Prevalence of infection differs, with highest rates reported in sub-Saharan African, including the country of Tanzania. In pregnancy, the hormonal changes and immune changes seem to facilitate HPV persistence, increasing the cancer risk and the risk of vertical transmission towards the placenta and the fetus. The burden of HPV infection is still high despite multiple screening and detection test available. The AmpFire® HPV assay is a novel nucleic acid isothermal amplification with real-time fluorescence detection assay that can test simultaneously 15 high-risk HPV. This nested cohort study aims to contribute evidence on the prevalence of HPV infection and persistence across two time points among pregnant women in Pemba island, Tanzania.1=385 and n2=187) and were tested with AmpFire® screening assay, for simultaneous detection of the HPV 16, 18 and other high-risk HPV genotypes 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68.vaginal swabs that were previously collected during pregnancy were stored in eNAT buffer (nthe AmpFire® HPV assay detected an 11% and 6% high-risk HPV prevalence at the two time points among pregnant women in Pemba island, consecutively. For the 133 women whose samples were tested at both time points, the persistence rate of high-risk HPV was 64%.novel isothermal HPV assay, such as the AmpFire®, might be feasible to use in low-income regions. Genital human papillomavirus (HPV) is the most common sexually-transmitted virus in the world. Prevalence of infection differs among regions, with the highest rates reported in South America and sub-Saharan Africa, including the country of Tanzania . In mostConsidering the significance of HPV in numerous pathologies, the interest to develop innovative diagnostic methods has grown. To date, there are several molecular diagnostic tests available to detect hrHPV genotypes . Even thAmpFire HPV assay was Conformité Européenne  marked in 2017 and received Chinese food and drug administration approval in December 2015. For self-collected vaginal samples, the AmpFire test has shown similar specificity 92.9%) and almost comparable sensitivity (92.9%) rates as the Roche Cobas 4800 2.9% and . BesidesSample collection: the vaginal samples were collected in the context of a previously established biobanking effort (AMANHI) with the support of the Bill and Melinda Gates Foundation [undation  initiateundation . The vagundation . For theDNA isolation: DNA was extracted from the collected vaginal swabs with the Chemagen  automated DNA extraction machine by using the buccal swab extraction kit according to the manufacturer´s instructions and were afterwards stored at 5°C [d at 5°C .AmpFire HPV screening assay: the AmpFire HPV screening assay kit  was used in this study following the information in the user manual provided in the kit. The kit includes reaction mix, primer mix, external positive control template and negative control template. Briefly, 12μl reaction mix was combined with 11μl primer mix in a 0.2ml optical 96-well plate. Two μl of processed DNA samples were added to the reaction tube to bring the total volume to 25μl. Real-time PCR was performed using the Applied Biosystems™ 7500 Real-Time PCR system with the isothermal reaction condition set at 60°C while taking fluorescence dye reading at the FAM™/HEX™/CY5™/ROX™ channels once every minute for a total of 75 minutes. The thermocycling software system automatically reports the results of the cycle threshold (Ct) values for each amplification curve in all fluorescence channels. For each sample, an exponential amplification curve in CY5™, ROX™, FAM™ and HEX™ channels indicates the presence of DNA of HPV 16, HPV 18, other hrHPV genotypes and internal control respectively. The lack of exponential amplification curve in the HEX channel was interpreted as an invalid result. The test results were used to determine: hrHPV point prevalence; co-infections of HPV 16 and/or HPV 18 with other hrHPV; persistence and incidence rate of hrHPV during pregnancy.Statistical analysis: dichotomous variables were generated as pregnant women were either negative or positive for hrHPV infection. Persistence of infection was considered when the vaginal samples of the same women tested positive for HPV infection at both timepoints. Chi-square test was used to determine whether the point prevalence changed significantly over time during pregnancy. A p-value of <0.05 indicated a significant difference.HPV prevalence: a total of 11.2% (43/385) of pregnant women tested positive for HPV infection at the first time and 5.9% (11/187) at the second time point .me point . Of the me point . Among 1For women tested only once at either time point, the overall hrHPV prevalence was 11.1%, HPV16 prevalence was 1.5% and other hrHPV prevalence was 10.5% (data not shown in the figures). Within this group of women, the overall hrHPV prevalence at the first time point was 12.7% and at the second time point 3.7%. The distribution of the infected women was not symmetrical within both time point . The hrHCo-infections: only at the first time point, the assay detected multiple hrHPV infections in two women. These two women tested positive simultaneously for HPV 16 and other hrHPV genotypes.The persistence rate and new incidences: among the 133 pregnant women tested at both time points, eleven were hrHPV positive at the first time point, and seven remained positive at the second time point. The persistence rate for hrHPV was 63.6% in this subgroup. Among the women who tested negative for HPV at time point one, two acquired a new HPV infection and resulted positive at the second time point. One of the women had a de novo HPV 16 infection and a different women was superinfected with other hrHPV during the course of her pregnancy.In this cohort of pregnant women in Pemba island Tanzania, the hrHPV point prevalence was between 5.9% and 11.2%. The overall hrHPV positive status was higher in an earlier pregnancy stage compared to a later stage, although the difference was borderline significant. There is no logical biological argument why the hrHPV prevalence should be higher at the start of the pregnancy or why it should clear during pregnancy. Moreover, when the comparison was made between the paired samples, no significant difference was observed between the two time points. Once the study is complete at all time points, a more definite answer can be drawn.Previous studies showed a higher hrHPV infection rate among pregnant women, compared to non-pregnant ones . UnexpecCompared to other pregnant cohort studies, differences in geographical regions with varying exposures to infection and risk factors might explain the dissimilar HPV prevalence. This might partially explain the broad range in hrHPV prevalence reported: among Ghanaian (21% and 46%), Lithuanian (7-9%), Brazilian (25%), Turkish (15%) and Indian 39%) pregnant women 9% pregna,23-26. HWith regard to the persistence of hrHPV, we found a maximum rate of 64% in this cohort. This AmpFire screen HPV assay does not distinguish between hrHPV genotypes other than HPV 16 or HPV 18. Therefore, the exact persistence rate of specific hrHPV genotypes might be lower since now those fall within the same other hrHPV group. In addition, some women might have had triple or even multiple HPV genotype infections. Nevertheless, the persistence rate during pregnancy found in this study is roughly comparable with previous findings among pregnant women in Uganda (50.4% and 71.8%), Brazil (53%) and the Netherlands (58%), but is higher than in human immunodeficiency virus (HIV) positive pregnant women in Spain (46%) ,15,29. HThe AmpFire assay was previously compared to the Roche Cobas 4800 HPV assay and showed comparable sensitivity and specificity . Also, wTo our knowledge, this study is the first that investigated the prevalence of HPV in pregnant women in Pemba island, Tanzania. The sample size included in this study is higher than other pregnancy studies conducted in Uganda, Kenya, Nigeria, Ghana, South Africa, Brazil, Lithuania and India.Among pregnant women in Pemba island the hrHPV prevalence is rather low, but not negligible and the persistence rate seems to be high. This study highlights the importance of monitoring this viral infection during pregnancy. Therefore, good diagnostics methods for the detection and screening of HPV in pregnancy are essential to manage the burden of infection, as well as further co-infections that might affect the mother, fetus or newborn.HPV is the most common sexually transmitted virus in the world, with the highest rates reported in South America and sub-Saharan Africa;In pregnancy, hormonal and immune changes seem to facilitate HPV persistence, therefore increasing the cancer risk and the risk of vertical transmission towards the placenta;High-risk HPV infection during pregnancy has been associated with various adverse pregnancy-related complications or outcomes, such as vaginal infection, preterm birth and preterm prelabor rupture of membranes.The prevalence of high risk HPV is between 5.9% and 11.2% in pregnant women in Pemba island, Tanzania;The persistence rate for high risk HPV during pregnancy was high (63.6%) in this Tanzanian cohort.


