Higher NRS 2002 was an independent adverse prognostic factor for OS and DFS in HPV-, but not in the HPV+ group. Regardless of HPV status, patients with OPC can develop malnutrition during RT/CRT. Therefore, nutritional support during RT/CRT is required in patients with HPV- and HPV+ OPC.The aim of this study was to assess and compare the nutritional status (NS) of patients with HPV-related (HPV+) and non-HPV-related (HPV-) oropharyngeal cancer (OPC) before and after radiotherapy (RT) or chemoradiotherapy (CRT). The analysis included 127 patients with OPC who underwent radiotherapy (RT) alone, or in combination with chemotherapy (CRT). In both groups, a significant decrease in all analyzed nutritional parameters was noted after RT/CRT. Overall survival (OS) and disease-free survival (DFS) were significantly better in patients with a higher BMI in the HPV- group; DFS was significantly better in patients with total lymphocyte count (TLC) >1.28/mmp < 0.01). CRT caused significant weight loss and decreases in BMI, albumin, total lymphocyte count (TLC), and hemoglobin concentration, as well as an increase in the Nutritional Risk Score (NRS) 2002, in HPV- and HPV+ patients. A significant decrease in prealbumin levels after CRT was noted only in HPV+ patients. RT caused a significant decrease in hemoglobin concentration and TLC in HPV- patients. There were no significant differences regarding other nutritional parameters after RT in either group. RT did not have negative impact on body mass index (BMI), weight, NRS, CRP, Alb, Prealb, or PNI. Overall survival (OS) and disease-free survival (DFS) were significantly better in patients with a higher BMI in the HPV- group ; DFS was significantly better in patients with C-reactive protein (CRP) < 3.5 g/dL in the HPV- (p = 0.021) and HPV+ (p = 0.018) groups, and with total lymphocyte count (TLC) >1.28/mm3 in the HPV+ group (p = 0.014). Higher NRS 2002 was an independent adverse prognostic factor for OS and DFS in HPV-, but not in the HPV+ group. Kaplan–Meier analysis showed that both OS and DFS were significantly better in HPV- patients with lower NRS 2002 scores. However, this relationship was not observed in the HPV+ group. Conclusions: Regardless of HPV status, patients with OPC can develop malnutrition during RT/CRT. Therefore, nutritional support during RT/CRT is required in patients with HPV- and HPV+ OPC.Background: Radiotherapy plays an essential role in the treatment of oropharyngeal carcinoma (OPC). The aim of this study was to assess and compare the nutritional status (NS) of patients with HPV-related (HPV+) and non-HPV-related (HPV-) OPC before and after radiotherapy (RT) or chemoradiotherapy (CRT). Methods: The analysis included 127 patients with OPC who underwent radiotherapy (RT) alone, or in combination with chemotherapy (CRT), in the I Radiation and Clinical Oncology Department of Maria Skłodowska-Curie National Research Institute of Oncology, Gliwice Branch, Poland. Patients were divided according to HPV status. Confirmation of HPV etiology was obtained from FFPE  tissue material and/or extracellular circulating HPV DNA. Basic anthropometric and biochemical parameters before and after RT/CRT were compared between the HPV- and HPV+ groups. The effect of NS on survival was also analyzed. Results: In both groups, a significant decrease in all analyzed nutritional parameters was noted after RT/CRT ( The incidence of oropharyngeal carcinoma (OPC) has been increasing recently, mostly in young patients, which could be associated with a relatively new risk factor—infection with the high-risk human papilloma virus (HPV). HPV infection is usually not associated with other well-known typical determinants of OPC such as smoking, alcohol abuse, diet, chemical irritants, or poor oral hygiene ,2. TypicMalnutrition is a common problem in OPC patients. It is caused by insufficient food intake due to dysphagia, odynophagia, and lack of appetite caused by the tumor. It is also secondary to RT/CRT as a consequence of mucositis, with dry mouth, loss of taste, and dysphagia. The severe dysphagia is the most serious challenge in patients with OPC, because in 20–30% of patients it leads to the definitive, total impossibility of eating through the mouth. These patients require permanent percutaneous gastrostomy tubes . AlthougThe aim of this study was to assess and compare the NS of patients with HPV+ and HPV- OPC before and after RT or CRT, using various anthropometric, clinical, and biochemical parameters.The analysis included 127 patients with OPC who received definitive radical RT/CRT at the I Radiation and Clinical Oncology Department of Maria Skłodowska-Curie Research Institute of Oncology, Gliwice Branch, Poland, in the period 2012–2016. Assessment of the NS was performed in patients before and after treatment (RT or CRT). There were 87 (68%) men and 40 (32%) women in the analyzed group, with a mean age of 60.5 years (range: 30–80 years). The following inclusion criteria were used: primary OPC, age > 18 years, regionally advanced cancer without distant metastases . Exclusion criteria included surgical treatment, cancer recurrence, and incomplete demographic and/or clinical data.This study was supported by a grant from the National Centre of Research and Development, Poland .All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee , and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.2 on irradiation days 1, 22, and 43, or at a dose of 40 mg/m2 administered weekly. In the case of induction chemotherapy, 2–3 cycles were used according to the PF regimen (cisplatin and 5-fluoruracil) or the TPF regimen .The patients were radically irradiated with the total dose, sterilizing the squamous-cell carcinoma tissue. Most of the patients received the conventionally fractionated 70 Gy dose. Accelerated schemes were also used, including continuous accelerated irradiation (CAIR) and simultaneous integrated boost (SIB). Therefore, the dose range was in the 66–72 Gy range. Despite the obvious differences in the total physical dose, it can be assumed that the total biological doses were not different. Simultaneously, during radiotherapy, cisplatin was administered at a dose of 100 mg/m2), and well-nourished patients (BMI ≥ 18.5 kg/m2) [3) [All patients were asked about deterioration of NS, body weight before disease and before treatment, loss of body weight, and food intake since the onset of disease. Information on smoking (including the amount and duration of smoking and/or smoking cessation after diagnosis) and alcohol consumption was collected. The patients’ height and weight were measured, and laboratory blood tests were performed before and after treatment. The selected blood count parameters and biochemical parameters , hemoglobin, C-reactive protein) were analyzed. The body mass index (BMI) and weight loss over the course of the disease were calculated. Percentage weight change during treatment was calculated using weight before RT as a baseline and weight after the end of RT. The patients were divided into two groups according to their BMI: malnourished patients (BMI < 18.5 kg/m5 kg/m2) . The nut5 kg/m2) ,14. Onod/m2) [3) .The patients were divided into two groups according to their HPV involvement: HPV-related and non-HPV-related groups. Confirmation of HPV etiology was obtained from FFPE  tissue material and/or extracellular circulating HPV DNA.The stage of OPC was classified according to the eighth edition of the American Joint Committee on Cancer (AJCC)’s TNM classification system: primary tumor (T), regional lymph node metastasis (N), or distant metastasis (M) ,7. TumorThe clinical, anthropometric, and laboratory parameters before and after treatment were compared between HPV- and HPV+ patients. The parameters before treatment were signified as “0”, and after treatment as “1”.The median follow-up was 74.58 (0.1–165.58) months. Overall survival (OS) and disease-free survival (DFS) were analyzed in both groups. OS was defined as the time from randomization to death from any cause. DFS was defined as the time from randomization to the first event of either recurrent disease or death.Comparisons between all HPV- and HPV+ patients, as well as between HPV- and HPV+ patients undergoing RT and CRT separately, were performed to exclude the concomitant chemotherapy as a confounding factor in the analysis of the whole cohort. HPV+ OPC patients were treated more predominantly with CRT than HPV- OPC patients.Confirmation of the HPV etiology was carried out before treatment using FFPE tissue material (if it was available) and/or extracellular circulating HPV DNA .Formalin-fixed, paraffin-embedded tumor samples were examined for high-risk HPV (HR-HPV) infection using a double-check algorithm, including immunohistochemical assessment of p16 (INK4A) protein expression, followed by the detection of HR-HPV DNA in tumor tissue using real-time PCR. Only cases with both p16INK4A expression and HR-HPV DNA amplification were classified as truly HR-HPV-positive.g and 1000× g, both at 4 °C for 10 min. DNA was extracted (according to the manufacturer’s instructions) from 1 mL of plasma using the Genomic Mini AX Body Fluids Kit . Each measurement consisted of a standard curve of three dilutions of a plasmid construct containing the HPV16 genome, a negative control, and a sample. For HPV16 detection, a reaction was performed using primers and a probe set for the HPV16 genome. PCR reactions were performed using the Bio-Rad CFX96 qPCR instrument . If HPV16 was found, its presence was confirmed with a second independent DNA isolation.Peripheral blood (12 mL) was collected in K3EDTA tubes . Plasma was separated within an hour by double centrifugation at 300× U test was used to compare the HPV+ and HPV- groups. The Wilcoxon test was used to compare pre- and post-treatment parameters in all patients and both HPV groups separately. Multiple testing correction was performed using Bonferroni correction. Cox regression analysis was used to calculate overall survival (OS) and disease-free survival (DFS). The log-rank test was used to assess the equality of survival distributions across different strata. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cutoff values for prognostic factors related to DFS and OS. Youden’s index was selected as the approximate cutoff value for each parameter. Kaplan–Meyer curves were constructed to determine the impact of selected nutritional parameters on OS and DFS in HPV- and HPV+ OPC patients. A p-value of less than 0.05 was considered to be statistically significant. The statistical analyses were performed using the Statistica® software program, version 13.0: Dell Inc. (2016). Dell Statistica , version 13. software.dell.com. .The categorical variables were presented as numbers and percentages. Continuous variables with normal distribution were expressed as the means and standard deviations. The Shapiro–Wilk test was used to determine statistical distribution in the analyzed patients. The Mann–Whitney The general clinical characteristics of the 127 patients are presented in 2; p = 0.009). Final BMI (after treatment) was similar in both groups (p = 0.419).Initial BMI (before treatment) was significantly lower in the HPV- group (72.38 ± 15.24 (44–104.80) vs. 79.41 ± 14.64 (46–111) kg/mp = 0.004, and 78.1% vs. 55.9%; p = 0.012, respectively). The compared groups were different with regard to the detailed tumor locations, histopathological grading, and lymph node invasion. The tonsil location was more frequently observed in HPV- patients, and palate locations were noted only in HPV- patients (p = 0.010). G3 grading was significantly more frequent in HPV+ patients compared to HPV- patients (p = 0.049). Lymph node invasion was significantly more common in HPV+ patients compared to HPV- patients (p = 0.029)  .p = 0.906) and weight loss (5 (7.40%) vs. 3 (5.10%); p = 0.724) were comparable in both groups . The analyzed groups were different in terms of the rate of neck tumor, which was significantly more frequent in HPV+ patients (29 (49.20%)) compared to the HPV- group (17 (25.00%)) (p = 0.008)  vs. 15 (25.40%); = 0.008) .p = 0.403). The treatment duration was significantly longer in HPV+ patients compared to HPV- patients, which was associated with more frequent use of CRT in these patients .The duration of medical history was similar in HPV- and HPV+ patients . There was no statistical difference in terms of weight after treatment between the two groups. The weight after treatment (M1) was also higher in HPV+ patients, but not significantly (72.96 ± 14.81 (45–110) vs. 70.56 ± 15.24 (42–94.70); p = 0.419) .The baseline weight (M0) was significantly higher in HPV+ OPC patients compared to those with HPV- OPC (79.41 ± 14.64 (46–111) vs. 72.38 ± 15.24 (44–104.80); = 0.419) . Loss of = 1.000 . It shouPost-treatment evaluations were performed depending on the treatment duration reported in p = 0.680). After treatment, NRS 2002 was not significantly higher in HPV+ patients (3.39 ± 1.10 (1–5) vs. 2.98 ± 1.09 (1–5); p = 0.074). This difference was nearly statistically significant. Thus, there was a clear trend of dependency   vs. 0.28 ± 0.64 (0–4); pendency . Compari 0.0001) .p < 0.0001). Thus, the percentage of patients with nutritional risk increased significantly after treatment in both groups. There was no difference in the proportion of HPV- and HPV+ patients in each of the NRS 2002 groups before and after treatment .In addition, the distribution of the NRS 2002 groups (<3 vs. ≥3) significantly changed after treatment. Before treatment, most patients (97.6% and 98.0%) were classified as NRS 2002 < 3, and significantly fewer patients (2.4% and 2.0%) had NRS 2002 ≥ 3 . These proportions changed significantly after treatment, as follows: NRS 2002 < 3 was reported in 35.7% and 25.5%, while NRS 2002 ≥ 3 was noted in 64.3% and 74.5% of HPV- and HPV+ patients, respectively . C-reactive protein (CRP) 0 was not significantly higher in HPV- than in HPV+ patients . CRP 1 was comparable in both groups (p = 0.179). A comparable CRP increase was noted after treatment in both groups (p = 0.431). The correlations between TLC and CRP before and after treatment were analyzed and excluded: for TLC 0 and CRP 0: R = 0.053 (p = 0.563); for TLC 1 and CRP 1: R = −0.116 (p = 0.214), using Spearman’s test.Most laboratory results were comparable in both groups. TLC 1 was significantly lower in HPV+ compared to HPV- patients (PNI was comparable in both groups before and after treatment. A comparable decrease in PNI after treatment was noted in both groups. The differences in laboratory parameters before and after treatment in both groups are presented in Due to the different treatment regimens (more frequent CRT in HPV+ patients), additional comparisons of nutritional parameters before and after treatment in patients undergoing RT and CRT separately were performed .Differences in weight, BMI, laboratory results , and PNI before and after treatment were similar in the HPV- and HPV+ groups.CRT caused significant weight loss, decreases in BMI, albumin, TLC, hemoglobin concentration, and increases in NRS 2002 score in HPV- and HPV+ patients. A significant decrease in prealbumin levels after CRT was noted only in HPV+ patients. RT caused a significant decrease in hemoglobin concentration and TLC in HPV- patients. There were no significant differences regarding other parameters after RT in either group. RT did not have a negative impact on BMI, weight, NRS, CRP, Alb, Prealb, or PNI.Comparison of differences in weight, BMI, NRS, PNI, and laboratory results before and after CRT and RT between HPV- and HPV+ patients did not show any significant differences between groups according to HPV status.p > 0.05)  among the study population were set as the border values to divide the high- and low-PNI groups in order to perform statistical comparisons of clinicopathological findings between both groups. Comparison using Holm–Bonferroni correction did not show any statistically significant differences between the low- and high-PNI subgroups in both HPV- and HPV+ patients ( > 0.05) .p = 0.011) and DFS (p = 0.028) were significantly better in HPV- patients with higher BMI. OS was comparable regardless of TLC in the HPV- (p = 0.294) and HPV+ (p = 0.501) groups. DFS in the whole cohort (p = 0.026) and DFS in the HPV+ group (p = 0.014) were better in patients with a higher TLC > 1.28. A similar, but non-significant result was noted in the HPV- group (p = 0.262). There was no clear impact of the hemoglobin (HB) level on OS in the HPV- (p = 0.337) and HPV+ (p = 0.661) groups. There was better DFS in patients with HB > 13.5 g/dL in the HPV- group (p = 0.017). There was better OS in patients with CRP < 3.50 g/dL in the whole cohort (p = 0.008). DFS was significantly better in patients with CRP < 3.5 g/dL in both groups . There was no influence of serum albumin levels on OS  and DFS  in either group. PNI did not influence OS in either group . There was significantly better DFS in patients with a higher PNI (>39) in the whole cohort (p = 0.042), and a non-significant increase for both HPV groups separately . There was no statistical difference between HPV- and HPV+ patients in terms of OS and DFS according to loss of weight (<5% vs. >5%) in either group (p > 0.05) (p > 0.05)  . Regardi > 0.05) . Signifi > 0.05) .p = 0.008), NRS 2002 , and alcohol abuse  were significant independent adverse prognostic factors, while female gender  was a good predictor for OS. There were no significant prognostic factors for OS in HPV+ patients , higher lymph node status , higher NRS 2002 , and alcohol abuse  were significant independent adverse prognostic factors for DFS, while higher hemoglobin concentration  was a good predictor for DFS. In HPV+ patients, only higher TLC  was a good, significant, independent prognostic factor for DFS , tonsil location , and alcohol abuse  were independent adverse prognostic factors, while higher BMI  and positive HPV status  were good prognostic factors for OS , higher lymph node status , and alcohol abuse  were independent adverse prognostic factors for DFS. Positive HPV status  was a good prognostic factor for DFS  at diagnosis compared to HPV+ patients ,17,18. GPatients with HPV+ OPC usually present with a more advanced stage of disease, but in spite of this their prognosis is better . This waMalnutrition is an important problem in patients with HNSCC, including OPC, and is associated with aggressive disease located in the oropharynx and severe side effects related to the treatment. Predominant acute side effects—such as xerostomia, mucositis, loss of appetite, odynophagia, dysphagia, nausea, and vomiting—usually lead to weight loss and dehydration. Moreover, many publications have proven that RT also decreases NS in these patients. In such patients, nutritional intervention is needed. Most frequently, a feeding tube (gastrostomy/jejunostomy) is used in order to feed these patients ,26,27,28p = 0.003). Critical weight loss (>5%) was noted in 86% of patients, with a higher percentage in the HPV+ group (p = 0.011). The authors noted that the mean percentage weight change in both HPV status groups in their study was at a critical level (≥5%) [Vangelov et al. ,28 noted2) before RT is positively associated with survival in patients with OPC. Moon et al. [2 was an independent predictor of cancer-specific survival (CSS) and OS in HNSCC patients who had undergone definitive CRT.In our study, OS and DFS were significantly longer in patients with higher BMI in all patients and in the HPV- group, and not significantly for the HPV+ group. The association between higher BMI and longer survival was presented in the literature. According to Ottosson et al. , higher n et al.  reportedThe importance of HPV status as a prognostic marker in OPC patients has been well established in the literature ,17,18. TAccording to the literature, age, smoking, tumor stage, and treatment also have an important influence on survival in patients with OPC . Our stuIt should be added that our study did not show any clear impact of nutritional status (including PNI) on survival in patients with OPC, regardless of HPV infection. This may be associated with other, stronger factors—including HPV status—that determine survival in patients with OPC. It is possible that positive HPV status has a stronger influence on survival than nutritional parameters. This hypothesis is supported by the fact that our study found a greater association between NS and survival in HPV- patients. Higher NRS 2002 was an independent adverse prognostic factor for OS and DFS in HPV- patients, but not in the HPV+ group. The significant impact of NRS 2002 on survival in HPV- patients was confirmed via Kaplan–Meier analysis, where both OS and DFS were significantly better in HPV- patients with lower NRS 2002 scores. This relationship was not observed in the HPV+ group. The impact of NRS 2002 on survival according to HPV status in OPC patients has been not reported in the worldwide literature. According to some authors, a higher PNI predicts better survival in patients with HNSCC ,39,40,41Our study confirmed that HPV+ OPC patients had better NS at diagnosis compared to those with HPV- OPC ,41,42,43The strength of this study is that it tackles a clinically relevant problem regarding the changes in nutritional status during CRT, and their impact on survival in OPC patients depending on HPV status. There are no similar articles in the global literature. To the best of our knowledge, this is the first study to conduct a comprehensive analysis of NS, including its impact on survival, in HPV- and HPV+ OPC patients. This study was conducted in a large tertiary oncological center with experienced, well-established, multidisciplinary teams.The single-center observation and retrospective analysis are limitations of this study. A prospective randomized multicenter study is needed to assess the influence of NS on survival in HPV- and HPV+ patients with OPC. To our knowledge, this research is one of the first studies in the worldwide literature to conduct a comprehensive comparative analysis of the nutritional status of HPV- and HPV+ OPC patients.Despite differences in local stage and treatment , NS was comparable in both groups. CRT adversely affected the NS of patients regardless of their HPV status. RT caused significant decreases in hemoglobin concentration and TLC in HPV- patients. There were no significant differences regarding other nutritional parameters after RT in either group. Generally, RT/CRT worsened the NS of patients with OPC regardless of their HPV status, because in both the HPV- and HPV+ groups, there were significant decreases in all analyzed nutritional parameters after treatment. Differences in weight, BMI, laboratory results , and PNI before and after RT and CRT were similar in the HPV- and HPV+ groups. Therefore, deterioration of NS after treatment was similar in HPV- and HPV+ patients. A greater association between NS and survival was noted in HPV- patients. This might be associated with a fact that HPV+ status is a stronger predictive factor for survival than nutritional parameters. Higher NRS 2002 was an independent adverse prognostic factor for OS and DFS in the HPV- patients, but not in the HPV+ group. The significant impact of NRS 2002 on survival in HPV- patients was confirmed via Kaplan–Meier analysis, where both OS and DFS were found to be significantly better in HPV- patients with lower NRS 2002 scores. However, this relationship was not observed in the HPV+ group. OS and DFS were significantly better in patients with NRS 2002 < 2 compared to NRS 2002 ≥ 2 for HPV- patients, and not significantly for the HPV+ group.Our study showed that both HPV- and HPV+ OPC patients can develop malnutrition during RT/CRT. Therefore, regardless of HPV status, nutritional support is required for OPC patients during RT/CRT. Further prospective multicenter observational studies regarding the impact of HPV status on NS and the influence of nutritional parameters on survival in OPC patients are needed.


