N = 40) and from those detected with HSIL from cytology and colposcopy (N = 40). Liquid-based cytology diagnosis, DNA HPV-detection test, and LC-MS analysis were carried out for all the samples. The same sample, in a customized collection medium, could be used for all the diagnostic techniques employed here. The metabolomic profile of cervical cancer provided by LC-MS was found to indicate unique molecular signatures for HSIL, being two ceramides and a sphingosine metabolite. These molecules occurred independently of women’s HPV status and could be related to the pre-neoplastic phenotype. Statistical models based on such findings could correctly discriminate and classify HSIL and no cervical lesion women. The results showcase the potential of LC-MS as an emerging technology for clinical use in cervical cancer screening, although further validation with a larger sample set is still necessary.Cervical cancer is the fourth most common neoplasia in women and the infection with human papilloma virus (HPV) is its necessary cause. Screening methods, currently based on cytology and HPV DNA tests, display low specificity/sensitivity, reducing the efficacy of cervical cancer screening programs. Herein, molecular signatures of cervical cytologic specimens revealed by liquid chromatography-mass spectrometry (LC-MS), were tested in their ability to provide a metabolomic screening for cervical cancer. These molecules were tested whether they could clinically differentiate insignificant HPV infections from precancerous lesions. For that, high-grade squamous intraepithelial lesions (HSIL)-related metabolites were compared to those of no cervical lesions in women with and without HPV infection. Samples were collected from women diagnosed with normal cervix ( Cervical cancer is the fourth most common neoplasia in women, and the seventh overall. In 2012, for instance, an estimative of 528,000 new cases and 266,000 deaths from cervical cancer were reported worldwide, accounting for 7.5% of all female cancer deaths. Almost 9 out of 10 cervical cancer deaths occur in the less developed countries due to poor effectiveness of screening, lack of early detection, and treatment of precancer (Infection with human papilloma virus (HPV) is recognized as the cause of invasive cervical cancer . There aThe primary goal of cervical cancer screening programs is, therefore, to prevent cervical cancer by detecting and treating cancer precursors . But, anThe subjective composition of cytology results among HPV-positive women calls for alternatives that include automated screening methods. Such automation would allow the development of integrated approaches using HPV testing and automated tests, minimizing the subjectivity of cervical cancer screening . Among tIn the search for diagnosis biomarkers for cervical cancer, investigations over proteomics content have been performed from cervical tissue, directly over the tumor and using cervical cytologic species –27. For via molecular signatures. We tested whether the LC-MS methodology would be able to differentiate clinically insignificant HPV infections from precancerous lesions. HSIL-related metabolites were also compared to those of no cervical lesions in women with and without HPV infection. Indeed, the LC-MS method, using the same cervical cytologic specimen employed for cytology and DNA tests, was found to efficiently screen for cytologic abnormalities and to detect for molecular signatures related to the pre-neoplastic phenotype, which helps in the understanding of the molecular mechanisms underlying cervical cancer predisposition.Herein, we tested the ability of LC-MS to perform an efficient metabolomics screening of cervical cancer Cervical cytologic samples were obtained from women from two groups: (i) 40 women undergoing gynecological examination in the family planning clinic (FPC) from the Women’s Hospital “Prof. Dr. José Aristodemo Pinotti”—CAISM-UNICAMP, previously diagnosed with normal cervix in the clinical evaluation and (ii) 40 women undergoing to a loop electrosurgical excision procedure (LEEP) due to previous detected HSIL from cytology and colposcopy, in the surgical service from CAISM-UNICAMP. Women in both group had their sample collected prior to cervical manipulation and following the same collection instruction, by trained personnel. For cervix diagnosis, the following information was considered: (i) clinical evaluation at collection; (ii) liquid-based cytology diagnosis; (iii) HPV test results; and (iv) histology evaluation of the excised tissue from HSIL patients. Inclusion criteria were clinically normal cervix with negative cytology for the “no cervical lesion (NCL) group” and HSIL confirmed lesion from histology evaluation of the transformation zone for the “HSIL group.”®, BD) and immediately immersed in the same collection vial containing the ethanolic solution. After homogenization, each sample was divided as follows: (i) 50% of the material was sent to chemical extraction for LC-MS analysis; (ii) 10% was dedicated to preparing liquid-based cytology slides to be diagnosed by a board-certified pathologist; and (iii) the remaining 40% was sent to the Laboratory of Molecular Biology at CAISM to screen for HPV DNA. Maximum interval of 72 h was considered between sample collection and preparation prior to further extraction procedures.This study was carried out in accordance with the recommendations of the Resolution no. 466/2012 of the Brazilian National Health Council (CNS), with written informed consent from all subjects and in accordance with the Brazilian National Research Ethics Commission (CONEP). The protocol was approved by the Research Ethics Committee of the University of Campinas (CEP/UNICAMP) under the number 1.181.203 from 08/25/2015. Specimens were collected in a laboratory-made, preservative-free liquid-based preparation  . CervicaCervical samples in solution were gently vortexed and a volume ranging from 0.5–1 mL was manually dripped on a silanized glass slide. The slides were allowed to dry, sent to the Cytology Laboratory at CAISM (UNICAMP), and then prepared using standard protocols for cytology. These steps were done in duplicate resulting in two slides per sample which were analyzed by a board-certified pathologist.All the slides evaluated by the pathologist had their number of squamous cells estimated according to the Bethesda System. This estimation was obtained by performing representative field cell counts. For that, four adjacent microscopic fields were counted per slide at ×40 magnification and their results were averaged. The correlation of 3.8 visualized cells for 5,000 estimated cells was used. Such estimative showed the adequacy of the specimens for cytology analysis. Correlation studies of cell counting and relevant clinical variables were also examined to investigate possible bias added to the results which could be related to the contraceptive method of choice, menstrual cycle phase, age, and body mass index of the subjects. Figures S1–S4 in Supplementary Material show box plots of cell counting correlating to these variables. None of the analyzed variables showed significant differences for this test set.oC until DNA extraction. DNA extraction and quantitation were performed as previously described, as well as β-globin amplification serving as internal control for quality and sufficiency of sample’s DNA  was centrifuged . The resulting pellet was transferred to a 1.5 mL microtube and centrifuged again . The cellular pellet was kept frozen at −20le’s DNA . HPV DNAle’s DNA , 39. Spele’s DNA .From the original cervical sample, 5 mL were vortexed and transferred to a new vial followed by the addition of 10 mL of water (Milli-Q). After this, vials were centrifuged  and the supernatant was discarded. Water (800 μL) was added to the pellet and the cellular content was then transferred to a 2 mL microtube, followed by the addition of methyl tert-butyl ether . Samples were then submitted to an automatic vortex mixer (10 min) and centrifuged . The upper layer was collected, sent to vacuum centrifugation until dryness and stored at −20°C until analysis, when these extracts were resuspended in 120 μL of a methanol:chloroform solution .oC and a flow rate of 600 μL min−1 was used. The composition of the mobile phase is shown in Supporting Table S1 in Supplementary Material. HPLC was coupled to a hybrid 6550 Quadrupole-Time-Of-Flight Mass Spectrometer  equipped with an electrospray (ESI) ionization source. Samples were analyzed in the positive ion mode, using the following instrumental parameters: VCap 2.800 V; fragmentor voltage at 175 V; skimmer voltage at 60 V; OCT 1RF Vpp at 750 V; gas temperature at 260°C; sheath gas temperature at 300°C; and drying gas at 12 Lmin−1. Mass spectra were acquired in centroid mode and the acquisition mass range was 100–1,500 Da. Samples were randomly analyzed. Raw data were converted to the file format .mzData at MassHunter Qualitative software (Agilent), using filters of ions for relative intensity of 5% of the most intense ion. Files were then imported to the software XCMS online (https://xcmsonline.scripps.edu/) for retention time alignment and for the extraction of ion chromatograms (EIC) . The injection volume was 10 μL, the oven was kept at 40ms (EIC) . Main pahttp://www.metaboanalyst.ca)  was used. For the supervised analysis through support vector machine (SVM), a total of 70% of the available data were used to build a statistical method of classification (training set), whereas about 30% of the available data was used as an independent test set. Receiver operating-characteristic (ROC) curves were calculated for the performance of SVM models and the models with the highest area under the ROC curve (AUC) value were selected as the optimal ones. Potential biomarkers were identified through supervised statistical analysis by SVM and the minimal number of ions which could discriminate the groups was selected to compose the statistical model.Statistical analysis was performed through multivariate models available in the MetaboAnalyst website (lyst.ca) . No datahttp://www.lipidmaps.org) and Metlin (https://metlin.scripps.edu).Ions indicated as those formed from putative biomarkers by the statistical analysis were selected for MS/MS experiments and for that, a Q-TOF 6550 (Agilent) instrument was used. For ion assignment, the exact mass of the ion and its fragments were considered and then compared to databases, such as Lipidmaps . This agreement shows the adequacy of sample preparation for cytology, which was adapted to use a preservative-free liquid-based medium for collection of specimens. HPV was detected in 32 (91%) of women from HSIL group compared with 23 women (58%) with normal cervix. The normal cervix group was composed of a sexually active young population, attending to a family planning clinic, thus justifying their risk factor for HPV infection (Table 23 women 8% with nnfection .NCL+ group and (ii) HSIL subjects screened positively for HPV, designed as the HSIL+ group. The aim of this comparison was to find whether the metabolomic profiles of these samples would reveal differences assigned to the precancer stage (HSIL). For that, SVM was applied for training and test sets.For the differentiation of HPV-positive subjects, two groups were considered for SVM analysis: (i) normal cervix subjects screened positively for HPV, designed as the Support vector machine is a multivariate classification algorithm using a non-parametric machine learning technique, which identifies important variables for the construction of both classification and regression models. It was developed to solve binary problems, such as case–control studies and, compared to other multivariate classification methods, SVM is less susceptible to outliers and over-fitting , 19.p-value for such differentiation, which is significant when it is less than 0.05. The resulting SVM model was applied to classify the test set and was fortunately found to correctly classify 8 out of 9 HSIL+ tested samples and 6 out of 6 NCL+ samples which had not been used before to build the statistical model, resulting in a positive predictive value (PPV) of 1.00, negative predictive value (NPV) of 0.86, specificity of 1.00, and sensitivity of 0.89 in a per-patient analysis. The medium probability of correct classification found for the test set was 81.3% for HSIL+ group and 86.4% for NCL+ group.Figure m/z 312.326 eluted with mean retention time (mRT) of 1.64 min and termed M312T2; (ii) the ion of m/z 468.440 eluted with mRT of 4.89 min and termed M468T5; and (iii) the ion of m/z 568.445 eluted with mRT of 5.22 min and termed M568T5. Figures m/z values and their fragmentation behavior . The feature M312T2 was assigned as the protonated molecule [M − H2O + H]+ of dimethyl sphinganine (d18:0), a sphingosine metabolite. The feature