TERTp) mutation is a potential candidate for pathogenesis and therapeutic target of tonsillar squamous cell carcinomas (TSCCs) in association with human papillomavirus (HPV). Their clinical relevance has not been validated under the new 8th American Joint Committee on Cancer (AJCC) staging system. We analyzed real-time peptide nucleic acid–mediated PCR and sequencing methods (TERTp mutation) and real-time PCR-based assay (HPV) in 80 surgically resected TSCCs. The 8th edition staging system improved the stratification of the early and advanced stages and between T or N categories for overall survival over the 7th edition. TERTp mutation was found in 7.5%, and HPV in 80.0% of the patients. The majority (83.3%) of TERTp mutation cases were HPV-positive TSCCs. Applying the 8th edition staging system, TERTp mutation was an independent factor of poor prognosis for disease-free survival (DFS) in TSCC patients, supporting the clinical significance of TERTp mutation in tonsil cancer. TERTp mutations were also negatively correlated with overall survival and DFS in HPV-negative TSCCs. Conclusively, TERTp mutation provides negative prognostic impact on survival of surgically managed tonsil cancers staged with the AJCC 8th edition.Telomerase reverse transcriptase gene promoter ( Tonsillar squamous cell carcinomas (TSCCs) account for 70–80% of the oropharyngeal cancers most prevalent for human papillomavirus (HPV) –3. In orTERT promoter (TERTp), a critically important regulatory element for telomerase expression harboring binding sites for a number of transcriptional activators and repressors, contributes to increased telomerase activity that leads to immortalization of cells, which is one of the hallmarks of cancer [TERT gene occur as a pathogenic mutation in human malignancies [TERTp mutation, alone and with the HPV oncogenes, plays an important role in oral and uterine cervical SCCs [TERTp mutation in tonsil cancers and the association to HPV have rarely been investigated in the series of their studies [The f cancer , 11. Twognancies , which agnancies –17. The cal SCCs , 18, 19.cal SCCs , 21. Alt studies , 23. FurTERTp mutation, HPV infection, and clinicopathological characteristics in 80 Korean primary TSCC patients. We sought to validate the 8th edition of the AJCC staging system compared with the 7th edition, with variation in patient location and therapy.Here, we investigated Formalin-fixed, paraffin-embedded (FFPE) tissues were obtained from 80 TSCC patients who underwent primary resection, with no prior treatment and complete medical records at our institution between 1997 and 2018. Clinical information was analyzed using medical records and radiological results. Heavy smoking was defined as > 20 pack-years . AlcoholDiagnosis and histological differentiation were evaluated according to the World Health Organization classification . PatientTERTp mutations (C250 and C228) were identified using the PNAClamp™ TERT mutation detection kit .  follows : forwardHPV status was evaluated by PANA RealTyper HPV genotyping kit and PANA RealTyper HPV screening kit (PANAGENE). This kit, approved for clinical use in Korea, detects a total of 40 HPV genotypes including 20 high-risk genotypes , 2 low-risk genotypes (6 and 11), and 18 other genotypes. Briefly, real-time PCR assays were performed in a 25 μl reaction mixture containing 19 μl of HPV mix, 1 μl of Taq DNA polymerase, and 5 μl of extracted DNA, positive control, or negative control. PCR was performed using the following conditions: 1 cycle of incubation at 50 °C for 2 min and Taq activation at 95 °C for 15 min; 45 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 45 s, and extension at 72 °C for 15 s; and a melting curve step at 95 °C for 5 min, 35 °C for 5 min, followed by increase in temperature from 35 °C to 80 °C for 5 min, with a gradual increment of 0.5 °C (every 5 s) to achieve fluorescence in all four channels .TERTp mutation and clinicopathological variables were assessed using the Chi-squared test or two-tailed Fisher’s exact test. Factors found to be significant in univariate analysis were included in subsequent binary logistic regression analysis to identify independent variables associated with TERTp mutation. Survival analyses were performed using the Kaplan-Meier method and were compared using a log-rank test. Overall survival (OS) was defined as the interval from the first day of surgery until death. Disease-free survival (DFS) was defined as