The Uygur is the fifth most populous ethnic group in China. Compared to other Chinese population, cervical cancer in them had high incidence, and HPV infection also was particular. Their HPV integration situation has never been reported. We aimed to investigate the integration situation of 20 subtypes of HPV gene into host cell genome in Chinese Uygur cervical cancer patients; meanwhile, we explored the influence of gene integration on PD-L1 expression.  40 frozen Chinese Uygur cervical cancer specimens with positive HPV infection were obtained from the cancer prevention and treatment institute of Tumor Hospital Affiliated to Xinjiang Medical University. The integration situation of HPV gene into host cell genome was detected by Agilent SureSelect™ Target Enrichment Chip and Next-Generation Sequencing. The related genes were analyzed by GO functional annotation and KEGG pathway enrichment. The expression levels of PD-L1 in cancer cells were tested by immunohistochemical assay (IHC). Meanwhile, the relationship between PD-L1 levels in cancer cells and gene integration were analyzed. P < 0.05). There were 13423 integration events in 40 specimens, involving 6867 human genes. These integration events were distributed on all human chromosomes, and chromosome 19 had the excessive concentration phenomenon of integration events. There were some integration hotspots in human genome such as PPP1R37, HECW2, EMBP1, ANKRD50, SPTBN4, LINC00895, LYRM4-AS1, LINC00374, RBFOX1, CSMD1, CDH13, and KLHL4. Insertion breakpoints can be found in all gene regions of the HPV genome. The actual observation of the integration times of E1 and E6 was much higher than the expected value, while the actual observation times of E5 were much lower than the expected value. The result of GO functional analysis showed that binding molecular function and cellular process biological process were the main ways to influence the cell biological behavior of HPV gene integration. The enrichment pathway analysis of KEGG showed that pathways in cancer were the most important enrichment pathways involved in the genomic integration of HPV. The positive PD-L1 rate was 62.5%. Logistic regression analysis showed that 9p24.1 existing integration sites and the number of all gene integration were risk factors for PD-L1 expression . Conclusions and Relevance. Most high-frequency sites of HPV integration in Chinese Uygur cervical cancer are related to cancer progression, and the gene integration hotspots may be potential HPV carcinogenic targets. The problem of multiple HPV infection in Chinese Uygur cervical cancer patients should be paid attention. L1 and E6 genes are inapposite as the target gene of commercial HPV type detection kit, because of high-frequency breakpoints in these genes. The gene integration especially the integration existing on 9p24.1 could affect the expression level of PD-L1. The HPV multiple infection rate by HIVID was as high as 92.5%, much higher than 35.0% by the commercial kit ( Cervical cancer was one of the most common malignant tumors in China, in which its prevalence rate was 7th in all cancers and its mortality was 8th in all female cancers . HPV infThe mainstream methods of HPV detection included two types according to the basic principle, such as the second-generation hybrid capture method (HC-2) and fluorescence PCR method, which could be used to differentiate the subtypes of HPV infection and evaluate the total amount of viral replication in the high-risk type HPV. But these tests only focused on whether HPV gene existed in host cells, which could not detect whether HPV genome was integrated into the human genome, could not provide the location of the HPV genome into the host genome, and could not find which segment of the viral genome could integrate into the host genome. Because HPV gene integrating into the human genome was the key step that led to the host cell pathological changes, therefore it needed to analyze the HPV gene integration status, in order to assess the potential effect of HPV infection on the infected cervical cells, and thus, it met the requirements of precision medical treatment.Though HPV genome integrating into human genome research had been reported, most of the studies only focused on certain genes of HPV16 or HPV18 type, such as the genetic integration of HPV16 E6 and E7 genes ; the intHPV infection played an important role in the process of cervical cancer progression, which was one of the important drivers of cervical cancer. The HPV gene integrated into the cervical cell genome, then changed the cervical cell genome state, eventually creating important changes in the biological behavior of cervical cells, such as changing the normal immune state of cervical cells, leading to the immune escape of cervical pathological cells. Many studies have confirmed that PD-L1 on the surface of cervical cancer cells was an important molecular marker of tumor immunity. Therefore, the relationship between HPV gene integration into the cervical cell genome and PD-L1 expression of cervical cells was explored, which could help further clarify the role of HPV in the evolution of cervical lesions and provide new ideas for the prevention and treatment of cervical cancer.The 40 cases frozen cervical cancer pathological specimens were collected from the specimen repository of the cancer prevention research institute in the Tumor Hospital Affiliated to Xinjiang Medical University. All cases were Chinese Uygur cervical cancer, all of have existing HPV infection which were proved by flow-through hybridization and gene chip (FHGC). The ethics committee of the Tumor Hospital Affiliated to Xinjiang Medical University approved the study and the consent procedure.The basic technique of the study was performed with an Agilent SureSelect™ Target Enrichment System and the second-generation sequencing technology (the HiSeq 2000 sequencer of Illumina).(1) Chip Design and Order. We designed the sequence-capture probes according to 20 types of HPV genome  sequences, and these probes were produced by Agilent Inc., USA. The enrichment chip was also ordered from Agilent Inc., USA.(2) DNA Extraction. The DNA of 40 cases were extracted by a Genomic DNA Extraction Kit. Agarose gel electrophoresis and Qubit fluorometer were used to evaluate the DNA quality.(3) Sample Test. DNA fragments 150–200 bp were obtained after ultrasonic fragmentation; then, these fragments were purified, end blunted, “A” tailed, and adaptor ligated. The products of ligation were amplified by PCR and constructed into a DNA sequencing library. Bioanalyzer 2100  was used to quantify the concentration of the DNA library. Next, hybridization was performed according to the instruction of Target Enrichment Protocol. This process was carried out at 65°C for 24 h, and the untargeted fragments were then removed by washing buffer. These eluted fragments were amplified by PCR and were further processed to paired-end 100 bp read-length for sequencing by the HiSeq 2000 sequencer .(4) Data Analysis. First, clean reads were obtained through removing low-quality and duplicated reads, as well as adaptor-contaminated reads. Burrows-Wheeler Aligner (BWA) was used to align clean reads onto human  and HPV genome. The paired-end reads that can be mapped to human or HPV reference genome with both ends were removed. The remaining reads were paired-end assembled to reconstruct fragment sequences, which was used to locate the position of breakpoints more precisely. Subsequently, reference mapping was performed again to remap the paired-end assembled reads onto human and HPV genome using BWA. The position of a breakpoint was defined as the junction of human and HPV sequence in a paired-end assembled read. To minimize the impact of different sequencing data amount of each sample, the normalized support-reads number  was introduced, which was equal to the supported reads number of each HPV breakpoint per million clean read pairs. The breakpoints with Norm value ≥ 2 were retained. The Sanger sequencing validation rate for the selected breakpoints was 82.5%. ANNOVAR was used to do the annotation for the integrated breakpoints.2O2, antigen repair, and dyeing. Phosphate-buffered saline (PBS), rather than the first antibody, was the negative control; the previous positive specimen was the positive control. For PD-L1 expression in cancer cells, we chose the nests of cervical cancer including more than 100 cancer cells and then calculated the proportion of positive cells ; the positive criterion is in the cytomembrane and cytoplasm or both at the same time appeared tan particles.Monoclonal antibodies of PD-L1 were bought from Golden Bridge Biotechnology Co. Ltd . H&E staining was used to assess the pathological slices. The detection method used was IHC streptavidin peroxidase and diaminobenzidine chromogenic detection. All slices were produced using the following procedures: washing in xylene, ethanol, steamed classification, boiling water to wash, dissolving endogenous peroxide physical activity by incubation with 3% Hhttp://www.webgestalt.org) and DAVID (https://david-d.ncifcrf.gov/) were used to complete the Gene Ontology categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene function pathway categories. Multivariate logistic regression analysis was used to evaluate the relationship between the number of gene integration, the integration in 9p24.1, and PD-L1 expression by SPSS 17.0 statistical software.The Genesis analysis tool of webgenstal , 22.5% (9/40), 22.5% (9/40), 22.5% (9/40), 22.5% (9/40), 22.5% (9/40), 20% (8/40), 20% (8/40), 17.5% (7/40), 17.5% (7/40), 17.5% (7/40), and 17.5% (7/40). In this study, these top 10 genes were referred to as hot integration gene sites of HPV gene integration.Among the 13423 integration events in the 40 samples, 5057 events were found to be caused by the integration of HPV16 gene into the host cell genome, which is from 35 samples. According to these 5057 integration events, the gene breakpoint distribution of HPV16 genome was analyzed, which is shown in In Taking the length of every functional gene of HPV16 genome as reference, the expected value of gene breakpoints that should exist in every functional gene region was obtained, then compared to the actual number of observed gene breakpoints. The results were shown in The results of Gene Ontology categories are showed in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on 58 human genes, whose integration sites were detected more than 20 times. The top 10 enrichment pathways of gene function pathway categories are showed in The positive and negative PD-L1 expression in cervical cancer cells was present in 25 cases (62.5%) and 15 cases (37.5%), respectively  included to one of the high-throughput viral integration detection (HIVID) had proved to be very useful test methods , 15. In At present, the current clinical detection methods of HPV type were FHGC, real-time fluorescence quantification, and so on, which were the principle of various commercial HPV type detection kits. For 40 specimens, the retrospective results of HPV type detection were given by commercial HPV type detection kits, which was approved by CFDA, and the detection method was FHGC. In the retrospective results, the HPV subtypes were mainly HPV16 and HPV18; the HPV multiple infection ratio was 35% (14/40), which was exactly consistent with the previous reports . But by By analyzing the specific integration sites of HPV genes, it was found that the integration sites involved all chromosomes of human genome, with a total of 6867 human genes, indicating the randomness of HPV gene integration. PPP1R37, EMBP1, HECW2, ANKRD50, SPTBN4, LINC00895, CSMD1, LINC00374, CDH13, RBFOX1, LYRM4-AS1, and KLHL4 were the high-frequency integration sites; compared to the hotspots reported by Zheng et al. , there wIn By the functional and pathway enrichment analysis, it could be found that the HPV gene integration could mainly affect the physiological process of host cells and could also affect pathways in cancer. The GO and KEGG categories analyzed the integration gene sites, which was the first comprehensive analysis about the integration landscape. The pathways in cancer were a cell signal transduction system which played an important role in cell carcinogenesis and cancer evolution. Because HPV gene integration had the deepest level of impact about the pathways in cancer, so it could be supposed that the HPV gene integration into the cervical cells could cause the occurrence and progression of cervical cancer.As for the relationship between HPV infection and PD-L1 expression in cervical cells, it is still a great controversy. Most reports showed that HPV infection promoted the high expression of PD-L1 –27. PrevThe chip enrichment combined with the second-generation sequencing technology could significantly improve the detection sensitivity of HPV integration sites; multiple infectious problem was worthy of in-depth study, the HPV gene integrated into the human genome was universal and random, but also, there were some hotspots in human genome, and the hotspots in Chinese Uygur cervical cancer women were different from the other Chinese population. The breakpoints of HPV genome could lead to missed detection of the commercial HPV type detection kits. The integration of HPV genes into the human genome could affect pathways in cancer, which was a key step in the development of cervical cancer. The gene integration especially the integration existing on 9p24.1 could affect the expression level of PD-L1. The VIB should regulate the high expression of PD-L1 in some cancer cells because of oncogenic virus such as HBV and EB.