HPV 16 is the primary etiologic agent of cervical cancer and the presence of L1 and E6 oncoproteins are largely responsible for its virulence. It was the objective of this study to identify HPV16 isolates from suspected cases of cervical cancer at Specialist Hospital Sokoto and Sir Yahaya Memorail Hospiatal Birnin Kebbi, Nigeria and also to identify potent HPV16's L1 protein inhibitor using in silico analysis.A total of 144 cervical samples consisting of 21 low grade squamous intraepithelial lesion, 6 high grade lesion and 117 negative pap smears were collected. The samples were subjected for molecular detection using PCR targeting E6 gene of the virus. Data generated for the molecular prevalence was statistically analyzed using Chi-square method. AutoDock Vina was used to carry out the molecular docking between 2hr5 and Chicoric acid, curcumin and Echinacoside.Out of the 144 samples, 24 samples were positive for the PCR representing 16.9% molecular prevalence rate. There is statistically significant association between cyto-diagnoses and presence of HPV16 (P < 0.05). Docking analysis showed that the Chicoric acid components of Echinacea purpurae have strong binding affinity  to the L1 protein of the HPV.This study provides data on HPV 16 epidemiology in northern Nigeria, and also provides novel evidence for consideration on certain interacting residues, when synthesizing Anti-HPV compounds in the wet lab. Human papillomavirus (HPV) is one of the most prevalent infections that are transmitted during sexual activities affecting both male and female genderEchinacea purpurae is one of the world's most common medicinal plant and belongs to the Astracea familyIn most parts of northern Nigeria, the prevalence of HPV is unknown, therefore the main objective of this study is to identify HPV16 isolates from suspected cases of cervical lesion at Specialist Hospital (SH) Sokoto and Sir Yahaya Memorial Hospital (SYMH) Birnin Kebbi and also to identify potential inhibitors for HPV16's L1 protein using in silico analysis of echinacoside, curcumin and chicoric acid against the viral protein.A total of 144 women with an average age of 33 years (range 20–60 years) participated in this study. The population was recruited from patients attending obstetrics and gynaecology units of SYMH Birnin Kebbi and SH Sokoto, Nigeria.Cervical smear was collected from each of the participants by an application of standard procedure using sterilized speculum and swab (cyto-brush).14. this study adopted the following cytological classification: Negative; for normal cytology, low grade intraepithelial lesion, high grade intraepithelial lesion and carcinoma in situ; for malignancy.Consented patients aged 18 years and above attending attending obstetrics and gynaecology units of the selected hospitals.Non-consented patients, pregnant women and participants with vagina bleeding were excluded from the study.DNA extractionDNA extraction was done using Viral Nucleic Acid Extraction kit II . Extraction was carried out following the manufacturer's instruction, stated as follows: Two hundred (200) µl of sample was dispensed ınto1.5ml tube, then 400 µl of VB lysis buffer was added into the tube containing the samples, mixture was vortexed, then incubated at room temperature for 10 minutes. Four hundred and fifty (450) µl AD buffer was added into sample lysate and vortexed vigorously, VB column was placed in 2ml collection tube, 600 µl of lysate mixture was added into the VB column and centrifuged at 16,000g for 1 minute, flow-through was discarded and VB was place back into the collection tube, the remaining mixture was transferred to the VB and centrifuged for 1 minute, collection tube was discarded with the flow-through, then VB column was transferred to a new 2ml collection tube. Four hundred (400) µl of W1 buffer was added to the VB column and centrifuged at 16,000g for 30 seconds, flow-through was discarded and VB was placed into collection tube, then centrifuged at 16,000g for 3 minutes to dry the column. Dried VB column was placed in a clean 1.5ml microcentrifuge tube, 50 µl of RNase-free water was added to the centre of VB column, mixture was allowed to stand for 3 mınutes, then centrifuged at 16,000g for 1 minute.Sequence of E6 gene of HPV 16 were retrieved from NCBI, the selected gene sequence were saved in FASTA format, sequence were imported to BioEdıt, sequence were aligned by running ClastalW from the accessory application Menu, conserved regions were selected as template, the specificities were checked using nucleotide Blast.The PCR was conducted using KOD-FX Neo following manufacturer instructions as follows, each PCR mix of 50µL contained, buffer 25µL, 1.3 µL of forward and reverse primer each, DNP of 10µL, molecular grade water of 10 µL, DNA template 1.4 µL, KOD of 1 µL. the following PCR conditions were used, 95°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, final extension at 72°C for 5 minutes, then held for 16°C.All computational simulation studies were performed using (Intel Core i5-2430M) 6.00GB RAM with processor 2.40 GHz on Windows 7 operating system. Docking analysis was carried out using bioinformatics software including PyRx virtual screening software (AutoDock Vina) and the visualization of structure was carried out using Pymol molecular graphic system. Online resources were also used in this study.For this present study, ligands were downloaded from zinc12 (zinc.org), and saved in mol2 format, they were converted into pdbqt format by autodock vina.www.rcsb.org/pdb). This was done by typing the PDB code, 2r5h on the protein data bank search engine. Furthermore, the receptor was saved in a receptor file folder, by selecting 2r5h→ download files → PDB format→ download. After saving, the file was opened by double clicking the folder containing the receptor structure which comes out in Pymol. The receptor was prepared by deleting protein and water via edit then delete.Preparation and Optimization of L1 Protein Structure Pentamer structure of L1 protein of human papillomavirus (PDB Code:2r5h) was retrieved from the protein data bank (AutoDock Vina was used to carry out the protein-ligand docking15. Both the ligands and receptor (2r5h) were converted from pdb files to pdbqt  files (Vina input file format). AutoDock Tools (ADT) was used to prepare L1 protein of the human papillomavirus by the complete addition of hydrogen atoms to the receptor's carbon atoms. Non-polar hydrogen were added to the docked ligands. For Lamarckian genetic algorithm, a maximum number of 15 × 105 energy evaluations, 27,000 maximum generations, 0.02 gene mutation rate and 0.8 crossover rate were used16. Each ligands had two hundred independent docking runs.AutoDock Vina cannot recognise any file format except a pdbqt files format, therefore ligands and protein have to be converted from the downloaded file format to pdbqt format following the procedure as shown in Calculation of the binding affinity was done as shown in The pymol was opened by double clicking its icon on the desktop of a laptop computer to lunch its work space. From the file menu, Open was clicked followed by macromolecule folder containing the docked results, all supported format was changed to all files, receptor was clicked followed by control + ligand. By clicking open ligand and receptor were exported to pymol.The ligand was selected, copied and pasted on the page containing the receptor.From the right upper tools 2r5h was selected.Action was right clicked Find→Polar contact→Within selection shown in figure.Wizard was clicked → mearsurement → Show distance.Statistics analyses were performed using Chi-square with aid of an SPSS version 16.0, and data were presented in tables and figures.The low grade squamous intraepithelial lesion (LGSIL) in this study was 21, with about 57% of the low-grade lesion been positive for HPV type 16 virus, none of the participants in this study has carcinoma in situ, only 6 subjects showed cytology of high grade squamous intraepithelial lesion (HGSIL) with all been positive for HPV type 16. The P value is statistically significant (<0.05).HPV type 16 E6 gene sets of oligonucleotides were used in amplifying HPV DNA using PCR, the PCR products were visualized by 1% agarose gel electrophoresis with Ethidium bromide. As shown in Molecular docking studies were carried out between the targets  which has 3 chains, and its inhibitors, active components of Echinacea  The best three docking scores is seen on compounds 2r5h_zinc_33737268, 2r5h_zinc_95098864 and 2r5h_zinc_899824 due to their low binding affinity  kcal/mol as depicted in HPV type 16 has been the major cause of cervical lesion in Africa, it causes 49% of cervical cancer in the continent, higher than any other HPV serotype. HPV type 16 is responsible for 70 percent of all cervical cancer alongside with type 1817. Of the 144 participants recruited in this study 16.9% were positive for HPV type 16, which is slightly higher than the study carried out in Kano, Nigeria where they reported prevalence of 15.8% for HPV type 16, though their study recruited fewer subjects (50) than this studyThis study shows there is an association between infection with HPV 16 and cervical lesions, this is consistent with a study where the strength of the association was even demonstrated with odds ratio of 182, showing a strong association between HPV 16 and cervical lesionsThe population awareness about HPV infections and its vaccine in Africa is low. The cost per person on HPV vaccination is beyond what an average citizen can afford, Herbal therapy such as Echinacea has been reported to not only minimize viral replication but also improve the immune system, with an easy accessibility and affordability prospects in NigeriaMolecular docking studies were carried out between the targets (pentamer structure of major capsid protein L1 of HPV type 16) which has 3 chains, and its inhibitors  to fulfil most of the requirements to be used as a therapeutic agent. One of the criteria for a successful therapeutic agent to be fulfilled is the binding energy, binding affinity depends on thform and quantity of bond between target protein (2r5h) and the ligands , the lower the binding energy, the stronger the relationships would be and the better the binding affinity. Also Root Mean Square Deviation (RMSD) values is primarily used to evaluate protein stability and predict protein conformational changes, ideally RMSD of less than 1.5 Å shows the best protein stability. In this study only RMSD of less than 1.5 Å was considered. All the compounds were found to inhibit strongly by completely occupying the active sites in the target protein (2r5h). In this study the inhibitors occupied the AA 50–60 of the L1 protein, which is situated in the DE-Loop of the HPV L1 protein. Most of the ligands were found to be having polar contact with the receptor (2r5h). The best two docking results were zinc_33737268 (chicoric acid) with a binding energy of -8.7 kcal/mol which is lower than the binding energy reported for Withaferin A docked against HPV E6 protein in a study by Kumar et al. (2014), though in the same study carrageenan showed a much lower binding energy when docked against E6 protein, but the present study targeted the L1 protein which is a more conserved region, the L1 also has the heparin binding site in its loop, which plays important role in high risk HPV infectionNumerous researches are currently ongoing in order to identify promising therapeutic agents for the management of HPV associated diseases, advancement in molecular modelling and bioinformatics are very important in validating those therapeutic agents using in silico analysis. Although, few researchers have worked on the inhibitory action of natural compounds against L1 protein. The L1 is a conserved region and its loops also have a heparin binding site that plays an important role in high risk HPV infection. This study provides promising data on how the plant Echinacea pupurae can be used to manage HPV-related infections by inhibiting the action of L1 protein when confirmed in the wet laboratory.


