Studies have shown that type-specific persistence of high-risk human papillomavirus (HPV) infection contributed significantly to cervical carcinogenesis.In this population-based study (on 24041 women), we report on the prevalent genotypes of HPVs and the prevalent genotypes of HPV persistent infection in the northeast of China.Our results showed that in HPV infected women , , 17.35%  suffered persistent infection. The most common high-risk HPV types in persistent positivity were HPV-16 , HPV-58 , HPV-18 , HPV-52  and HPV-33 . The prevalence of persistent infections with HPV-16,–58, −18, −52 and 33 in cervicitis were lower compared to those in CIN . HPV-58, −33 and multiple HPV persistent positivity were significantly associated with older age . HPV-18 persistent positivity was significantly associated with adenocarcinoma and lymphatic metastasis . HPV-18 persistent positivity was associated with cervical cancer prognosis (P <0.0001). Multivariate analyses showed that HPV-18 persistent positivity,  and lymphatic metastasis  were independent predictors for 3-year survival in cervical cancer.we provided extensive results of HPV genotype prevalence and distribution in the northeast of China. HPV genotyping is worthwhile to perform because of its independent prognostic value in cervical cancer Human Papillomavirus (HPV) infection usually proceeds for the development of virtually all invasive cervical cancers, their associated precancerous lesions, and genital warts ,2. To daIt is expected that the next-generation approach of HPV vaccines may more efficiently prevent cervical cancer, however, efforts to implement and evaluate a vaccination strategy are dependent on our understanding of the behaviors of HPV type-specific infection. Meanwhile, the geographic variation in the prevalence and distribution of HPV genotypes had been reported in different countries, even different regions in the same country. It’s necessary for us to study the prevalence and role of human papillomavirus genotypes in primary cervical screening in different geographic regions ,9. We haWe performed a population-based study (24041 women) to investigate the prevalent high-risk genotypes of HPVs and the results in HPV infected women in the northeast of China. After they first visited, we randomly selected 15257 from those 24041 women, and followed them up to test their HPV persistence as well as their pathological changes, in order to study the association between the persistent HPV infections and cervical lesions.From 2007 to 2010, 24041 women between the ages of 18 and 60 years who were permanent residents in the northeast of China were eligible to participate. All women were composed of healthy Chinese women who had underwent cervical cancer screening in our Health Check Center or at the Department of Obstetrics and Gynecology, Shengjing Afiliated Hospital of China Medical University. These women were not pregnant during their first visit and also had no intention to be pregnant during the first year of follow-up. Moreover, we excluded patients who had a history of hysterectomy, or were being treated with vaginal medicine at that time or will be treated for cervical diseases in 6 months. To obtain specific data about HPV persistent infection or re-infection, 24041 from 26126 eligible women (92%) were successfully followed-up, patients who died for non-cancer related diseases were excluded from this analysis. As for women with HPV DNA positive were followed-up every 4 months during the first year and subsequently twice a year until 31 Dec 2010; as for women with HPV DNA negative were followed-up once a year until 31 Dec 2010. To analyze the correlation between HPV persistent infection and cervical lesions, HPV screening was applied to the 24041 women. Meanwhile, 15257 women were enrolled for cervical biopsy done by professional pathologists . SubjectAll HPV tests were performed without the knowledge of cytological or histological interpretation. Human papillomavirus DNA was amplified with the L1 consensus HPV primers. Biotin was used for negative control and an internal control set of biotinylated primers simultaneously amplifies a 268-bp fragment of the human A-globin gene was used for positive control in each reaction as previously described [Human papillomavirus genotyping was performed using the HPV GenoArray test kit (HybriBio Ltd), which was used in both DNA amplification and HybriBio’s proprietary flow-through hybridization technique as previously described. HPV Blot contains 21 types of genotypes, including 5 low-risk types , 14 high-risk types , and 2 intermediate-risk types (CP8304 and 53). HPV L1 and an internal control of the human A-globin in each reaction confirm absence of HPV DNA contamination15257 eligible women, corresponding to a 63% response rate, were chosen for a colposcopic examination and cervical biopsy at every subsequent visit. Multiple punch biopsy specimens were taken under colposcopy. Lesions in the transformation zone were assessed by 5% acetic acid and iodine solution. If the colposcopy results proved unsatisfactory for a definitive diagnosis, further exploration of the endocervix was systematically carried out.178 cervical cancer biopsies were fixed in formalin buffer and embedded in paraffin. Sections of 5-mm thickness were obtained from the paraffin blocks, placed on siliconized glass, and left at 37°C overnight. The antibody used to label the HPV-18 E6 protein was the polyclonal antibody (Santa), . Color development was achieved with the chromogen diaminobenzidine. Sections were counterstained with hematoxylin. Staining was scored by estimating the percentage of cells that had cytoplasm staining for DKK4 and multiplying by the assessment of the intensity of the stain on a 1+, 2+, or 3+ scale. The theoretical limits of the scores ranged from 0 (0% of cells staining) to 300 (100% of the cells staining at 3+ intensity), as described above . All of χ2 test was used to evaluate the association between covariates. Cumulative the overall survival time was calculated by the Kaplan–Meier method and analyzed by the log-rank test. Multivariate analyses were based on the Cox regression model. All tests were two sided, and P <0 .05 was considered statistically significant.The data were analyzed using the SPSS version 13.0 statistical package. Their hazard ratio’s (HRs) and 95% CIs were calculated using the Wald test. Pearson’s In this study, persistent positivity was defined as the detection of the same HPV genotypes over three subsequent visits, whereas transient positivity was defined as the detection of the same high-risk HPV genotypes no more than three visits. A total of 24041 participants were successfully enrolled for HPV screening from 2007 to 2010 as mentioned above. Our results found that 45.6%;  was positive for HPV DNA. Among those HPV positive women, 17.35%;  suffered persistent infection and 28.25%;  suffered transient infection. Furthermore, we studied the prevalent genotypes of HPV persistent positivity. Among the women who were HPV persistent positivity , 14.16%;  were found to have a single HPV genotype. On the other hand, two HPV genotypes were 2.77%;  and 0.34%;  infected with three HPV genotypes and 0.08%;  infected with more than three HPV genotypes, 3.19%;  women were found more than one HPV type at the same or different times;.All of the 21 different HPV genotypes were found in the persistently infected samples. Among these types, the most common high-risk HPV types in persistent positivity were HPV-16 , HPV-58 , HPV-18 , HPV-52  and HPV-33  . Our results showed that, the prevalence of HPV persistent infection in cervicitis, CIN I, CIN II/III, and CC was 2.5%; , 13.68%; , 88.9%;  and 88%;  respectively. The prevalence of HPV in persistent infection in cervicitis was significantly lower than that in CIN I. The HPV prevalence in CINI was significantly lower than that in CIN II/III. However, there was no significant difference between HPV persistent infection in CIN II/III and that in CC. The prevalence of common HPV types in persistent infection was HPV-16,-58,-18,-52,-33 (as mentioned above). In the women with HPV-16 infection, the prevalence in cervicitis  was lower than that in CIN I , (P < 0.0001). HPV-16 in CIN I was significantly lower than that in CIN II/III , (P < 0.0001). Same as in the multi-high-risk HPV infection, there was no significant difference between HPV-16 persistent infection in CIN II/III and that in CC , (P = 0.829). However, there was some difference in HPV-58 and HPV-33 infection. Although it was significantly lower in cervicitis with HPV-58  and HPV-33  compared to in CIN I infected with HPV-58 , (P < 0.0001) and HPV-33 , (P < 0.0001), there were no significant differences when comparing between in CINI and in CIN II/III with infection of HPV-58 , (P = 0.522), as well as HPV-33 , (P = 0.094). It was similar in the groups of CIN II/III and CC. There were no significant differences in HPV-58 , (P = 0.571) versus in HPV-33 , (P = 0.378). HPV-18, -52, and multiple HPV infection had alike patterns. In details, HPV-18 persistent positivity in cervicitis  was significantly lower as compared to that in CIN I , (P < 0.0001), HPV-52 persistent positivity in cervicitis  was significantly lower as compared to that in CIN I , (P < 0.0001), and multiple HPVs persistent positivity in cervicitis  was also significantly lower as compared to that in CIN I , (P < 0.0001). The prevalence of HPV-18 in CINI was significantly lower than that in CIN II/III , (P = 0.016), HPV-52 in CINI was significantly lower than that in CIN II/III , (P = 0.031) and multiple HPVs in CINI was also significantly lower than that in CIN II/III , (P < 0.0001) . Exceptionally, the prevalence of HPV-18 persistent infection in CIN II/III was significantly lower than that in CC , (P = 0.013). However, there was no significant difference between HPV-52 persistent infection in CIN II/III and that in CC , (P = 0.157) as well as multiple HPV infection in CIN II/III to in CC , (P = 0.271).Among 15257 eligible women chosen for cervical biopsy, 1108 women were diagnosed to have CC. We studied the associations of HPV persistent infection with various clinicopathologic characteristics . The results were shown in Table Our results found that there were no significant differences between HPV persistent positivity and clinicopathologic characteristics . The associations between the genotypes of HPV persistent positivity and various clinicopathologic characteristics were also studied. The results showed that there were no significant differences between HPV-16, -52 persistent positivity and clinicopathologic characteristics . HPV-58, -33 and multiple HPV persistent positivity were significantly associated with older age . HPV-18 persistent positivity was