the interval from the first day of surgery until tumor recurrence. OS and DFS were analyzed until February 2019. Univariate and multivariate analyses using the Cox proportional hazard regression model were applied to determine the hazard ratio (HR) and 95% confidence intervals (CI) for specific variables related to OS and DFS. SPSS version 18  was used for all statistical analyses. P values < 0.05 were considered statistically significant.Correlations between the According to the 8th edition staging system, 18 (22.5%) tumors were classified as T1, 31 (38.8%) as T2, 20 (25.0%) as T3, and 11 (13.7%) as T4. Of the 80 patients, 17 (21.3%) were categorized as N0, 37 patients (46.3%) as N1, 11 (13.7%) as N2, and 15 (18.7%) as N3. Combining the T and N categories, the overall stages of 31 patients were diagnosed as stage I (38.8%), 18 (22.5%) as stage II, 11 (13.7%) as stage III, and 20 (25.0%) as stage IV. The median follow-up period was 64 months . The 5-year OS and DFS rates were 53.2% and 43.8%, respectively.P = 0.063 and P = 0.471, respectively). Only the T category was well discriminated according to clinical outcomes (P = 0.043). In contrast, the 8th edition provided statistically significant stratification for the T category, N category, and overall staging . Therefore, the 8th edition was applied in our study.We performed Kaplan–Meier survival analyses of OS in 80 patients who had previous TNM information according to AJCC 8th vs. AJCC 7th staging system Fig. . We compHPV was identified in 64 tumors (80.0%) analyzed by real-time HPV genotyping kit. All 80 cases were interpretable. There were only high-risk HPV genotypes including HPV 16 , HPV 18 , HPV 58 , and concurrent HPV 16 and HPV 18  Fig. . We alsoTERTp in 80 TSCC patients  of 80 tumor samples. The observed point mutations were hot spot nucleotide changes G to A at position − 124 within TERTp in 6 cases , low alcohol consumption (P = 0.021), pN-positive status (P < 0.001), lower AJCC stage (P = 0.020), and presence of ipsilateral lymph node metastasis (P = 0.004). HPV positivity was not associated with TERTp mutation. There were no statistical associations between TERTp mutation and clinicopathologic features of TSCCs.We analyzed the associations of HPV or Cs Table . The preTERTp mutation occurred in 5 cases (6.3%) of 80 TSCCs: a total of 5 cases (83.3%) among the 6 TERTp mutations were in HPV 16-positive tonsil cancers and one TERTp mutated tumor was HPV-negative. TSCCs showed the most frequently HPV-positive/TERTp-wild-type , followed by HPV-negative/TERTp-wild-type , HPV-positive/TERTp-mutated , and HPV-negative/TERTp-mutated   Fig. .Fig. 3a TERTp mutation was found to be statistically associated with shorter DFS rates than those of TERTp wild-type (P = 0.007), whereas no statistical difference was observed for OS between TERTp mutation and TERTp wild-type (P = 0.983)    Fig. . We furtly) Fig. . Howeverly) Fig. .P = 0.006), higher T category (P = 0.007), and base of tongue (BOT) invasion (P = 0.015) were associated with shorter OS rates, while TERTp mutation (P = 0.007), higher T category (P = 0.004), BOT invasion (P = 0.002), and soft palate invasion (P = 0.005) were associated with shorter DFS rates. Multivariate analyses confirmed that older age and higher T category were independent negative prognostic factors for shorter OS in patients with TSCCs . TERTp mutation was identified to be the only independent prognostic factor for DFS in tonsil cancers .We analyzed the OS and DFS through univariate and multivariate analyses Table . In the TERTp mutation in tonsil cancers has been rarely reported. Applying the new AJCC 8th edition, we confirmed the negative prognostic impact of TERTp mutation on survival of surgically managed tonsil cancers staged with the new AJCC 8th edition, especially on HPV-negative TSCCs. TERTp mutation has been reported as a predictor of poor prognosis in laryngeal cancers but not in oral SCCs [TERTp mutation serves as an adverse prognostic factor in any cancer regardless of organ [TERT by its promoter mutation representing late events of the oncogenic process may increase the self-renewal capacity of cancer stem cells and induce poor clinical outcomes [TERTp mutation might biologically sustain how TERTp mutation is related to poor prognosis.In the present study, the new staging system improved stratification of early and advanced stages and between T or N category concerning overall survival based on Korean