10.0%) and 62 (8.0%) were the most prevalent. The highest prevalence (42.0%) of HPV infection occurred in women diagnosed with high-grade squamous intraepithelial lesion. There was a statistically significant association between HPV infection and the detection of cytological abnormalities in all age groups except in women over 60 years. There was a statistically significant association between the municipality of origin and the number of partners with HPV infection.It is important to incorporate new cervical cancer screening techniques incorporating the cervical-vaginal cytology. For further studies, it is necessary to determine the level of knowledge of Quilombola population on health-related issues including HPV infection and cervical cancer. This will contribute to the continuous improvement of healthcare coverage among the population and enhance the implementation of cancer care in the state of Maranhão. Cervical cancer (CC) is the third most common cancer among women worldwide, with approximately 528,000 new cases per year, being responsible for the death of 266,000 women each year . AccordiPapillomaviridae family, genus Papillomavirus. It is an icosahedral virus, non-enveloped, and about 55 nm in diameter. The genome consists of a double-stranded circular DNA, containing approximately 7900 base pairs ’ (ASC-H), 11 (33.3%) women with ‘ASC-US, 10 (30.3%) women with HSIL, and 7 (21.2%) women with LSIL.p < 0.001).The overall prevalence of HPV was 12.6% (50/395). In Table Among the HPV types, the most prevalent genotypes were 68 26.0% - 13/50), 58 and 52 with 20.0% (10/50) each, 31 (10.0% - 5/50) and 62 (8.0% - 4/50). Only 6.0% (3/50) of the cases were positive for HPV-61  compared with the low oncogenic risk type (22.0% -11/50). Among those diagnosed with an abnormality, the prevalence of HPV infection was 24.0% (12/50). The highest prevalence was reported with HSIL (42.0%), followed by LSIL (21.0%) and ASC-US (21.0%). In all diagnoses, simple infections were more frequent than multiple (Table 9/50 compp = 0.02), 31–40 years (p = 0.0009), 41 to 50 years (p = 0.03) and 51 and 60 years (p = 0.0003). Only among HPV-positive women, aged over 60 years, were there no statistically significant association with the detection of cytological abnormalities (p = 0.12).Table p < 0.0001). The remaining sociodemographic variables  showed no significant association with HPV positivity.In Table p = 0.01).The behavioral predictors of HPV, at the univariate level, are shown in Table In Brazil, the Black population experience higher cancer mortality rates compared to the general population. This is probably a consequence of inequities in the social, economic, political, and health factors. This population is faced with unequal experiences at birth, in life, during illness and in death . QuilombThe overall prevalence of HPV can vary depending on the technique used, as well as the population and the region studied. In this study, the prevalence of HPV infection in Quilombola women was 12.6% by hybridization technique. Different results were found in various studies including Akarolo-Anthony et al. (2014), which showed an overall prevalence of 37% in Nigerian women , and WatSome women had multiple HPV infections in this study, and these occurred more frequently with HPV 68, with a total of 71 HPV types among HPV positive women, and among these, 50 (69.4%) were of high oncogenic risk. Of the 25 HPV types found in this study, HPV 68 was the most prevalent (26.0%), followed by HPV 58 and 52 (20.0%) and HPV 31 (10.0%).A study conducted in France showed HPV 16 to be most prevalent, followed by HPV 53 and 31 . Study iGoldman et al. (2013) observed that the presence of HPV 68 occurred at a rate four times higher, than expected, in multiple infections. Thus, this indicates the existence of an interaction between HPVs of high oncogenic risk, especially between types 31/68, 51/68 and 33/58. These authors suggested that some virus genotypes act as co-factors in the infection by other types, demonstrating a new possible risk factor .Considering the prevalence of HPV in women with different cytologic findings in this study, age group (particularly less than 30 years) showed important influence on HPV positivity and the detection of cytologic abnormalities. Thus, these results indicate that HPV testing is useful in women over 30 years, who are more likely to be carriers of significant cervical lesions. According to Akarolo-Anthony et al. (2014), the age group most affected by HPV infection were those aged less than 30 years, while a decline were shown in women over 45 years of age, thus corroborating the findings of this study . In FijiIn a study by Foliaki et al. (2014), the positivity for at least one HPV genotype was 24.0% among 1244 women studied, however, HPV 16 was the most prevalent genotype, while HPV 68 was present in only 3 (0.2%) of the infected women. From the HPV positive cases, 70.1% had only one viral genotype, while 29.9% had multiple infections. HPV genotypes most often involved in multiple infections were of the high oncogenic risk .p < 0.001). Marks et al. (2015) found a much higher prevalence (43.8%) of HPV infection among women diagnosed with inflammation, 36.5% diagnosed with ASC-US, 80.9% of women diagnosed with LSIL and 71.5% of women with the diagnosis of HSIL [Among the 33 women who had cytological abnormalities, 36.4% were HPV positive. Among those diagnosed with inflammation, 10.4% were HPV positive. For ASC-H, ASC-US, HSIL and LSIL, HPV infection rate were 40.0%, 18.2%, 60.0% and 