significantly associated with adenocarcinoma and lymphatic metastasis . Meanwhile, immunohistochemical staining found that HPV-E6 protein was positively correlated with adenocarcinoma and lymphatic metastasis (P < 0.001) . The mean ± SD of the overall 3-year survival time for CC patients with HPV-18 persistent positivity (43.294 ± 2.978months) was significantly lower than that for CC patients with HPV-18 transient positivity or negativity (59.271 ± 0.415months), with P 0.0001. HPV-18, lymphatic metastasis, pathological grade, FIGO stage and age were chosen for Cox regression. Multivariate analyses showed that the HPV-18,  and lymphatic metastasis  were independent predictors for 3-year overall survival time.Cervical cancer remains the second most common malignancy in women in the world . PersistOur results showed that in HPV infected women , , 17.35%  suffered persistent infection. A total of 21 HPV genotypes were detectable in this study, including 14 high-risk types , 2 intermediate-risk types (CP8304 and 53), and 5 low-risk types . Among of these types, the most common HPV types in persistent positivity were HPV-16, HPV-58, HPV-18, HPV-52 and HPV-33.Furthermore, our results also showed that the prevalence of HPV in persistent infection was increased with the severity of CIN. However, there were no significant differences between HPV persistent infection in CIN II/III and that in CC. The prevalence of HPV-16,-58 and multiple HPV persistent infections in normal cervicitis were significantly lower than those in CIN. There were no significant differences between HPV-16,-58 and multiple HPV persistent infection in CIN II/III and that in CC. Then we hypothesis that HPV-16,-58 and multiple HPV persistent infection may be involved in cervical intraepithelial neoplasia while have little contribution to the carcinogenesis from CIN to cervical cancer. The prevalence of HPV-18 persistent infection was increased with the severity of cervical lesion. The prevalence of HPV-52, -33 persistent infections in normal cervicitis were significantly lower than those in CINI. However, there were no significant differences between HPV-52,-33 persistent infections in CINI and that in CIN II/III and that in CC.Additionally, HPV-58, -33 and multiple HPV persistent positivity were significantly associated with older age. Recently, some studies have confirmed our results ,18. SomeIn conclusion, we provided extensive results on HPV genotype prevalence and distribution in the northeast of China. HPV genotyping is worthwhile to perform because of its independent prognostic value in cervical cancer, and the predicting models for death could be useful for counseling the individual patient and stratifying study patients in HPV type-specific vaccines.The authors declare they have no competing interests.SW carried out the HPV DNA detection, participated in the sequence alignment and drafted the manuscript. HW carried out the HPV DNA detection,and participated in collected cases. NW carried out the immunohistochemistry analysis,and participated in collected cases. SZ participated in the sequence alignment and drafted the manuscript,statistical analysis and experimental guidance. YZ carried out the colposcopic examination. QR carried out the HPV DNA detection and human papillomavirus genotyping analysis. WJ carried out the immunohistochemistry analysis. QX participated in collected cases. XL participated in collected cases. XQ participated in collected cases. LZ participated in collected cases. XG participated in collected cases. XS participated in collected cases. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/160/prepub


KIR, HLA class I and II, and single-nucleotide polymorphisms (SNPs) of cytokines with HPV-related cervical disease. We selected 79 non-related, admixed Brazilian women from the state of Paraná, southern region of Brazil, who were infected with high carcinogenic risk HPV and present cervical intraepithelial neoplasia grade 3 (CIN3), and 150 HPV-negative women from the same region matched for ethnicity. KIR genes were genotyped using an in-house PCR-SSP. HLA alleles were typed using a reverse sequence-specific oligonucleotide technique. SNPs of TNF −308G>A, IL6 −174G>C, IFNG +874T>A, TGFB1 +869T>C +915G>C, and IL10 −592C>A −819C>T −1082G>A were evaluated using PCR-SSP. The KIR genes were not associated with HPV, although some pairs of i(inhibitory)KIR-ligands occurred more frequently in patients, supporting a role for NK in detrimental chronic inflammatory and carcinogenesis. Some HLA haplotypes were associated with HPV. The associations of INFG and IL10 SNPs potentially reflect impaired or invalid responses in advanced lesions. The genetic variability of the host contributes to the risk of human papillomavirus (HPV)-related cervical disease. Immune response genes to HPV must be investigated to define patients with the highest risk of developing malignant disease. The aim of this study was to investigate the association of polymorphic immune response genes, namely  Human papillomavirus (HPV) infections occur frequently in healthy individuals, and high carcinogenic risk (HR) HPV types are a major causal factor for cervical cancer. Persistent infection with one among approximately 15 genotypes of carcinogenic HPV causes almost all cases of cervical cancer; type 16 and HPV-18 account for more than 70% of the cervical cancers detected worldwide. Despite being considered a preventable disease, cervical cancer remains the second most common malignancy among women worldwide, with a higher incidence in underdeveloped countries .The major mechanisms by which HPV contribute to neoplastic initiation and progression involve the activity of two viral oncoproteins, E6 and E7, which interfere with the critical cell cycle tumor suppressive proteins p53 and retinoblastoma (Rb). However, HPV infection alone is not sufficient to induce malignant transformation. The multistep process of tumor formation requires the contribution of other significant cofactors, such as individual genetic variations, intratypic HPV variability, and environmental factors , 2. The The innate immune system comprises the first line of defense following HPV infection. It provides nonspecific protection and enhances the adaptive immune response . InflammKIR genes, HLA classes I and II, and SNPs of cytokines, with HPV infection in Brazilian women. To define patients with the highest risk of developing malignant diseases, the interaction between the host immune response and HPV infection must be investigated. The goal of the present study was to investigate the association of the polymorphic immune response genes, namely, the KIR locus comprises an approximately 150 kb region located on chromosome 19q13.4, which encodes a group of inhibitory and activating KIR molecules. KIRs are key receptors of human natural killer (NK) cells, a subset of lymphocytes that trigger early innate immune response against infection and tumors [The d tumors .The major histocompatibility complex (MHC), also known as the human leukocyte antigen (HLA) complex, located on chromosome 6p21.3, is the most polymorphic genetic system in mammalians and has been studied with regard to a wide variety of diseases of distinct etiology. The fundamental role of the different molecules within the MHC is antigen processing and presentation to the T-cell receptor (TCR), which is crucial for the cell interactions in cell-mediated immunity .IL10 region [TNF) [(IL6) [IFNG) [TGFB1) region [Polymorphisms of regulating regions of cytokine genes have been correlated with its production and can confer flexibility in the immune response to the viral infections and cancer biology. Five independent regions were investigated: chromosome 1: 0 region , chromos) [(IL6) , chromos) [IFNG) , and chr) region .Patients comprised 79 nonrelated, admixed Brazilian women from the state of Paraná in the southern region of Brazil, who were infected with HR-HPV and present cervical intraepithelial neoplasia (CIN) grade 3 (CIN3) and 150 women from the same region matched for ethnicity who were HPV-negative/normal cytology. The study protocol was approved by the ethics committee, and all selected patients signed the free and informed consent form.In the Paraná state, the degree of the European ancestry is high (80.6%), with a small but significant contribution of African (12.5%) and Amerindian (7.0%) genes according to Probst et al. , and theThe patients were diagnosed with high-grade squamous intraepithelial lesion (HSIL) by cytologic smears, CIN3 by histopathology, and also with HR-HPV. in situ or invasive adenocarcinoma (ISCC); or invasive squamous cell carcinoma (SCC). The cervical and endocervical material was collected with the aid of an Ayre spatula and a cytobrush for cervical smears and for PCR amplification . The cytological smears were evaluated and reported according to the Bethesda system as atypical squamous cells of undetermined significance (ASC-US); atypical squamous cells of undetermined significance, which cannot exclude a high-grade squamous intraepithelial lesion (ASC-H); low-grade squamous intraepithelial lesion (LSIL); high-grade squamous intraepithelial lesion (HSIL); in situ or invasive adenocarcinoma. The histological criteria for CIN were the failure of maturation of the squamous epithelium, nuclear hyperchromasia, and an increased nucleus/cytoplasm ratio. The intensity of these criteria was used to stage the degree of CIN or carcinomas [The cytological criteria for HSIL diagnosis adopted were squamous cells, either isolated or present in small fragments with fewer than ten cells. The cells were the length of the metaplastic cells, showing an increase in the proportion in the nuclear area. The nuclear irregularities, including hyperchromasia, chromatic clustering, irregularity, thickening, or multinucleation, were also used as important cytological criteria . The hisrcinomas . The HSIμg/mL ethidium bromide, and photodocumented under UV light (approximately 450 bp). Coamplification of the human beta-globin gene (approximately 268 bp) was performed as an internal control, using primers GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′) and PC04 (5′-CAACTTCATCCACG TTCACC-3′) under the same conditions as the HPV PCR. Two types of controls were also included in each reaction series: “no DNA” (negative control) and “HPV-positive DNA” (positive control) [For HPV molecular detection, genomic DNA was extracted using DNAzol . HPV polymerase chain reaction (PCR) amplification for HPV was carried out using MY09 (5′-CGTCCMAARGGAWACTGATC-3′)/MY11(5′-GCMCAGGGWCATAAYAATGG-3′), and the PCR product was electrophoresed on a 1.5% agarose gel, stained with 1 control) .HpyCH4V . The following HPV genotypes were determined for this genotyping method: HR , UR—undetermined risk (26 and 53), and LR—low-risk . The genotypes were grouped according to the International Agency for Research on Cancer (IARC) based on the carcinogenic potential and evolutionary branch [Genotyping was carried out using PCR-based restriction fragment length polymorphism analysis using μL of the buffy coat obtained from 5 mL of EDTA anticoagulant peripheral blood using the EZ-DNA Kit . The DNA concentration was then determined using a Qubit fluorometer .Genomic DNA samples were extracted from 150 All genotyping methods were validated using previously typed and tested reference samples. Positive and negative controls were included in all genotyping method.KIR genes and one pseudogene  were studied using an in-house polymerase chain reaction using the sequence-specific primer method (PCR-SSP) according to Martin et al. [Fourteen n et al.  and adapn et al. . PrimersHLA classes I and II allele typing was conducted using the reverse sequence-specific oligonucleotide technique  with Luminex xMap technology . HLA groups 1 (C1) and 2 (C2) of HLA-C and group Bw4 of HLA-B were defined according to Carrington and Norman  and PeteTNF −308G>A  (rs1800629), IFNG +874T>A (rs2430561), IL6 −174G>C (rs1800795), IL10 −1082G>A (rs1800896), IL10 −819C>T (rs1800871), IL10 −592C>A (rs1800872), TGFB −509T>C (rs1800469), and TGFB1 +915G>C (rs1800471)  was performed to genotype the following SNPs: 1800471) , accordiKIR, HLA, and cytokines were calculated by direct counting. Fisher's exact test and the chi-square test with Yates' correction were used for statistical comparisons. P ≤ 0.05 were considered significant, and P  values were adjusted by means of the Bonferroni correction to enable multiple comparisons. The odds ratio (OR) was calculated based on the cross product ratio and the exact 95% confidence intervals (CI) using the SISA statistical package (http://www.quantitativeskills.com/sisa/index.htm). Hardy-Weinberg equilibrium [http://cmpg.unibe.ch/software/arlequin3/).Allele, genotype, and haplotype frequencies of ilibrium  was deteKIR haplotype frequencies were in Hardy-Weinberg equilibrium. The distributions of allele frequency ratios for all analyzed genes and for KIR genes frequencies in patients and controls  and in the combination of KIR-HLA ligands with the HPV disease . The number and type of inhibitory KIR-HLA pairs were evaluated , and theTwenty-six KIR haplotypes were observed in HPV patients, 10 of them were not found in controls, and, otherwise, 13 others haplotypes were present only in controls . There wNo differences were observed in the distribution of HLA-A, B, and DRB1 allele groups between patients and controls . HLA-A∗02-HLA-B∗51 haplotype showed a reduced frequency in HPV patients  than in controls; the other six haplotypes were more frequent in the patients, but a large CI was obtained due to the small number of patients and controls , and for an intermediate producer of IL-10  defined by the haplotype GCC/ACC and GCC/ATA . There were significant differences for the low producer phenotypes of INF-otype AA , which h GCC/ATA . The GCCIt is widely accepted that cofactors, including endogenous hormones and genetic factors, such as HLA and other genes related to the host immune response, may have important roles in the development of HPV-cervical lesions . InheritKIR genes encode molecules that convey either inhibitory or activating signals (iKIR and aKIR) to NK cells and to a subset of CD8+ T cells. Binding of iKIR (designated 2DL and 3DL) to specific HLA allotypes has been clearly demonstrated and correlated to the ability to inhibit NK cytolysis of target cells bearing these HLA molecules. These interactions are remarkably complex, and synergistic relationship between these polymorphic loci may regulate NK cell-mediated immunity against viral infections [NK cells play an important role in innate immunity against infected and transformed cells as part of the immune surveillance process. fections . KIR2DS3, KIR2DS5, and KIR2DL5 were more frequent in the patient group. KIR2DL5, an inhibitory receptor, possesses a combination of genetic, structural, and functional features that make it unique among the KIR [KIR3DS1 can induce a persistent, weak inflammatory reaction to HPV that results in continuous tissue injury, similar to HBV-susceptible genes [KIR3DS1 is related to an increased risk of neoplasia, particularly in the absence of protective inhibitory KIR-HLA. In contrast, Arnheim et al. [KIR3DL1 is associated with increased risk of CIN.No relationship was found between KIR genes and HPV-related cervical disease in Brazilian patients, consistent with the findings of Song et al. in Korean patients . Althoug the KIR  and potele genes . Carringle genes  found thm et al.  indicateThe frequencies of the ligand groups  did not differ between patients and controls (data not shown). However, Madeleine et al.  demonstrIn the present work, KIR2DL1-C2 was more frequent in patients (70%) than in controls (38.5%). The strength of NK inhibition varies according to the receptor and the ligand: KIR2DL1-C2 provides a stronger inhibition than other iKIR-HLA , 35. TheHLA-A∗02-B∗51 haplotype was associated with resistance to disease. Susceptibility to HPV infection or cervical cancer and precancerous lesion development was associated with the HLA class II: HLA-DRB1 alleles [HLA-DQB1∗03 increases and DRB1∗13 decreases the risk of cervical cancer. In other Brazilian populations, Maciag et al. [DRB1∗15:03, DRB1∗04:05, and DQB1∗06:02 alleles.HLA class I and class II proteins are central to host immune responses to viral infections and other pathogens. They are the most polymorphic genes in the human genome, and variations in the peptide binding groove of these proteins influence antigenic specificity. Numerous studies have evaluated the association of HLA with HPV infection and the importance of HLA in the pathogenesis of cervical neoplasia . Similar alleles , 39–51;  alleles , 49–51;  alleles , 52, 53. alleles , 55. In g et al.  found thP = 1.6 × 10−18); the second is between HLA-DRB1 and HLA-DQA1 ; and the third is at HLA-DPB2 . Previously reported associations of B∗07:02 and DRB1∗15:01-DQB1∗06:02 with susceptibility to DRB1∗13:01-DQA1∗01:03-DQB1∗06:03 with protection against cervical cancer were confirmed. A genome-wide association study of 731.422 SNPs was performed in cervical cancer patients and controls . Three iThe variable results for the association between HLA and disease could be related to the differences in the distribution of HLA in the population; the disease phases ; and HPV types. The effects of HLA polymorphisms on cervical carcinogenesis and their biological mechanisms remain unknown. Previous findings suggest a strong link between an inefficient immune response, particularly inefficient cell-mediated and innate immunity, both of which involve classes I and II HLA alleles, and susceptibility to HPV infection. HPV infections are more prevalent and more likely to persist in immunosuppressed individuals. γ and TNF-α can induce a cell-mediated immune response, whereas Th2 cytokines such as IL-6 and IL-10 induce predominantly a humoral immune response and immunomodulation of the cellular response. The Th2 cytokine profile is associated with progression to cervical cancer [Accumulating epidemiological evidence suggests that polymorphisms in cytokine genes may be involved in the etiology of cervical carcinoma . Th1 cytl cancer . TNF, IFNG, IL6, IL10, and TGFB1 which are multifunctional cytokine that have been implicated in inflammation, immunity, and cellular organization and have been proposed to play important roles in infection and cancer biology.In the present study, we genotyped SNPs of IFNG +874A/A genotype, which characterized the low producer phenotype of IFN-γ and was more frequent among patients compared with controls. According to Telesheva et al. [Susceptibility to infection was observed in patients with the a et al. , the outIL10 GCC/ACC and GCC/ATA genotype, which characterized the intermediate producer phenotype of IL-10, were less frequent in patients, suggesting protection against disease. The IL-10 high producer phenotypes was more frequent in patients, although this increased frequency was not significant and might be related to an immunosuppressive response and development of HPV-positive cervical cancer. Serum levels of IL-10 and its expression in tumor cells are elevated in patients with cervical cancer [IL10 −1082G>A was not associated with susceptibility to the development of cervical cancer or HPV infection [In the current study, the l cancer . IL-10 pl cancer . The SNPnfection . β is well known for its antiproliferative effects; however, neoplastic cells often lose their sensitivity to TGF-β. Iancu et al. [β signaling pathway might contribute to the malignant progression of cervical dysplasia. In the present study, SNPs of TGFB1 +869, +915 were not associated with HPV infection. TGF-u et al.  showed tα, which is secreted mainly by activated macrophages, is an extraordinarily pleiotropic cytokine that has a central role in immune homeostasis, inflammation, and host defense and could be involved in protection against HPV infection by modulating viral replication. Dysregulated TNF expression within the tumor microenvironment appears to favor malignant cell tissue invasion, migration, and ultimately metastasis [TNF −308G>A and HPV infection or cervical cancer. However, our findings differ from those reported for the Argentina population, among whom the high producer allele TNFA −307A was associated with an increased risk for the development of cervical cancer [TNF-tastasis . Our fintastasis , who deml cancer .IL6 encodes a cytokine that plays important roles in the risk for cervical carcinogenesis. In the present study, there was no significant association between IL6 and HPV-related cervical disease. However, a previous report has shown a significant association between the IL6-rs2069837 SNP and an increased risk of cervical cancer [l cancer .KIR genes were not associated with HPV, although some pairs of iKIR-ligands were more frequent in patients, suggesting that NK cells play a role in detrimental chronic inflammatory conditions and in carcinogenesis. HLA was associated with HPV and participated in the immune response, although its function in carcinogenesis remains unclear. The polymorphic INFG and IL10 genes were associated with the outcome of HPV infection and might be indicative of impaired or invalid immune responses in patients with advanced stage lesions. Additional studies of the immune response to HPV are needed to better define the risk of developing malignant diseases associated with HPV infection.The genetic variability of the host contributes to the risk of HPV-related cervical disease.