Family caregivers may be at a higher risk for several chronic diseases, including cancer. Cervical cancer is one of the most common causes of cancer death among women. Despite family caregivers’ vulnerability, the status of their HPV awareness, knowledge, and preventive health behaviors, including cervical cancer screening, has been understudied. Thus, this study aimed to examine the sociodemographic factors associated with HPV awareness and knowledge and adherence to the cervical cancer screening guidelines among caregivers in the U.S.Nationally representative cross-sectional survey data were obtained from the Health Information National Trends Survey . Female caregivers aged 21–65 were included (N = 1190). Weighted multivariable logistic regression was performed to identify factors associated with HPV awareness (heard of HPV), knowledge , and adherence to the United States Preventive Service Task Force 2018 cervical cancer screening guidelines by sociodemographic factors  and the intensity of caregiving.aged 21–50 vs. 51–65), Hispanics of race/ethnicity compared with Black/African Americans , with a high school education or less , and with intense caregiving duty (spending 35 h/week or more on caregiving) compared with light-duty  had poor adherence to the cervical cancer screening guidelines. Caregivers who were older, racial minorities , and less educated showed lower HPV awareness (Heard of HPV) than their counterparts.An estimated 79% of female caregivers were aware of HPV and 84% adhered to the cervical cancer screening guidelines. Caregivers who were older than 50 (OR = 3.62, 1.91–6.85, adherence of There are caregiving populations whose HPV awareness and cervical cancer screening adherence are low. To improve their awareness and knowledge of HPV and support their cervical cancer screening behaviors, we need to consider interventions that target those specific populations. A family caregiver is defined as “any relative, partner, friend or neighbor who has a significant personal relationship with, and provides a broad range of assistance for, an older person or an adult with a chronic or disabling condition” . ApproxiThe majority of caregivers are female (61%) , and theIn 2022, 14,100 new cervical cancer cases and 4280 cervical cancer death were reported despite effective prevention and treatment options . In the While multiple studies have shown disparities in HPV knowledge and cervical cancer screening behavior in women in the U.S., studies focused on family caregivers’ negative health behaviors due to the caregiving burden and the receipt of preventive clinical services, including cancer screenings, are still scant with inconsistent results –42. StudDespite the inconsistent findings, there has been a longstanding concern that caregivers are less likely to receive cancer screenings because the burden of caregiving may hinder them from obtaining care for themselves. This is alarming as their health status is already vulnerable –8, 33–36This study used publicly available cross-sectional data from the Health Information National Trends Survey (HINTS) . HINTS iCaregiving status was identified by one question; “Are you currently caring for or making health care decisions for someone with a medical, behavioral, disability, or other condition?” Those who affirmatively responded ‘yes’ were defined as caregivers.The following question was used to investigate caregivers’ awareness of Human Papillomavirus (HPV): “Have you ever heard of HPV? HPV stands for Human Papilloma Virus. It is not HCV, HIV, or Herpes.” Among respondents who answered, ‘yes’ to HPV awareness, HPV knowledge was assessed with the question, “Do you think HPV can cause cervical cancer?” In this study, HPV knowledge was defined as having knowledge of the causal effect of HPV on cervical cancer.To distinguish the caregivers who were compliant with the USPSTF 2018 cervical cancer screening guidelines, the question, “How long ago did you have your most recent Pap test to check for cervical cancer?” was used. Those who answered, “I have never had a Pap test,” were re-coded to “Never.” Caregivers with all other answers were divided into “within guidelines” if they had a pap test within the past 3 years or “outside guidelines” if they had a pap test more than 3 years ago. As only 18 women reported never receiving a Pap test, we only included ‘within guidelines’ and ‘outside guidelines’ in our analysis, and ‘Never’ was excluded.The social determinants of health framework from the Healthy People 2030 was used to determine the sociodemographic predictors for this study : Age .The prevalence of HPV awareness, HPV knowledge, and adherence to cervical cancer screening guidelines were reported . To examine the association between characteristics and the three outcomes of interest (1) HPV awareness (heard of HPV), (2) HPV knowledge , and (3) adherence to the cervical cancer screening guidelines (a Pap smear within 3 years), weighted multivariable logistic regression was performed to obtain odds ratios (ORs) and 95% CIs. Models for HPV awareness and knowledge were adjusted by sociodemographic factors, and the model for screening uptake was additionally adjusted for time spent on caregiving. The statistical significance was determined at a Among female caregivers aged 21–65, 39% were aged 51–65, 63% were non-Hispanic White, 46% reported less than $50,000 annual household income, 24% had a high school education or less, 60% were employed, 64% were married, 67% were insured by an employment-based or private plan while 33% were insured by a government health plan , and approximately 98% were urban residents. An estimated 33% spent 35 h or more on caregiving activities weekly  heard of HPV, and the majority of participants thought that HPV could cause cervical cancer (99%) , HPV knowledge , and adherence to the cervical cancer screening guidelines (had a pap test within 3 years) among female caregivers in the U.S. from 2017 to 2020, using nationally representative survey data . Female caregivers who were older, racial/ethnic minorities, and those with less education had lower HPV awareness and cervical cancer screening adherence. Intense caregiving duty (spending 35 h/week or more on caregiving) was also associated with low adherence to the cervical cancer screening guidelines. Compared to the general population in the U.S., there were some similarities and differences. Similar to women in the general population, women caregivers who were older, racial/ethnic minorities, and those who were less educated were less likely to be aware of HPV and less likely to adhere to the cervical cancer screening guidelines –51. HoweOlder age was associated with low HPV awareness and low uptake of cervical cancer screening among caregivers. Plausible explanations could include that HPV vaccination campaigns and recommendations target young girls and/or healthcare professionals may focus more on younger age groups (11–12 years rather than 21–26 years) to promote HPV vaccine uptake and series completion –54. ThisWe found that HPV awareness was lower among caregivers who were racial/ethnic minorities. Specifically, Hispanic caregivers were less likely to obtain preventive cancer screening behaviors compared with their Black/African American counterparts. Low HPV awareness and knowledge and low cervical cancer screening uptake has been previously reported among Hmong, a growing Southeast Asian American population, and Korean Asian Americans, and Hispanic women , 58, 59.We also found that HPV awareness was lower among caregivers who had less educational achievement. Individuals with higher education and higher income are more likely to have higher health literacy and greater access to health resources, which can possibly increase their knowledge and awareness of HPV. However, HPV knowledge was not different by socioeconomic status in our study. A potential explanation could be that only those who were already aware of HPV were asked further HPV knowledge questions. Thus, it would have been challenging to identify sociodemographic disparities in HPV knowledge in our sample. Our findings support the need for tailored interventions that take into consideration the individual  , 60, socLong caregiving hours (35 h or more weekly) were associated with low adherence to cervical cancer screening guidelines among caregivers. Several studies that examined the associations between caregiving, the caregiving burden, and low engagement in preventative cancer services reported inconclusive results –38, 65. A recent study reported that the most common reason for not receiving timely screening was lack of screening knowledge . AddressThe current study has several limitations. First, HINTS is a cross-sectional survey and cannot provide knowledge of temporality or causal associations. Second, as HINTS data is self-reported, we need to acknowledge the possibility of respondent biases. Respondents may have mis-reported the dates of their last Pap Smear test, especially if it happened 2–3 years ago . Moreover, respondents might not always honestly report their knowledge and behaviors, which potentially could lead to either an increase or decrease in screening rates (reporting bias). Third, the small sample size may be related to some of the non-significant results. Fourth, there is a possibility of mislabeling the screening guideline-compliant individuals as non-compliant due to the potential confusion between hrHPV test and a Pap smear. As HINTS only asks about a Pap smear as a preventative screening service for cervical cancer, we might have underestimated the percent who were screening-compliant, as we did not include those who received hrHPV testing every 5 years. Since there is an increasing trend of using hrHPV test over a Pap smear due to convenience in recent years , we woulWe found that sociodemographic factors, particularly age, race/ethnicity, educational attainment, and the intensity of caregiving, were associated with disparities in HPV awareness and adherence to the cervical cancer screening guidelines among female caregivers in the U.S. The value of this study is the suggestion that targeted interventions should focus on female caregivers who are aged 51–65, racial/ethnic minorities , have low educational attainment (high school graduate or less) and spend 35 h or more on caregiving weekly. Therefore, targeted interventions to improve HPV awareness and cervical cancer screening adherence are urgently required to mitigate existing inequities among these vulnerable subgroups of caregiving women in the U.S.