The frequency of HPV18 increased from 0% for women with normal cytology to 2.6% for CIN1, 3.4% for CIN2, 5.4% for CIN3, and 21.4% for CC (p = 0.03). HPV40 was only found in one patient with CC (p = 0.04). There was no relation between HPV genotype and women’s age. In Kunming (Southwest China), the frequency of HPV infection was 74.0% among women who underwent CC screening. HPV16 and HPV18 were the two most frequent genotypes. SPR could be of value for the screening of HPV infection.No study examined the frequency of human papillomavirus (HPV) genotypes by surface plasmon resonance (SPR) in Southwest China. This was a cross-sectional survey  in 150 patients who were hospitalized or volunteered for cervical cancer (CC) screening. A HPV typing kit was used to detect 24 types of HPV by the SPR technique. The HPV-positive rate was 34.8% in women with normal cytology and 92.9% in women with CC. The frequency of HPV16 increased from 9.4% for women with normal cytology to 28.9% for cervical intraepithelial neoplasia (CIN)1, 41.4% for CIN2, 54.1% for CIN3, and 71.4% for CC ( The human papillomavirus (HPV) is the causative agent for cervical cancer (CC) . Nearly The worldwide prevalence of HPV infection is estimated at 11.7%, but it varies widely among geographical regions and age groups ,12,13. RCC screening aims to decrease the morbidity and mortality of the disease in women. A systematic review showed that CC screening appears to reduce the incidence of invasive CC . It has Surface plasmon resonance (SPR) is a novel technology for a direct and label-free analysis of organic compounds and DNA. SPR can detect 24 HPV genotypes in one reaction ,24.No study examined the epidemiology of HPV by SPR in Southwest China up until now. Therefore, the present study aimed to identify HPV infection of female patients in Kunming by the SPR technique and explore the correlation with CC.This was a cross-sectional survey that was carried out at the Gynecology Outpatient Clinic and Gynecology Inpatient Department of the Second Affiliated Hospital of Kunming Medical University in 150 patients who were hospitalized or volunteered for CC screening, between October 2010 and December 2011. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Kunming Medical University. All women provided written informed consent.The inclusion criteria were: 1) adult women; 2) women who had sex without pregnancy; 3) no previous treatment for cervical and vaginal diseases; 4) no vaginal operation or sexual intercourse within 72 h before examination; and 5) informed consent was obtained.The cell brush was inserted into the external os of the cervix, at the junction of the squamous and columnar epithelium; it was rotated evenly 3–5 times using the external os of the cervix as the center. The cervical brush was taken out, and the brush head was put into the cervical exfoliated cell preservation solution and evenly mixed to dissolve the specimen into the preservation solution . All subjects were tested for cervical lesions using cervical sedimentation type ThinPrep liquid-based cytology (LBC). The remaining cells in the preservation solution were extracted by the SPR technique for the detection of HPV. Cervical biopsy was performed under colposcopy for subjects whose LBC ≥atypical squamous cells of undetermined significance (ASCUS) or for those with high-risk HPV detected by the SPR method.A HPV typing kit  was used for detecting 24 HPV types, including 16 high-risk types  and 8 low-risk types , according to the manufacturer’s instructions . The kitin situ (CIS), CIN2, and CIN3, showing abnormal cells with an increased ratio of nucleus to cytoplasm. “Positive” LBC included all results other than NILM.The Liquid-based Cell Processing, Smearing and Staining Kit from Taipu Bioscience Co., Ltd.  was used according to the manufacturer’s instructions. Preservation buffer was vortexed for 30 s to resuspend cells. Gradient centrifugation buffer (4 ml) was added to a centrifuge tube together with 8 ml of cell preservation buffer. The mixture was centrifuged at 1080 rpm for 2 min. The top 8 ml were discarded and the tube was centrifuged again at 2000 rpm for 10 min. After the supernatant was discarded, buffer solution (500–800 µl) was added to the bottom and vortexed for 15 ± 5 s. Then, 30 µl of this suspension was transferred to a slide, and the cells were left to adhere for 10 min. The slide was rinsed, stained twice with 500 µl of hematoxylin for 90–120 s each time, and rinsed with rinsing solution after each staining. The slide was then stained with 500 µl of Papanicolaou staining solution for 90–120 s. The slides were mounted with optical resin and observed under a microscope. The results were classified as: 1) no intraepithelial lesion or malignant lesion (NILM), which meant that no abnormal squamous epithelial cells or abnormal glandular epithelial cells were found; NILM also included non-neoplastic findings such as inflammatory or reactive changes caused by various microbial infections; 2) low-grade squamous intraepithelial lesions (LSILs), which referred to mild squamous epithelial cell morphological abnormality; the majority of the patients were diagnosed with squamous epithelial dysplasia or CIN1 and there might also be cervical CIN2, CIN3, or some reactive changes; and 3) high-grade squamous intraepithelial lesions (HSILs), which referred to moderate to severe dysplasia and carcinoma The high-risk group found by screening was examined by colposcopy and cervical biopsy. The specimens were fixed with 10% formalin, routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and observed by light microscopy.LSIL (CIN1) presented as nuclear pleomorphism and deep staining involving the lower one-third of the epithelium. Nuclear chromatin was irregular, and the nucleoli were not conspicuous. The mitotic activity was increased in the lower one-third of the epithelium. Most cells displayed the HPV effect. Flat condyloma acuminatum was considered to be LSIL.HSIL (CIN2 or CIN3) and CIS presented as nuclear pleomorphism and deep staining involving the lower two-third of the epithelium  or the full layer . Nuclear chromatin was irregular and the nucleoli were not conspicuous. There was a high nuclear-cytoplasm ratio with the presence of atypical mitosis. Binuclear and multinuclear cells were common but less than in LSIL. HPV was occasionally present in lesions and HPV was more common near lesions.Chlamydia trachomatis status, and HPV status) of patients were collected.Demographic characteristics , reproductive health characteristics , birth history (parity), and clinical characteristics . A value of p < 0.001] (p = 0.03). HPV40 was only found in one patient with CC (p = 0.04). There were no differences among lesion types regarding the remaining 21 HPV genotypes .The frequency of HPV16 increased from 9.4% for women with normal cytology to 28.9% for CIN1, 41.4% for CIN2, 54.1% for CIN3, and 71.4% for CC [< 0.001] . The frep = 0.40).Up until now, no study has examined the frequency of HPV genotypes by SPR in Southwest China. Therefore, our objective was to identify HPV infection of female patients in Kunming by the SPR technique and explore the correlation with CC. The results indicated that HPV16 and HPV18 were the two most frequent genotypes. SPR may be useful for the detection of HPV infection in women undergoing CC screening.In the present study, the frequency of HPV infection was 74% among 150 women who underwent CC screening in Kunming. This rate is considerably higher compared to previous studies from China -16 and cIn the present study, HPV16 and HPV18 were the two most common genotypes, with increasing frequencies from normal cervix to CC. A cross-sectional 5-year study of non-vaccinated women from Chongqing (Southwest China) showed that HPV16 and HPV18 were indeed the most frequent genotypes and that their frequency increased with lesion severity . A revieA significant proportion of women were HPV-positive but had negative cytology. This probably reflects low viral loads in the setting of early infection. As SPR is a sensitive method for the detection of HPV ,24, thesThe present study has limitations. It was a single-center study of a small number of women seeking HPV screening. In addition, the SPR results were not confirmed by sequencing, because it is a time-consuming and expensive method.In Kunming (Southwest China), the frequency of HPV infection was 74.0% among women who underwent CC screening, and HPV16 and HPV18 were the two most frequent genotypes. SPR could be useful for detection of HPV infection in women undergoing CC screening.


Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6. Human papillomavirus (HPV) is a small double-stranded DNA virus family that infects mucosal and cutaneous epithelia. Currently, about 400 types of HPV have been identified . This faHowever, there are questions about the oncogenic potential of β-HPV in the general population. In most immunocompetent individuals, β-HPV infections are transient . FurtherAmong β-HPV proteins, the E6 or β-HPV E6 is best characterized. This report focuses on the E6 from β-HPV 8 (β-HPV 8E6), and its ability to disrupt DNA repair ,17,18,19When HR fails, non-homologous end joining (NHEJ) serves as a backup repair mechanism. NHEJ is not restricted to any portion of the cell cycle, but tends to occur when HR is not available  ,28,29,30Since limitations in HR are addressed with increases in NHEJ, it was reasonable to hypothesize that β-HPV 8E6 increased repair by NHEJ. However, this report presents contrary evidence to this idea. Furthermore, β-HPV 8E6 reduced NHEJ repair at a defined genomic location and reduced DNA-PKcs autophosphorylation. This culminated in more persistent DNA-PKcs foci and diminished pDNA-PKcs-dependent signaling events (phosphorylation of Artemis and XRCC4 repair complex formation). Interestingly, β-HPV 8E6 appears to diminish NHEJ activity, through both p300-dependent and -independent mechanisms.We have previously shown that β-HPV 8E6 disrupts HR by destabilizing p300, a transcription factor for two HR genes (BRCA1 and BRCA2) . NHEJ coβ-HPV 8E6 prevents the repair of UV lesions and completion of HR by reducing the abundance of key repair factors  ,17,18. T2O2) was used to activate NHEJ in U2OS LXSN, U2OS β-HPV 8E6 and U2OS β-HPV Δ8E6 cells. Unlike Zeocin, which induces breaks by intercalating into base pairs and causing cleavage, H2O2 generates DSBs by generating reactive oxygen species (ROS) [2O2  ,55. Thises (ROS) . β-HPV 8OS) [2O2 .To facilitate repair, pDNA-PKcs must localize to a DSBs. This localization results in complexes that are detectable as foci by immunofluorescence (IF) microscopy. These foci are indicative of ongoing repair. pDNA-PKcs foci were readily detected in untreated HFK LXSN cells, but less frequent in HFK β-HPV 8E6 cells A,B. PrioTo further determine the ability of β-HPV 8E6 to alter pDNA-PKcs signaling, DSBs were induced with Zeocin (10 μg/mL), then observed with immunofluorescence microscopy. pDNA-PKcs foci appeared rapidly in HFK LXSN cells and reached their maxima approximately one hour after Zeocin exposure A,B. TwenHaving seen β-HPV 8E6 impair DNA-PKcs autophosphorylation and repair complex resolution, the ability of β-HPV 8E6 to hinder other DNA-PKcs-dependent steps in NHEJ was determined. Published reports indicated that Artemis is a DSB-induced target of DNA-PKcs phosphorylation at Serine 516 (pArtemis) ,47. ThisTo better understand the extent that NHEJ was impaired by β-HPV 8E6 hindered NHEJ, the ability of XRCC4 to localize to sites of damage was assessed. This occurs downstream of Artemis activation and is required for the DNA ligation step in NHEJ ,61,62. LThe data above suggest that p300 is required for DNA-PKcs-dependent NHEJ. To confirm this relationship, NHEJ and DNA-PKcs signaling was assessed in previously described p300 competent (p300 WT) and p300 knockout (p300 KO) HCT116 cells . p300 KOBecause β-HPV 8E6 attenuated the repair of DSBs by HR , we initβ-HPV 8E6 hinders NHEJ, at least in part, by binding and destabilizing p300. p300 functions as a transcription factor for repair gene expression ,69. By rThe evidence provided here shows that β-HPV 8E6 diminishes essential NHEJ events, including DNA-PKcs phosphorylation at S2056. However, our efforts fall well short of resolving the role of β-HPV in NMSC development. Granted, the reduced DNA repair potential associated with β-HPV 8E6 would not be desirable in cutaneous tissue, as our skin protects against external mutagens. Supporting this assertion, previous studies have shown that pharmacological inhibition of DNA-PKcs increases mutagenesis . FurtherAccumulating evidence shows that β-HPV E6 increases the mutagenic potential of UV. This includes increasing the frequency with which UV causes DSBs and hindering repair of these deleterious lesions. Both error-free HR and error-prone NHEJ are impaired when β-HPV 8E6 is expressed. However, β-HPV 8E6 does not appear to limit their initiation, as evidenced by the formation of both RAD51  and pDNAImmortalized human foreskin keratinocytes (HFK), provided by Michael Underbrink , were grown in EpiLife medium , supplemented with 60 µM calcium chloride (Gibco), human keratinocyte growth supplement (Gibco), and 1% penicillin-streptomycin . U2OS and HCT116 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Zeocin  and H2O2 were used to induce DSBs. NU7441 (Selleckchem) was used to inhibit DNA-PKcs phosphorylation. KU55933  was used to inhibit ATM kinase activity.After being washed with ice-cold PBS, cells were lysed with RIPA Lysis Buffer , supplemented with Phosphatase Inhibitor Cocktail 2  and Protease Inhibitor Cocktail . The Pierce BCA Protein Assay Kit  was used to determine protein concentration. Equal protein lysates were run on Novex 3–8% Tris-acetate 15 Well Mini Gels  and transferred to Immobilon-P membranes . Membranes were then probed with the following primary antibodies: GAPDH , DNA-PKcs , phospho DNA-PKcs S2056 (abcam), Artemis (abcam), phospho Artemis S516 (abcam), XRCC4 (Santa Cruz Biotechnologies), Ligase IV (abcam), CD4 (abcam), and DYKDDDDK (FLAG) Tag (Invitrogen). After exposure to the matching HRP-conjugated secondary antibody, cells were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).Cells were seeded onto either 96-well glass-bottom plates (Cellvis) or coverslips, and grown overnight. Cells treated with Zeocin for specified time and concentration were fixed with 4% formaldehyde. Then, 0.1% Triton-X solution in PBS was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin in PBS for 30 minutes. Cells were then incubated with the following antibodies: phospho DNA-PKcs S2056 (abcam), XRCC4 (Santa Cruz Biotechnology). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 goat anti-rabbit (Thermo Scientific A11012), Alexa Fluor 488 goat anti-mouse (Thermo Scientific A11001). After washing, the cells were stained with 30 µM DAPI in PBS, and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described [The reporter assay used a previously described protocol , with tht-test. p-values in all experiments were considered significant, at less than 0.05.All values are represented as mean ± standard error (SE) from at least three independent experiments. Statistical differences between groups were measured by using Student’s Accumulating evidence shows that β-HPV 8E6 reduces genome stability by disrupting DNA damage response. Particularly, β-HPV 8E6 disrupts homologous recombination, which is a major DSB repair pathway in the S phase and G2 phase of the cell cycle. The data presented here show that β-HPV 8E6 diminishes NHEJ, which can occur throughout the cell cycle. This expands β-HPV 8E6′s influence over DSB repair throughout the cell cycle. Finally, this work demonstrates that β-HPV 8E6 uses p300-dependent and p300-independent mechanisms to disrupt DNA repair. This suggests that there are considerable evolutionary forces driving β-HPV to hinder cellular responses to damaged DNA.


Radiomics-based risk-stratification of HPV-associated OPSCC was significant for PFS and OS. Similar trends were observed in HPV-negative OPSCC. In conclusion, radiomics imaging features extracted from pre-treatment PET/CT may provide complimentary information to the current AJCC staging scheme for survival prognostication and risk-stratification of HPV-associated OPSCC.Accurate risk-stratification can facilitate precision therapy in oropharyngeal squamous cell carcinoma (OPSCC). We explored the potential added value of baseline positron emission tomography (PET)/computed tomography (CT) radiomic features for prognostication and risk stratification of OPSCC beyond the American Joint Committee on Cancer (AJCC) 8th edition staging scheme. Using institutional and publicly available datasets, we included OPSCC patients with known human papillomavirus (HPV) status, without baseline distant metastasis and treated with curative intent. We extracted 1037 PET and 1037 CT radiomic features quantifying lesion shape, imaging intensity, and texture patterns from primary tumors and metastatic cervical lymph nodes. Utilizing random forest algorithms, we devised novel machine-learning models for OPSCC progression-free survival (PFS) and overall survival (OS) using “radiomics” features, “AJCC” variables, and the “combined” set as input. We designed both single- (PET or CT) and combined-modality (PET/CT) models. Harrell’s C-index quantified survival model performance; risk stratification was evaluated in Kaplan–Meier analysis. A total of 311 patients were included. In HPV-associated OPSCC, the best “radiomics” model achieved an average C-index ± standard deviation of 0.62 ± 0.05 ( Over past decades, the incidence of oropharyngeal squamous cell carcinomas (OPSCC) has continuously increased, which is attributed to a marked rise in prevalence of sustained high-risk human papillomavirus (HPV)-infection in the oropharynx ,2,3,4. DAdvancements in high-throughput computing and machine-learning led to emergence of the “-omics” concept, referring to collective characterization and quantification of pools of biologic information, such as genomics, proteomics, or metabolomics. Radiomics refers to automated extraction of high-dimensional, quantitative descriptor (“feature”) sets from medical images for various applications, including survival modelling, treatment guidance, and biomarker design ,15,16,17[18F]Fluorodeoxyglucose positron emission tomography (PET) and computed tomography (CT) are a mainstay of OPSCC staging, treatment planning, and surveillance. We applied machine-learning algorithms to devise prognostic radiomics biomarkers for OPSCC using baseline PET and/or CT scans from a multi-institutional cohort. Then, we compared the radiomic biomarkers’ performance with AJCC staging for prognostication and risk-stratification of PFS and OS in HPV-associated and HPV-negative subgroups. For the PFS study arm, 311 OPSCC patients (235 HPV-associated and 76 HPV-negative) met inclusion criteria; 94 (30.2%) experienced events. For the OS study arms, 306 OPSCC patients (233 HPV-associated and 73 HPV-negative) met inclusion criteria; 58 (19.0%) died. The