M468T5 was assigned as the protonated molecule [M − H2O + H]+ of the Ceramide CE (29:1). The feature M568T5 could not be firmly characterized, but it was assigned as the protonated molecule [M + H]+ of a derivative of a ceramide, since its fragmentation profile is similar to the one observed to M468T5.The model was built based on three molecular features: (i) the ion of N = 3, HSIL− group), the same three molecular features mentioned above were also able to correctly discriminate the groups with an excellent average accuracy of 100%. This comparison confirmed the previous obtained results for HPV-positive subjects, since the same set of molecular features was used to discriminate the groups containing or not the precancer lesion.A comparison among HPV-negative subjects, containing or not HSIL lesions, was also performed. Figure S7 in Supplementary Material shows the results and, although in a reduced number of samples for HSIL/HPV-negative subjects , unsupervised analysis of the data was also performed through PCA . The analysis shows reproducibility among samples considering the site of collection as a variable. The procedures carried out at different sites, therefore, were equivalent and failed to interfere with the results.HSIL group, allowed us to build an efficient model using the metabolomic data of the samples. This model was found to discriminate and correctly classify 93% of HPV-positive subjects presenting HSIL from those with no cervical lesion for a test set of 15 samples. Three molecular features, assigned to sphingolipids, were found to be the most discriminant and, therefore, were selected to build the model. We used this set of biomolecules also to discriminate HPV-negative subjects presenting HSIL from those with no cervical lesion. When performing a blind test using only LC-MS to differentiate histologically confirmed HSIL and NCL women, a high discrimination power was found: that is 25 out of 28 samples were correctly classified. Although our analysis was performed using LC-MS, which could provide quantitative assessment of the molecular markers, in our approach we used statistical analysis to obtain a diagnosis based on the detected levels of the predictive features, which provided high accuracy for cancer detection. These results showcase the potential of this technology for clinical use in cervical cancer screening, since it can correctly detect and diagnose HSIL in patients, independently of their HPV status or cytological evaluation, although further validation with a larger sample set remains necessary.In this study, we found that there is a proper collection medium for liquid-based cytology that enables to analyze (i) cytology, (ii) HPV DNA screening, and (iii) metabolomics through LC-MS, all performed over the same sample, without the need of extra sample collection and without loss of quality for cytology and HPV screening. The correlation of the results of these three techniques, summed to the clinical evaluation of the subjects and to the histology results of excised specimens from This study emphasizes the importance of interdisciplinary investigations and translational studies in finding prognostic markers that could solve the insufficient specificity and sensitivity of present cervical cancer screening programs from the molecular point of view , 44, 45.In this study, the concurrent use of these two main screening techniques with clinical examination  and histological evaluation of excised specimens (for HSIL subjects) ensured the proper diagnosis of samples . The LC-HSIL+ and NCL+ group were composed of HPV-positive subjects. The model has been already demonstrated to be able to classify unknown samples assigning the correct diagnosis with good statistical results. The minimum number of characterized molecular features was selected to build the model, so as to ensure that these features had some biological relevance and that they were not contaminants or artifacts of the technique. To study the mechanism of disease progression, most if not all the detected features should be considered and characterized, thus generating a panel of molecules which could enable the illustration of the biological processes taking part in HSIL formation and/or HPV infection, which was not the aim of this study . HPV clearance could also be a possible reason for HPV-negative patients who had HSIL. Viral clearance occurs prior to viral integration into the host genome (carcinogenic stage) in 80% of HPV-infected women , therefolearance .The last comparison is a proof of concept, which was performed without considering information regarding HPV status or cytology results of the samples, therefore, simulating the status of unknown samples. The use of LC-MS as a stand-alone technique is not suggested but certainly, after proper validation, it could be implemented in large cohort as part of cancer prevention strategies, since we showed here that the use of the very same sample used for other screening techniques is achievable, without the need of extra collection. After further studies with a larger sample set, LC-MS could, therefore, be used as a co-test for cervical cancer screening and, when LC-MS indicates any alteration, confirmation could be done for HSIL by colposcopy or biopsy. The full development of LC-MS as a method for screening routines could, therefore, bring the specificity and sensitivity that are still missing for HPV and cervical cytology screening.via LC-MS, as shown here, could provide new classification models that, when aligned with other screening techniques, would enhance the knowledge of molecular signatures that could better elucidate and discriminate disease phenotype rather than its genotype. After proper method validation and optimization focused on known predictive markers, LC-MS could also achieve the qualities of being fast, automated, and less-expensive than it is on initial research stages. By performing a target analysis, this method would be compatible to sample’s preservatives generally employed in HPV and cervical cytology collection medium, thus allowing it to be used concomitantly, without major logistic changes in routine exams and biological sampling.There is clearly a need for improved specificity in HPV screening programs that could discriminate women who, after HPV infection, will progress to precancer status or will clear without clinical intervention . There iThis study was carried out in accordance with the recommendations of the Resolution no. 466/2012 of the Brazilian National Health Council (CNS), with written informed consent from all subjects and in accordance with the Brazilian National Research Ethics Commission (CONEP). The protocol was approved by the Research Ethics Committee of the University of Campinas (CEP/UNICAMP) under the number 1.181.203 from 08/25/2015.AP: methodology, data analysis and interpretation of the data, and drafting of the manuscript. FN: methodology and drafting of the manuscript. GT: MS/MS data interpretation. DP: DNA-HPV determination. EC: DNA-HPV determination. DM: cytology analysis (pathologist). AM: drafting and revision of the manuscript. ME: supervision on mass spectrometry, study design; SD: study design, clinical supervision, sample collection, analysis of the data, and revision of the manuscript.All authors have read the journal’s policy on disclosure of potential conflicts of interest. All authors have disclosed any financial or personal relationship with organizations that could potentially be perceived as influencing the described research.


Nasopharyngeal carcinomas (NPC) are endemic in Far East Asia and commonly harbour Epstein-Barr virus (EBV) which is known to serve as a key oncogenic promoter. Human papillomavirus (HPV) is known to contribute to the pathogenesis of NPC. However, in Ghana these two viruses have not been linked to NPC prevalence. This study was designed to determine the HPV genotypes and EBV involved in NPC tissue biopsies. A retrospective study design involving 72 formalin-fixed paraffin-embedded tissue (FFPET) samples of NPC from 2006 to 2012 were retrieved from the Department of Pathology, University of Ghana School of Biomedical and Allied Health Sciences. Sections were taken for histological analysis and for DNA lysate preparation. The DNA lysates were subjected to polymerase chain reaction (PCR) analysis to determine the presence of HPV genotypes and EBV. HPV specific primers were used to type for fourteen HPV genotypes . Out of the 72 NPC biopsies analyzed by PCR, EBV DNA was present in 18 (25%) cases and HPV DNA in 14 (19.23%). High risk HPV (HR-HPV) genotypes 18 and 31 were associated with the NPC. There were 3 (4.2%) cases of coinfection by both viruses. The EBV DNA present in the undifferentiated variant of the NPC and the histopathology of the NPC in Ghana is similar to the type described in endemic areas. Nasopharyngeal carcinoma (NPC) is classified as a malignant neoplasm, arising from the mucosal epithelium of the nasopharynx . NPC hasNPC is a commonly occurring tumour in individuals in the southern regions of China , the EskMen are twice as likely to develop NPC as women, and the incidence rate generally increases from ages 20 to approximately 50 with signs and symptoms presenting themselves as painless, enlarged cervical lymph nodes, nasal obstruction, epistaxis, diminished hearing, tinnitus, recurrent otitis media, cranial nerve dysfunction, sore throat, and headache , 12. OncEBV has been implicated in the pathogenesis of several human tumours such as the African form of Burkitt lymphoma, B-cell lymphomas in immunosuppressed individuals, nasopharyngeal and some gastric carcinomas, rare forms of T-cell lymphomas, and natural killer (NK) cell lymphomas . Anim etHPV on the other hand has also been detected in a variety of head and neck tumours including NPC . It is aStudies suggest that normal human pharyngeal epithelial cells, especially nasopharyngeal cells, could be susceptible to persistent HPV and EBV coinfections and that EBV and HR-HPV coinfections may play an important role in the initiation of neoplastic transformation of these cells . Other sIn Ghana, studies on the prevalence of EBV and HPV genotypes and their involvement in NPC have not been carried out. Hence, this research work investigates the presence of these viral oncogenic DNAs associated with the NPC using molecular probes. Molecular analysis will help identify the viral genotypes responsible for the transformation of the nasopharyngeal epithelial cells to aid preventive and therapeutic strategies.With the current prevalence rate of 29% of all head and neck cancers  and 1.2–The study was a retrospective cross-sectional design with laboratory analysis to investigate the presence of the DNA of EBV and HPV genotypes from primary NPC archived tissue samples obtained from the Department of Pathology, University of Ghana, School of Biomedical and Allied Health Sciences (SBAHS), from January 2006 to December 2012. The inclusion criteria required a patient's age to be greater or equal to ten (≥10) and histopathologically diagnosed with primary tumour of NPC.A total of 72 formalin-fixed paraffin-embedded tissue (FFPET) samples from patients with histopathologically confirmed primary NPC at the Pathology Department, Korle-Bu Teaching Hospital (KBTH), Accra, Ghana, were used. The department serves as the main pathology unit of the hospital and receives referral cases from all over Ghana and other West African countries. It also serves as a teaching unit in pathology  for SBAHS and the School of Medicine and Dentistry. The patients in this study originated from various social and ethnic groups as well as geographically distinct areas from the Greater Accra region and the southern part of Ghana.The clinical information on gender, age, and diagnosis of the NPC cases was recorded.