population. Under the new AJCC 8th edition, the prognostic implication of ral SCCs , 25. A mof organ . Overexpoutcomes , 28. TheTERTp mutations (< 5%) because mutations affecting the telomerase coding region are very uncommon in the cancer setting [TERTp mutation in TSCCs, which is a relatively high frequency according to Vinagre et al. [TERTp mutation is variable in head and neck SCCs, which could be because the TERTp mutation can also result from environmental factors such as ultraviolet radiation and chemical carcinogens [TERTp mutation [Vinagre et al.  divided  setting . We obsee et al. . The frecinogens , 30. Thumutation , 22, 23;mutation .TERTp mutation, found in the present study, has been mainly described in SCCs in head and neck or other sites including skin, lung, and uterine cervix [TERTp mutations occurred exclusively in the HPV16-positive TSCCs, although there was no statistical association between TERTp mutation and HPV. The considerable presence of TERTp mutation in HPV-positive tumors but a lack of statistical association between them have been previously described in the oral, oropharyngeal, and uterine cervical cancers, where all HPV16-positive oral SCCs and 70% of HPV-positive uterine cervical cancers harbored TERTp mutations [TERTp activations would be functionally different from the consequences of TERTp mutation in the absence of HPV [The lack of specific clinicodemographic features related to e cervix , 25, 31.utations , 22. Some of HPV , 18–20.This study thoroughly investigated HPV status and genotypes in clinical specimens and stratified patients according to HPV status, with long-term follow-up data. In the present study, only high-risk HPV was dominated (80%) with the majority 92.2%) of HPV16. High-risk HPVs are the cause of approximately 31% of oropharyngeal SCCs .2% of HP, 32, 33.HPV16 is the dominant virus in oropharyngeal SCC accounting for 82–87% of all HPV-positive cases . The fraTERTp mutation is present in a subpopulation of patients with TSCC and emphasizes the negative prognostic impact of TERTp mutation in tonsil cancers under the newly 8th edition of the AJCC staging system for oropharyngeal cancers, especially on HPV-negative TSCCs. This may be utilized to determine clinical aggressiveness of TSCCs.In conclusion, the present study reveals that the


The purpose of this study was to compare cervical cancer screening by pap smear (PS) versus preliminary HPV testing based on self-collected samples (SC-HPV).Interventional study among underprivileged women from 25 to 65 years old in four French cities. The control group (CG) was referred for a PS. The experimental group (EG) conducted a SC-HPV test followed by a PS in case of positivity. Differences on screening completion and cytological abnormalities were analysed by logistic and Cox regression.p < 0.001). The proportion of cytological abnormalities was 2.0% in the CG and 2.3% in the EG . The proportion of participants lost to follow-up was 60.5% in the CG and 63.2% in the EG HPV positive (p = 0.18).383 women were assigned to the EG and 304 to the CG. The screening completion proportion was 39.5% in the CG compared to 71.3% in the EG (HR = 2.48 (CI 95% [1.99–3.08]; Providing an SC-HPV-test increased the participation of underprivileged women in CCS. Nevertheless, the significant number of lost to follow-up in both groups can undermine the initial benefits of the strategy for HPV positive women.Registration: Clinicaltrials.gov: NCT03118258. Human papillomavirus (HPV) is the most common sexually transmitted infection . In mostCervical cancer is the most frequently diagnosed type of cancer among women in 28 countries and the leading cause of cancer-related mortality in 42 countries, most of which are located in Sub-Saharan Africa and South-East Asia .In France, cervical cancer affects nearly 3000 women and causes around 1100 deaths every year . CurrentThe French national cervical cancer screening program relies directly on health professionals providing gynaecological services to the women concerned, mainly gynaecologists, general practitioners and midwives. The strategy is accompanied by communication campaigns and individual invitations for target population (women between 25 and 65 years old). Cytology exams and HPV tests are covered 100% for insured women. This strategy ostracises women without health coverage or experimenting health care access difficulties. Actually, it is estimated that women with limited access