AJCC staging, demographics, treatment, and imaging characteristics are summarized in OPSCC random survival forest (RSF) models combining radiomics features and AJCC staging  yielded higher averaged Harrell´s C-index scores than AJCC models in the PFS and OS study arms for both HPV-associated and HPV-negative cohorts in the majority of permutations . Similarp = 0.02) compared to 0.54 ± 0.06 (p = 0.32) from AJCC staging; and the best OS radiomics model (using PET consensus VOI radiomics features) yielded 0.63 ± 0.08 (p = 0.06) compared to 0.55 ± 0.08 (p = 0.34) from AJCC staging  radiomics features) achieved an average C-index ± SD of 0.62 ± 0.05 ( staging . p = 0.25) compared to 0.50 ± 0.06 (p = 0.51) from AJCC staging, and the best OS radiomics model (using CT primary tumor radiomics features) yielded 0.60 ± 0.09 (p = 0.17) compared to 0.50 ± 0.08 (p = 0.53) from AJCC staging. Combined models utilizing radiomics features and AJCC variables yielded similar performance  achieved an average C-index ± SD of 0.55 ± 0.07 , whereas AJCC staging was not significant , while the 2-year OS p-value approached significance , while the 2- and 3-year OS p-values approached significance , which is likely attributable to low event rates and relatively small cohort sizes. On the other hand, in the HPV-associated subgroup, a PET/CT radiomics model using the full set of consensus VOI features for PFS prognostication produced an averaged C-index ± SD of 0.62 ± 0.05 (p = 0.02), and a PET model using consensus features for OS prediction achieved 0.63 ± 0.08 (p = 0.06). We observed similar trends in HPV-negative OPSCC, despite using a smaller cohort. To enhance generalizability and model robustness against heterogeneity in imaging and reconstruction protocols and scanning equipment, we acquired a multi-institutional dataset provided by cancer centers in the United States and Canada. Overall, AJCC models had modest prognostic accuracy in HPV-positive and HPV-negative subsets, achieving an averaged C-index ± SD of up to 0.55 ± 0.08  cohorts with a heterogenous array of testing methods. We retrospectively acquired clinical and imaging data from (1) Yale’s Smilow Hospital cancer registry from 2009–2019, and (2) two publicly available TCIA collections : the “HePatients with (1) histopathologically confirmed OPSCC, (2) known HPV-status, (3) pre-treatment PET and non-contrast CT scans, and (4) complete follow-up information were included. We excluded patients (1) presenting with distant metastases upon initial staging, (2) receiving palliative therapy and/or denying treatment, (3) recurrent OPSCC at presentation, (4) with CT artifacts affecting >50% of the primary gross tumor volume on visual evaluation , and (5)Patients from Yale were regularly followed up for cancer surveillance with physical examinations, endoscopy, and imaging; additional tissue sampling was performed at oncologists’ discretion. Disease progression or recurrence was ascertained by biopsies or unequivocal imaging evidence; the latter was confirmed by additional tissue sampling or documented response to anticancer therapy. For TCIA cohorts, annotations provided within the datasets were utilized to determine study endpoints. HPV association was determined by high-risk HPV-specific  testing To facilitate radiomics feature extraction, we defined separate PET and CT VOI for primary tumors and individual metastatic cervical lymph nodes. Each lesion was manually contoured (“segmented”) on PET axial slices, and segmentations were transferred to the co-registered CT and adapted to exclude uninvolved bone, air, and preserved fat planes. CT axial images with streak artifacts affecting the VOI were excluded from analysis on the basis of visual assessment, and lymph nodes with artifacts in >50% of the VOI were entirely disregarded . SegmentAn automated image pre-processing pipeline facilitated homogenized radiomics analysis . As detaSubsequently, we extracted 1037 PET and 1037 CT radiomics features per primary tumor or lymph node: 18 first-order and 75 texture-matrix features from each lesion’s representation in the original and derived PET and CT images, and 14 volumetric shape features from the original series  clinical “AJCC” staging, i.e., overall stage, T-stage, and N-stage; (2) “radiomics” signatures; and (3) “combined” models using AJCC staging and radiomics signatures. Survival models were fitted on the combined dataset including all subjects and were evaluated in HPV-associated and HPV-negative study cohorts. AJCC features were concatenated with HPV status and were included as categorical variables, including overall stage with seven levels , and T- and N- stage with eight levels each . Since patients with distant metastasis were excluded from analysis, no HPV-associated stage IV patients were included. We compared several approaches to generate optimized radiomics signatures for radiomics and combined models. Three feature dimensionality reduction techniques were compared to the prognostic performance of the full feature set , radiomics feature standardization, dimensionality reduction, and RSF training were consecutively performed on the training folds, and RSF performance was evaluated in the validation fold. This strategy yields accurate estimates of models’ prognostic capability in new cohorts, as “information leakage” between folds is rigorously avoided. Harrell’s C-index ,55,56 qut-test  was Uno’s estimator of cumulative/dynamic area under the curve (AUC) for right-censored survival data ,60 was cWe used the R “Hmisc” package  for C-inTo investigate the potentials of quantitative imaging for risk-stratification in HPV-associated and HPV-negative OPSCC, we utilized radiomics features for binary classification (high-risk vs. low-risk) and subsequently subjected cohorts to Kaplan–Meier analysis. For risk-stratification, we used random classification forest (RCF) models (“ranger” package version 0.12.1)  configurp < 0.05. For comparison, AJCC overall stage groups, T-stage, and N-stage were applied for risk-stratification. All classified patients were subjected to Kaplan–Meier analysis with their radiomics risk group, and a log-rank test ascertained statistical significance defined as The radiomics-only datasets of survival models selected for further evaluation were used as RCF input without feature reduction applied, and risk-stratification models were trained and evaluated separately in each study cohort and study arm. To label subjects for Kaplan–Meier analysis, cutoffs corresponding to 2, 3, 4, and 5 years of follow-up were used; patients experiencing events before a given cutoff were labeled as positive instances, subjects lost-to-follow-up before a cutoff were excluded, and all remaining patients were labelled negative and censored at the cutoff. RCF models were trained for each cutoff, and separate Kaplan–Meier plots were generated using radiomics risk groups and AJCC variables for risk-stratification. This approach allows supplying “dense” survival data to RCF algorithms  while enabling accurate comparison with AJCC stratification. Pre-treatment PET/CT radiomics biomarkers may provide complementary prognostic value for OPSCC beyond AJCC/UICC 8th edition staging via systematic quantification of tissue density, texture patterns, lesion geometry, and metabolic properties. Our results suggest an added value of radiomics biomarkers for survival prognostication and risk-stratification in HPV-associated OPSCC, with similar trends in HPV-negative cancers. Pending careful development and rigorous validation in larger multi-institutional/multi-national datasets, radiomics markers may improve prognostication and risk-stratification in a clinical setting and pave the road for personalized treatment and targeted therapy.


The study aimed to examine and characterize human papilloma virus (HPV) cytological grade trends and genotypes among HPV/HIV co-infected/cases and HPV monoinfected/control women attending Kisii Teaching and Referral Hospital, Kenya.® HPV system (GXHPV-CE-10) alongside pathological cytology analysis of cervical tissue samples.HIV positive co-infected with HPV (HPV/HIV) and HIV negative women monoinfected with HPV profiled as co-infected/cases and monoinfected/control arms respectively were enrolled. HPV subtypes were assayed by XpertLow grade intraepithelial lesion (LSIL) was the most predominant cytological grade across cases and controls with a prevalence of 32 (38.1%) while high grade squamous intraepithelial lesion (HSIL) was highest among HPV/HIV co-infected with a prevalence of 23 (27.38%). Among the monoinfected (controls) the predominant lesion was low grade intraepithelial lesions (LSIL) with a prevalence of 23 (27.38%). HPV type 16 had the highest prevalence 26 (30.8%) among the VIA positive women in the overall study participants followed by combinations of HPV types  at 19 (22.6%).High risk HPV types 16 and 18/45 were the most predominant in the established cytological grades and among the co-infected women. Routine screening using both cytological and HPV testing should be embraced and/or reinforced as early screening and preventive strategies in the covered geographical region population. Provision of the currently available vaccines to these women at an early age would provide effective protection since the HPV type profiles in this population are covered by such vaccines. Human Papillomavirus (HPV) is classified as high risk or low risk depending on its association with infection progression to cancer . Human IAccording to data at Kenya Medical Research Institute (KEMRI) which serves as regional cancer registry, approximately 80% of reported cases of cervical carcinoma are diagnosed at advanced stages, when little can be achieved in terms of curative treatment , 7. EvenStudy design and setting: the case-control study was conducted at Kisii Teaching and Referral Hospital (KTRH) located in Kisii County, Kenya. The hospital being a referral hospital receives between 1400-2000 HIV patients per month with a population of over 900 HIV positive women of reproductive age . The reproductive Health clinic attends to an average of 300 women of reproductive age per month with an average of 80 women of reproductive age being HIV positive .Sample size and participants: the study targeted women within reproductive age, between 18-59 years who attended the reproductive health care clinic at KTRH between July 2018 and July 2019 who met the inclusion criteria. The age bracket was also within the prevalence peak period when most reproductive age