μm sections of the tissue were taken using a microtome. The sections were then stained with haematoxylin and eosin (H&E), using an automated H&E staining system (Leica Auto Stainer XL). The H&E stained slides were reviewed under the light microscope at the Department of Pathology, SBAHS, to confirm the previous diagnosis. The histopathological types of NPCs were determined according to the WHO 1991 classification and consisted of type I (keratinizing squamous cell carcinoma), type II (differentiated nonkeratinizing carcinoma), and type III (undifferentiated nonkeratinizing carcinoma). The H&E stained tissue slides were also used primarily as microscopic controls to ensure that the tissue sections used in the PCR contained malignant tissue. Photomicrographs of the NPC subtypes were then captured using the digital binocular light microscope, OLYMPUS 500 microscope .After trimming of the 72 FFPET samples of NPC blocks obtained, 5 μm) sections of the tissue were taken using a microtome. To avoid carry-over of the samples and contamination, the microtome blade was changed after each section and all surfaces were also cleaned with xylene after each tissue section. The 10 μm sections were placed into a sterile 2.0 mL microcentrifuge tube. The tissues were then digested in 250 μL of digestion buffer by incubation for 16 hr at 55°C on a heat-block using the method described previously [μg/mL Proteinase K , 50 mM Tris-HCL (pH 8.5), 1 mM EDTA, and 0.5% Tween-20. After 16 hr of incubation, the proteinase K was inactivated at 100°C for 5 min on a heat-block. After cooling the samples to room temperature, they were centrifuged at 13000 rpm for 5 min in a microcentrifuge. The residues containing DNA lysate were pipetted out and the supernatant containing paraffin was discarded. The digested product of each sample was stored in a freezer at −20°C for future use. The residue obtained (DNA lysate) contained the DNA extract that was used as a source of DNA template for the PCR analysis.Ten micrometre , 1.5 mM MgCl2 , 200 μM of each of the four oligonucleotide triphosphates (dNTPs) , 3.75 pmol of each consensus primer and 0.625 units of Taq polymerase enzyme , and 1 μL or 4 μL of the DNA lysate, the tissue digested product (source of HPV DNA template). Nuclease-free water  was used to make up the volume to 25 μL. A master-mix was made without the DNA template, vortexed to mix, and then pulse centrifuged for 30 sec before being aliquoted out into the samples contained in the PCR tubes. The positive control was HPV-18 DNA-containing plasmid and negative control (25 μL of primer mix). The controls were also taken through the same PCR reaction conditions as per samples. The PCRs were performed by the following cycling conditions for the first nested PCR: an initial denaturation at 94°C for 4 min, followed by 40 PCR cycles consisting of 1 min denaturation step at 94°C, annealing step at 40°C for 2 min and elongation step at 72°C for 2 min, and a single final elongation step at 72°C for 10 min. As an internal control for the first nested PCR, the positive and the negative control PCR products were analyzed on a 2% agarose gel for every batch of the first PCR. The presence of a 630 bp band of the positive control gel lane and a DNA absence in the negative gel lane signifies that that batch of PCR is fit to be continued for the second nested PCR.In the first nested PCR amplification reaction, a single consensus forward primer (GP-E6-3F) and two consensus reverse primers (GP-E7-5B and GP-E7-6B) were used. For a total volume of PCR reaction mix of 25 μL, the reaction conditions were as follows: 2 μL from PCR products of the first nested (source of HPV DNA template) and 3.75 pmol of each of the genotype specific primers (forward and reverse). The HPV genotype specific primer sets of the following HPV genotypes; 6/11, 16, 18, 31, 33, 35, 44, 42, 43, 45, 56, 52, and 58, were used in three cocktails. Nuclease-free water was used to make up the volume to 25 μL. The parameters for the first nested PCR reaction was maintained for the second nested PCR. 10 μL of the PCR products was then separated on 2% agarose gel  thereafter in 1x TAE running buffer  by electrophoresis at 80 volts  using 2 μL of blue/orange DNA loading dye (6x)  and stained with 0.5 μg/mL ethidium bromide . A hundred bp nucleotide sequence marker  was run alongside the PCR products on the gel.For the second nested PCR, a total volume of PCR reaction mix of 25 The EBV DNA sequence was accessed using NC_007605. Amplified product size of 229 bp for EBV was used for identification of EBV DNA using agarose gel electrophoresis. The same reaction mixture was carried out using a modified one-step PCR. The PCR cycling conditions for the one-step reaction are as follows: an initial denaturation and polymerase activation at 95°C for 10 min, followed by 35 PCR cycles consisting of a denaturation step at 95°C for 30 sec, an annealing step at 60°C for 30 sec, an elongation step at 72°C for 30 sec, and then a final extension step at 72°C for 5 min, as described previously .μg/mL ethidium bromide. 10 μL of each sample was added to 2 μL of orange G (5x) gel loading dye for the electrophoresis. 100 bp DNA molecular weight marker  was run alongside the PCR products. The gel was prepared and electrophoresed in 1x TAE buffer using a minigel system at 120 V for 40 min and was optically visualized and photographed over a UV transilluminator , followed by image analysis.The amplification products (for both HPV and EBV) were analyzed by gel electrophoresis on 2% agarose gel and stained with 0.5 The study was approved by the College of Health Sciences Ethical and Protocol Review Committee, University of Ghana .P value) was used to measure the statistical significance of the calculated odds ratio. The P value obtained as is deemed statistically significant when value of the corresponding association is less than or equal to 0.05.The data obtained from the tests were analyzed using SPSS version 16 . Qualitative variables were summarized by proportions and percentages while means, medians, and standard deviations were used to summarize quantitative variables. A test of association between EBV and HPV infection and subject's clinical and demographic data were summarized by Fishers' exact test with corresponding 95% confident interval (CI) and Fisher' exact test's probability value , comprising 38 males and 34 females given a 1.12 : 1 male to female ratio.All NPC were regrouped into three histopathological types according to the WHO 1991 classification. This classification consisted of type I (keratinizing squamous cell carcinoma), type II (differentiated nonkeratinizing carcinoma), and type III (undifferentiated nonkeratinizing carcinoma) . WHO typP = 0.178) and also between histological subtypes of NPC and HPV infection (P = 0.595).The distribution of EBV DNA in the 18 EBV positive NPC cases  showed tOut of the 14 HPV DNA positive cases, 13 (92.9%) were infected with HPV-18 genotype, and 1 (7.1%) was infected with HPV-31 . There wβ-globulin PCR analysis, all the 72 NPC samples were positive for human β-globulin DNA.All the 72 archival tumour tissues were microscopically confirmed to contain malignant cells by H&E staining and were successfully regrouped under the WHO types I, II, and III of the NPC classification. From the human Results showed a male preponderance with a male : female ratio of 1.12 : 1. This falls within the range of results obtained in Ghana , 28 and For gender, females had higher (15.3%) EBV positive NPC tumour cases and a corresponding (OR = 2.12) than males 9.7%). Again, in contrast, males had a higher HPV positive frequency (13%) compared to females (5.6%). Thus, women were more likely to have EBV positive NPCs than men, with male subjects being at a higher risk of HPV infection as compared to women, even though the differences in prevalence estimates were not statistically significant. This disparity of viral tropism in gender group could be due to the route of entry and life style of the groups. Our result affirms other research works carried out elsewhere, where males had higher HPV positive cases (17), compared to females (7) %. Again,. This spThe observed results showed no significant differences between the oncogenic viral status with either age or gender in the NPC cases. These findings are in agreement with other studies carried out previously , 32, 33.In endemic areas, the majority of the nasopharyngeal carcinomas are classified as nonkeratinizing WHO type II or undifferentiated type III tumours . In contResults from recent studies on NPC elsewhere pointed out that the nonkeratinizing type, particularly the undifferentiated carcinoma (WHO type III), was the most common histopathological type, followed by differentiated nonkeratinizing carcinoma (WHO type II) and then the keratinizing type (WHO type I) , 29, whiIn the present study, the observed results revealed that among the 72 cases, 56 (77.8%) were classified as type III, 13 (18.1%) as type II, and only 3 (4.1%) as type I.The majority of our cases were of type III, followed by type II, whereas only three cases were found to be the type I variant. This suggest that the pathology of nasopharyngeal carcinomas in Ghana is indicative of the endemic type. This is in accordance with previous results from Anim et al. and Adam et al., who noted that the pathology of NPC in Ghana resembles that seen in endemic areas , 28.The predominance of the undifferentiated type in most of the Ghanaian population demonstrates EBV genome (18/56) only in the type III NPC. Meanwhile, the relatively high prevalence of NPC cases in the younger Ghanaian subjects suggests that exposure to the EBV during the early ages of life could be a major contributing factor to NPC in Ghana. This confirms the earlier findings made by Anim et al., who stated that the close association between EBV and NPC on the one hand and Burkitt's lymphoma on the other could provide a common denominator in explaining the greater prevalence of NPC among young patients in Ghana .The overall prevalence of EBV DNA in the studied NPC tissue samples was 25% (18 out of 72). Out of the 18 EBV DNA positive cases, results showed that the EBV DNA was only present in the NPC type III, thus 32.14% (18/56) of the total number of type III EBV positive cases obtained. None was seen in both types I and II. Although molecular analysis revealed that all the EBV positive NPC biopsies were of the type III, HPV testing revealed the presence of HPV DNA in all 3 histological subtypes. These figures are similar to those reported in Greece, where among 63 FFPET samples of NPC examined using PCR, EBV specific sequence was amplified in 20 (32%) and HPV in 12 19%) of the samples % of the . HundredThe nonkeratinizing type (types II and III), particularly the undifferentiated histopathological type, has the strongest association with EBV infection , 28. ResA total of fourteen (14) specific HPV genotype primers  were used in this study. This included both HR-HPV  and LR-HPV .Out of the 14 HPV DNA positive cases, 13 (92.9%) were infected with HPV-18, and only 1 (7.1%) was infected with HPV-31; these are HR-HPV genotypes. This is in line with the research reported by Attoh et al. , where tAlthough the study found only HPV-18 and HPV-31 in the NPCs, other research work reported on other HPV genotypes such as HPV-59, 16, 33, 35, 45, and HPV type 6/11 in NPC cases , 21. TheIn our study, EBV and HPV coinfection was detected in 3 (4.2%) of the 72 cases and was detected in the undifferentiated variant of the NPC. In USA, among 30 FFPET samples of NPC studied using the ligation-dependent polymerase chain reaction (LD-PCR), 2 samples were coinfected with HPV and EBV . Also inResearch carried out in China revealed that, among 88 fresh NPC specimens from the Chinese population, coexistence of EBV and HPV DNA was observed in 42% of NPC samples , and simIn terms of histological types, EBV and HPV positive cases were seen mostly in the undifferentiated type of the NPCs , followed by 21.4% and 7.5% for HPV only in type II and type I, respectively. However, there was no statistically significant association between histological subtypes of NPC and EBV infection and also between histological subtypes of NPC and HPV infection. Although some studies had no significant association to both EBV and HPV using FFPET samples , most stFrom the observed results, EBV/HPV had a relative risk of 1.2, which means that the NPC subjects have 1.2 times high risk of being infected with EBV than HPV. This fact is supported by the high number of EBV positive cases (25%) realized, as compared to HPV positive cases (19.4%) in the study.Generally, we observed no statistically significant association between the oncogenic viral (EBV and HPV) infection and age, gender, or the tumour type. From the observed results, there appears to be a broad profile in the relationship between HPV, EBV, and NPC histological subtypes. In totality, this study would be the first to demonstrate EBV and HR-HPV (HPV-18 and HPV-31) genotypes in NPC cases in Ghana. Since no research data exists currently in the country on NPC and its association with the oncogenic viruses, it is hoped that the results of this study would act as the baseline for putting in place measures to monitor and control the disease.Out of the 72 archival NPC biopsies screened, EBV DNA PCR amplification was achieved in 18 (25%) NPC cases and that of HPV DNA, in 14 (19.23%) cases. The HPV genotypes associated with the cancers were of the high risk types; HPV-18 and HPV-31. There appears to be a geographical difference between our results and those obtained from high risk areas. In conclusion, the histopathology of nasopharyngeal carcinoma in Ghana is of the type described in areas endemic for the disease, and they are associated with HPV and EBV in a minority of cases.