to the healthcare system, women of low socio-economic status, and women living in economically depressed areas are less likely to have access to CCS .Data from Médecins du Monde (MdM) indicated that nearly 67% of women between the ages of 25 and 65 attending to MdM programmes in France had never performed a PS . This fiHPV testing has been shown to be more sensitive than the PS exam –16. SelfThe purpose of this study was to compare the proportion of screening test completion and the proportion of cytological abnormalities detected using two different strategies within underprivileged women in France. These strategies included an individual preventive medical consultation followed by either a referral to a partner facility for a PS or preliminary SC-HPV testing.Interventional, multicentre, comparative, and randomised research focused on four types of programmes: CASO , CAOA , Squats/Slums, and Sex Worker programmes (SWP) in four cities .MdM's CASO/CAOA are structures conceived to facilitate access to healthcare rights and prevention for people experimenting socioeconomic difficulties. They have permanent and voluntary staff, including doctors, nurses, psychologists, social workers and community agents. Users are mainly newly arrived migrants, but also other profiles with social factors of vulnerability . People come spontaneously to the centres through multiple community communication channels.The other programs include outreach actions, particularly in vulnerable settlements (squats and slums) and in sex workers place of practice. In the slums, MdM offers on-site medical and prevention consultations accompanied by referrals to other healthcare structures and sensitization about contraception, family planning and prenatal care. Programs with sex workers address the topics of sexually transmitted infections, hepatitis, HIV, unwanted pregnancies, violence, psychological suffering and advocacy.Programmes participating in the study were selected to have representative profiles and locations according to the flux of women in the targeted age range and to the presence of sexual and reproductive activities in the programmes’ current line-up of services.The study was conducted from March 2017 to December 2018.All women between the ages of 25 and 65 who had not had a PS in the past three years were included in the study. Exclusion criteria included study participation refusal, having a history of a complete hysterectomy or the absence of sexual intercourse in the past.Eligible women were offered an individual sexual and reproductive health (SRH) consultation which included an adapted and individual consultation informing about HPV infections, cervical cancer, the importance of gynaecological care and screening strategies. An interpreter or healthcare mediator was also available. At the end of the consultation, women were separated into two groups: (1) The “PS” control group (CG) (in which study participants were referred towards partner associations and institutions to have a PS); (2) The “SC-HPV” experimental group (EG) . EG patients were provided with a SC-HPV kit and given the choice of either collecting the sample on site in a private room or at home (in the case of menstruation or if the patient declined to collect the sample on site). Patients were given their HPV results two weeks later, and they were scheduled for a follow-up appointment. If results were positive, patients were directed to partner centres to get a PS. Women whose HPV tests were positive received a reminder phone-call. Patients assigned to CG benefited systematically from a HPV test when cytological abnormalities were detected in the PS.Women who had not completed an HPV test or PS four months after their appointment were considered lost to follow-up.Sample size calculation was based on risk a = 5% and power of 80%, a proportion of CCS completion of 80% in the EG and 40% in the CG, and a proportion of cytological abnormalities of 2.5% in the CG and 4% in the EG. The number of participants was estimated at 1258 to answer to both objectives.Participants in each programme were randomly assigned over one-month periods. Each month in which participants were offered an SC-HPV was followed by a period in which participants were not offered this option.The test selected for the study was the ABBOTT Real Time High Risk HPV test . All partner facilities conducted liquid-based cytologies.Participants’ medical and social data were collected through