L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples. Marinomonas mediterranea, catalysing the stereospecific oxidative deamination of L-amino acids to the corresponding α-keto acids with the release of ammonium and H2O22O2All the described L-amino acid oxidases  are flavoenzymes, except the lysine oxidase of 2O22O2 production constitute attractive assays. In particular, the horseradish peroxidase (HRP) which acts as H2O2 sensitive probes is the most widely used for the detection of H2O2 due to its reasonable simpleness, sensitivity and reproducibility While a range of methods are applicable to the detection of LAAO activity, including measurement of production, such as ammonia 2O2 and other easily oxidizable compounds. It was also applied to biosensors for transition of metal hexacyanoferrate 2O2 based on the visualization of Prussian blue-forming reaction. Recently, Prussian blue agar medium assay for the detection of H2O2 was preliminarily reported to screen the LAAO activity 2O2 concentration. Compared with HRP assay, Prussian blue agar assay shows the benefits, including more simpleness, cost-effectiveness and convenience. Another key advantage of Prussian blue assay over HRP assay is that Prussian blue assay can be directly used for in-gel determination of the numbers and molecular weights of LAAO on the coupled SDS-PAGE after visualization of Prussian blue-forming reaction.Prussian blue has been known since 1704, and has been definitely one of the most ancient coordination materials and an important pigment for color uses 2O2 in electrochemical reactions because iron (III) and potassium hexacyanoferrate (III) can be oxidized to yield the blue precipitate of Prussian blue by the sequential reactions as shown below. It should be noted that in this assay H2O2 acts as electron donor.2O2. In addition, it was found that without peptone and yeast extract the resultant Prussian blue agar plate was opaque and greenish-white in color and had also no effect on color mature of Prussian blue.Prussian blue assay has been widely used in sensors to detect the presence of H−, followed by the reduction of Fe3+ to Fe2+ by CN− and subsequent formation of Prussian blue (either “water-insoluble Prussian blue” FeIII4[FeII(CN)6]3 or “water-soluble Prussian blue” KFeIIIFeII(CN)6). In addition, the bigger diameters of Prussian blue holes were generated by the lower pH of background solution, probably due to the stronger degradation of hexacyanoferrate. On the other hand, the background solutions with pH values from 10 to 14 all generated fade-white holes, probably due to the white precipitation of Fe(OH)3. Next, the standard 5 mM H2O2 solutions with the same pH of 7.5 were prepared for Prussian blue agar test. As indicated in row 2, all standard H2O2 solutions presented uniform and reproducible blue holes with almost the same size. In contrast, the 5 mM H2O2 solutions with different pH values ranging from 1 to 14 yielded different resultant blue holes  with the increase of H2O2 from 0 to 20 mM, but not significant (P>0.05) with the increase of H2O2 from 20 mM to 30 mM (2O2 with concentrations from 100 µM to 500 µM. As displayed in 2O2), all the H2O2 solutions with different concentrations gave blue holes. The diameters of blue holes expectedly increased with the increase of H2O2 concentration. However, it was difficult to very accurately measure the diameters of blue holes caused by H2O2≤400 µM. To observe the correlation between H2O2 concentration from 0 to 20 mM and Prussian blue diameter, the data in 5.611, where x is the diameter of blue hole and y the H2O2 concentration. Further plotting in 2O2≤20 mM the change in diameter of blue hole was a function of logarithm of H2O2 concentration with linear fits under the equation y = 1.772x–1.890, where x is the diameter of blue hole and y the logarithm of H2O2 concentration. To estimate the LAAO activity described as H2O2 concentration fashion using the above extracted equations, the culture supernatants with different LAAO activities from strain B3 and strain R3, two LAAO-producers, were used to oxidize the substrates L-Leu and L-Met, respectively, in separate reactions, followed by Prussian blue agar (without peptone and yeast extract) assay. The results in 2O2 concentrations of 1.65 mM and 1.40 mM, respectively, based on the above extracted equations in 2O2 concentrations of 3.32 mM and 1.72 mM, respectively. To confirm the reliability of fitted curves or equations, the standard H2O2 with concentrations of 1.65 mM, 1.40 mM, 3.32 mM and 1.72 mM were used for the assay and finally gave the blue holes with diameters of 1.19 cm, 1.16 cm, 1.35 cm and 1.20 cm, respectively, all perfectly agreeing with our calculated concentrations based on the fitted equations. Therefore, the LAAO activity of B3 supernatant with L-Leu and L-Met as substrates had 0.330 U/ml and 0.280 U/ml, respectively, whereas the LAAO activity of R3 supernatant with L-Leu and L-Met as substrates had 0.664 U/ml and 0.344 U/ml, respectively, indicating that R3 supernatant has higher LAAO activity than B3. All these findings indicate that Prussian blue agar assay is feasible to distinguish the LAAO activity. Moreover, the extracted equations in the present study are reliable to quantitatively determine the LAAO activity. To confirm the method with another enzyme source using a purified enzyme, the commercial Crotalus adamanteus venom LAAO  was applied to Prussian blue assay with L-Leu as substrate. 2O2 based on the extracted equation. When 6.11 mM standard H2O2 was applied to Prussian blue assay, a blue hole with 1.50 cm diameter appeared, indicating great reliability of Prussian blue assay for determination of LAAO activity.To investigate the sensitivity of Prussian blue agar to Hto 30 mM . To push2O2 with different concentrations from 0.5 mM to 20 mM, after oxidation by R3-LAAO, L-Leu, L-Lys, L-Phe, L-Asn, L-Trp, L-Met, L-Arg, L-Ile, L-Tyr, L-cystine, L-Hpa, L-Phg and L-Val yielded blue holes. The diameters of blue holes further showed that R3-LAAO had different activities to different substrates with an order of L-Leu>L-Lys≈L-Phe>L-Asn≈L-Met≈L-Trp>L-Ile≈L-Arg>L-Tyr≈L-cystine>L-Hpa≈L-Phg>L-Val, an overall agreement with the results obtained from another detection method using 2, 4-dinitrophenylhydrazine (DNP) which can react with carbonyl group of a-keto acids derived from corresponding amino acids to generate dinitro-phenylhydrazone with a brown-red color  assay. It was found in ed color . On the Pseudoalteromonas sp. R3 was precipitated by adding ammonium sulfate to 40% saturation. After centrifugation, the supernatant and precipitate, namely 40% supernatant and 40% precipitate, respectively, were collected for Prussian blue agar assay. As shown in A simple and sensitive method for in-gel determination of LAAO numbers and their approximate molecular weights is highly promising. To achieve this purpose, we tried to develop an SDS-PAGE coupled Prussian blue assay. First, the harvested supernatant from 2O2 solutions or H2O2 produced by LAAO activity. The diameters of the formed blue holes is perfectly a function of H2O2 concentration with exponential fits or logarithm of H2O2 concentration with linear fits varying from 0.5 mM to 20 mM, thus making Prussian blue agar assay quantitatively determine the LAAO activity as H2O2 concentration based fashion. Moreover, coupled with SDS-PAGE, Prussian blue agar assay can be directly used to not only sense the presence of LAAO activity, but also determine the numbers and approximately molecular weights of the LAAO protein in one assay, showing significant advantages in comparison to usual spectrophotometric and fluorometric detection methods for LAAOs.In this communication, we demonstrated that Prussian blue agar assay can be used to detect the standard H3+ precipitation. Fortunately, common fermentation solutions produced by microorganisms or direct enzymatic reaction solutions have pH values in the range of 4∼12, thus giving broad applicability of this method.The pH of detection solution is a critical factor to Prussian blue agar assay. There is no significant influence of detection solutions with pH 5 up to 12 on color development of Prussian blue agar. However, it was not recommended to use this assay under pH either below 4 or above 13 because both situations can shield the coloration of Prussian blue. Most probably, the peracid condition can cause the degradation of hexacyanoferrate, while peralkaline situation will make Fe2O2 solutions or H2O2 already produced by LAAO activity using Prussian blue agar assay, it is unnecessary to add peptone, yeast extract, etc. The simplest way to make Prussian blue agar plate is just to mix agar with iron (III) and hexacyanoferrate (III). However, to directly determine LAAO activity without oxidation reaction beforehand, it is necessary to supply substrates for LAAO, such as amino acid and peptone, into Prussian blue agar. Therefore, LAAO can oxidize its substrates present in Prussian blue agar to release H2O2 and trigger the coloration of Prussian blue agar. Similarly, to screen the LAAO-producing microorganisms, it is also necessary to add substrates of LAAO to Prussian blue agar plate.When using Prussian blue agar assay, we