28.2% respectively. There was a statistically significant association between the presence of cytological abnormalities in cervical smears and the presence of HPV  than those of low oncogenic risk (22.0%). This has important implications for the clinical care of women with the high oncogenic risk. For those diagnosed with an abnormality, the prevalence of HPV infection was 24.0%, and was highest with HSIL (42.0%).p < 0.0001). Akarolo-Anthony et al. (2014), in studying Nigerian women, also found no association between sociodemographic factors  and HPV infection [In relation to sociodemographic factors and HPV infection, only the municipality of origin was significantly associated with positivity for HPV (nfection . This map = 0.01). Work carried out in Guinea by Keita et al. (2009) also demonstrated a significant association between the number of partners and HPV infection [In this study, there was significant association with HPV positivity only in relation to the number of partners (nfection .A limitation of the study is the approach used for the collection of data on risk factors used to determine how HPV was acquired and maintained, since the statistical associations were made solely based on the information reported by women. Therefore, another limitation is that the study lacks an adequate comparison group.This study provides a regional estimate on the prevalence of HPV infection among Quilombola women. It highlighted the higher prevalence of HPV type 68. This virus type can contribute to an increase in cervical carcinogenesis related to specific HPV genotypes.It is believed that the findings of this study can foster the development of strategies aimed at enhancing the health of the Quilombola women towards the prevention and management of specific HPV infection in view of the particularities of this group. Greater knowledge, on the part of women, on the mode of HPV acquisition, risk factors and the frequency of HPV infection in different age and population groups are crucial. This is because, such knowledge would contribute to a greater perception of their risk of developing cervical preneoplastic lesions and therefore influence their continued adherence to screening strategies. It may also encourage changes in HPV risk behavior and lifestyle.


The global burden of disease caused by both human immunodeficiency virus (HIV) and human papillomavirus (HPV) is the greatest in the developing world, with the highest rates in sub-Saharan Africa. South African women not only have high rates of infection with HPV, but also have high rates of multiple concurrent infections with two or more HPV genotypes, and are among the world’s most vulnerable to developing invasive cervical cancer. HIV co-infection increases these risks. Understanding clustering patterns of concurrent HPV infections in this population has important implications for HPV screening and will help define vaccination strategies in the future as vaccines continue to be developed to target more HPV genotypes. Latent class analysis was used to identify four distinct patterns of HPV co-infection: individuals with at least one low risk HPV genotype, but no high-risk HPV (HR-HPV) infections; individuals with a disperse pattern of HR-HPV infections; individuals infected with members of the alpha-7 group, but not HPV-18; and individuals infected with HPV-16, but not HPV-18. In this analysis, although alpha-7 HPV infections were more prevalent among HIV-infected adolescents than their HIV-uninfected counterparts, overall clustering patterns were not different based on HIV status. The high-risk human papillomavirus genotypes (HR-HPV) cause cervical cancer and are targets of vaccines preventing HPV infection and subsequent malignant transformation. While most HPV infections are transient, they are more likely to persist and progress to invasive disease when multiple concurrent infections are present and/or there is co-infection with human immunodeficiency virus (HIV) . It has South African adolescent women have high rates of infection with multiple concurrent HPV genotypes and HIV, but only 15.4% of South African adolescents were infected with the two HPV genotypes typically covered by current vaccine strategies (HPV-16 and HPV-18) . DespiteThis study used a novel approach to the question, employing latent class analysis (LCA), to better understand clustering patterns of HPV. LCA is a parametric modeling procedure to determine discrete patterns of subgrouping within a population with multivariate categorical data. Compared to previous methods, such as hierarchical or k-means cluster analysis, LCA allows for statistical and conceptual comparison of multiple grouping patterns to ensure that data-clustering patterns most accurately reflect potential biological clustering, whereas previously employed methods rely largely on arbitrary stopping rules to decide the optimal number of groups, clusters or classes.A cross-sectional study was conducted in which self-collected vaginal swabs for HPV DNA testing were collected from 100 sexually active HIV-infected and HIV-uninfected South African adolescent females aged 17 to 21 years between October 2012 and October 2014. Participants were recruited from the youth community center and clinic in two urban disadvantaged communities in Cape Town, South Africa. Exclusion criteria included a history of HPV vaccination and/or cervical surgery.Informed consent was obtained from all participants aged 18 and above. Adolescent assent and parental consent were obtained for participants aged 17 years. The Research Subjects