a questionnaire administered by trained professionals. These data were entered into a secure “electronic patient record” tool. MdM’s partner structures submitted the results of the PS exams via confidential mail.Screening completion rate was defined as the proportion of women who have a PS done in the CG and the proportion of women who had a negative HR-HPV test or had a PS done if the HR-HPV test was positive in the EG.As cytological abnormalities we considered ASC-US, LSIL and HSIL.The descriptive analysis was conducted based on the demographic, socio-economic, and clinical variables in each procedure group. Comparability of the groups was evaluated using Chi-2 tests, ANOVA tests for categorical variables whereas t-Student tests for continuous variables. The comparison between the rate of screening completion and the number of cytological lesions was conducted using logistic and Cox regressions. All analyses were repeated with another sample that only included data from CASO/CAOA programmes. Statistical analysis was done using the software Stata 15 .This research project was approved by the Île de France IV Institutional Review Board (IRB). The study was conducted in compliance with the ethical principles of the Helsinki Declaration. All participants were informed of the study’s objectives and design, and their participation was voluntary after providing verbal informed consent. The protocol was registered in clinicaltrials.gov.The study flowchart is depicted in Fig. In the CG, out of the 304 women who were referred for a PS, 120 (39.5%) followed through with the exam and 184 (60.5%) were lost to follow up. Out of the 120 PS conducted, 11 cytological abnormalities were detected: 8 ASC-US (3 of which were caused by an HPV infection), 2 LSIL, and 1 HSIL.In the EG, out of the 383 women, 365 (95.3%) submitted SC-HPV, 15 (3.9%) were lost to follow-up, and three (0.8%) declined to submit a self-sample. Out of the HPV tests that were completed, 226 were negative, 127 were positive, 9 could not be interpreted, and 3 were missing. 34.4% of the HPV-tests done were positive. Out of the 136 women who tested positive for HPV or whose test could not be interpreted, 47 (34.6%) had a PS and 9 cytological abnormalities were detected .In the CG, 184 women were lost to follow-up after they were given a referral for a PS (60.5%). In the EG, 18 women were lost to follow-up before they could submit an SC-HPV (4.7%). In addition, out of the 136 women whose HPV results were positive or could not be interpreted, 35 were lost to follow-up after they were referred for a PS (63.2% of women with an indication to have a PS).Table p < 0.001), with a risk ratio (RR) of 1.80: CI 95% [1.55–2.10]; p < 0.001.With respect to the proportion of screening completion, the participants in the EG were approximately twice as likely to have access to the test as the CG .Figure p < 0.001). The analysis of the sub-groups shows that the effect was not modified by the study’s other covariables.The hazard ratio (HR) for the screening test completion rate for the EG compared to the CG was 2.48 . The effect was not modified by other study’s covariables.Figure All analyses were also conducted using a sample that only included participants from programmes with more consistent results (CASO/CAOA). Both groups were well balanced and their results do not differ from the results of the entire sample in terms of screening performance, number of participants lost to follow-up, screening test completion, and cytological abnormalities detected . This number might have been even higher if women needed to receive additional tests, undergo a colposcopy, or receive treatment for their lesions. In the EG, the two-stage screening strategy , shortened the screening process for women whose HPV results were negative but added an extra visit in the event of positive HPV results. As a result, one of the most notable findings of our study was the high number of women lost to follow-up. It should be noted that this took place in both study groups, regardless of whether or not the women knew their HPV results. Results delivery delays (10 days for the HPV tests and a few weeks for the PS exams) added another barrier. The high number of participants who were lost to follow-up underscores how difficult it is to treat these women and could undermine the benefits gained by increasing primary screening coverage by using SC-HPV.On the other hand, it is clearly beneficial for