found that prior to autoclave it was a key step to adjust the pH of Prussian blue agar solution to alkaline below 13 by adding NaOH. Otherwise, the Prussian blue agar plate will be very sensitive to the pH of detection solution. Most probably, NaOH can render Prussian blue agar buffering capacity. The key components of Prussian blue agar are iron (III) and hexacyanoferrate (III). Actually, these two chemicals are kind of flexible to mix with a variety of mediums, thus allowing wide applicability of this Prussian blue assay to screen LAAO-producing microorganisms. To detect the standard H2O2 ranging from 0.5 mM to 20 mM in concentration. In fact, H2O2 with concentration of around 100 µM still can cause the formation of Prussian blue holes  to determine the numbers of LAAO and estimate its approximate molecular weights, showing clear advantages in comparison to commonly used spectrophotometric and fluorometric LAAO detection methods. Knowing directly the numbers and molecular weights of LAAO can be very helpful for further purification and characterization of this enzyme. In particular, the sliced target bands with LAAO activity could be directly analyzed with different techniques, such as protein sequencing and LC-MS/MS analysis.A variety of methods have been developed to detect LAAO activity. Among them, the spectrophotometic and fluorometric methods are the most widely applied to detect LAAO activity through HRP involved reactions. A spectrophotometric 96-well microtiter plate is suitable for processing of large numbers of samples, although fluorometric measurement is more sensitive than spectrophotometric measurement. In many laboratories absorption-detecting micro-plate readers are more available than fluorescence-detecting ones. In these spectrophotometric assays, although HRP is hydrogen peroxide sensitive probes, it might be easily inactivated, unstable and expensive. Moreover, most of the HRP substrates are mutagenic, carcinogenic or extremely toxic compounds. In contrast, our Prussian blue agar assay doesn't need to rely on any detection instrument and can detect a large number of samples in the same reaction condition. Other advantages of our assay over spectrophotometic and fluorometric methods include convenient preparation, long-term storage at room temperature, simple operation and feasibility for direct determination of LAAO numbers and molecular weights. Rau et al. Pseudoalteromonas sp. B3, was isolated from the intertidal zone sludge sample  located at Dinghai, Zhoushan, China. Phylogenetic analysis of 16S rDNA indicated that it belongs to the genus Pseudoalteromanas and its closest neighbor is the strain of Pseudoalteromonas viridi (98.5% identity). Previous detections of the produced α-keto acids 4+ (pH paper method) and H2O2  all revealed that marine bacterial B3 can produce LAAO (data not shown). The pH paper method for detection of NH4+ was performed as follows: 3 ml of 1 M NaOH was added to 50 ml of fermentation supernatant in 250 ml glass flask; then the color change of special pH paper indicator on glass flask was monitored by eye to trace the NH3 released by NaOH from solution. A spontaneous red-pigmented strain of B3, designated as Pseudoalteromonas sp. R3, showed higher LAAO activity, which was confirmed by measuring the concentrations of the produced α-keto acids 2O2  (data not shown).A yellow-pigmented LAAO-producing bacterial strain, designated as Strain R3 or B3 was grown in a marine medium (MM)  for 2 days at 25°C with shaking at 160 rpm. Next, 1 ml of the above culture was added to 250 ml conical flask containing 50 ml MM for fermentation at 28°C with shaking at 160 rpm. After 96 h fermentation, the culture supernatant was harvested with a centrifugation at 8000 rpm for 10 min at 4°C.3·6H2O and potassium hexacyanoferrate (III) in separate 50 ml water to make solutions A and B, respectively; (2) prepare 900 ml MM medium with 2% agar; (3) mix 50 ml solution A with 50 ml solution B to give a 100 ml reddish brown solution C (Prussian brown); (4) pour the 100 ml solution C into 900 ml MM agar medium and adjust pH using NaOH to around 7.5 with resultant formation of a deep green precipitate; (5) autoclave the mixture at 115°C for 30 min and pour into glass Petri dish to make agar plate; (6) make circular wells with diameter 6 mm on plate; (7) for detection, add 50 µl reaction solution to each well and wait for 30 min at room temperature for color change; (8) visualize the Prussian blue forming and measure the blue hole size.Prussian blue agar test was performed according to the report 2O2/h at 37°C.Amino acid was dissolved in 10 ml harvested culture supernatant harboring LAAO activity to have a final concentration of 5 mM. This mixture was incubated for 30 min at 37°C for oxidation reaction. Unless otherwise stated, the mixture pH after oxidation was adjusted to around 7.5. Then, 50 µl of solution was subjected to Prussian blue agar assay. One unit (U) is defined as the amount of enzyme that catalyses the formation of 1 mM HAs carbonyl derivatives, a-keto acids was detected by using 2, 4-dinitrophenylhydrazine (DNP) which can react with carbonyl group to generate dinitro-phenylhydrazone with a brown-red color according to the report Pseudoalteromonas sp. R3 to give 40% saturation and kept on ice for 5 h. After centrifugation at 10000 rpm for 30 min at 4°C, the supernatant (40% supernatant) and precipitate were collected. The precipitate was dissolved in 10 ml of 0.02 M sodium phosphate buffer (pH 7.4) and dialyzed three times against the same sodium phosphate buffer overnight at 4°C to have a final volume of 20 ml (40% precipitate). The 40% supernatant was further precipitated by slowly adding solid ammonium sulfate to 60% saturation on ice. After incubation on ice for 5 h, the resultant mixture was centrifuged at 10000 rpm for 30 min at 4°C to have the supernatant (60% supernatant) and precipitate. Then, the precipitate was dissolved in 10 ml of 0.02 M sodium phosphate buffer (pH 7.4) and dialyzed three times against the same sodium phosphate buffer overnight to have a final volume of 20 ml (60% precipitate). Finally, 50 µl of each 40% supernatant, 40% precipitate, 60% supernatant and 60% precipitate was used for Prussian blue agar assay to track the LAAO activity.Solid ammonium sulfate was slowly added to 500 ml culture supernatant produced by The solutions with LAAO protein were mixed in a 4∶1(v/v) ratio with 4-fold sample loading buffer . Then, without heating, 20 µl of the resultant mixture was resolved in each well of an SDS-polyacrylamide gel (SDS-PAGE) with a stacking gel of 5% and separation gel of 12%, as described by Laemmli Figure S12O2 with concentration from 100 µM to 500 µM on Prussian blue agar.Measurement of H For all detections, 50 µl solutions were added to 6 mm circular well. CK: negative control, without H2O2.(TIF)Click here for additional data file.Figure S22O2 produced by Prussian blue agar measurement of HCrotalus adamanteus2O2 (right). LAAO (caLAAO) activity with L-Leu as substrate (left) and 6.11 mM standard H The diameters of the blue holes were indicated under the holes.(TIF)Click here for additional data file.Figure S3Tracking of LAAO activity after step-by-step precipitation by solid ammonium sulfate. The harvested different samples  and 5 mM H2O2 standard solution were separately added to the wells at room temperature for 30 min before photographing. As expected, the MM medium without LAAO enzyme did not give any blue hole, whereas the others all gave the blue holes with different diameters.(TIF)Click here for additional data file.Figure S4SDS-PAGE coupled in-gel Prussian blue agar assay for determination of LAAO activity. After electrophoresis, different lanes on SDS-PAGE were sliced out for different treatment and subsequently put together on Prussian blue agar for color development. Lane M: standard protein marker stained with CBB; lane 1: 66 kDa bovine serum albumin (BSA) stained with CBB; Lanes 2 and 3: duplicate 60% precipitate samples from an LAAO producer Pseudoalteromonas sp. R3 without CBB staining; lane 4: a replicate of lane-2 and lane-3 with CBB staining; lane 5: the sliced protein bands from a lane-4 replicate as indicated by arrows which are near the formed blue band. The results showed that 60% precipitate sample had only one active protein band which can form Prussian blue band and its molecular weight was around 65 kDa.(TIF)Click here for additional data file.Figure S5SDS-PAGE coupled in-gel Prussian blue agar assay for detection of caLAAO activity. After electrophoresis, different lanes on SDS-PAGE were sliced out for different treatment and subsequently put together on Prussian blue agar for color development. Lane 1: R3-LAAO of 60% precipitate without CBB staining; lane 2: caLAAO without CBB staining; lane 3: a lane-2 replicate with CBB staining; lane 4: the sliced protein band from a lane-3 replicate as indicated by arrow. Results showed that caLAAO had only one active protein band and its molecular weight was larger than one of R3-LAAO.(TIF)Click here for additional data file.Table S12O2 concentrations by ANOVA.Statistical analysis of dependent variable blue hole diameters from different H(DOC)Click here for additional data file.