TTN has a high mutation rate in the LUAD, which encodes a large abundant protein of striated muscle. However, the value of TTN in prognosis and the immune environment are poorly understood.Lung adenocarcinoma (LUAD) is the most common type of lung cancer, accounting for around 40%. Despite achievements in the treatment approach, the prognosis is still dismal, with overall survival of fewer than five years. Thus, novel prognostic biomarkers are needed to predict the clinical outcomes of individual patients better. TTN gene in LUAD patients through bioinformatics analysis.We investigated the clinicopathological characteristics, transcriptional and protein level, prognostic value, biological function, and its relationship with immune infiltration of TTN expression was significantly lower in LUAD than that in normal lung tissue. Lower TTN expression was associated with worse survival. Besides, TTN is highly expressed in alveolar type 2 cells which were surmised as the origin of LUAD.TTN and its role as a biomarker for determining the immune infiltration levels in patients with LUAD.Our findings indicated the potential prognostic value of  Lung adenocarcinoma (LUAD) accounts for a majority of cancer-related death worldwide. In the last decade, breakthroughs in immunotherapy research have dramatically improved survival rates for several tumor types –4, and rSeveral clinical trials showed that the amounts and characteristics of immune cells in the immune microenvironment are key predictors of ICBs response. KEYNOTE-086 revealed a positive correlation between the number of TILs and the response to pembrolizumab . An inflTTN-AS1 in diverse cancers (TTN-AS1) has been regarded as a tumor-promoting lncRNA in numerous cancers, such as LUAD, hepatocellular carcinoma, cervical cancer, papillary thyroid cancer, and gastric cancer (TTN missense mutation plays a favorable prognostic role in lung squamous cell carcinoma(LUSC)  . TTN is TTN in LUAD, aiming to provide a new biomarker for predicting prognosis and a new immunotherapy target for advanced LUAD patients.Although immunotherapies have become the standard of care in patients with advanced LUAD, a fraction of patients still have a poor prognosis. Herein, we conducted comprehensive research to elucidate the prognosis value and function of TTN gene in different types of cancers was analyzed in the Oncomine database (https://www.oncomine.org/resource/login.html) and TIMER (http://timer.cistrome.org/). The threshold was determined according to the following values: P-value of 0.0001, fold change of 2, gene ranking of top 10% and data type of all.The expression level of the database  . The correlation between TTN expression and survival in various cancer types was investigated in the PrognoScan database (http://www.abren.net/PrognoScan/). The threshold was adjusted by a log-rank p-value of <0.05. Hazard ratio (HR) with 95% confidence intervals (CI) was also calculated.The overall survival and progress-free survival curves correlated to  plotter , 26 . CBioPortal database contains somatic mutation and copy number variation data from the cancer genome atlas (TCGA) database . The data of alteration frequency  of TTN was also visualized and downloaded from the official website. Besides, we excluded samples that are not profiled for all queried genes in all queried profiles.Clinicopathological characteristics and the mutation information of database   in the Human protein atlas (www.proteinatlas.org). Specificity and distribution classification were performed to determine the number of genes elevated in different cell types. The genes expressed in each of the cell types were explored in interactive UMAP plots and bar charts. Color-coding is based on groups of cells, and each cell type has common functional features. The functional state of TTN in various cancer types was analyzed by CancerSEA (http://biocc.hrbmu.edu.cn/CancerSEA/). Correlations between the gene of interest and functional state in different single-cell datasets were filtered by correlation strength > 0.3 and P-value < 0.05.in atlas , 31 . The gene expression level was displayed with log2 RSEM. The significantly correlated genes in TIMER were validated in GEPIA (http://gepia.cancer-pku.cn/index.html). The Spearman method was used to analyze the correlation coefficient. TTN was used for the x-axis, and other genes of interest are represented on the y-axis. The tumor and normal tissue datasets were used for analysis.The relevance of in GEPIA   was downloaded from the GEO database. GSE116959 contains transcriptome profiling information of 57 LUAD samples and 11 peritumoral normal lung tissues, with gene expression measured by Agilent-039494 SurePrint G3 Human GE v2 8x60K Microarray 039381 in GPL17077. The TTN. A549, HCC827, H3255 and H1975 cell samples belong to lung adenocarcinoma cell lines, H1703 belongs to lung squamous cell line and HPAEpiC belongs to human pulmonary alveolar epithelial cell line. The mRNA expression of TTN was detected by real-time PCR, and GAPDH was the internal control gene. Real‐time PCR was performed with TB Green qPCR Master Mix  using the QuantStudio 6 Flex Real-Time PCR System . The expression values were analyzed by using 2−ΔΔCt relative quantitative methods. Primer sequences for the real-time PCR were listed as follows:Two tumor tissue samples and paired tumor-adjacent tissues, five human lung tumor cell lines and one human pulmonary alveolar epithelial cell line (HPAEpiC) were used to detect transcriptional expression of TTN forward: 5′- ACCCTTCTTTGACATCCGT -3′, reverse: 5′- TACTTTCCGCCACTTCGT -3′;GAPDH forward: 5′- ACAACTTTGGTATCGTGGAAGG -3′, reverse: 5′- GCCATCACGCCACAGTTTC -3′.Clinical samples were obtained from 1 patient with LUAD who was surgically treated at shanghai pulmonary hospital. The LUAD tissue and the paired adjacent tissues were prepared into 5 μm paraffin sections and incubated with mouse polyclonal antibodies of titin  at 4° overnight in a refrigerator. The sections were coupled with the secondary antibody labeled with horseradish peroxidase  at room temperature for 1.5 h, then each section was stained with DAB reagent, and counterstained with hematoxylin. IHC sections were independently reviewed by two pathologists .Protein from five lung cancer cell samples and one normal epithelial cell sample were extracted for used in the following western blotting (WB) experiments. WB experiments were performed according to the detailed protocol previously reported , with anP-values. The results generated in Oncomine are displayed with P-values, fold changes, and ranks. The results of Kaplan Meier plots and GEPIA are displayed with HR and P or Cox P-values from a log-rank test. The correlation of gene expression was evaluated by Spearman’s correlation and statistical significance, and the strength of the correlation was determined using the following criteria: 0.00–0.19 “very weak,” 0.20–0.39 “weak,” 0.40–0.59 “moderate,” 0.60–0.79 “strong,” 0.80–1.0 “very strong.” P -value<0.05 was considered statistically significant.Survival curves were generated by the PrognoScan and Kaplan Meier plots. The results generated in PrognoScan were performed with the hazard ratio (HR), 95% confidence interval (CI), and Cox TTN in various types of cancer and normal tissues. mRNA expression of TTN was significantly lower in lung cancer  and progress-free survival (PFS).    and OS  while male patients with high TTN expression level can benefit in OS . Thus, these results provided a theoretical basis for the poor prognosis of patients with TTN mutations.We investigated the relationship between TTN had a higher expression level in alveolar cells type 2 c-1, c-6  and endothelial cells c-9 . Consequently, TTN function in tumorigenesis was investigated in nine organs or tissues at a single-cell level, including LUAD and other tumors .We investigated the transcriptomic expression level in diverse cell clusters of lung tissue , CD4+ T cells , B cells , neutrophils , T cell regulatory , macrophages , monocytes , myeloid dendritic cells  and mast cell activated . The results showed that TTN and different tumor-infiltrating immune cell subsets were weakly to moderately correlated. Biomarker sets of immune cells were significantly associated with TTN in LUAD , including 204 samples, showed that a lower TTN expression level was significantly associated with poor prognosis  cells are precursors of the alveolar epithelium, which can transform into AT1 cells, maintain self-renewal during normal homeostasis and recovery after injury –37. SomeTTN expression and tumor-infiltrating lymphocytes. TTN expression level was positively correlated with the infiltration levels of CD8+ T cells, CD4+ T cells, and other immune cells  and the Cancer Research Funding of CSCO-Hausen .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Human papillomaviruses (HPVs) are etiologically associated with various benign and malignant neoplasms of cutaneous and mucosal epithelia. We describe an improved diagnostic protocol for comprehensive characterization of causative HPV types in common warts, in which broad-spectrum PCRs followed by Sanger sequencing, two previously described and seven newly developed type-specific quantitative real-time PCRs (qPCRs) coupled with the human beta-globin qPCR were used for: (i) diagnosis of HPV infection in warts; (ii) estimation of cellular viral loads of all HPV types detected; and (iii) determination of their etiological role in 128 histologically confirmed fresh-frozen common wart tissue samples. A total of 12 different causative HPV types were determined in 122/126 (96.8%) HPV-positive warts, with HPV27 being most prevalent (27.0%), followed by HPV57 (26.2%), HPV4 (15.1%), HPV2 (13.5%), and HPV65 (7.9%). The cellular viral loads of HPV4 and HPV65 were estimated for the first time in common warts and were significantly higher than the viral loads of HPV2, HPV27, and HPV57. In addition, we showed for the first time that HPV65 is etiologically associated with the development of common warts in significantly older patients than HPV27 and HPV57, whereas HPV4-induced warts were significantly smaller than warts caused by HPV2, HPV27, HPV57, and HPV65. Alphapapillomavirus (Alpha-PV), Betapapillomavirus (Beta-PV), Gammapapillomavirus (Gamma-PV), Mupapillomavirus (Mu-PV), and Nupapillomavirus (Nu-PV) . Sa. Sa18]. The study was conducted in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of the Ministry of Health of the Republic of Slovenia (consent references 63/10/13 and 0120-87/2020/3). All patients signed an informed consent form before participating in the study.Total DNA from fresh-frozen tissue samples of common warts was extracted using the QIAamp DNA Mini Kit  according to the manufacturer’s protocol for tissue DNA extraction . SubsequThe integrity of the extracted DNA and absence of PCR inhibitors were verified in all samples by qPCR amplification of a 150 base-pair (bp) fragment of the single-copy HBB gene using the specific qPCR primer-probe set , Table 14 to 1 × 10−1 HBB gene copies per reaction. The detection limit of the HBB qPCR assay was established at 10 HBB gene copies per reaction. The calculated coefficient of determination of the HBB qPCR standard curve was 0.999, and the amplification efficiency was estimated at 97.2%.The analytical sensitivity of the HBB qPCR assay was determined as described previously ,25 by teTo determine the presence of the most common cutaneous HPV types, each sample was first tested using the HPV2/27/57 multiplex type-specific qPCR assay , Table 1Alpha- and Gamma-PV genera was tested in all HPV qPCR-negative samples and in samples with minute HPV viral loads using two sets of broad-spectrum primers: (i) LR-α-HPV FW/RW, targeting an approximately 190-bp fragment of the E1 genomic region of at least 23 different low-risk HPV types from the Alpha-PV genus ; and (ii) Gamma-PV-E1F/E1R, targeting an approximately 500-bp fragment of the E1 genomic region of at least eight different HPV types from the Gamma-PV genus , as described previously  following the manufacturer’s instructions. Sanger sequencing of the purified HPV-positive PCR products on an automated ABI3500 Genetic Analyzer sequencing instrument  and HPV type determination were performed on both DNA strands, as described previously [Gamma-PV-E1F/E1R PCR products and used for HPV type assignment was approximately 130 and 440 bp, respectively.The presence of a wide spectrum of HPV types from the eviously  [26,27,2eviously . The sizhttps://bioinfo.ut.ee/primer3-0.4.0/, accessed on 20 February 2022) [http://www.premierbiosoft.com/netprimer/, accessed on 20 February 2022) web-based applications, respectively. The nucleotide sequences of reference HPV genomes used as templates for the design of type-specific primers and double-quenched probes were obtained from the Papillomavirus Episteme [https://www.ncbi.nlm.nih.gov/genbank/, accessed on 20 February 2022), under the accession numbers X74462 (HPV3), X70827 (HPV4), X74463 (HPV7), X74465 (HPV10), U31783 (HPV28), U31784 (HPV29), X70829 (HPV65), and AJ620210 (HPV95). To avoid potential cross-reactivity with non-targeted sequences, the specificity of the designed primers and probes was additionally verified using the NCBI Nucleotide BLAST web-based service . Due to the differences in genomic nucleotide sequences and their chemical and thermodynamic properties, it was more convenient to develop type-specific qPCR assays targeting different genomic regions of individual HPV types to achieve efficient HPV DNA PCR amplification.According to the results of sequencing-based HPV typing, HPV3-, HPV4-, HPV7-, HPV10-, HPV28-, HPV29-, HPV65-, and HPV95-specific qPCR primer-probe sets  were desry 2022) ,32 and NEpisteme  and GenBTo test the presence of identified HPV types by Sanger sequencing, all samples were subjected to qPCR amplification of 122 and 130 bp fragments of the HPV4 and HPV65 L2 genomic regions, respectively, using the newly developed HPV4- and HPV65-specific qPCR primer-probe sets . The HPV9 to 1 × 10−1 copies of viral DNA per reaction, and reference plasmids, containing the target HPV10 genomic region at concentrations of 1 × 108 to 1 × 10−1 copies of viral DNA per reaction, showed that all the type-specific qPCR assays mentioned had a sensitivity of at least 10 viral genome equivalents per reaction. The calculated coefficient of determination of all qPCR standard curves was 0.999, and the amplification efficiencies were estimated at 94.2% for HPV3, 95.0% for HPV4, 96.7% for HPV7, 97.0% for HPV10, 92.3% for HPV28, 96.0% for HPV29, 97.2% for HPV65, and 95.7% for HPV95.Testing triplicates of 10-fold serially diluted reference plasmids, containing the target HPV3, HPV4, HPV7, HPV28, HPV29, HPV65, and HPV95 genomic regions, respectively, at concentrations of 1 × 10A reaction mixture with no DNA template was used in all PCR runs as a reference control for potential amplicon carryover contamination.Reference plasmids containing partial or complete HPV genomic DNA were kindly provided by the International HPV Reference Center –E.-M. de Villiers, German Cancer Research Center, Heidelberg, Germany , M. Favre, Pasteur Institute, Paris, France , and T. Matsukura, National Institute of Health, Tokyo, Japan (HPV65).To determine the cellular viral load of HPVs in common warts, HPV type-specific qPCR assays were used in combination with the qPCR assay targeting the HBB gene described above. HBB gene and HPV concentrations (copies/μL) were calculated from the quantification cycle (Cq) value of sample DNA using standard curves generated by plotting Cq values against known concentrations of input human genomic DNA (Roche Diagnostics) or HPV reference plasmids. In the calculations, it was assumed that 3.3 pg of human genomic DNA contained one copy of the HBB gene . Ratios U test was used to evaluate differences in age distribution between patients of both sexes and to compare estimated viral loads between two different groups of causative HPVs. The Kruskal–Wallis test was used to compare patients’ age, wart size, and estimated viral loads between different causative HPV types. The Fisher’s exact test was used to determine associations between patients’ sex and the number of common warts caused by HPV27. Results were considered statistically significant at p-value ≤0.05.Data management, analysis, and graphic presentation were performed with RStudio v1.2.5033 software . The descriptive statistics are presented as number and percentage for categorical variables. For numerical variables, the minimum, maximum, arithmetic mean, standard deviation, and median were calculated. The Mann–Whitney p = 0.094, Mann–Whitney U test; The main study population consisted of 90 immunocompetent patients , including 47/90 females (52.2%) and 43/90 males 47.8%; . The medAt study entry, a single common wart was obtained from 57/90 patients (63.3%), two warts from 28/90 patients (31.1%), and three different warts from 5/90 patients 5.6%; . The medThe 150 bp fragment of the HBB gene, serving as an internal control, was successfully amplified from all 128 fresh-frozen tissue samples of histologically confirmed common warts included in the study, indicating adequate quality of the sample DNA for subsequent HPV testing.Alpha-, Gamma-, and Mu-PV, and six viral species–Alpha-PV species 2, 4, 8 , Gamma-PV species 1 (Gamma-1), and Mu-PV species 1, 2  were identified. HPV27  and HPV57  were the most frequently detected HPV types in common warts, followed by HPV4 , HPV2 , HPV65 , HPV1 , and HPV10 . HPV63 and HPV28 were detected in three and two different common wart samples, respectively, and HPV3, HPV7, HPV29, and HPV95 were each identified in a single wart sample.Using type-specific qPCR assays and broad-spectrum PCR assays followed by Sanger sequencing analysis, HPV DNA was detected in 126/128 (98.4%) samples of histologically confirmed common warts, with single and multiple HPV infections detected in 88/126 (69.8%) and 38/126 (30.2%) samples, respectively . As showBased on the estimation of cellular viral loads by the type-specific qPCR assays and applying the cutoff value for determination of the causative HPV type of one viral copy per cell , a causaAlpha- and Gamma-PV, respectively. In addition, in a 41-year-old male patient  had a complete concordance of the determined causative HPVs. The exceptions were a 15-year-old male patient  was significantly higher than the viral load of HPV2 , HPV27 , and HPV57 . Meanwhile, the estimated viral load of HPV65  was significantly higher than the viral loads of HPV2 (p < 0.001), HPV4 (p = 0.014), HPV27 (p < 0.0001), and HPV57 (p < 0.0001). The mean of HPV10 and HPV28 viral loads in the caused common warts was estimated to be 3.54 × 103 and 4.17 × 105, respectively, and the viral loads of HPV1, HPV3, HPV7, HPV29, and HPV95 were estimated to be 2.53 × 104, 2.17 × 104, 1.75 × 101, 1.02 × 104, and 5.87 × 102 copies/cell, respectively .The proportion of HPV2 as the etiological agent of common warts was the same in patients of both sexes, whereas HPV4, HPV57, and HPV65 were identified as the causative HPV types in more female than male patients . In contp = 0.047) and HPV57 , whereas there was no significant difference with the age of patients with HPV2  and HPV4-induced warts  than HPV27 , followed by HPV57 , HPV4 , HPV2 , HPV65 , HPV28 , HPV1 , HPV7 , HPV10 , HPV29 , and HPV95 , HPV27 , HPV57 , and HPV65 , HPV27 , and HPV57 , whereas the size of warts caused by HPV65 did not differ significantly  than HPV2  from the Gamma-PV genus, and HPV1 (3.3–31.3%) from the Mu-PV genus [Gamma-PV genus has been previously reported to be more prevalent in common warts on the hands in Japan (in 14.1% of samples) [Alpha-PV genus, HPV63 from the Mu-PV genus, and HPV95 from the Gamma-PV genus, which were also rarely detected previously [The overall prevalence of HPVs detected in fresh-frozen tissue samples of histologically confirmed common warts in our main study population was 126/128 (98.4%), which is more or less consistent with previous studies in which HPV positivity ranged from 82.4 to 100% ,15,16. MPV genus ,10,11,12samples) . Other Heviously ,16.To identify causative HPV types in common warts based on the estimation of cellular viral loads of all 13 HPVs detected, we used existing type-specific qPCR assays targeting five HPV types in combination with seven newly developed type-specific qPCR assays targeting eight different HPV types. Consistent with the “one virus, one lesion” concept, the single causative HPV type was reliably identified in 96.8% of HPV-positive common warts in this study, whereas the causative HPV type remained undetermined in four samples. The proportion of