women to know that they tested negative for HR-HPV. Not only is it a source of relief, but it also means they can complete their screening process more quickly. Moreover, the high level of acceptability and logistical simplicity of this technique could also increase long-term participation in screening programmes.In this sense, the benefits of HPV test approach are evident and the endpoint “participation” is absolutely clear, as the self-collected HPV test offer much more protection.Several studies have shown that the long duration of the screening process represents a barrier to completing it within underprivileged women , 37. In Even though some of the barriers are directly related to the access to healthcare facilities, also essential for completing the screening programmes are socio-cultural determinants, understanding of the message plus the individual’s own risk perception. This reality highlights the importance of counselling and interpreting services.This study has a certain number of limitations. First, monthly randomisation was selected in collaboration with teams on the field together with the scientific committee. Choosing to alternate between one-month periods was easier to follow and helped to limit bias related to seasonal variations. In the SWP in Paris, where the sex worker community is highly cohesive, offering SC-HPV during specific periods encouraged the women to visit the healthcare structures. Nevertheless, this pattern does demonstrate the benefit of this screening strategy for this population.In addition, there were low participation rates in the Squat programmes and SWP in Rouen. This fact can be due to the different approach of the activities .To limit these potential sources of bias, we decided to conduct a supplementary analysis that only included data from the CASO/CAOA (77% of the sample), obtaining very similar results to the full dataset.The study was underpowered for the outcome “proportion of cytological abnormalities”, as the study had to stop before sample size completion due to budget constraints.In addition, it would have been better to measure the completion rate based on the number of retrieved results, which would have ensured that the women knew their status. Unfortunately, this information was available for the results of the HPV tests (which were conducted and collected within MdM facilities), but not for the results of the PS exams (which were conducted and collected in a variety of partner facilities).Finally, it would be beneficial to offer a holistic SRH strategy to these populations, including vaccinations and testing for other STIs, such as HIV and hepatitis.Our study is the first to provide information about different CCS strategies for underprivileged women in France.SC-HPV strategy presented a higher proportion of screening test completion and cytological abnormalities detection .Providing participants with a SC-HPV kit improved the participation of underprivileged women in CCS. Nevertheless, the significant number of lost to follow-up in both groups can undermine the initial benefits of the strategy for HPV positive women.It is important to better understand the barriers to screening encountered within this population and thus to design more adapted, differentiated, and appropriate strategy to reduce inequalities.


Clinacanthus nutans Lindua (C. nutans), a strong antiviral traditional medicine, can be used to treat condyloma acuminata (CA) caused by the human papillomavirus (HPV). However, its molecular mechanism for CA elimination is unknown. Herein, we conducted a randomized clinical trial to evaluate the effectiveness of C. nutans and its molecular mechanism compared with podophyllin, the gold standard treatment. Using a randomized block design, six patients were treated with C. nutans and podophyllin for four weeks. Efficacy of drugs was assessed by size reduction of the warts and HPV viral load quantification using droplet digital PCR. The gene expression profiling of CA was analyzed using NanoString Technology. After the podophyllin and C. nutans treatments, CA lesion sizes were reduced to 97.0% and 84.4% clearance, and the HPV viral loads were reduced by 74.0% and 46.6%, respectively. The gene expression pattern of immune profiling showed that 23 genes  were significantly differentially expressed by podophyllin, whereas 2 genes (IFNL1 and IRF2) were remarkably expressed by C. nutans. In inflammatory profiling, 108 genes  were highly