To evaluate hr-HPV persistence and associated risk factors in a prospective cohort of young unscreened women. Additionally, the relation between hr-HPV status and cytology/histology results is examined.10LiPA for HPV detection. Only women hr-HPV positive at sample month 12 were invited for a second year of follow-up. After study follow-up, available cytology/histology data were requested from PALGA . These data were compared with available cytology/histology data of the month 12 hr-HPV negative women from the same cohort. 44.1% of the hr-HPV types detected at study month 12, persisted during follow-up. HPV types 45, 31, 16 and 18 were most likely to persist with percentages of 60.0%, 56.8%, 54.4%,and 50.0%, respectively. Compared to newly detected infections at month 12, infections present since 6 months or baseline had an increased risk to persist . Other co-factors influencing persistence were, multiple HPV infections, smoking and multiple lifetime sexual partners. The percentage of women with a HSIL/CIN2+ (12.1%) in the persistent HPV group, was not significantly different (p = 0.107) from the 5.3% of the women who cleared the hr-HPV infection, but was significantly (p 0.000) higher than to the 1.6% of women in the hr-HPV negative control group.Two year follow-up of 235 out of 2065 young women (18–29 years), participating in a large, one year epidemiological study, with questionnaires, self-collected cervico-vaginal samples (Vibabrush), and SPFWe showed that HPV genotype, multiple infections, smoking, and multiple lifetime sexual partners are co-factors that increase the risk of hr-HPV persistency. Most importantly, we showed that hr-HPV infections are more likely to persist the longer they have been present and that women with a persistent hr-HPV infection have a high risk of HSIL/CIN2+ development. It has been well established that a human papillomavirus (HPV) infection is a necessary cause in the development of cervical intraepithelial neoplasia (CIN) and cervical cancer Most investigators define a persistent HPV infection as detection of the same HPV type, or group of types, on two consecutive visits, but these could be from 2 months up to 72 months apart Several risk factors have been identified that are associated with HPV persistence. Especially viral characteristics like viral load, and HPV genotype are linked to persistence In this study, a prospective cohort of young unscreened women (18–29 years) in the pre-vaccine era was followed for 2 years, to examine the influence of viral factors , and co-factors  on hr-HPV persistence. Additionally we examined the relation between hr-HPV status and follow-up cytology/histology results.A large prospective cohort study on HPV prevalence, incidence and clearance was performed in the Netherlands, in 2007–2010. At study entry, 2065 unscreened women 18 to 29 years of age were included The women provided five self-samples with a 3-month interval  during the first year of the study. In the second year of follow-up, women provided two additional self-samples with a 6 month interval (month 18 and 24). All women received the self sample kit and the additional questionnaires by mail.tm, Tripath Imaging®, Inc., Burlington NC, U.S.A.), as described previously The self sample kit contained an explanatory letter, a questionnaire, an illustrated instruction form on how to perform the cervico-vaginal self-sample, a small brush in a sterile cover , and a collection tube containing medium (SurePath10primer sets 10 amplimers were used on SPF10-DEIA-positive samples to identify HPV genotype by reverse hybridization on a line probe assay (LiPA) , which detects the following HPV genotypes: low-risk HPV types: type 6, 11, 34, 40, 42, 43, 44, 53, 54, 55, 66, 70, 74, and “X” (DEIA positive and LiPA negative samples); and high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82.Broad-spectrum HPV DNA amplification was performed using a short-PCR-fragment assay. Extracted DNA was used for PCR amplification with the SPFAn HPV infection was considered cleared when a woman had two consecutive HPV negative samples nd study year was 6 months and persistence is generally defined as two consecutive HPV positive samples. Thus, a types–specific hr-HPV infection was considered persistent when it was detected for at least 6 months. Women who were positive at sample month 18 and/or month 24 were therefore considered to have a persistent type-specific hr-HPV infection in the 2nd study year , sample month 6 and sample month 12  prior to sample month 12 influences persistence in the 2h 12 see . This annd study year, a control group was selected from the women who were hr-HPV negative at sample month 12. These women were 27–29 years at study baseline, and had received an invitation of the Dutch national cervical cancer screening program. In order to relate cytology/histology results to the hr-HPV status, information of all sample points in the 1st and 2nd study year had to be available. The four women who were treated for abnormal cytology before sample month 18, were also excluded in the analysis of persistence related to cytology. This resulted in 224 women hr-HPV positive at sample month 12 and 336 women hr-HPV negative at sample month 12 (control group). PALGA data on gynecologic histo- and cytopathology, test date, and age of the woman at time of the test, were available for 166 (74.1%) hr-HPV positive women and 246 (73.2%) women of the control group. Due to privacy legislation, identification data were made anonymous by a Third Trusted Party . To relate these anonymous gynecologic histo- and cytopathology test results with the hr-HPV status, women were grouped.The study protocol was based on self-sampling for HPV detection. Gynecologic histo- and cytopathology data were thus not available at the end of the study. To be able to evaluate the relation between HPV status and cervical lesions, we requested information from PALGA  Month 12 hr-HPV positive women:nd study yeartype-specific hr-HPV type persistent in the 2nd study yearCleared type-specific hr-HPV type in the 2Control group: Month 12 hr-HPV negative womennd study year. To analyze the relation between hr-HPV status and histo- and cytopathology test results significance was calculated with the Fischer's exact test. All statistical analyses were performed using SPSS version 16.0 .The baseline characteristics of the study population were analyzed and presented in frequencies, mean and standard deviation (SD). The percentage and 95% confidence interval (95% CI) of the hr-HPV prevalence at sample month 12 and persistence in the second year of follow-up were calculated using the number of infections instead of the number of women. Data from the questionnaires were used to determine, with simple logistic regression analysis, whether co-factors like OCP use, smoking and sexual behavior influenced the persistence in the 2The 235 hr-HPV positive women included in this analysis had a mean follow-up time of 25.3 months (SD 1.5) and were all born in the Netherlands. At study baseline these women were 18-29 years of age with a mean age of 24 years (SD 3.2), and 233 (99.1%) women reported to ever have had sexual intercourse. Sixty six (28.1%) women reported that they were current smokers, and there were 165 (70.2%) current oral contraceptive pill (OCP) users. These baseline data are shown in nd study year, women had to be hr-HPV positive at sample month 12. Of the 235 women, 163 had one hr-HPV type, 54 had two hr-HPV types, 15 had 3 hr-HPV types, two had 4 hr-HPV types and one woman had 6 hr-HPV types at sample month 12. This resulted in a total of 330 type-specific hr-HPV infections detected at sample month 12. HPV 16 had the highest prevalence of 30.2%, followed by HPV 51 (19.1%), HPV 31 (16.6%), and HPV 52 (14.9%). HPV 18 was prevalent in 11.1% of the women. , HPV 16 (54.4%), and HPV type 18 (50.0%). Notably, HPV type 45 was only number 10 in prevalence with 11 (4.7%) type-specific infections at month 12, whereas HPV type 51  was one of the least persistent infections with a persistence rate of 25.0%.The percentage of the type-specific hr-HPV infections, present at sample month 12, persisting in the 2nd study year. Overall, a type-specific infection detected since baseline had an odds ratio (OR) of 4.99 (95% CI: 2.67-9.32) to persist in the 2nd study year, compared to infections newly detected at month 12. Infections first detected at sample month 6 also had an increased risk, OR of 3.09 (95% CI: 1.74-5.51), to persist in the 2nd study year, compared to newly detected infections. . . See Furthermore, Twelve of the 15 women with 1 lifetime sexual partner at baseline reported to have a new sexual partner during the study follow-up. Together they had 16 type-specific hr-HPV infections detected at sample month 12, of whom 10 were newly detected, four were first detected at month 6, and two at baseline. Because most hr-HPV types were newly detected at month 12 and newly detected hr-HPV infections are less likely to persist, this might be a confounding factor. However, confounding could not be ruled out with multiple logistic regression analysis because ‘duration of hr-HPV detection’ and ‘number of lifetime partners’ are based on different dependent variables, ‘number of infections’ and ‘number of women’, respectively.nd study year. . In the control group, 4 of the 246 women (1.6%) developed HSIL/CIN2+. Women with a persistent hr-HPV type were significantly more often diagnosed with HSIL/CIN2+ compared to women in the control group (p 0.000).In the study group 1a; 11 (12.1%) of the 91 women with a persistent hr-HPV infection were identified with HSIL/CIN2+. Four of the 75 women (5.3%) in group 1b, who cleared their type-specific hr-HPV infection in the 2In this 2-year prospective cohort study among young unscreened women, we showed that an already persistent hr-HPV infection has an increased risk to persist during follow-up compared to newly detected infections. Hr-HPV types persistent for 6 and 12 months or longer had a respective, threefold or fivefold, increased risk of persistence. Other studies also reported that HPV infections are more likely to persist the longer they have been present Persistence was also influenced by the genotype. The overall persistence of type-specific hr-HPV in this study is 44.1%, HPV types 45, 31, 16, and 18, had the highest rate of persistence and the highly prevalent HPV type 51 had a low tendency to persist. In agreement with our findings, other studies also showed that HPV 16, 18, 31, and 45 have an increased risk to persist compared to other HPV types Having multiple HPV infections is a risk factor for hr-HPV persistence. The risk to have a persistent type-specific hr-HPV infection during follow-up increased almost twofold in women with multiple (high-risk and/or low-risk HPV) HPV types at month 12. These results are in agreement with other studies showing that women with multiple infections have a higher risk to have a persistent type-specific HPV infection during follow-up Only two co-factors, smoking and number of lifetime sexual partners, were found to correlate with the likelihood that a hr-HPV type would persist. Smokers had a two-fold increased risk of hr-HPV persistence. Smoking is a known immune suppressor and a risk factor for cervical carcinoma Women with 1 lifetime sexual partner at baseline were at lower risk for a persistent infection than women with multiple lifetime sexual partners. The highest risk was found for women with 6–10 lifetime sexual partners at baseline. It should be noted, however, that the majority of the women with 1 lifetime sexual partner at baseline, had a new sexual partner during study follow-up and newly identified hr-HPV types. Newly detected hr-HPV types are less likely to persist, so this might be a confounding factor. In literature, the number of lifetime sexual partners is not a consistent risk factor for hr-HPV persistency.The use of OCP's is also a potential risk factor for the development of cervical carcinoma p 0.000) compared to women who were hr-HPV negative at study month 12 (1.6%).Previously it has been shown that women with a persistent type-specific hr-HPV infection are significantly more likely to have or develop CIN than those who were sequentially infected by different hr-HPV types or who cleared their infection Due to the anonymous histo- and cytopathology data, we were not able to correlate the histo- and cytopathology results to the type-specific hr-HPV types, nor if the woman had multiple HPV types. Therefore we could not confirm whether the infections with HPV types 45, 31, 16, and 18 were more likely to cause HSIL/CIN2+ and whether the number of infections was correlated to the severity of the lesion as described previously There is considerable interest in the possibility of using HPV testing as a primary cervical cancer screening tool Based on our results in this population of young women, we suggest that this interval should be a minimal of 6 months, because a 6 month persistent hr-HPV type had a threefold increased risk to persist during follow-up compared to a newly detected hr-HPV infection. However, this interval may be extended to 12 months, because 68.3% of the hr-HPV types already persistent for 12 months or longer continued to persist during follow-up. Therefore a 12 month interval for detection of type-specific hr-HPV persistency will select a group of women that needs close surveillance for HSIL/CIN2+ development in the following year(s).In conclusion, we showed that co-factors increasing the risk of hr-HPV persistency are, genotype specific , multiple infections, smoking, and multiple sexual lifetime partners. Most importantly, we showed that hr-HPV infections are more likely to persist the longer they have been detected and that women with a persistent hr-HPV infection have a higher rate of HSIL/CIN2+ detection in the following year. Thus, women with a persistent hr-HPV infection should be monitored for HSIL/CIN2+ development.