women are sexually active [y active , 13.Inclusion criteria: all consented sexually active women between 18-59 years of age; non pregnant women; no previous diagnosis of cervical cancer; had not used vaginal medication for the previous two days; HIV positive women with HPV infections for cases and HIV negative women with HPV infections for controls.Exclusion: those who failed to give written consent; pregnant women; women who are < 18 and > 59 years; women with other medical conditions such as mental incompetence, had hysterectomy, or had treatment on the cervix less than 6 months prior to visit or on menstruation; HIV positive women without HPV infections and HIV negative women without HPV infections. According to needs assessment data obtained from Kisii Teaching and Referral Hospital records in the year 2017, a total of 4676 women were screened for cervical cancer using Visual inspection with Acetic Acid and Visual Inspection with Lugol's Iodine (VIA/VILI). Out of those screened, 846 (18%) were VIA/VILI positive but HIV negative and 2151 (46%) were HIV and VIA/VILI positive . In reference to a clinical study in Kenya, the prevalence of HPV amongst HIV infected women was 49% while HPV prevalence in HIV negative women was 17% [ was 17% . Going b was 17%  while adThe study participants were recruited by invitation. Three female nurses and one female clinical officer with prior knowledge and experience on visual inspection of the cervix with acetic acid (VIA) were trained on recruitment process of the participants and how to obtain satisfactory cervical smears. They also worked in conjunction with a residence gynecologist who oversaw routinely the process of sample collection as is recommended . A reseaCervical specimens for cytology and High-Risk (HR) HPV genotyping: all VIA positive women had cervical cell samples exfoliated and preserved for confirmatory testing with Pap and HPV genotyping [® Solution (Hologic Corp.) per manufacturer protocols [notyping -24. Brienotyping , 23 To gnotyping , 21. Immnotyping , 19, 25.rotocols -29. Therrotocols , 31. Therotocols . The womrotocols , 19.® HPV assay system (GXHPV-CE-10). Based on the manufacturer's instructions, samples were shaken for several seconds, and 1 ml total volume of PreservCyt was poured into an Xpert® HPV cartridge and subsequently loaded into Cepheid Xpert Diagnosis instrument that uses a second generation real-time PCR [® HPV assay specifically identifies types HPV 16 and HPV 18/45 in two distinct detection channels, and reports 11 other high risk types  in a pooled result [For HR-HPV genotyping, all samples were analyzed by Xperttime PCR , 28. Inttime PCR -29. The d result -29. The d result -29.® rapid test kit , and the positive results were confirmed by Uni-Gold® . Those found to be HIV positive were counselled and referred to the relevant unit for initiation of treatment.HIV testing was done at the reproductive health clinic at KTRH according Ministry of Health, National AIDS & STI Control Program (NASCOP) 2018 national testing guidelines using the DetermineData management and analysis: the data collected was cleaned, coded, and entered into a Microsoft Excel database developed for this purpose. Statistical analysis was performed using SPSS software, version 20. Cervical lesions and HPV characterization data was analysed using categorical variables which were summarised by descriptive statistics using prevalence and the significance of their difference tested by one sample chi-square statistics. The significance level was set at P < 0.05.Ethics approval and consent to participate: ethical clearance was obtained from the Registrar, Research and Extension Kisii University, Scientific Ethical Committee of University of Eastern Africa Baraton, National Commission for Science, Technology and Innovation (NACOSTI) and Research Authorization given from the office of County Director of Education Kisii County. Written informed consent was given by participants who consented to participate in the study.Clinical characteristics of the participants: clinical characteristics and screening of eligible participants in recruitment from preliminary stage to desired sample size attainment in the enrollment process are as shown in Cytological grades analysis and prevalence: the 410 samples that were VIA positive were evaluated for cytological grading, 398 (97.07%) had satisfactory smears for cytology which were evaluated for cytological abnormalities. The overall cytological categories in both co-infection (HPV/HIV) and monoinfections (HPV) were analyzed, graded and classified according to the revised standardized Bethesda 2001 [sda 2001 , 33. TheHPV genotypes: whereas there was no significance difference in the prevalence of HPV type's combinations (16) and  between HPV/HIV co-infections and HPV monoinfections p = 0.067 and 0.05 respectively, all the other type combinations had significant difference between HPV/HIV co-infections and HPV monoinfections P < 0.05  with HR-HPV prevalence being high among women with abnormal cytology 34% compared to normal cytology 1.46%. In relation to HIV status, the cytological grade that had the highest HPV prevalence was low grade squamous intraepithelial lesions (LSIL) in controls with type categories (16) and  each category having a prevalence of 5 (6.0%). In HSIL of controls the HPV type (16) had the highest prevalence 14 (16.67%) followed by HPV type category  with a prevalence of 6 (7.14%). In cases, HSIL cytological grade had second highest total HPV prevalence of 30 (35.7%) in type categories 16 with a prevalence of 9 (10.7%) followed by type categories  with a frequency of 6 (7.1%). In these cytological grades the most prevalent HPV types were (16) and . This study shows an increase in prevalence of HPV type 16 from normal cytology to LSIL and HSIL having 4 (4.8%), 6 (7.2%) and 14 (16.6%) respectively. ASCUS grade in co-infections was having a combination of HPV types  with a prevalence of 5 (6.0%). With increase in severity of cervical lesions, HPV 16 takes the dominance with a prevalence of 14 (16.6%) followed by  combinations with a frequency of 6 (7.1%). It was noted that in the cytological grades, type combinations , (18/45),  with a prevalence of 4 (4.8%) and 2 (2.4%) respectively had types 35 and 52 appearing quite often in the early cytological grades. Other types that appeared adversely in the advanced grades include , (18/45), , , ,  with prevalence of 4 (4.8%), 2 (2.4%), 1 (1.2%), 1 (1.2%), (1.2%) and (1.2%) respectively. All the other HPV type prevalence in the cytological grades by HIV status together with their p- values indicating whether there is a significance difference between cases and controls are as profiled in The study examined and characterize HPV cytological grade trends and genotypes among VIA positive HPV/HIV co-infected and HPV monoinfected women attending Kisii Teaching and Referral Hospital, Kenya in a quest to generate and enhance regionally adapted insights for better public health cues and planning information for instance thus, bringing to the fore understandings that would prove valuable in deciding on the most appropriate screening strategies and HPV vaccination programs approach. Overall, there was 27.38% prevalence of high grade squamous intraepithelial lesions (HSIL) among the HIV positive HPV co-infected women which was the predominant lesion. The overall prevalence of HR-HPV in the VIA positive women in this population was 20.48% while the highest prevalence of high risk HPV genotypes was type 16 while genotypes (16), (18/45) and  which accounted for 66.67% of the cases. In the cytological grading of participants who were HR-HPV positive regardless of HIV status, the prevalence increased in the order; f normal cytology, ASCUS, HSIL to LSIL albeit, there was marginal difference between LSIL and HSIL cytological grade prevalence. There was diverse variance in prevalence trends of the predominantly prevalent HPV types in the cytological grades ASCUS, LSIL, HSIL across numerous findings reported this far but outstandingly, normal cytology was the least observed cytological grade in regard to HR- HPV infection -36. It hRelated findings have also noted that HPV types did not influence severity of dysplasia rather the duration of HPV infection and immunological responses to infections by the host determine cervical tissue transformation to ICC after HPV infections . There cThe overall prevalence of HR-HPV in the VIA positive women was lower than that observed by Ghedira et al  with womIn the present study, HPV type 52 has a low prevalence while type 53 was not detected. This trend is amenable with Bangkok Metropolitan women findings which indicated that the rate of HPV detection was related to severity of cytological grades with the rate following an ascending order from 13.0%, 30.8%, 39.5%, 56.3% and 100.0% for ASC-US, ASC-H, LSIL, HSIL and SCC respectively . In our High risk HPV types 16 and 18/45 were the most predominant in the established cytological grades and among the co-infected women. There was a higher prevalence of HR-HPV genotypes in high grade lesions in co-infections which was twice that in mono-infections. Routine screening using both cytological and HPV testing should be embraced and/or reinforced as early screening and preventive strategies in the covered geographical region population. Such routine screening services would substantially mitigate progression of the pre-invasive lesions to cervical carcinoma since most women were within the low grade squamous intraepithelial ranges especially for the HIV positive. Provision of the currently available vaccines to these women at an early age would provide effective protection considering the presented findings ascertain the HPV type profiles in this population are covered by such vaccines.HPV genotypes has been reported to be highly prevalent and widely varied among HPV monoinfected and HIV/HPV co-infected women in the country and beyond;Regional variations in the prevalence of HPV types exist in the country.The findings provide comprehensions on genotype specific HPV trends on the most common HPV type categories in the study population in Kenya not reported before to the best of our knowledge;The results further provide a basis for effective screening and vaccination strategies success monitoring in the geographical setting on the basis of applying the insights in post vaccination evaluation of the control and prevention policies rolled out in Kenya on HPV vaccination.The authors declare no competing interests.