The VIL imaging revealed surprising insights into the understanding of the iconography and the technology of this rare painting. Previously undetected elements of the painting include rays of light that were discovered emerging from the body of Christ and illuminating the other figures in the painting. Although this motif is an important part of the Transfiguration narrative and appears in most of its scenes depicted elsewhere, it had not been previously identified in this painting as it was undetectable by any other inspection technique. Another important result is the identification of Egyptian blue as a common blue pigment used at Shivta during the Byzantine period, when it is considered to be very rare.The Transfiguration scene depicted in a Byzantine church at Shivta, Israel, is one of two figurative examples of the scene from the early Christian period. The use of Egyptian blue pigment in the wall painting was investigated with various analytical methods. Visible Induced Luminescence (VIL) imaging was used  The Transfiguration is the moment when Christ revealed his divine nature to his disciples, Peter, John and James . The scein-situ Transfiguration images have survived from the period before Iconoclasm . In. In-1, t of Cu2+ ,16,17.The VIL showed positive identification of areas that were painted with Egyptian blue, which were clearly observed from its very intense emission in the near IR spectrum that appeared as very bright areas in the VIL images ,16 Fig .The identification of Egyptian blue is significant for the understanding of the use of the pigment during the Byzantine period. Although the pigment was extensively employed during the Roman period between the 1st century BCE and the early 4th century CE ,30,31,32The findings of this study show that Egyptian blue was still in use in the eastern Mediterranean after the Roman period, as reflected in the extensive presence of the pigment in large areas in the painting. Therefore, the identification of Egyptian blue in the Transfiguration painting at Shivta adds an important landmark that fills a gap in the historical chronology of the pigment's use during the first centuries after the Roman period.in-situ in areas where no paint could be detected by the naked eye , which is considerably paler compared to the darker lower part of the apse . The difThe light rays are a prominent motif of the Transfiguration scene in the St. Catherine mosaic and other later depictions of the Transfiguration ,40. FormThe use of Egyptian blue for depicting the rays of light, although it was painted underneath other colors and not visible to the naked eye , Fig 7, Thus, this special use of Egyptian blue in the Shivta Transfiguration wall painting might have a religious meaning and symbolism.When discussing the meaning of the color blue in Byzantine art, Gage  suggestsThe Gospel of John differs from the Synoptic account of the Transfiguration by identifying Jesus as “the light” , and by announcing that “In him was life, and the life was the light of all people” (v. 4) . The disThe Transfiguration scene involves as a central element a bright, divine light gleaming from Christ‘s face and clothes, a light that orthodox Christianity has referred to as the “uncreated light” . The TraAnother key element in the Transfiguration scene is the mandorla surrounding the figure of Christ, which is not well preserved and hard to define in the Shivta painting. A blue color is not visible inside the mandorla, but through VIL imaging, remains of Egyptian blue were discovered in different parts in the outer strip of the mandorla. It is likely that the outer strip of the mandorla was based on Egyptian blue. In addition, the VIL images enabled us to note the two thin lines of a dark color that were painted above the Egyptian blue and define the width of the outer mandorla strip (about 12 cm). This phenomenon appeared, in areas where the rays cross the mandorla outer border, e.g. in ray 2, where the two dark lines are emphasized by the bright background of the Egyptian blue emission Figs  and 8.The use of blue color for depicting elements of the mandorla around Christ is documented in various other works of art of the Transfiguration scene, including the mosaics of St. Catherine, Ravenna and other later Byzantine works of art: in the Carolingian wall painting of Müstair in Switzerland, of 800 CE  and in tThe use of VIL imaging was highly important in revealing Egyptian blue in the depiction of the different figures of the scene as shown in Figs This area is covered with thick incrustation that does not allow the determination of a clear pattern of decoration, although some traces of color appear where the incrustation is missing. VIL imaging revealed part of the original decoration at the edge of the apse which appears as a geometric pattern of parallelograms depicted in Egyptian blue .There is a niche in the lower part of the center of the apse with an arch decorated with triangles in alternating colors. Only traces of colors can be seen today. These decorations were described by Woolley and Lawrence in 1914 [VIL imaging shows Egyptian blue in one of the triangles, at the center of the arch of the niche , correspAn initial survey of the two other apses at Shivta’s southern church revealed very slight remains of wall paintings that, together with a literature survey, indicate that these apses were also painted.In the northern apse, except the red triangle decoration, no clear painting or colors were visible. As mentioned above, the VIL imaging technique also detected clear remains of the decoration, corresponding well with the description of Woolley and Lawrence , and conOther parts of the northern apse also reveal patterns of painted areas of Egyptian blue, such as remains of possible rays. Traces of Egyptian blue throughout the entire surface of the apse indicate that it was probably painted in a similar technique and style to the southern apse.On the contrary, in the central larger apse, only traces of a very small fragment of blue-painted plaster (about 5x5 cm) were found. This blue color was analyzed with VIL imaging, indicating that it was probably painted with a different blue pigment than Egyptian blue. This supports the fact that this area was painted on plaster, using a different technique than the southern apse of the Transfiguration scene.VIL Imaging was also used to study the application techniques of the Transfiguration painting: although the original distribution of the blue color was not visible, the restricted area in which it should have been painted was well planned ahead. The Egyptian blue layer that was painted below other colors , createdVIL imaging provided important information on areas where the painting was damaged or no longer visible, such as in the southern half of the apse , where cAn important discovery by the VIL imaging is related to repairs of a stone block in the southern apse, indicated by the presence of modern cement. The VIL image of Ray 4, shows a clear break in the line of the ray, which might indicate that during former restoration works the stone was probably put back in the wrong orientation . In our This study used VIL imaging to investigate the presence of Egyptian blue pigment in the early Christian wall painting of the Transfiguration scene in Shivta. It showed that the pigment was available to the artists of Shivta in the early Christian period. This technique proved to be a very efficient tool for identifying Egyptian blue and mapping its distribution on the painted surface as well as an under-drawing.The results of the study clearly show that Egyptian blue was found in areas that were not previously identified as being painted by any other method, and also show the pattern of painted areas that were impossible to interpret before.The most intriguing outcome of this study was the detection of the light rays that emerge from the body of Christ to the other figures in the scene, a result that has highly significant iconographic and theological importance.The significance of the findings is mainly in the new and exciting information, which provides very important highlights to our understanding of the oldest known wall painting of the Transfiguration scene. VIL imaging is thus shown to be an efficient technique for detailed studies and surveys of wall paintings in archaeological sites where there was extensive use of Egyptian blue.S1 Text"The Wilderness of Zin ". Palestine Exploration Fund DS111.A1P28, Vol. 3 copy 1. Harrison.(PDF)Click here for additional data file.S1 Figa: Exposure time– 1/10 s. f-number 8. No stray light–all references are black. b: Exposure time– 1/6 s. f-number 8. Very little stray light at the bottom of the photograph, references of 5% and 18% are exposed. c: Exposure time– 1/4 s. f-number 8. Stray light appears–references of 5% up to 50% are exposed. d: Exposure time– 1/2 s. f-number 8. More stray light is seen–references 5% up to 50% are exposed. .(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe geometric pattern of parallelograms in the outer border of the apse is depicted with Egyptian blue and thus revealed only by the VIL technique through the thick incrustation .(TIF)Click here for additional data file.S4 FigThe arch of the inner niche of the northern apse showing that the downward pointing triangles are red in regular photography (a) and the upward pointing ones have traces of Egyptian blue shown only in the VIL image (b) .(TIF)Click here for additional data file.S5 FigAstone block that has traces of ray 4 revealed by VIL imaging, showing that it was formerly put in place wrongly (a). A digital reconstruction shows its correct position (b) .(TIF)Click here for additional data file.