common warts with an identified etiological agent was thus much higher than in our previous study, in which etiological agents were successfully assigned in 79.8% of the warts analyzed . In agreAlpha-4 species are the most frequent etiological agents in common warts [Gamma-1 species are also important causative HPV types with high prevalence. The least common causative HPV types in this study were HPV3, HPV10, HPV28, and HPV29 from the Alpha-2 species, which were previously predominantly described in flat warts (verrucae planae) [Alpha-8 species, which is most often seen in the “butcher’s wart” variant of common warts in individuals whose hands are chronically exposed to moisture and cold [Gamma-1 species [Mu-1 species is frequently identified in clinically normal skin and in skin warts of patients in primary healthcare, especially in children under 12 years of age, with a distinct clinical profile because HPV1-induced warts resolve relatively quickly without treatment  [Based on the cutoff value for the determination of the causative HPV type of one viral copy per cell , this ston warts ,20 and h planae) ,12, HPV7and cold ,39, and  species . Previou months) ,37. In tGamma-PV genus was not tested and the cellular viral load of only six detected HPV types was estimated  [Gamma-HPVs and estimation of the cellular viral load of 13 HPV types, we determined the etiological agents in 7/9 (77.8%) previously “HPV-negative” common wart samples and in 18/25 (72.0%) HPV-positive warts without previously assigned causative HPV types [In this study, the causative HPV type was not determined in only six of 128 samples of common warts in which either HPV DNA was not detected or the estimated viral load of the HPV types present was less than one viral copy per cell. In our previous study, using the initial diagnostic protocol, HPV DNA was not detected in 9/185 4.9%) common warts, and no etiological agent was found in 25/124 (20.2%) randomly analyzed HPV-positive common warts, which was primarily a consequence of the suboptimal methodological approach because the presence of HPVs from the  HPV204) . Using tPV types , with HP.9% commo0 to 5.45 × 105 copies per cell, which is in line with similar previous studies [2 to 6.75 × 105 copies per cell, and, to our knowledge, were estimated here for the first time in common warts using type-specific qPCR assays. This study showed that the estimated cellular viral loads of HPV4 and HPV65 were significantly higher than the viral loads of HPV2, HPV27, and HPV57 in the caused common warts. The possible reason for the relatively higher estimated viral loads of HPV4 and HPV65 compared with HPVs from the Alpha-4 species could be the differential impact of their viral proteins in regulating viral replication, as seen in HPV1, where the viral replication protein E4 is highly abundant in cytoplasmic and nuclear inclusion granules, which supports the initiation of HPV1 genome amplification immediately in the parabasal cell layer of the cutaneous epithelium during the presumed inhibition of cellular DNA replication and cell proliferation in the G2 phase of cell division, resulting in high viral particle synthesis [The estimated cellular viral loads of HPV2, HPV27, and HPV57 in the caused common warts ranged from 1.12 × 10 studies ,20, wherynthesis ,42. The ynthesis , even inynthesis ,44.Gamma-PV genus have been shown to persist in healthy skin and cause mainly chronic asymptomatic infections associated with long-term viral replication supported by keratinocyte differentiation and viral shedding from the skin surface, minimizing the risk of immune clearance [In contrast to similar research in Japan , a compalearance ,45,46. Ilearance ,48.Whereas HPV4- and HPV65-induced common warts occurred primarily on the hands, HPV2, HPV27, and HPV57 frequently caused warts at various anatomical sites, including the hands, arms, legs, and head. Comparison of the size of common warts at different anatomical sites showed that HPV4 was etiologically associated with the development of significantly smaller common warts than HPV2, HPV27, HPV57, and HPV65. Previously, HPV4 was shown to be associated with the small, endophytic, punctate variant of common warts whose size did not exceed 5 mm, mostly on the hands , whereasIn this study, HPV2, HPV4, HPV27, HPV57, and HPV65 have been characterized as the five most frequent etiological agents in common warts of immunocompetent patients referred to the tertiary level of dermatological care. This may cause bias, and thus a larger number of wart biopsies obtained across all age groups would probably need to be studied to determine the causative HPV types in common warts in the general population. Nevertheless, data generated in persistent and treatment-resistant common warts could be essential to determine the most appropriate target HPV types for future cutaneous HPV vaccines, which would be particularly beneficial for immunocompromised patients . FurtherGamma-1 species were estimated in common warts for the first time in our study and were significantly higher than the viral loads of HPV2, HPV27, and HPV57 from the Alpha-4 species. In addition, we showed for the first time that HPV65 was etiologically associated with the development of common warts in significantly older patients than HPV27 and HPV57. HPV2, HPV27, and HPV57 frequently caused common warts at various anatomical sites, whereas HPV4- and HPV65-induced warts primarily occurred on the hands. Furthermore, HPV4-induced warts were significantly smaller than warts caused by HPV2, HPV27, HPV57, and HPV65.This study describes an improved diagnostic approach for the comprehensive detection and characterization of causative HPV types in histologically confirmed common warts in immunocompetent patients based on the estimation of HPV type-specific cellular viral loads, thereby importantly improving the knowledge of the diversity and pathogenesis of causative HPV types in common warts. To evaluate the etiological role of all HPV types detected in the development of common warts, we used seven newly developed type-specific qPCR assays targeting eight different HPV types. Using an improved diagnostic strategy to systematically estimate the cellular viral load of each HPV detected and according to the applied cutoff value for determining the causative HPV type, an etiological agent was reliably identified in our main study population in 96.8% of HPV-positive common warts, including several warts with multiple HPV types, whereas the causative HPV types could not be reliably assigned in the four remaining warts. Furthermore, 87.9% of patients from whom multiple samples of common warts were collected had a complete concordance of the causative HPVs identified, confirming that a single HPV type is typically involved in the development of multiple warts in an individual patient. Of the 12 different causative HPV types identified, HPV27 had the highest prevalence rate, followed by HPV57, HPV4, HPV2, and HPV65, which together have been etiologically associated with the development of 89.7% of common warts. The improved diagnostic protocol allowed reliable determination of causative HPV types in the great majority of common warts being HPV DNA-negative or HPV-positive but without a reliably determined causative HPV type in our previous study . To the


Lesack et al. recently published a cross-sectional study that focused on human papillomavirus (HPV) self-sampling in the screened population, a population not conventionally thought of for HPV self-sampling. They found 52% of well-screened, highly educated women who participated in the Human Papillomavirus For Cervical Cancer (HPV FOCAL) screening trial in British Columbia, Canada, would be willing to self-collect an HPV sample. We published a similar study in 2021 on well-screened, highly educated women affiliated with a family medicine clinic in Edmonton, Alberta, Canada, and found that 60% of these women preferred to have the option of HPV self-sampling. Our findings reinforce Lesack et al.’s results and together provide evidence for offering HPV self-sampling as an option for the well-screened population. We read with interest the recently published study by Lesack et al. “Willingness to Self-Collect a Sample for HPV-Based Cervical Cancer Screening in a Well-Screened Cohort: HPV FOCAL Survey Results ”.We published a similar, albeit smaller, study in the Journal of Lower Genital Tract Disease in 2021 [n = 212) in a family medicine clinic in Edmonton, Alberta, to answer a survey on their attitudes toward human papillomavirus (HPV) self-sampling as they were waiting for their appointment. Similar to Lesack et al.’s study, our survey included a brief description of self-sampling; however, we also included an illustration of the self-sampling process. Ninety-four percent of women took the survey, and, as in Lesack et al., these women were highly educated.We invited female patients  Whether they should have the option for HPV self-sampling and (2) Which option  they would select for future cervical cancer screening and why. We found that 60% of women agreed or strongly agreed with having the option of HPV self-sampling available to them, and 54% of women would select a Pap test, 24% of women would select HPV self-sampling, and 22% of women were undecided. Women who selected the Pap test had concerns that centered on taking an HPV self-sample correctly and the accuracy of HPV self-sampling. Given these reasons, it is likely a greater percentage of women would have selected HPV self-sampling had they been given more than a brief description of the process as well as its accuracy.In addition, Lesack et al. found a significant association between a higher level of education and willingness for HPV self-sampling. Our multivariate analysis did not find a significant association for this variable but did find a trend toward significance ; the lack of significance could be due to the smaller sample size in our study.The similarity in findings between our study and Lesack et al.’s study is especially noteworthy given that the studies were conducted in different geographic locations and independently of one another. We hope that these synergistic findings will encourage policy makers to consider HPV self-sampling as an option for well-screened women in their cervical cancer screening programs.