HPV genotypes are the most common etiological factor for genital neoplasia. It would appear that sexually transmitted infections accompanied with HPV genotypes might have synergistic interactions in cancer progression. The genetic polymorphisms are involved in metabolizing carcinogens which may contribute to the susceptibility of developing genital cancers by less efficient or overly down metabolic pathways and cell signaling. MTHFR polymorphisms are related to several metabolic disorders and human cancers. We investigated the contribution of MTHFR 1298 and MTHFR 677 polymorphisms as potential risk factors for outcomes with HPV genotypes and STIs in Iranian population.As a case–control study, MTHFR A1298C and C677T were assessed for SNPs analysis using a PCR–RFLP assay in 50 cervical intraepithelial neoplasia (CIN) cases, 98 HPV-positive subjects and 47 non-cancerous/non-HPV patients as healthy controls.P ≤ 0.05) in women with CIN as compared to non-cancerous/non-HPV subjects. There was not a significant difference of MTHFR 677 between outcomes.Finding suggested a significant association between the MTHFR 1298 CC polymorphisms (OR = 3.5, 95% CI = 1.13–10.82, It would seem MTHFR 1298 CC is more likely to be a potential risk factor for HPV–cervical cancer progression. Consequences support further attempts to understand the clinical manifestations of neoplasia related to genital infections and gene mutations. Cervical malignancies are one of the most common disorders in females worldwide. Approximately 86 percent of these cases occur in developing communities, where careHPV, prophylactic screening and a nationally organized program are not commonly available. Lifestyle, environmental conditions, coinfections, genetic patterns and epigenetic characteristics accompanied with HPV genotypes are major risk factors for genital malignancies and disorders –3.Most sexually active women and men are infected at some points in their lives. Additional microbial pathogens such as sexually transmitted infections (STIs) could increase the risk of cervical neoplasia induction . Some siMethylenetetrahydrofolate reductase, folate-metabolizing enzyme, is a critical component in complex process of folate cycle. SNPs in the MTHFR gene are arguably associated with thrombotic events and may increase or decrease the activity of cancer suppressor genes. Homozygosity for the substitution (C → T) at nucleotide 677 and for the substitution (A → C) at nucleotide 1298 in the MTHFR gene is suggested to be associated with an increased risk of cervical cancer .Chlamydia trachomatis, HSV-2 and Mycoplasma genitalium in Iranian women suffering from STI coinfections.Few studies are found on the association between polymorphisms and STIs related to cervical cancer. The need for identifying genetic susceptibility factors for CC related to HPV and STI is a matter of debate . TherefoIn this case–control study, 148 women who were referred to Moheb Yas Hospital and other private laboratories in Tehran, Iran with or without genital infections and cervical intraepithelial neoplasia were enrolled in the survey. Forty-seven age- and sex-matched healthy controls were consecutively registered for this study from April 2012 to October 2015. Informed consent was obtained from subjects prior to their registration. Cervical scrapping and genital lesions with known cervical intraepithelial neoplasia , HPV-positive subjects and non-cancerous/non-HPV women as healthy controls were 50, 98 and 47, respectively. These liquid-based cytology samples (LBCs) were transported to a molecular biology laboratory where they were stored in proper condition until genomic analysis.Extraction of infectious agents and genomic DNAs from LBCs and lesions was performed using a high pure PCR template preparation kit , following the kit protocol. Extracted DNAs were kept at − 20 °C until experiment.®, Denmark), 10 pmol/μl of forward and reverse specific primers and 10 μl of the extracted DNA as a template in separate reactions was used for evaluation of MTHFR1298 and 677 mutations. The reactions were amplified in a thermal cycler  by using the following conditions: primary denaturation of 94 °C for 5 min, 32 cycles with 94 °C for 1 min, 63 °C for 1 min and 72 °C for 1 min, plus an additional extension at 72 °C for 10 min. In RFLP analysis, 15 μl of 1298 A > C PCR products were digested with 20 μl of restriction enzyme reaction mixture containing 16 μl of sterile distilled water, 3 of μl blue buffer and 1 μl of MboII (5 U/μL) enzyme (Thermo Scientific®). In addition, 10 μl of 677 C > T PCR products were digested with 20 μl of restriction enzyme mixture containing 16 μl of sterile distilled water, 3 μl of Tango buffer and 1 μl of HinfI enzyme (10 U/μL) (Thermo Scientific®). The PCR–RFLP products were separated by electrophoresis on a 3% agarose gel, stained by SYBR Safe, and were visualized under ultraviolet light along with a molecular weight marker (50 bp). In a MTHFR 1298, PCR–RFLP was expected to produce 145 bp as undigested, 77, 37 and 29 bp as wild type, 108, 77, 37 and 29 bp as heterozygous and 108 bp as homozygous. In addition, in a MTHFR 677 PCR–RFLP was expected to produce 200 bp as undigested, 200 bp as wild type, 200 and 175 bp as heterozygous and 175 bp as homozygous  Comparison of the MTHFR 1298 and 677 polymorphisms between each of the cervical intraepithelial neoplasia subjects, HPV-infected cases and non-cancer/non-HPVs control indicated a significant difference in some genotypes. There was a significant association between the MTHFR 1298 CC polymorphisms  in women with cervical intraepithelial neoplasia and non-cancerous/non-HPV control group.We performed a detailed HPV genotyping and pathogen detection of STIs such as  studies , 14. TheThere was no significant difference between MTHFR 677 genotypes and population study. The heterogeneity, odds ratios and Hardy–Weinberg results are shown in Table C. trachomatis were the most common infections among the cervical intraepithelial neoplasia patients and healthy controls. However, HPV 6 is the most common genital infection that might not be an etiological factor for cervical cancer, but is a major cause of genital warts. It seems no previous study has reported the MTHFR A1298C and MTHFR C677T mutations in cervical cancer and STIs pathogens in Iranian women and west Asian regions. Extensive evidence propose that methylenetetrahydrofolate reductase (MTHFR) includes MTHFR A1298C and MTHFR C677T genotypes which may be implicated as potential risk factors in developing cervical cancer. It would appear that gene polymorphisms are implicated in cancer progression and they can be used for screening early stage of malignancies [The association between the MTHFR 1298 A > C and 677 C > T polymorphisms and susceptibility to cervical malignancies and genital disorders has been investigated in the case–control study in Iranian women. We found that HPV 6 (LR), HPV 16 (HR) and gnancies . As a regnancies , 20–24.C. trachomatis, M. genitalium and HSV2 in genital disorders has not been extensively studied in developing countries. Multiple sexually transmitted infections are significant causes of infertility, PID, ectopic pregnancy, congenital infections and cancer. Morbidity, mortality and long-term complications of these infections are increasing public health concerns worldwide [The function of SNPs in the pathogenesis of cervical cancer coexisting with HPV genotypes and other microorganisms such as orldwide , 25–30. orldwide –33. In oorldwide , 30–32.The MTHFR A1298C allele frequency was also appraised in Korea, China and India . In the M. genitalium were prevalent in cervical intraepithelial neoplasia patients and healthy women (non-cancerous/non-HPV).The allele’s frequencies can have significant relationship with cervical cancer and genital disorders such as Iran , China , India aethnic groups and use of approved and modern technologies such as whole-genome sequencing are needed to confirm the association between mutations and STIs. Finding a specific molecular biomarker can be helpful in detecting early stage of genital cancers along with diagnosis of sexually transmitted infections.This can be the first study to investigate the association between MTHFR 1298 A > C and 677 C > T in Iranian women suffering from genital disorders. We concluded that some of SNPs were associated with cervical intraepithelial neoplasia related to HPVs and other STI pathogens. We suggest that studies with larger sample size, different


To determine obstetrician-gynecologists’ (OBGYNs) practice patterns regarding human papillomavirus (HPV) testing in cervical cancer screening. Secondly, we aimed to examine OBGYNs’ adherence to guidelines in the management of women with HPV-positive test results.The study was a cross-sectional survey conducted in Antalya and Istanbul provinces in Turkey using a self-reported questionnaire. A 12-item questionnaire form was administered to the participants in face-to-face interviews. Of the targeted participants, 343 OBGYNs completed the questionnaire.The majority of participants, (81.0%) stated that they offered/used HPV testing in cervical cancer screening. Of those, most OBGYNs (89.9%) preferred to use HPV testing concomitant with cervical cytology (co-testing) whereas only 10.1% preferred to use HPV testing alone (primary HPV testing). The most preferred screening intervals for women with HPV-negative results were 5 years (53.4%) and 3 years (19.9%), respectively. In compliance with the guidelines, the rate of participants who recommended “referral directly to colposcopy” for women who were HPV16/18-positive and cytology-negative; and “co-testing at 12 months” for women who were positive for HPV genotypes other than HPV16/18 and cytology-negative was 53.1%. Multivariate analysis revealed that the “professional working setting” was the sole independent determinant of the adherence to the guidelines. OBGYNs working in private settings had the worst adherence rate (42.4%).Primary HPV testing is not yet widespread among Turkish OBGYNs. Moreover, adherence to practice guidelines in the management of HPVpositive test results is relatively low. There is a need for continuing medical education regarding screening programs and the management of women with positive screening results. PRECIS: Using a 12-item self-reported questionnaire, we have evaluated obstetrician-gynecologists’ practice patterns regarding HPV testing in cervical cancer screening in Turkey.. Persistent high-risk human papillomavirus (HPV) infection plays a key role in cervical carcinogenesis. The estimated absolute risk for cervical intraepithelial neoplasia of grade 3 (CIN3) or cancer within 12 years following a persistent HPV16 infection has been estimated as high as 47%. Turkey, though it is a developing country, has a relatively low incidence (4.5/100000) of cervical cancer in line with its low prevalence (3.5%) of high-risk HPV infection,4.Cervical cancer is the third most common cancer in women worldwide. Approximately 85% of cases occur in developing countries where cervical cancer is a public health problem. However, the false-negative rate of cytology (>50%) is still very high, particularly in endocervical adenocancers and postmenopausal women,7. Studies on the detection of high-risk HPV nucleic acid in cervical epithelial cells (HPV testing) have revealed that HPV testing had a sensitivity of 90% and a specificity of 90% in the detection of CIN2 or worse (CIN2+) lesions, including glandular lesions. As a result of studies demonstrating the high diagnostic performance of HPV testing, there have been changes in the joint American Cancer Society (ACS), American Society for Colposcopy and Cervical Pathology (ASCCP), and American Society for Clinical Pathology (ASCP) screening guidelines in 2012, and the use of HPV testing together with cytology (co-testing) every five years started to be recommended as the screening method for cervical cancer in women over the age of 30 years.Cervical cancer can be prevented, primarily with HPV vaccines, and secondarily with screening programs. Population-based screening programs using cervical cytology has successfully decreased cervical cancer incidence and mortality, and lower than that following co-testing every five years. Subsequent to these results, many national and international societies made changes in their guidelines,14,15,16. In 2015, the Society of Gynecologic Oncology and ASCCP issued interim clinical guidance recommending primary HPV testing every 3 years for women aged ≥25 years. One year later, the American College of Obstetricians and Gynecologists and the United Kingdom