Expressions of TLR4, NF-κB p65, and iNOS in CaSki, HeLa, and C33a cells were determined by Western blot. NO concentration in cervical canal of CC group was significantly higher than in other groups (P < 0.05). Positive rates of iNOS in cervical tissues were 72.1%, 28.2%, and 3.1% in the CC group, HR-HPV group, and controls, respectively (P < 0.05). Levels of TLR3, TLR4, TLR7, TLR8, NF-κB p65, and iNOS in cervical epithelial cells were higher in CC group than in other groups (P < 0.05). Both mRNA and protein levels of TLR4, NF-κB p65, and iNOS were higher in HPV-positive HeLa and CaSki cells than in HPV-negative C33a cells (P < 0.05). Together, these results suggest that TLR/NO signaling pathway may be involved in pathogenesis of cervical cancer caused by HR-HPV.Human papillomavirus (HPV) can activate Toll-like receptor (TLR)/nitric oxide (NO) signaling pathways; however, whether the TLR/NO pathway is involved in cervical cancer caused by high-risk HPV (HR-HPV) remains unclear. In this study, 43 HR-HPV-positive patients with cervical cancer (CC group), 39 HR-HPV-positive patients with a healthy cervix (HR-HPV group), and 33 HR-HPV-negative controls were recruited. NO concentration in cervical canal and expression of inducible NO synthase (iNOS) in cervical tissues were detected. Expressions of key TLR/NO pathway genes (TLR3/4/7/8, NF- Cervical cancer is one of the most common gynecologic malignant tumors. The incidence of cervical cancer is the third highest among all female malignant tumors worldwide, and its mortality rate is high , 2. HighHPV can be eliminated by autoimmunity in 90% of HPV-infected patients. Among the remaining chronically infected patients, only 1% will finally develop cervical cancer , 6. As HToll-like receptors (TLRs) are the most distinctive pathogenic pattern recognition receptors involved in the defense against infection in humans . In addiIn recent years, the tumor microenvironment has become a hot topic in oncology research. Nitric oxide (NO) is one of the key components in the formation of the tumor microenvironment caused by chronic infection . InducibκB), regulate iNOS, and mediate NO secretion through the myeloid differentiation factor 88 (MYD88) or TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway [The binding of TLR with its specific ligand has been shown to trigger signal transduction cascades, activate nuclear factor kappa B  . MeanwhiE6 and E7 mRNA kits  were used for the HR-HPV detection, which can detect 14 kinds of HR-HPV , according to manufacturer's instructions. For detection of the NO concentration in the cervical canal, those patients with local cervical bleeding were excluded and specimens of cervix secretion with cleanliness ≤ I degree were selected. This research was approved by the ethics committee of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. Written informed consent was obtained from all subjects for participation in the study.κB p65 monoclonal antibody, rabbit anti-human iNOS polyclonal antibody, and mouse anti-human GAPDH monoclonal antibody were from Cell Signaling Technology .CaSki (HPV16+), HeLa (HPV18+), and C33A (HPV−) cell lines were purchased from the Chinese Academy of Sciences Typical Culture Preservation Committee Cell Bank . The NO detection kit was from Beyotime Biotechnology . The streptavidin-peroxidase (SP) immunohistochemical staining kit was from Mingrui . The mRNA isolation kit, TRIzol kit, fetal bovine serum (FBS), and DMEM were from Thermo Fisher . Rabbit anti-human TLR4 polyclonal antibody, mouse anti-human NF-Patients lay in the lithotomy position. The cervix was exposed with a speculum, and the cervical secretion was wiped. A cotton swab was placed into the cervical canal about 1 cm for 20 s and then washed in the glass tube containing 1.5 mL double-distilled water for 2 min. The NO concentration was indirectly detected using the Griess method, as described in the instructions for the NO detection kit. The absorbance at 540 nm was determined with an iMark/xMark microplate reader . A standard curve was drawn , and then the concentration of the sample was calculated.μm slices. After deparaffinization, each specimen was incubated for 5 min with 3% hydrogen peroxide, followed by overnight incubation with anti-iNOS polyclonal antibody (1 : 50) at 4°C. The specimens were incubated with biotinylated secondary antibodies for 10 min, followed by treatment with horseradish peroxidase-labeled streptavidin for 10 min. A section was considered negative or positive according to the absence or presence of positive yellow or brown staining. The staining based on positive cells was scored as follows: no positive cells (0), 1%–10% (1), 11%–50% (2), 51%–80% (3), and >81% (4). The staining based on color was scored as follows: no positive cells (0), light yellow (1), brownish-yellow (2), and brown (3). The final score was the multiplication of the above two scores. The positive or negative expression was determined by the final score of 0–2 (−) and >2 (+). The positive rate was then calculated.Expression of iNOS protein was detected by the SP immunostaining kit, according to manufacturer's instructions. Briefly, the histologic material fixed in 10% formalin and embedded in paraffin was cut into 3 μL SYBR Green Master Mix, 0.2 μL each of forward and reverse primers, and 2 μL cDNA and was taken to a final volume of 20 μL with water. Quantitative PCR was performed for 40 cycles of denaturation (95°C for 45 s), annealing (62°C for 30 s), and extension (72°C for 30 s); dsDNA was measured at 86°C after each cycle. All the data of the triplicate experiments were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Fold change in the relative gene expression to control was determined by the standard 2Ct−ΔΔ method.The expression of key genes in the TLR/NO signaling pathway in CaSki, HeLa, and C33a cells was detected by quantitative RT-PCR. Total RNA was extracted using TRIzol reagent from cells. cDNA was then synthesized from a total of 100 ng extracted RNA, followed by PCR using a quantitative PCR kit according to the manufacturer's instructions. All primers used are listed in 6 cells were lysed in 200 μL cell lysis buffer  for 30 min at 4°C and then clarified by centrifugation at 8000g for 10 min. Protein concentration of each sample was determined using a protein assay kit , with bovine serum albumin as the standard. Western blotting was performed as previously described [μg of cell protein underwent SDS-PAGE, followed by transfer to a polyvinylidene difluoride membrane . After blocking with 5% nonfat milk powder in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20, 1-2 μg/mL of primary antibody was added and incubated with the blots overnight at 4°C. After washing and incubation with horseradish peroxidase-labeled secondary antibodies for 2 h, the membrane was imaged using a VersaDoc MP 5000 system (Bio-Rad). GAPDH was used as an internal reference using an anti-human GAPDH polyclonal antibody .Approximately 1 × 10escribed , 19. Brit-test was used to compare values between the two groups; one-way analysis of variance was used when comparing more than three groups. Statistical significance was set at a level of P < 0.05.Every experiment was repeated three times with duplicates, and data are reported as the mean ± SD. Statistical significance of the results was calculated using SPSS v.20 software . Student's According to the FIGO staging criteria, 17 cases (39.5%) of the 43 cervical cancer patients were phase I, 8 cases (18.6%) were phase II, and 18 (41.9%) cases were phase III. Sixteen cases (37.2%) had lymph node metastasis, 21 cases (48.8%) had lymphatic vascular invasion, and 6 cases (14.0%) had both. The detailed information was shown in μmol/L in the cervical cancer group, significantly higher than that in the HR-HPV group  and the control group . In addition, there was significant difference between the HR-HPV group and the control group .Immunohistochemical results showed that no tan or brown staining was observed in the cervical tissue of most healthy controls and that iNOS protein expression was negative . A smallκB p65, and iNOS mRNA expression levels in the cervical tissue epithelial cells of the three groups and found that all mRNA levels were higher in the cervical cancer group than in the HR-HPV group and control group , HeLa (HPV18+), and C33a (HPV−) cells. First, we detected mRNA expression levels with quantitative RT-PCR and found that the TLR4, NF-In this study, we investigated the role of the TLR/NO signaling pathway in HR-HPV-positive cervical cancer. We found that the expressions of key genes and proteins in the TLR4/NO signaling pathway were upregulated in HR-HPV-positive cervical cancer tissues and cells, suggesting that TLR4/NO signaling pathway activation may participate in the pathogenesis of cervical cancer caused by HR-HPV infection.κB activation in tumor cells, immune cells, and other cells, thus synthesizing a large amount of NO [The expression of iNOS occurs during inflammation, cancer, and other pathological conditions . Its expnt of NO , 16. Prent of NO . This sunt of NO . Furthernt of NO . TogetheκB p65, and iNOS mRNA in the cervical squamous epithelial cells were significantly higher in the cervical cancer group than in the HR-HPV patients and healthy controls. In addition, although there was no obvious difference in TLR expression levels between the HR-HPV and healthy control groups, the NF-κB p65 and iNOS mRNA expression levels were increased significantly in HR-HPV patients. This suggests that HR-HPV may mediate the activation of NF-κB p65 and iNOS pathways through receptors other than TLRs.Chronic inflammation is closely related to the pathogenesis of tumors . PathogeThe TLR3 pathway can be activated by many viruses, such as influenza A (H1N1), simian immunodeficiency, and measles –28. In tκB p65, and iNOS) were significantly upregulated in HR-HPV-positive cervical cancer tissues and were also significantly higher in HR-HPV-positive cervical cancer HeLa and CaSki cell lines than in the HPV-negative cervical cancer C33a cell line. Together, these results indicate that TLR4/NO signaling pathway activation in cervical cancer is associated with HR-HPV infection and may be involved in the regulation of the local cervical immune microenvironment and the pathogenesis of cervical cancer.In the current study, we found that the expression levels of key molecules in the TLR4/NO signaling pathway , the precursor of cervical squamous carcinoma, were not included, and more fully designed studies including patients with CIN should be carried out in future; second, this study is a cross-sectional research, and the cause and consequences relation between TLR/NO signaling pathway and cervical cancer should be validated by further studies. For example, the expression of NO and iNO and activtion of TLR/NO signaling pathway should be investigated after elimination of HPV .In conclusion, the expression levels of key genes in the TLR4/NO signaling pathway are upregulated in HR-HPV-positive cervical cancer tissue. Based on our results, we speculate that TLR4 pathway activation is associated with HR-HPV infection and may participate in the regulation of the local cervical immune microenvironment and the pathogenesis of cervical cancer. This is a novel immunoregulatory mechanism in the local cervical microenvironment and provides new strategies for the development of cervical cancer immunotherapies and for the elimination of HPV. Despite this, the role and mechanism of the TLR4/NO signaling pathway in other types of HPV infection, as well as the role of other receptor pathways in HPV infection and cervical cancer, require further investigation.