expressed by podophyllin, but none of genes were observed to change expression by C. nutans. These results suggested that podophyllin may reduce the HPV infection through a mechanism related to proinflammatory response. In addition, C. nutans was found to suppress the HPV infection through mechanism related to the activation of immune response. This study shows novel therapeutic mechanisms of podophyllin and C. nutans. It is suggested that C. nutans might be used as an alternative treatment for CA treatment. Human papillomavirus (HPV) is etiologically associated with the development of genital warts. More than 100 different types of HPV have been classified as either low risk (LR-HPV) or high risk (HR-HPV), based on their oncogenic characteristics . Up to dClinacanthus nutans Lindau (C. nutans) is an important traditional medicine from Acanthaceae family that has been used as a vital medicine in tropical Asia. C. nutans has been proved to have anti-inflammatory, antioxidant, immune response and antiviral activities [C. nutans compound inhibits HPV infections by interfering at the early step of an HPV16 infection, and it may have a TLR4-transcriptional inducible effect, which was a key regulatory molecule to stimulate the immune response and inflammatory cytokines secretion [C. nutans could affect alteration in the nonspecific cell-mediate immune response, which relates to the releasing of IL4 from peripheral blood mononuclear cell [C. nutans cream has been included in the National Lists of Essential Medicines for skin inflammations treatment from virus infection [tivities , 5. Furtnfection .C. nutans cream for CA treatment has not yet been studied in clinical trials nor has molecular mechanisms been addressed. Hence, this study aims to investigate the efficacy of C. nutans treatment on HPV-infected CA patients in comparison with podophyllin. Size reduction of the warts together with the amount of HPV DNA and gene expression profiling in response to drug treatments were subjected in the study.To date, efficacy of Six Thai male patients (aged >18 years) with clinical CA diagnosis at their first visit were enrolled into this study. Each patient must have at least two CA, with at least one centimeter apart between each wart. Patients had no medical history of immunomodulator or systematic antiviral drugs usage. Patients with documented HIV or autoimmune diseases were excluded. Written informed consent according to the Declaration of Helsinki approved by The Institution Review Board (IRB), Faculty of Medicine Siriraj Hospital, Mahidol University (COA no: Si 629/2016) were obtained from all patients and were registered in Thai Clinical Trials Registry (TRTC: 062 875 7627).C. nutans cream  or podophyllin . Patients were followed up every week for one month, and sample swabs were collected at week 0 and week 4. All sample swabs were preserved in DNA/RNA shield™ collection tubes at −20°C. Following extraction, DNA and RNA samples were subjected to HPV genotyping, HPV viral load, and gene expression profiling analysis.CA specimens were obtained from Dermatology Outpatients Unit at Siriraj Hospital, Mahidol University. Sample swabs were collected from both pretreatment and posttreatment visits. Samples were arranged in randomized block design. Each block was arranged in the block of four. Following random separation of the warts as “A” or “B,” they were treated with μL prior to ddPCR. RNA concentration was also measured, and its purity was approximately 2 by NanoPhotometer® before gene expression analysis was immediately performed.Genomic DNA and total RNA were extracted from the swab samples using the Allprep DNA/RNA Mini Kit . Concentration of the purified DNA was measured by a Nanodrop™ 8000 spectrophotometer and stored at −20°C until use. DNA samples with 260/280 nm absorbance ratio between 1.8 and 2.0 were diluted to approximately 5 ng/Linear array HPV genotyping assay was performed by the Molecular Microbiology Laboratory, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University using the linear array HPV genotyping test .