National Screening Committee recommended this strategy as an effective screening option,14. At the same year, the American Society of Clinical Oncology endorsed primary HPV testing every 5 years for women aged ≥25 years as one of several screening strategies.In the most recent studies, however, it has been shown that the 3-year risk following HPV screening alone (primary HPV testing) was identical to that following co-testing every three year. According to this program, women aged between 30 and 65 years are invited for screening by primary care physicians every 5 years. Women who are HPV16/18-positive are referred to centers specialized in colposcopy. In women who are positive for HPV genotypes other than HPV16/18, reflex cytology is performed. Women with negative cytology are invited for repeat HPV testing after 12 months, and women with cytologic abnormalities are immediately referred to colposcopy centers.The Turkish Ministry of Health implemented a population-based screening program in 2014, which included primary HPV testing as the screening method, with a nationwide centralized diagnostics laboratory and a well-defined screening algorithmIn the current study, we primarily aimed to determine obstetrician-gynecologists’ (OBGYNs) knowledge, attitudes, and practice patterns regarding HPV testing in cervical cancer screening in Turkey. Secondly, we aimed to examine OBGYNs’ adherence to the national and/or international guidelines in the management of women with HPV-positive test results.. According to the most recent report on health education and health manpower in Turkey, which is prepared jointly by the Turkish Ministry of Health and Turkish Council of Higher Education, there are 5,227 actively working OBGYNs in Turkey. Based on these data, the optimal sample size required for the study was calculated as 358. The study was approved by the local ethics committee and it was performed in accordance with the ethical standards described in an appropriate version of the 1975 Declaration of Helsinki, as revised in 2000. Informed consent was obtained from all participants.The study was a cross-sectional survey conducted in Antalya and Istanbul provinces in Western Turkey between May and September 2018 using a structured self-reported questionnaire. The sample size was calculated using a random sample calculator with 5% margin of error and 95% confidential intervals (CI)From a list of all members of the Turkish Association of Obstetricians and Gynecologists stratified by region, 500 representative OBGYNs were selected at random for participation. All participants were given verbal instructions and written information about the study, and all were informed about confidentiality measures and their rights to withdraw. A 12-item questionnaire form was administered to the participants through face-to-face interviews. Of the targeted participants, 343 OBGYNs completed the questionnaire, yielding a response rate of 68.6%. The margin of error at 95% CI was calculated as 5.1%.The survey questionnaire had two sections. The first section included six questions on the participants’ demographic characteristics such as sex, age, the number of years in specialty practice, whether they had a subspecialty, professional working setting, and the type of practice. The second section included six questions that assessed the OBGYNs’ knowledge, attitudes, and practice patterns regarding HPV testing in cervical cancer screening. These questions were as follows:1. Do you offer/use HPV testing in cervical cancer screening? (No/Yes)2. If your response to the first question was “yes”, how do you prefer to use HPV testing in cervical cancer screening? Primary HPV testing  vs Co-testing (high risk-HPV testing concomitant with cytology)3. If your response to the first question was “yes”, do you prefer an age threshold for beginning HPV testing? (No vs ≥21 vs ≥25 vs ≥30 vs others)4. If your response to the first question was “yes”, what is your preferred screening interval for women with an HPV-negative test result? 5. What is your recommendation for women who are HPV16/18-positive and cytology-negative ?6. What is your recommendation for women who are positive for HPV genotypes other than HPV16/18 and cytology-negative?Two separate binary logistic regression models were developed to investigate the determinants of OBGYNs’ use of the HPV testing in cervical screening and their adherence to the guidelines in the management of women with HPV-positive test results. In univariate analyses, Pearson’s chi-square test was used because all the variables were categorical. Categorization of the age and years in specialty practice was performed according to the median value. Validities of median values were tested using receiver operating characteristic curve analysis. Variables with a p-value <0.20 in univariate analyses were included in the multivariate analyses. The effects of variables on the use of HPV testing and adherence to the guidelines were reported as adjusted odds ratios (OR) and 95% CI.The mean age and the years in specialty practice of the participants were 43.3±9.2 years and 11.2±8.0 years, respectively. The rate of women (53.4%) was slightly higher in sex distribution. The majority of the participants were general OBGYNs (84.5%), working at secondary-care (public/private) settings (70%), and had no academic position (88.9%) .The practice behaviors of OBGYNs regarding HPV testing are shown in The determinants of OBGYNs’ use of HPV testing in cervical cancer screening are presented in Practice behaviors of OBGYNs regarding the management of women with HPV-positive test results are summarized in The determinants of OBGYNs’ adherence to the practice guidelines in the management of women with HPV-positive test result are presented in The current study investigated the OBGYNs’ practice patterns regarding HPV testing in cervical cancer screening in Turkey. The study demonstrated that the majority of OBGYNs (81%) in Turkey used/offered HPV testing in cervical cancer screening; most (89.9%) preferred to use HPV testing as part of co-testing, a significant proportion (43.2%) used no age threshold for beginning HPV testing, and OBGYNs working at secondary-care public hospitals and those with >10 years of practice experience used HPV testing less often. The study also implied that the “professional working setting” was the sole independent determinant of the adherence to the guidelines in the management of HPV-positive test results. OBGYNs working at private settings had the worst adherence rate.,11. Wright et al. compared the 3-year results of primary HPV testing, co-testing, and cytology, and found that the sensitivity for CIN3+ of cytology alone was 47% compared with 61% for co-testing and 76% for primary HPV testing. On the other hand, the specificity for CIN3+ was 97%, 94%, and 93% for cytology, co-testing, and primary HPV testing, respectively. The authors also noted that 3-year incidence rate for CIN3+ was lower in HPV-negative women (0.3%) than in cytology-negative women (0.8%), but was identical to that in co-testing-negative women. Gage et al. compared the risk of CIN3+ for the three strategies among approximately one million women and reported that the 3-year risk of CIN3+ following an HPV-negative result (0.069%) was lower than the 3-year risk following a cytology-negative result (0.19%) and 5-year risk following a negative co-test result (0.11%).Accumulating evidence in the literature indicates that cervical screening with primary HPV testing is superior to screening with cytology alone, and is as effective as co-testing in the detection of CIN3+ lesions reported that almost all (95%) of OBGYNs offered HPV testing in cervical screening, mostly as part of co-testing, and in women aged over 30 years. The rate of participants who recommended HPV testing in women younger than 30 years was only 14%. In another study from the US, which was conducted in 2015, Cooper and Saraiya reported that co-testing was recommended by 95% of OBGYNs; however, the rate of participants who recommended HPV testing for women aged under 30 years was 22%. Caglioti et al. investigated the practice behaviors of Italian OBGYNs in terms of HPV testing in 2015. The authors reported that the vast majority (94%) recommended HPV testing in women aged ≥30 years, but 42% stated that it is always preferable to perform HPV testing as part of co-testing. In women with an HPV-negative test, 44% recommended subsequent HPV testing at 5-year intervals, and 33% preferred a shorter interval, mainly 3 years.In a survey study conducted in the United States (US) in 2013, Darwish-Yassine et al.Although the tendencies in our study, to some degree, are similar to the tendencies reported from the US and Italy, the frequency of using HPV testing in cervical screening  and the rate of using HPV testing as a stand-alone screening method is relatively lower than in other countries . Nevertheless, the data from all three countries reveal that primary HPV testing is not yet widespread among the OBGYNs. One notable finding of the current study was that OBGYNs working at secondary-care public hospitals and those with >10 years of practice experience used HPV testing significantly less often as compared with those working at tertiary-care hospitals and private settings. In Turkey, the national cervical screening program is conducted by primary-level health staff trained by the Ministry of Health for sample collection and referral of women based on a national screening algorithm. According to the Turkish Ministry of Health Screening Algorithm, women with an HPV16/18-positive test result are referred to colposcopy centers, most of which are at tertiary-care public hospitals. Secondary-care public hospitals, therefore, have a relatively small role in this program. Besides, limitations in access to HPV tests in laboratories of secondary-care public hospitals can explain why physicians working at these hospitals use HPV testing less frequently. On the other hand, the decrease in the use of HPV testing as the years in specialty practice increased can be explained by the fact that HPV testing is a relatively new method, that it started to be included in the guidelines in 2012, and that it is difficult and requires time to replace settled practices with new ones.Our findings also suggested that the “professional working setting” is the sole independent determinant of the adherence to guidelines in the management of women with HPV-positive test results. Although OBGYNs working at secondary-care public hospitals use HPV testing at lower rates, they reveal more compliance with the guidelines compared with those working at tertiary-care hospitals and private settings. The Cancer Control Department of Turkish Ministry of Health holds educational workshops periodically for physicians working in primary and secondary public healthcare regarding cervical screening and colposcopy. These workshops, however, do not include physicians working at private healthcare institutions. The lack of coverage of continuing medical education in physicians working in private settings is responsible for the lower rates of adherence.The main limitation of the study is that the survey was conducted in two of the most developed cities of Western Turkey, which limits the generalizability of findings. As with all survey studies, this study may also be a subject to non-response bias. Additionally, the inclusion of gynecologic oncologists might have affected the results. However, the number of gynecologic oncologists included in the study was limited (only 20 of 343 participants), and being a gynecologic oncologist does not guarantee for adherence to guidelines, as is evident (55%) in our results. On the other hand, the strengths of the study include surveying nationally representative samples of OBGYNs and a high response rate of 68.6%. The study provided insights into the attitudes of Turkish OBGYNs towards HPV testing. The findings of the study are important for designing appropriate educational interventions to improve the knowledge of physicians about screening and the management of cervical premalignant and malignant lesions.Primary HPV testing is not yet widespread among Turkish OBGYNs. Most OBGYNs continue to prefer using co-testing as the primary tool for cervical cancer screening. Moreover, adherence to the guidelines in the management of women with HPV-positive test results is relatively low, particularly in OBGYNs working in private settings. There is a clear need for continuing medical education in terms of cervical screening programs and the management of women with positive screening results.