Highlighted text in the Internet  is predominantly blue and underlined. The perceptibility of these hypertext characteristics was heavily questioned by applied research and empirical tests resulted in inconclusive results. The ability to recognize blue text in foveal and parafoveal vision was identified as potentially constrained by the low number of foveally centered blue light sensitive retinal cells. The present study investigates if foveal and parafoveal perceptibility of blue hypertext is reduced in comparison to normal black text during reading.A silent-sentence reading study with simultaneous eye movement recordings and the invisible boundary paradigm, which allows the investigation of foveal and parafoveal perceptibility, separately, was realized . Target words in sentences were presented in either black or blue and either underlined or normal.No effect of color and underlining, but a preview benefit could be detected for first pass reading measures. Fixation time measures that included re-reading, e.g., total viewing times, showed, in addition to a preview effect, a reduced fixation time for not highlighted (black not underlined) in contrast to highlighted target words (either blue or underlined or both).The present pattern reflects no detectable perceptual disadvantage of hyperlink stimuli but increased attraction of attention resources, after first pass reading, through highlighting. Blue or underlined text allows readers to easily perceive hypertext and at the same time readers re-visited highlighted words longer. On the basis of the present evidence, blue hypertext can be safely recommended to web designers for future use. The Internet plays an important role in our daily life. One of the first but also most critical advantages of the Internet is the use of hypertext. Hypertext allows the web designer to efficiently link important snips of text to additional information. Thus, hypertext works by replacing referencing in printed text and eliminating effortful literature searches. The most common implementation of hypertext, embedded as blue underlined text, was prominently criticized . It was Recently, The finding in the first experiment from To differentiate between these hypotheses the present study realized an invisible boundary paradigm . This paIn case parafoveal bottom-up processing of blue stimuli is limited, a reduced parafoveal preview benefit in contrast to black words is expected. Limited foveal bottom-up processing of blue text would result in higher fixation times of blue vs. black target words. This should be the case irrespective of underlining or parafoveal preview. The underlining aspect is crucial here as longer fixation durations for blue text in contrast to underlined black target words would exclude the possibility that such a difference would be the result of highlighting. Both parafoveal and foveal findings would indicate a hampered bottom-up processing of blue hypertext. Alternatively, if top-down processes that originate from highlighting influence the reading behavior, than the un-highlighted condition  should receive less attention. In contrast, the highlighted words, blue not-underlined, blue underlined and black underlined targets, should receive additional attention reflected in longer fixation duration measures. If color or underlining results in independent highlighting effects , one can even recommend text underlining or coloring independently for hypertext use.Forty native German–speaking students  with normal reading speed measured by the unpublished adult version of the Salzburger-Lese-Screening . Participants were seated about 52 cm in front of a CRT monitor  and a forehead and chin rest stabilized their heads. The display change latency of the experimental setup was below 15 ms (for details see N = 320) allowed the presentation of each sentence in one of the eight conditions  were assigned to each of the eight versions of the sentences (Latin square design). The parafoveal preview manipulation was realized by randomly replacing 45% of the black or blue pixels of the presented letters .The manipulation of color and highlighting was realized with five letter target words embedded in sentences, which were matched on the most important word characteristics .A 3-point calibration of the eye tracker preceded the experiment. Fixating between two vertical lines in the left margin of the monitor triggered sentence presentation in such a way that the participants’ fixation was at the center of the sentence’s first word. The students read silently for comprehension. Fixating an ‘x’ in the lower right corner of the screen terminated the trial and removed the sentence from the screen. After the presentation of about 50 randomly selected sentences, a question mark appeared on the screen and the experimenter orally presented comprehension questions, which the participants almost always answered correctly  for the skipping probability  with the lme4-package (https://osf.io/8c57w/).Skipping probabilities, first fixation durations, gaze durations, go-past times and total viewing time are reported. First fixation durations, gaze durations and total viewing times shorter than 80 ms were removed from the data (for each measure < 1% of the data). Data analysis was administered with linear mixed effect models (LMMs) for the -package  in R. G/As expected skipping probability, presented in In contrast, eye movement measures based on fixation durations during first pass reading indicated a strong preview benefit but no effect of color or highlighting. This was shown by the reliably lower first fixation durations and gaze durations for un-degraded parafoveal presentation in contrast to degraded previews see . No relit = 2.29; no underlining effect for blue targets; estimate = 0.002; SE = 0.016; t = 0.12). Both the interaction in total viewing times and the underlining effect in go-past times indicate that highlighting influences processing after first pass reading. The comparison of the two measures showed that re-readings after the target word was passed  reduced the effect of underlining for blue written words. This indicates that highlighting either by color or underlining increases the total reading times reflecting the allocation of additional attentional resources to highlighted words after the target word was first passed. Therefore, the reduced skipping probability of blue target words, described by t = 2.27).The total viewing times, including all re-fixation times after the first encounter  and the go-past times , showed, in addition to a reliable degradation effect, a reliable interaction of word color and underlining and a reliable effect of underlining, respectively. In sum, the present study demonstrated that reading was not significantly hampered by blue text presentation. Thus, the current findings do not indicate a bottom-up perceptual disadvantage of blue underlined hypertext in foveal and parafoveal processing. In contrast, the increased total viewing time for highlighted stimuli indicates an additional allocation of attentional resources triggered by top-down processes. These processes might reflect the learned association of hypertext to informative snips of texts in the Internet. Using blue underlined stimuli effectively highlights hypertext without hindering parafoveal and foveal perceptual processes during reading. In addition, the present study shows that text underlining and color results in highlighting, which indicates that either could be used independently to optimally implement hypertext. In conclusion, blue underlined hypertext allows effective reading and, therefore, can be safely recommended to web designers for future use.


Meetings are a common occurrence in academic and medical life. However, most of these meetings will be under-productive and inefficient uses of time. In this article, we provide valuable tips on how best to plan a meeting and get the most out of the people in attendance. This includes how to assess whether a meeting is necessary and what form this meeting should take. In addition to this, guidance is divided into before, during, and after the meeting. This guide will provide structure to your meetings and improve the output you and your team gain from them. Around the world, millions of meetings are being held every day—most of them unproductiveA meeting for meeting’s sake is unlikely to be productive. Before you call a meeting, think about whether there is any novel information or updatesIf there is new information, how much communication is required to find an appropriate course of action for the news? Can the update be provided in 10 to 15 minutes? If so, perhaps an email bulletin would be sufficient? Other similar problems can be solved without a meetingDo you need a question answered? PHONE CALLAre there difficult/sensitive issues? MEET ONE TO ONEA meeting is perhaps only necessary when the subject matter(s) require at least an hour of your time, any less can usually be resolved with alternative meansDetermine the structure and purposeWhat is the objective?Who needs to attend?Will an external speaker be required?How much time is needed?What preparation will help?What is your role?Communicate in advanceDevelop a written agenda—assigning ownership to each item.Send an agenda and supporting materials in advance.Set expectations for attendance.Set context/framing for the meeting—tell people why the meeting is being held.Ensure appropriate rooms are booked in advance, with extra time allocated for seating rearrangement.If special items are required  make sure that this is arranged well in advance.For communication in advance of meetings, online surveys, for example, surveymonkey.com are commonly used tools. They can allow you to gauge expectations of topics to cover in the meeting, as well as help you to organize a convenient date for the involved parties.On the occasions that external speakers will be required for organization meetings, it is important to contact them early and make it clear to them the reason that you have involved them in this meeting, so that they may prepare appropriately.Start and finish on time.Assign a note taker and time keeperProvide context for meeting—again remind people why the meeting is being held.If people have not been properly introduced, let everyone be acquainted to encourage further productive discussion.Manage the discussionAsking for action from someone? Do it early, and be specific.Off-topic ideas coming up? Bring people back to the agenda—future discussion can be held on these topics.People talking for too long? Set time limits, move on to other topics and if there is time at the end come back to the topic.Want attendees to stay engaged? Use active listening strategies and keep it interactive.Want attendees to feel invested in the outcome? Acknowledge their mind-sets and interests verbally.Stick to the agenda.Review next steps and establish accountabilityEnd early.Send brief notes to both meeting attendees and those absent with decisions made (or unmade) and further action items and owners.Review what worked and what did not, and note these for next timeThis can be done in person, or online survey platforms and evaluation forms can also be used.Most meetings can be improved. First, question if a meeting is even needed. Then follow the steps provided for before, during, and after the meeting to get the most out of your own and your colleague’s time.