Purpose. To describe the acquisition, persistence, and clearance of HPV infection in women with CIN 2 followed up for 12 months. Methods. Thirty-seven women with CIN 2 biopsy, who have proven referral to cervical smear showing low-grade squamous intraepithelial lesions or atypical squamous cells of undetermined significance and tested for HPV, were followed up for one year with cervical smear, colposcopy, and HPV test every three months. HPV DNA was detected by the polymerase chain reaction and genotyping by reverse line blot hybridization assay. Results. CIN 2 regression rate was 49% (18/37), persistence as CIN 1 or CIN 2 was 22% (8/37), and progression to CIN 3 was 29% (11/37). Multiple HPV types were observed at admission in 41% (15/37) of cases. HPV 16 was detected at admission in 58% (11/19) of the cases that persisted/progressed and in 39% (7/18) of the cases that regressed. HPV 16 was considered possibly causal in 67% (10/15) of the cases that persisted or progressed and in 10% (1/10) of the cases that regressed (P = 0.01). Conclusion. Multiple HPV infections were frequently detected among women with CIN 2 at admission and during the followup. The CIN 2 associated with HPV 16 was more likely to persist or to progress to CIN 3. Cervical intraepithelial neoplasia (CIN) and cervical cancer derive from cellular transformations of the cervix epithelium after HPV infection. High-risk HPV (HR-HPV) persistent infections represent a necessary cause of cervical cancer, but not sufficient . AdditioFirstly, it was thought that cervical squamous cell carcinomas (SCC) would always evolve from HPV infected normal cervical epithelium via a continuum, long-lasting consecutive CIN 1, CIN 2, and CIN 3 lesions , as is tCIN 2 thus is a borderline lesion, can show clinical behavior similar to CIN 1 or to CIN 3, and occurs frequently in young women . The 201The pattern of HPV infection and reinfection in women and the outcome of CIN 2 are not well known. A better discrimination of CIN 2 that trends to progression or regression could help in choosing the best clinical management. This study therefore aimed to describe the acquisition, persistence, and clearance of HPV infection in women with CIN 2 confirmed by biopsy followed for 12 months under expectant management.This cohort study was a part of a research for evaluating expectant management of women with CIN 1 and CIN 2, carried out between January 2007 and December 2009 at the State University of Campinas (UNICAMP), Campinas SP, Brazil. It was approved by the Institutional Review Board of the Faculty of Medical Sciences of UNICAMP.Women aged 18 to 46 years old were considered eligible for the study if they fulfilled the following criteria: (1) referral cervical smear showing atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL); (2) biopsy showing CIN 2 reviewed by senior pathologist who was unaware of the DNA HPV test results ; (3) lesThe planned followup was visits every three months during one year with cervical smear, colposcopy, and sample collection for HPV detection. The colposcopic image showing a worsening of the lesion margem, color, and vascular pattern was submitted to biopsy. When the biopsy revealed CIN 2 or a less severe diagnosis, the woman was maintained in the follow-up plan. When the biopsy revealed CIN 3, immediate treatment by excision of the lesion was performed. At one year of followup, all the women who still showed cytological or colposcopy abnormalities were submitted to complete evaluation, and they were treated according to the final diagnosis. No ablative procedures were carried out.During followup, 13 women were discontinued due to the following: five women diagnosed with persistence in the intermediate visit did not show up for the final visit; one woman tested positive for HIV; one woman missed three consecutive visits; one woman became pregnant; five women were without at least one HPV test during followup. After 12-month followup, 37 women had final diagnosis established.Progression: biopsy showing CIN 3 detected at any time during the followup. No lesion worse than CIN 3 was revealed.Persistence: biopsy showing CIN 1 or CIN 2 at twelve-month followup.Regression: concomitant negative cervical smear and negative colposcopy or negative biopsy observed at any time during the followup and confirmed at twelve-month followup. If the patient had a HLSIL cervical smear and negative biopsy, it was not considered regression and the patient was submitted to excisional treatment.The final outcome of CIN 2 was classified as progression, persistence, or regression, according to the following criteria.The HPV type possibly associated with the CIN 2 lesion was considered the possibly causal type. For the CIN 2 lesions that regressed, possibly causal HPV was considered when HPV type was detected at admission and persisted up to the follow-up visit immediately before the regression. If the lesion persisted, either as CIN 1 or CIN 2, possibly causal HPV was considered when detected at admission and during the follow-up visits. Among the cases that progressed, possibly causal HPV was considered when detected at admission and at least until the follow-up visit when progression was detected. The possibly causal HPV type was considered undefined if the test was negative at admission or if it was not possible to associate the HPV type with the CIN 2 lesion as described previously.μL of Universal Collection Medium UCM  were taken for polymerase chain reaction (PCR) testing and were centrifuged for 10 minutes at 13,000 g. The supernatants were immediately removed, and split cellular pellets were stored at −80°C before nucleic acid extraction and HPV detection. The cellular pellets were resuspended in 200 μL of digestion solution  and digested at 55°C for 2 hours. The digestion was followed by 5-minute incubation at 95°C to inactivate the proteinase K. Nucleic acids were purified by phenolchloroform extraction followed by ethanol precipitation. The DNA pellet was dried and was dissolved in 100 μL of TE. Nucleic acids were stored at −80°C before HPV detection.Aliquots of 200 HPV amplification and genotyping  were performed by the Roche Linear Array (LA) HPV genotyping test, according to manufacturer's instructions.Tables were constructed showing the HPV type at admission and for every three-month followup, grouped according to the clinical outcome. The association between two categorical variables was tested using Fisher's Exact Test. The cases with undefined HPV possibly causal type were not considered for the analysis.Eighteen cases of CIN 2 regressed, of which 39% (7/18) had HPV 16 detected at admission, 16% (3/18) had HPV 56, and 16% (3/18) were HPV negative at admission. There were 33% (6/18) of the cases with multiple HPV types detected at admission and during followup 61% (11/18) presented new HPV type detected, one case with three new HPV types and five cases with two new HPV types. The HPV test was negative at admission or at least at one visit during followup in 61% (11/18) of cases. The HPV 16 was considered possibly causal in 6% (1/18) of cases.Eight cases persisted, four as CIN 1, and four as CIN 2. At admission, HPV 16 was detected in 50% (4/8) of the cases that persisted, HPV 58 in 38% (3/8), and HPV test was negative in one case. Multiple HPV types were detected in 38% (3/8) of the cases at admission and new HPV types were detected in 50% (4/8). HPV 16 was considered possibly causal in 38% (3/8) of cases and HPV 58 in 38% (3/8).Eleven cases (29%) progressed to CIN 3 up to twelve-month followup. HPV 16 was detected at admission in 64% (7/11) of the cases, HPV 52 in 27% (3/11), and HPV 68 in 18% (2/11). Multiple HPV types were detected at admission in 54% (6/11) of cases and new HPV type was detected during followup in 45% (5/11). Among the cases that progressed there was no negative HPV test. HPV 16 was considered possibly causal type in 64% (7/11) of cases.P = 0.20). HPV 16 was considered possibly causal in 67% (10/15) of the cases that progressed or persisted and in 10% (1/10) of those that regressed, and the difference was statistically significant (P = 0.01). The cases with undefined HPV possibly causal type were not considered for the latter analysis.According to this cohort study, multiple HPV infections were frequently detected in women with CIN 2, as new HPV types were also frequently detected during twelve-month followup. The CIN 2 lesions associated with HPV 16 were more frequent among those lesions that progressed to CIN 3.Cuschieri et al.  showed tCastle et al.  found thHPV infection can be considered productive when the expression of viral gene products remains regulated, not leading to significant host cell changes. Transformation infections arise where the productive infection cannot be properly supported due to increased activity of viral protein E6 and E7, which leads to genomic instability in the infected cell, accumulation of oncogene mutations, further loss of cell-growth control, and ultimately precancer lesions and cancer , 17.There was no negative HPV test in those cases that progressed, regardless of the HPV type. Among the eight cases that persisted, three cases presented negative HPV during followup, but in two cases the lesion persisted as CIN 1. Among the 18 cases that regressed, 11 presented negative HPV during followup, indicating transient infections. Only 4 out of 37 cases were HPV negative at admission, which could be a false negative or a true negative test, but none progressed.The regression rate found is not different from the reported regression rate of CIN 2 in adult women, which ranges from 15 to 55% regression over 4–6 years of followup , 18. MosThis study has limitations due to small sample subject. The inclusion criteria were women with cervical smear referral showing ASCUS or LSIL, CIN 2 proven biopsy, and HPV test at admission and at least at one follow-up visit, which limited the number of selected women. Besides, multiple HPV infections were frequent in these women and, therefore, the indication of possibly casual HPV type for the CIN 2 was not always obvious. Despite the lack of reproducibility of CIN 2, the HPV types did not differ significantly between CIN 1, CIN 2, and CIN 3 . False nFor clinical practice, the findings of this study suggest that the HR-HPV testing would have limited utility to indicate those women with greater risk of CIN 2 progression since the rate of HR-HPV positive was high among women with CIN 2 regression. Nevertheless, expectant management, which may be proposed for young women, should be considered cautiously when HPV 16 infection is detected. In conclusion, infections with multiple HPV types were frequently detected at admission and follow-up visits as well as infections with new HPV types during followup. CIN 2 lesions with HPV 16 alone or in combination with other HR-HPV types are more prone to progression to CIN 3.