The aim of this research is to analyze knowledge of adults between 18 and 45 years of age and living in the Northern Cyprus about Human Papilloma Virus (HPV) along with their attitudes and beliefs towards HPV vaccine.The research, which was planned as a descriptive and cross-sectional, was executed on the web. The research was completed with 1108 women and men adults between 18 and 45 years of age, living in the Northern Cyprus and volunteered to participate in the study.p < 0.05). There was a statistically significant and negative correlation between HPV-KQ scores, questions on Current HPV Vaccination Program and the perceived barriers sub-dimension of the HBMS-HPVV whereas there was a statistically significant and positive correlation between the HPV-KQ scores, questions on Current HPV Vaccination Program and the perceived benefits and perceived susceptibility sub-dimensions of the HBMS-HPVV (p < 0.05).51.90% of the adults participating in the study were found to be women, 8.84% had a Sexually transmitted disease (STD) before and 63.27% of the individuals who had a sexually transmitted disease before also had had HPV and they knew it, 77.55% had undergone a treatment for their disease, 59.18% were found to be actively infected with HPV. Statistically significant and positive correlations were determined between the overall scores of the participants from the Human Papillomavirus Knowledge Questionnaire (HPV-KQ) and their scores in the perceived severity, perceived benefits and perceived susceptibility sub-dimensions of the Health Belief Model Scale for Human Papilloma Virus and Its Vaccination (HBMS-HPVV) (It has emerged that the participants do not have enough information about HPV, they do not know the ways and symptoms of protection from HPV, they do not have enough information about early diagnosis and screening, and they know very little about the HPV vaccine. Health policies should be developed to increase the awareness of individuals about HPV, to increase education and to provide free vaccines. HPV is a double-stranded DNA virus with more than 200 recognized sub-types . Low-risHPV, which is a sexually transmitted disease common all over the world, is the primary cause of cervical cancer . Sexual It was further reported in the literature that the level of prior knowledge of women about HPV varies between 11.9 and 16.1%, moreover HPV-positive people were found to be more well-informed compared to HPV negative individuals , 13. FurDespite the fact that the developments related to HPV have become more visible all over the world compared to the past, unfortunately they are still not at the desired level. Health professionals assume important roles particularly with regard to informing individuals and generally informing the society and protecting public health. No research was found in the literature review examining the level of knowledge of the people about HPV and their attitudes about the HPV vaccine in the northern region of Cyprus. The aim of this study is to determine the level of knowledge of individuals living in the northern region of Cyprus about HPV and their attitudes towards HPV vaccine, thereby to contribute to developing future health policies, raising awareness and protection of the society in cooperation with the necessary health institutions and organizations and even non-governmental organizations.The research, which was planned as a descriptive and cross-sectional, was executed on the web between 01.01.2022 and 30.03.2022. A purposeful sample selection was made in line with the study objectives and only individuals aged between 18 and 45 and living in the northern region of Cyprus were invited to the study. The research was completed with 1108 adults women and men who volunteered to participate in the study.Data collection process was executed online. Research data, all of which are quantitative in nature, were collected via a web-based online questionnaire within the specified time period. The invitation announcement for participating in the study was shared on the social media web pages  of the researchers. Individuals who clicked on the link shared in the informative announcement about the study were first welcomed with an informed consent form. Individuals who approved the informed consent form were eligible to participate in the study and to fill in the questionnaire. Mean time required to complete the online questionnaire is 5 min.The online questionnaire consists of 3 parts. The first part is the Personal Information Form (PIF), the second part is the Human Papillomavirus Knowledge Questionnaire (HPV-KQ) aiming to determine the level of knowledge on HPV and the third part is the Health Belief Model Scale for Human Papilloma Virus and Its Vaccination (HBMS-HPVV).The first part of the Personal Information Form (PIF) aims to determine the demographic characteristics of the participants such as age, gender, marital status and socio-economic characteristics. The second part of the PIF include questions such as whether the individual has been diagnosed with the disease before, the age of first sexual activity and the sexual partner in order to determine the medical history of the individual with regard to HPV. The questionnaire, consisting of 18 questions in total, was reviewed by taking the opinions of 2 faculty members from the Department of Midwifery and an academic staff from Obstetrics and Gynecology as an external observer.HPV-KQ was developed by Waller et al. in 2013 to measure the level of knowledge of individuals about HPV, HPV vaccine and screening tests . The scaHBMS-HPVV was developed by Kim . The scaResearch data were analyzed using the descriptive and parametric statistical analysis methods of the Statistical Program for Social Sciences 20.0 (SPSS). Frequency analysis was used for the distribution of the participants’ prior sexual experience and whether they had been infected with HPV before on the basis of their socio-demographic characteristics; the scores of the participants in the HPV-KQ and the HBMS-HPVV were interpreted with descriptive statistics; whether the data collected have met the normality assumption was analyzed by Kolmorogov Smirnov tests and skewness/kurtosis values and it was determined that they fit normal distribution. Accordingly, parametric tests such as independent sample t-test and ANOVA were used to compare the scores in the HPV-KQ on the basis of their socio-demographic characteristics, their prior sexual experience and whether they had been infected with HPV before. The relationships between the scores of the participants on the HPV Knowledge Scale and the Health Belief Model Scale for HPV and its Vaccine were examined with the Pearson test.Being literate, being married/single, men and women, living in the northern regions of Cyprus and willing to participate in the research, being between 18 and 45 years of age constituted the eligibility (inclusion) criteria of the study, while failing to fill in the questionnaire completely, not being in the appropriate age range and not living in the northern region of Cyprus constituted the exclusion criteria of the research.Table Table Table p < 0.05). Mean scores of the women participants from the HPV-KQ was found to be statistically significantly higher compared to the scores of male participants. Mean scores of the married participants in the HPV-KQ was found to be statistically significantly higher compared to the scores of single participants (Table Table ts Table .Table 4Cp < 0.05). Accordingly mean scores of the participants graduated from a primary school in the HPV-KQ were found to be statistically significantly lower compared to the scores of participants graduated from a secondary school, high school, participants with a bachelor’s degree and a post-graduate degree and mean scores of the participants with a low income in the HPV-KQ were found to be statistically significantly lower compared to the scores of participants with a moderate and high income . Accordingly, mean scores of the participants who have had a sexual intercourse before from the HPV-KQ was found to be statistically significantly higher compared to the scores of participants who did not have a sexual intercourse before and mean scores of the participants who first had a sexual intercourse before 18 years of age and younger from the HPV-KQ was found to be lower compared to the scores of participants who first had a sexual intercourse between 18 and 20 years of age. There was a statistically significant difference in the HPV-KQ scores of participants in terms of whether they have had multiple sexual partners (p < 0.05). Accordingly, mean scores of the participants who did not have multiple sexual partners from the HPV-KQ was found to be statistically significantly higher compared to the scores of participants who had multiple sexual partners . Accordingly, mean scores of the participants who had a STD before from the HPV-KQ was found to be statistically significantly higher compared to the scores of participants who did not have a STD. There was a statistically significant difference between the participants scores in the HPV-KQ on the basis of whether they have been infected with HPV before (p < 0.05). Mean scores of the participants who had been infected with HPV before in the HPV-KQ was found to be statistically significantly higher compared to the scores of participants who had not been infected with HPV before  before (re Table .p < 0.05). Accordingly, mean scores of the participants who had a family member diagnosed with cervical cancer from the HPV-KQ was found to be statistically significantly higher compared to the scores of participants who did not have a family member diagnosed with cervical cancer  (Table Table 5) Table .Table 6Cp < 0.05)  Table .p < 0.05)  Table .p < 0,05) and Perceived Barriers  sub-dimensions of the HBMS-HPVV were found to be statistically significantly and negatively predicted the HPV-KQ scores whereas the scores of the participants in the Perceived Benefits  and Perceived Susceptibility  sub-dimensions of the HBMS-HPVV were found to be statistically significantly and positively predicted the HPV-KQ scores (Table Analyzing the data presented in Table s β = 0,2;p < 0,05The society's knowledge about a disease, the attitudes thereof along with the care practices towards that disease provide an important opportunity with regard to the prevention of that disease and the development of control strategies. Differences with regard to the knowledge, attitudes and barriers towards a certain disease may be observed even between individuals living in the same geographic area due to socio-cultural characteristics. Although HPV vaccination and/or screening programs play a significant role in the early diagnosis of cervical cancer in developed and developing countries, cervical cancer has unfortunately been considered as an important health problem in underdeveloped countries and by individuals who cannot reach health services adequately .In a study conducted by Onan et al. in Turkey with the participation of 1808 female subjects, it was reported that 24.8% of the participants had heard of HPV infection and 24.0% had heard of the HPV vaccine . A similIn a study conducted by Özyer et al., the percentage of the individuals who had already known that HPV caused genital warts was reported as 14.2% and the percentage of the individuals who had already known that HPV could cause cervical cancer was reported as 33.3% . In a stResults of a research conducted by Altıntaş et al. with the participation of the students studying in the Faculty of Health Sciences revealed that mean score of the students in the perceived severity sub-dimension of the Health Belief Model Scale for HBMS-HPVV was 2.82 ± 0.70, mean score in the perceived susceptibility sub-dimension was 2.70 ± 0.70, mean score in the perceived benefits sub-dimension was 2.68 ± 0.68 and mean score in the perceived barriers sub-dimension was 2.42 ± 0.65 . In a siThe results of the research that as the level of knowledge of the participants about HPV infection and vaccine increases, they more likely think that HPV infection poses a serious problem, that the vaccine will provide benefits against infection, hence their perceived susceptibility towards this issue increases.Accordingly, we suggest that training programs should be prepared and scheduled with the aim to ensure compliance of individuals living in Northern Cyprus with protection and early diagnosis methods against HPV, to raise their awareness about HPV vaccination and to create a behavioral change in this direction. The demographic characteristics such as the age, educational background and gender of the individuals should be taken into consideration while organizing these trainings. Adequate and accurate information should be provided to midwives and other health care professionals involved in the health care services offered to individuals in line with the purposes of protecting and improving social health care indicators.The country's health care authorities should develop brochures on the subject as they are highly visible, easily accessible, easily memorable and it gives the opportunity to be easily shared with others. They should also make use of more interesting methods such as public service announcements, media and social media while developing educational instruments. We further suggest that legal regulations should be enacted to reduce the cost of HPV vaccine with the aim to ensure more individuals to easily access the HPV vaccine. In fact, it would be highly beneficial to ensure the HPV vaccine to be administered to individuals free of charge within the scope of the Ministry of Health's national vaccination program.