Review Board at the University of Rochester and the Human Research Ethics Committee at the University of Cape Town approved the study.HIV status was confirmed upon enrollment. Fifty study participants were HIV-infected and 50 were HIV-uninfected. However, for the purpose of this analysis, only adolescents with at least one HPV infection at enrollment (n=64) were included. Results of HPV DNA testing of self-collected specimens were used in this analysis. Participants were instructed to twirl a Dacron swab high in the vagina for 10 s. Specimens were transported in Digene transport medium. The MagNA pure compact nucleic acid isolation kit (Roche) was used to extract the DNA. The Roche Diagnostic Linear Array HPV test was used for HPV genotyping. This research-only test identifies 37 HPV genotypes including the 13 oncogenic HR-HPV genotypes in the alpha-papillomavirus genus as designated by the International Agency for Research on Cancer: types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 .Patterns of infections were examined using LCA. Analyses were performed using Mplus 7.11 and the best fitting model was determined by comparing the Akaike Information Criteria (AIC) and the Lo-Mendell-Rubin adjusted likelihood ratio test (LMR aLRT) across solutions with differing numbers of classes . LCA proBased on a meta-analysis of HPV infections in African women with invasive cervical cancer in which HPV-16, HPV-18, and HPV-68 were among the top ten HR-HPV genotypes detected , these gp = 0.05) indicating statistically better fit than the 3-class model. The first class included adolescents without a HR-HPV infection (termed Uninfected) and comprised 35% of the population. Class 2 represented the largest group of HR-HPV-infected individuals, 34% of the population, and comprised individuals with a disperse pattern of infection (termed Disperse Infections), though none of these individuals had an HPV-68 infection. These individuals had the highest conditional probabilities across classes of a non-HPV-16 alpha-9 infection (51% chance of infection), while also being the only individuals with a non-zero probability of an HPV-18 infection (37% chance of infection). Class 3 represented 18% of the population and was limited to individuals with moderate conditional probabilities of concurrent infections with HR-HPV alpha-7 genotypes  other than HPV-18 . These individuals demonstrated the highest probability across classes of a non-HPV-18 alpha-7 infection (60% chance of infection). Class 4 represented 14% of the population and consisted of individuals infected with HPV-16, but not HPV-18  and had the highest probability of HPV-68 infection (57% chance of infection). There were no differences across classes in HIV status, lifetime sexual partners, frequency of condom use, use of contraceptive injections, or use of contraceptive pills . Analysis of HIV-infected adolescents as compared to HIV-uninfected adolescents revealed no differences in clustering patterns. However, further analysis revealed that alpha-7 infections were the only pattern of infection significantly more prevalent among HIV-infected adolescents .The demographic and behavioral characteristics of the cohort are shown in Infection with multiple HPV genotypes and/or co-infection with HIV increases the risk of progression to invasive cervical disease . MultiplNot surprisingly given the uniqueness of this study population and the broad variations in clustering patterns in previous studies, LCA analysis revealed four new distinct patterns of infections. By using LCA rather than the non-parametric methods previously employed, this study was capable of providing statistical support for asserting that the patterns identified most accurately reflect potential biological clustering.In a study of US women, HPV-16 was the most frequently detected genotype in both single infections and in infections with multiple HPV genotypes . It alsoIn the current study, HPV-16 infections clustered in one of two classes with other disperse HR-HPV infections or with individuals who were not co-infected with HPV-18, but had a relatively high probability of infection with HPV-68. HPV-18 only clustered in individuals with a substantial probability of a disperse pattern of co-infections. This suggests that while HPV-16-18 co-infections may be found at increased frequencies amongst US women, in South African adolescents, this specific HPV-16-18 relationship may not persist. Specifically, there was only a single case of HPV-16-18 co-infection (representing 8% of HPV-16 infections and 13% of HPV-18 infections). Additionally, HPV-18 infection alone (n=3) occurred at rates almost as high as in co-infections (n=5).Our analysis found that not only were HPV-16 and HPV-18 co-infections weakly linked, but HPV-18 and HPV-68 infections  were mutually exclusive . HPV-68 South African women experience a disproportionate number of both HPV and HIV infections. In a meta-analysis that examined HPV infections in women with HIV, HIV-infected women were ten times more likely to be infected with multiple HPV genotypes than their HIV-uninfected counterparts . HIV co-This study is limited by its relatively small sample size. Although this is the first study to analyze HPV-clustering patterns in a particularly vulnerable population that should be the target of future vaccination programs in South Africa, larger studies are needed to strengthen the findings presented here. Understanding the patterns of HPV infection among high-risk groups is informative to HPV vaccination and screening strategies.