Objective: Cervical cancer is a leading cause of cancer mortality in South Africa. However, little is known about oral human papillomavirus (HPV) infection in high human immunodeficiency virus (HIV) seroprevalence settings.Method: Thirty-four adult heterosexual couples attending an HIV testing center in Soweto, South Africa were enrolled. Each participant provided an oral rinse sample and genital swab, which were tested for 37 types of HPV DNA, and completed a risk behavior survey.Results: Median age was 31 years and 9% (3/34) of men and 29% (10/34) of women enrolled tested HIV-positive; median CD4 count was 437 cells/mm3. Oral HPV prevalence was similar in women and men , and was non-significantly higher in HIV-infected vs. HIV-uninfected  subjects. Most men (82%) and women (84%) reported ever performing oral sex. Median number of lifetime sexual partners was “2–5” while median number of lifetime oral sex partners was 1. Oncogenic HPV subtypes were detected in 4% of oral, 26% of penile, and 74% of vaginal samples, including HPV16 in 1, 12, and 21% of these samples respectively. Genital HPV prevalence was significantly higher than oral HPV prevalence . Thirty-five percent of couples (12/34) had at least one type-specific concordant vaginal-penile HPV infection but only one of nine couples with oral HPV had concordant oral–oral infection. However, 67% (4/6) of men and 25% (1/4) of women with oral HPV infection had partners with concordant genital HPV infection.Implications and Impact: Oral–oral HPV concordance between couples is low, but oral-genital and genital–genital HPV concordance is higher, including concordance of male oral HPV infection with their partners’ vaginal HPV infection. This data is consistent with possible transmission of vaginal HPV infection to the oral cavity of sexual partners performing oral sex. Human papillomavirus (HPV) is a sexually transmitted infection that causes squamous cervical cancer and some vaginal and anal cancers , 2. MoreIn South Africa, HIV prevalence is estimated at 17.3%, and 5.6 million people are living with HIV as of 2011 , 9.Cervical HPV infection has been well studied in South Africa, where cervical cancer is one of the leading causes of cancer mortality in women . The preWe therefore evaluated oral and genital HPV prevalence, risk factors and concordance among couples attending the Perinatal HIV Research Unit at Chris Baragwanath Hospital in Johannesburg, South Africa.In 2011 we recruited 38 couples attending the Zazi Center at the Perinatal HIV Research Unit, Chris Baragwanath Hospital in Soweto, South Africa for HIV testing. The Zazi Center provides free, walk-in, voluntary HIV counseling and testing for individuals and couples. Clients who test positive for HIV receive CD4 count testing to assist in appropriate referral to care. To be included in this study, participants were required to be 18 years or older and be able to read English. A convenience sample that included all couples coming to the Zazi HIV testing clinic during the enrollment period were asked by their HIV testing counselor if they were interested in participating in the study. Roughly 33% of the individuals who came to the HIV clinic during this time period came without a partner and were therefore not eligible for this study. Sexually active couples who reported being together for at least 1 month were eligible for enrollment as long as both partners were willing to participate and provide informed consent. All 38 couples referred to the study coordinator enrolled in the study.A couple was defined as two individuals (regardless of gender) who self-identified as current sexual partners. Exclusion criteria included an inability to provide consent, or an unwillingness to provide an oral rinse sample and survey data. Individuals could opt out of providing a genital sample. The study was approved by the Johns Hopkins Bloomberg School of Public Health institutional review board and the University of Witwatersrand human research ethics committee.Each study subject provided an oral rinse and gargle sample, a genital swab and completed a risk behavior survey. Oral rinse and gargle specimens were obtained by having a participant gargle 10 ml of Scope mouthwash for a total of 30 s . Specimens were collected in a sterile cup and stored at 4°C while awaiting transport to the laboratory. Women were asked to provide a self-collected vaginal sample, obtained using a Dacron applicator  and stored at room temperature while awaiting transport to the laboratory. The penile swab was collected from participating men by a clinical study team member and penile cells were collected using two Dacron HC (Qiagen) swabs. One swab sampled the coronal sulcus and glans penis, while the other sampled the penile shaft. Both were combined into one collection vial and stored at room temperature . InitialAll study participants were asked to complete a self-administered risk factor questionnaire. Individuals from the same couple were asked to fill out the survey in separate rooms to ensure privacy of responses. The questionnaire included questions about risk factors for oral HPV infection including demographics, recent, and lifetime sexual behavior including performing oral sex, alcohol, tobacco, and drug use. Ever regular tobacco use was asked as: “Have you ever smoked cigarettes regularly?” with response options of: yes/no. Ever regular alcohol use was asked similarly. Responses to many of the survey questions were categorical, such as number of oral sexual partners, which was asked as: “In your lifetime, how many different people have you performed oral sex on?” with response options of: 0, 1, 2–5, 6–10, >10.All study participants were tested for HIV as part of their clinical care and if HIV-positive CD4 cell count by the Zazi clinic as part of their clinical visit. Women with oncogenic vaginal HPV infection were referred for Pap smears.Characteristics of participants were compared by gender and by HIV status using chi-squared for categorical and test of medians for continuous variables. Chi-squared was used to compare oral and genital HPV prevalence by HIV-status. Fischers exact test was used is comparisons of any group with five or fewer subjects. McNemar’s test was used to compare oral and genital HPV prevalence among the paired male and female couples. Agreement in HPV infections between partners was considered separately by site  and was described as the proportion of couples who had one or more type-specific concordant infections overall and when limited to subgroups who had one type of infection present. All statistical analyses were performed using STATA version 11.0 . There were 4/38 couples recruited in which one of the partners opted out of providing a genital sample; therefore all analyses were restricted to the 34 couples with complete oral and genital HPV data. Results were similar when these individuals were not excluded (results not shown).3 [Interquartile range (IQR): 161–575 cells/mm3]. Median age was 31 years (IQR: 28–36). Women were more likely than men to test HIV positive  and women were significantly younger and less likely to use tobacco or alcohol than men in the study , 45 and 52 (each 11.8%), 61 (10.3%), 66 (8.8%), 6, 18, 35, 72, 84, 89 (each 7.4%) as the most common types. For oral HPV the most common infections detected were HPV 62 , 72 (5.9%), 35, 52, 33, 58 (each 2.9%), and 16, 74, 66 (each 1.5%).p ≤ 0.001) (Table p = 0.48), and was higher, but not statistically different, among HIV-infected vs. HIV-uninfected individuals . Genital HPV prevalence was significantly higher in women vs. men  and HIV-infected vs. HIV-uninfected . Oncogenic HPV subtypes were detected in 4% of oral, 29% of penile, and 74% of cervical samples, including HPV 16 in 1, 12, and 21% of these samples respectively.Oral HPV prevalence was significantly lower than genital HPV prevalence  Table  but oralp = 0.04) and multiple HPV infections . For oral samples, there was a trend for higher oncogenic HPV prevalence  and multiple HPV infections  in HIV-infected vs. HIV-uninfected individuals, but this finding was not statistically significant.Comparing HIV-infected vs. HIV-uninfected individuals, HIV-infected individuals displayed a statistically significantly higher prevalence of both genital oncogenic HPV subtypes .Concordant type-specific oral-genital HPV infection was detected in 4/34 12%) couples .At least one type-specific concordant vaginal-penile HPV infection was observed in 35% of couples Figure . The proIn multivariate analysis, oral HPV prevalence was higher, although not statistically significantly different, among HIV-positive individuals , males , and those with ≥2 lifetime oral sex partners compared to those with <2 lifetime oral sex partners , Table This is one of the first studies to explore oral-genital HPV concordance in couples. We found that 12% of couples displayed oral-genital concordance and among these couples the majority had male oral HPV infection concordant with female vaginal HPV infection. This is consistent with higher likelihood of vaginal-to-oral transmission as compared to penile-to-oral transmission, and may help explain previously reported findings of a lack of significant association between oral-penile contact and incident oral HPV infections amongst women . Oral–orOur study demonstrated oral HPV prevalence of 15%, consistent with previous studies from South Africa, which showed an oral HPV prevalence of 20–25% , 23. RisVaginal HPV was detected in >90% of females in our study and the majority of these individuals were found to have oncogenic HPV subtypes. Direct comparisons to previously reported estimates of vaginal HPV amongst women in South Africa are difficult as previous studies report HPV prevalence based on degree of dysplasia present, however population based estimates previously reported for any HPV subtype in women with normal cytology were roughly 21% , 26 and Penile HPV was detected in 59% of males and 29% were found to have oncogenic HPV subtypes. The prevalence of high-risk HPV subtypes amongst males in South Africa has been previously reported as 15% for circumcised males and 22% for uncircumcised males . In our While this study was cross sectional, the agreement in oral-genital HPV infection between couples is consistent with transmission by oral sex. Additionally, in three out of the four couples with concordant oral-genital HPV infection, the partner with oral HPV reported having performed oral sex, a finding consistent with previous reports of an association between oral HPV prevalence and reported oral sex . The remThis study had several limitations. The small sample size limited the analysis of risk factors for infection. Because data was collected in categories exact medians could not be recorded. Additionally, our study provides cross-sectional data only so could not demonstrate transmission over time from one partner to another. However this study provides some of the first estimates of oral–oral and oral-genital HPV concordance in couples in Africa.This research demonstrates high prevalence of genital HPV infection and moderate prevalence of oral HPV infection in a South African population with a modest number of sexual partners. The HPV vaccine has recently been shown to be effective in preventing oral, as well as cervical and anal HPV infection , and thiGypsyamber D’Souza has research support for Merck Inc. The other co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.