Limited national information is available in Morocco on the prevalence and distribution of HPV-sub-types of cervical cancer and the role of other risk factors. The aim was to determine the frequency of HPV-sub-types of cervical cancer in Morocco and investigate risk factors for this disease.Between November 2009 and April 2012 a multicentre case-control study was carried out. A total of 144 cases of cervical cancer and 288 age-matched controls were included. Odds-ratios and corresponding confidence-intervals were computed by conditional logistic regression models.Current HPV infection was detected in 92.5% of cases and 13.9% of controls. HPV16 was the most common type for both cases and controls. Very strong associations between HPV-sub-types and cervical cancer were observed: total-HPV (OR = 39), HPV16 (OR = 49), HPV18 (OR = 31), and multiple infections (OR = 13). Education, high parity, sexual intercourse during menstruation, history of sexually transmitted infections, and husband’s multiple sexual partners were also significantly associated with cervical cancer in the multivariate analysis.Our results could be used to establish a primary prevention program and to prioritize limited screening to women who have specific characteristics that may put them at an increased risk of cervical cancer. Cervical cancer is the second most common cancer among women worldwide, with an estimated 529,409 incident cases and 274,883 deaths in 2008 . More thSome sub-types of human papillomavirus (HPV) are the central and necessary cause of cervical cancer . HPV infIn Morocco, cervical cancer is the second most common cancer for women . In 2008There is a lack of information on the prevalence and distribution of HPV sub-types of cervical cancer in Morocco as well as on the role of other risk factors for cervical cancer –8. In MoTo assess the role of HPV infection and other associated factors in the development of cervical cancer, we have carried out a case-control study of invasive cervical cancer in Morocco.Between November 2009 and April 2012, we performed our case-control study in three cities of Morocco: Rabat, Casablanca and Fez. A total of 144 patients with cervical cancer and 288 controls were included. The cases were recruited in three centers: the National Institute of Oncology of Rabat, the Oncology center of the Ibn Roshd University Hospital of Casablanca and the gynecologic centers of Hassan-II University Hospital of Fez. Inclusion criteria for cases included having a newly diagnosed, histologically confirmed cervical cancer, and having no previous cervical cancer treatment.Controls were selected from both public outpatient gynecological hospitals and primary health-care centers with gynecological units. They were selected using an individual age-matching (± 5 years). Exclusion criteria for controls included: diagnosis or history of cancer, history of hysterectomy, cervical conization or cervical cytologic abnormality.Protocols were approved by Ethical Review Committees of the Fez Faculty of Medicine and the Hassan-II University Hospital. Because of the high illiteracy rate among this population, we have not been able to administer a written information sheet. Instead, an explanation of its content has been given to the patients and their verbal consent have been gathered.A trained nurse administered a standardized questionnaire to all participants. The questionnaire items concerned the socio-demographic characteristics, sexual behavior, genital hygiene, history of sexually transmitted diseases, reproductive and contraceptive history and smoking.All patients received a pelvic examination performed by an oncologist or a gynecologist. For the cases, a tumor biopsy specimen was taken for HPV-DNA detection and typing and it was performed by an oncologist. For the controls, cervical exfoliated cells were obtained and followed by PAP-smear preparation. Besides, cervical specimens were placed in tubes with PBS for HPV-DNA detection and typing.The nested PCR-amplification of a conserved region of the HPV-L1 gene-DNA with the consensus MY09/MY11 and GP5+/GP6+ primers followed by genotyping with direct DNA-sequencing, generally accepted as a scientific tool in research , 11 was https://www.ncbi.nlm.nih.gov/blast/).For DNA-sequencing, the nested PCR products, if positive, were purified by PCR purification ExoSaP-IT clean-up system (USB-USA) and sequenced directly using GP6+ primer as the sequencing primer and BigDye®Terminator v3.1 Cycle Sequencing Kit , according to manufacturer’s protocol, on an ABI3130XL-DNA analyzer. Nucleotides sequences were aligned and compared with those of known HPV-sub-types available through GenBank by using the online BLAST 2.0 software server . In our population, the distribution of cases and controls by residence area was different. Thus, we consider it as a major confounding variable.P = 0.05 was the level of statistical significance. Statistical analyses were performed with SAS® software 9.1.To assess the role of factors, other than HPV, we used a multivariate conditional logistic regression with reduction strategy. All variables statistically associated to cervical cancer in bivariate analysis, with the exception of HPV infection and residence area, were used in the multivariate analysis. p = 0.8). Among 144 cases, 96.4% were squamous cell carcinoma and 4.7% were adeno/adenosquamous carcinoma.The mean age of the cases was 51.9 ± 11.9 years while the mean age of the controls 51.3 ± 12.6  9.2%, pregnancy control 8.1%, diabetes 7.1%, rheumatology diseases 6.0%, respiratory diseases 4.2%, dermatologic diseases 3.9%, and other diagnosis 9.2%.HPV infection was detected in 92.5% of all cases (92.2% of squamous cell carcinomas and 100% of adenocarcinomas) and in 13.9% of control women. Eight different HPV sub-types were identified in either single or multiple infections. HPV 16 was by far the most common type among cases as well as control women. Only HPV 16 infections were found among the 6 adenocarcinoma cases. Four controls and no cases were infected with low-risk HPV sub-types  for women with HPV infection of any type compared to women without HPV Table . The oddIn the bivariate analysis Table , an incrIn the bivariate analysis Table , women bIn the bivariate analysis Table , the ageRegarding smoking status, only 2.1% of cases and 1.8% of controls had a smoking history ≤1 packets/years  while 2.8% and 0.3% of controls had a smoking >1 packets/years . We found no statistically significant associations between cervical cancer and family history of cancer.In the multivariate analysis Table , an incrThis case-control study provides information on the risk factors for cervical cancer and the infection of HPV in Morocco. The results confirm the finding of the investigators indicating that HPV DNA is present in the vast majority of cervical cancers (92.5%) , 13. OddAll HPV infections in cervical cancer cases were a high risk. This finding is similar to that found in other worldwide  or MorocThe frequency of HPV infection (13.9%) among control women in Morocco was intermediate, between the low prevalence found in Europe  and the Because of the infection by high risk HPV is a necessary, but not a sufficient, cause for cervical cancer , it has Our results suggest also that high parity increases the risk of cervical cancer. The explanatory mechanism is not clear  but a knThe number of sexual partners was not a determinant in the development of cervical cancer, as reported in other World populations. This could be explained by the fact that most of the women in our study reported a unique lifetime sexual partner. In Morocco, polygamy is not a frequent practice among women, in this study 79.2% of the cases and 88.6% of controls women said that they were monogamous. However, polygamy is a common practice among men in our study . Geographic clusters of cervical and penile cancers , as wellWomen who most likely belong to lower social classes, in which education and incomes are quiet low, are mainly subjects to cervical cancer , 27. AlsIn this study, sexual intercourse during menstruation was a determinant of cervical cancer. The explanatory mechanism is not clear, but a related finding was noted in Sichuan, China, where a strong protection was observed in women who abstained from sexual intercourse during menses .In our study, history of sexually transmitted infections represents a risk factor for cervical cancer in Moroccan women. This result is consistent with other studies. Indeed, markers of exposure to other sexually transmitted infections have been found associated with cervical cancer repeatedly , 32.Our study has many strong methodological points. We conducted a multicenter case-control study which improves the representativeness of cases of cervical cancer of the study compared to the cases of cervical cancer in Morocco. The two cancer centers of Rabat and Casablanca represent the first and largest oncology centers in Morocco. Before 2007, they were the only public centers for oncology in Morocco . In our There are some limitations in the design of our study. The protocol was intended to identify a group of controls individually matched by age groups to the age distribution of the cases. The selection of cases was made in cancer centers; which attracts patients from a wide, ill-defined reference population. This is particularly true for cases of cervical cancer, which are referred for radiotherapy from distant areas in Morocco. Controls were derived from public outpatient gynecological hospitals and communal health-care centers with gynecological unit resulting in a group of predominantly urban residency compared with the cervical cancer cases. In Morocco, the residence area is an important indicator of the socioeconomic and educational levels  Women liIn Morocco, HPV is the central cause of the cervical cancer cases. Currently, two HPV vaccines are available, a bivalent vaccine against HPV 16 and 18, and a tetravalent vaccine against HPV 6, 11, 16, and 18. In Morocco, the vaccine in theory could prevent 86.5% of cervical cancer cases. Despite the development of vaccination, screening programs remain a mainstay in the secondary prevention of cervical cancer. Identifying the factors, other than HPV, that contribute to the development of cervical cancer is essential.The results of this study indicate that certain factors, such as low educational level, male sexual behavior, multiparity, sexual intercourse during menstruation, and history of sexually transmitted infections are the main lifestyle risk factors for cervical cancer in Morocco. This information could be used to install a program based on health education and to prioritize limited screening and treatment services to the benefit of women who have these specific characteristics putting them at an increased risk of cervical cancer.


The role of human papilloma virus (HPV) in oral lichen planus (OLP) is controversial.The primary aim of the current study is to calculate the pooled risk estimates of HPV infection in OLP when compared with healthy controls.Bibliographic searches were conducted in three electronic databases. Articles on the association between HPV and OLP were selected from case-control studies or cross-sectional studies, following predefined criteria. Pooled data were analyzed by calculating odds ratios (OR) and 95% confidence interval (CI).Of the 233 publications identified, 22 case-control studies met the inclusion criteria. Collectively, 835 cases and 734 controls were available for analysis. The summary estimate showed that OLP patients have significantly higher HPV prevalence  than healthy controls. In subgroup analyses, the association of HPV and OLP varied significantly by geographic populations. The ORs ranged from 2.43 to 132.04. The correlation of HPV and erosive-atrophic oral lichen planus (EA-OLP) (OR: 9.34) was comparable and well above that of HPV and non-EA-OLP (OR: 4.32). Among HPV genotypes, HPV 16 showed an extremely strong association with OLP (OR: 11.27), and HPV 18 showed a relatively strong one (OR: 6.54).In conclusion, a significant association was found between HPV and OLP. The strength of the association varied across geographic populations, clinical types of OLP, and HPV genotypes. The results suggest that HPV might play an important causal role in OLP and in its malignant to progression. Oral lichen planus (OLP) is a common chronic autoimmune disorder, which may present epithelial thickening or atrophy with or without ulceration . ClinicaAlthough the etiology of OLP is still unknown, it is generally accepted to be a T-cell-mediated inflammatory disease . The reaAs early as 1987, the association between HPV and OLP was reported . In one However, results have been conflicting: some studies seemed to show that HPV did play an important role in OLP –10, but Two English databases (PubMed and Web of Science) and one Chinese database (CNKI) were screened in order to collect more articles. Finally, 22 case-control studies were included to perform this systematic review and meta-analysis. The primary aim of the current paper was to clarify the magnitude of the association between HPV and OLP, and to get a further understanding of it concerning with different geographical populations, different OLP clinical types and HPV genotypes.www.ncbi.nlm.nih.gov/pubmed), Web of Science (SCI) (http://webofknowledge.com/) and CNKI (http://epub.cnki.net/) to screen relevant literature, until the last search update on September 21, 2015. The search terms were  and (human papillomavirus or HPV). The reference lists of selected papers were searched to identify additional studies.A systematic search was performed in PubMed (The screening process was carried out by two investigators (J. Ma and J. Zhang) independently under the same criteria . DisagreStudies included in the meta-analysis had to meet the following criteria: (i) address the relationship between HPV and OLP; (ii) full-text papers; and (iii) original case-control or cross-sectional studies. Studies were excluded if they included any of the following: (i) reviews; (ii) incomplete data; and (iii) republished articles or reused data.et al. [Results of observational studies are greatly influenced by the design of the research. To guarantee the quality of the meta-analysis, each included paper was carefully assessed using the standard proposed by Chalmers et al. , includihttp://www.cochrane-net.org/revman). Heterogeneity among different studies was measured by calculating χ2 and I2, if P < 0.05 and I2 > 50% were considered statistically significant. The primary outcome was reported as pooled odds ratios (OR) with 95% confidence intervals (CI), calculated using the random-effects model when heterogeneity existed or the fixed-effects model when no heterogeneity was found. To investigate potential for publication bias, the funnel plot asymmetry of the included studies was evaluated. Subgroup analysis was undertaken for geographic differences, clinical types of OLP, and HPV genotypes.The meta-analysis was conducted using RevMan5, a copyrighted freeware developed by the Cochrane Collaboration, for preparing and maintaining reviews  were excluded to investigate potential selection bias. Articles with different methods of HPV detection were analyzed to assess detection bias.The process of search strategy is showed in On the basis of the criteria described previously , 20 studχ2 and I2 were 35.81 (P = 0.02) and 41%, respectively, suggesting heterogeneity. The random-effects model was used to analyze the data. The pooled OR was 6.83 (95%CI: 4.15–11.27), and the overall effect Z value was 7.54 (P < 0.00001), which indicated a strong association between HPV and OLP. The visual examination of the symmetry of the funnel plot did not suggest a large publication bias  . Only Raet al.  and Sand [et al.  reportedThe exclusion of low-quality studies did not change the summary estimate significantly, OR was 6.60 . CompariOLP is a chronic inflammatory mucocutaneous disease whose pathogenesis is still the object of much speculation. Different mechanisms by which OLP may develop have been hypothesized in recent years. The association between OLP and viral infections is one of the most controversial positions. The most widely studied viruses in OLP are HPV and hepatitis-C (HCV) .HPVs are small, double-stranded, and circular DNA viruses. There are approximately 40 genotypes known to infect the oral cavity and urogenital tract . The incet al. in 2011 [This meta-analysis showed a significantly strong association between OLP and HPV. The pooled OR showed that OLP patients have about a 7-fold higher risk of HPV infection than controls, which was consistent with results reported by Syrjänen  in 2011 . Besideset al. [The current study showed there to be wide variations in the association of OLP and HPV with regard to different geographic populations, which was consistent with results reported by Lodi G et al. . Among tet al. –13. In cThe most common clinical forms of OLP are EA-OLP and non-EA-OLP. In the current meta-analysis, the association between HPV and EA-OLP (OR: 9.34) was comparable and stronger than the association of HPV and non-EA-OLP (OR: 4.32). The risk of progression to malignancy for non-EA-OLPs was approximately 0.5%, but in EA-OLPs it was at least 3.5–4.0% during similar follow-up periods . All thiHere, the association strength between HPV 16 and OLP was extremely high OR: 11.27), and HPV types16 has been classified as high-risk (HR) type and reported to be associated with malignancy . Besides7, and HPRecent data from case-control and meta-analytic studies suggested that HPV is a causal factor of the development of several types oropharyngeal and oral cavity squamous carcinomas –39. HoweThe major limitations of the current meta-analysis are the detection/diagnostic bias and selection bias of the case-control studies that were included. The 22 articles evaluated in this meta-analysis used three kinds of HPV detection methods: PCR, ISH and IHC. PCR is exquisite sensitive and widespread used, it is generally considered as the gold standard in most meta-analysis . HoweverIn conclusion, this systematic review and meta-analysis showed a significant association between HPV and OLP. All sub-analyses strongly and consistently indicated this association. Because of the lack of prospective cohort studies, it was not possible to take a position on the relationship between HPV infection and oral malignancy. More prospective cohort studies are needed to formally confirm the role of HPV as an etiological agent of OLP, and a uniform research standard should be established to produce get more convincing results.S1 PRISMA Checklist(DOC)Click here for additional data file.


There is limited data on the genotype distribution of human papillomavirus (HPV) in the Taihu River Basin, home to 1.29 million people on the coast of eastern China. This study evaluated the prevalence and genotypes among different female age groups in this region.Twenty-six HPV strains  were detected using Tellgenplex™ HPV technology in samples obtained from three clinical hospitals located in different regions of the Taihu Lake Basin.The results showed that 1855 samples  were found to be HPV-positive. Of these, 1375 samples  were found to have a single HPV infection. Age-specific prevalence showed two peaks, one that corresponded to the group of 21–30 year-old women and the other peak that corresponded to the group of women over 51 years old. The three most prevalent genotypes were HPV52 , HPV16 , and HPV58 . Mixed strains HPV58 + HPV33 and HPV58 + HPV52 were most commonly found in females infected with multiple HPV types.This investigation reveals that HPV infection in the Taihu River Basin varied significantly among different age groups. The most prevalent genotypes are included in the nonavalent vaccine, V503, however this vaccine is not licensed for use in mainland China. The most frequently occurring genotypes should be considered in the development of next-generation HPV vaccines for optimal protection of public health.The online version of this article (doi:10.1186/s12879-017-2328-6) contains supplementary material, which is available to authorized users. Cervical cancer (CC) is a major cause of morbidity and mortality among women in China, and also represents an important global public health problem. Persistent infection by high-risk human papillomavirus (hrHPV) is required for the development of pre-invasive and invasive carcinogenesis , 2. To dEarly studies demonstrated that the distribution of HPV genotypes in CC varies with geographic location , 5, 6. HThe purpose of this investigation was to obtain data on the prevalence of HPV genotypes, including age-specific prevalence differences and the distribution of multiple HPV genotypes. This investigation analyzed samples obtained from 3 clinical hospitals located in different regions of the Taihu River Basin. From November 2013 and June 2015, a total of 9012 women aged 21–70 years old  attended a gynecological outpatient clinic and expressed interest in cervical cancer screening. Participants met the following selection criteria: a) had not had sexual intercourse, had not used vaginal medications, and had not washed in the previous 48 h; b) not currently pregnant; c) with no history of total uterus or cervix resection; d) a permanent resident of the region; and e) agreed to undergo the HPV test and participate in this study.The cervico-vaginal cells at the transformation zone of the uterine cervix were collected from all participants by a gynecologist using a standard cytobrush according to the standard sampling procedure at the recruitment sites. Cells were resuspended in 3 ml sample transport medium (STM) for the Tellgenplex™ HPV DNA Test  and stored at 4 °C. According to the kit instructions, samples can be stored in STM for 1 month at 2–8 °C and 6 months at −20 °C. We stored all samples at 4 °C for 1 week prior to testing to obtain the most accurate results.This investigation was approved by the Ethical Committee of Wujiang First Hospital affiliated with Nantong University (permit number: 201405). All the specimens and their corresponding information were obtained with written informed consent from the participants involved in the project.The Tellgenplex™ HPV DNA Test uses one multiplex PCR test combined with Luminex technology to identify the 26 most common HPV types . The procedure includes DNA extraction, PCR amplification, and bead-coated hybridization and was performed according to the manufacturer’s guidelines [All statistical analyses were performed using SPSS version 19.0 . All genotypes from single and multiple infections were computed individually. A multiple-type infection was defined as one that was positive for at least 2 types. These data were also stratified by age .Among the 9012 individuals included in this study, 29 specimens were found to be negative for β-globin, and 138 cases could not be grouped because the analysis was restricted to women 21–65 old. Therefore, 8845 subjects (the median age was 42 and the quartile range was 32–47 years) were included in the analysis. As shown in Fig. Among the age groups, the HPV infection prevalence ranged from 16.3% for women aged 31–40 years to 25% for those 51–60 yeas old Fig. . The freHPV infection is the most common sexually transmitted infection. The majority of HPV infections are temporary, especially among young women, but CC usually develops after 10 years of persistent hrHPV infection . RegularIn this study, a rate of 20.97% HPV infection was observed in patients ranging from 21 to 65 years old. Most patients had a single infection. Of these HPV-positive patients, 77.09% were hrHPV infections, and there were two peaks in this analysis corresponding to women 21–30 years old and 51–60 years old. HPV is a sexually transmitted disease, and the higher rate of infection in younger, single women may be because these women may have more frequent sexual intercourse, particularly with more than one partner. Older women also show a higher rate of infection due to effects of physiological and immunological disorders associated with hormone fluctuations during the menopausal transition. These two peaks were also observed in the frequency analysis of multiple infections. Additionally, the frequency of dual and 3+ HPV infections was highest in individuals over 50 years old. These trends are consistent with the results of other researches that reported a second peak for the prevalence of multiple infections in females who are ≥50 or 60 years of age , 9, 12.Of the three most dominant HPV genotypes, HPV 52, HPV 16, and HPV 58, 70.3%, 54%, and 59.5%, respectively, were single-infections and the remaining cases of these genotypes were present together with other HPVs. Wang et al.  reportedThis analysis of HPV genotype distribution in the Taihu River Basin in China has potential limitations. First, this study determined the distribution of the prevalence of HPV genotypes in women aged 21–65 years who attended a cervical screening, however, the absence of cytological data of cervical pre-cancer or cancer remains a major concern. Second, there was no sampling of tissue specimens in this study. Comparison of HPV detection rates between cervical scraping and biopsy methods may help to clarify the pathological mechanism of HPV infection and the evolution of CC. Overall, this study provides a description of the genotype-specific prevalence in women from the Taihu River Basin on the coast of eastern China.Lifestyle and health are dramatically affected by economic conditions, cultural habits, and changing population dynamics. Therefore, it is urgent to pay attention to changes in the prevalence of HPV infection, particularly at a regional level. In the Taihu River Basin, the coast of eastern China, we found that the three top genotypes were HPV 52 (19.95%), HPV 16 (13.48%), and HPV 58 (11.32%). Mixed strains HPV58 + HPV33 and HPV58 + HPV52 were most frequently found in women with multiple infections. Considering the specific genotypes present in this geographic location, a cost-effective vaccine, such as the nonavalent vaccine V503, may provide optimal prevention of cervical cancer in this area.