β-globin gene as a reference gene. Reactions were set up according to the manufacturer's instructions. All primers and ddPCR conditions were shown in μL of the PCR reaction for the HPV target gene in comparison with β-globin gene.HPV L1 gene was determined using QX200 ddPCR system (Bio-Rad) in comparison with human NS_CANCERIMMUNE_V1.1) and a custom designed set of 256 genes in inflammatory panel (NS_INFL_HS_V2_C2534). A background correction was made by subtracting the background thresholds of negative control from raw counts. Adjusted raw counts were normalized using the combination of positive controls and reference gene normalization. The positive control normalization was computed by the geometric mean normalization factor, whereas reference gene normalization was calculated by the geometric mean of 11 genes, SF3A3, MRP55, TLK2, HDAC3, PPIA, TBP, ZNF143, DNAJC14, ABCF1 SAP130, and EDC3, in immune panel and four genes, CLTC, CUSB, HPRT1, and PGK1, in inflammatory panel.Immune and inflammatory panels were selected for gene expression analysis using NanoString nCounter® Platform (NanoString Technology). The reactions were set up according to the nCounter XT Gene expression protocol. Gene expression analysis was performed using nSolver Analysis Software v4.0 (NanoString Technology) involving in 770 genes using immune panel  method for multiple comparison (Benjamini–Hochberg) [SPSS Statistic version 18 Software  was used for clinical data and the viral load measurement. Pre- and posttreatments using podophyllin or ochberg) . FDRs lePrevalence of HPV genotypes from six patients revealed six different genotypes, which were classified into LR-HPV and HR-HPV. The most common HPV genotype was LR-HPV. Only one out of six patients had both LR-HPV and HR-HPV genotypes. Regarding to the pattern of the HPV infections, four patients (66.66%) had single HPV genotype, whereas two patients (33.33%) revealed multiple HPV genotypes. Of four patients with single HPV genotype, HPV-6 was found in three patients (75%), whereas HPV-11 was found in only one patient (25%). In contrast, two out of the six patients had multiple HPV genotypes. One patient had two HPV genotypes (HPV-11 and HPV-40), and another had four HPV genotypes . In case of multiple infections, HPV-11 was dominantly detected, with minor infection from other HPV genotypes. Thus, it is demonstrated that HPV-6 and HPV-11 were dominantly detected .C. nutans treatment were classified into 2 groups; responder  and nonresponder . Based on the CA clearance, median of the total clearances for responder of podophyllin and C. nutans treatment groups were 97% and 84.85% size reduction area respectively . However, one patient (ID 03), was classified as nonresponder showed that lesion were not completely cured.Baseline clinical outcome and HPV viral load clearances were described in p=0.043, Wilcoxon signed-rank test; n = 5). As to responder of C. nutans treatment, median values of HPV viral load were reduced from 1.74 to 0.71 HPV copies/cell, and difference was statistically significant . For nonresponder group, which has viral load reduction less than 75%, only one patient (ID03) showed a slight decrease in HPV viral load after podophyllin treatment, but the HPV viral load greatly increased after C. nutans treatment. The rates of HPV viral load clearances after podophyllin and C. nutans treatments were 74% and 46.58%, respectively. These data were found to be significantly different   .C. nutans were significantly different    . Among tge >1.2) . It is shange>2) . These thange>2) . ChanginC. nutans treatment group using Venn diagram  . However, none of the inflammatory genes were found to be differentially expressed if p < 0.05 was adjusted  . This imadjusted . It is oHPV is etiologically associated with the development of virtually all CA. It is widely accepted that HPV-6 and HPV-11 are dominant LR-HPV in the pathogenesis of CA . Our resC. nutans showed compelling effects in preclinical in vitro and in vivo studies, suggesting that it has multifaceted properties, involving in antiviral, anti-inflammation and antiproliferative activities [C. nutans cream is useful and has high efficacy in Herpes genitalis treatment [C. nutans cream (w/w) is commercially available, and it is recommended to apply 4 times daily [C. nutans-treated CA. This is the first study to evaluate the clinical effectiveness of C. nutans cream in a well-controlled clinical design. It was found that clearance rate of podophyllin treatment was similar to those observed by other clinical studies [C. nutans in LR-HPV-infected CA, but low clearance rate (50%) was found in HR-HPV-infected CA. These results suggested that C. nutans might be more effective in the clearance of LR-HPV infection than HR-HPV infection. In summary, podophyllin showed 74% reduction of HPV viral load in all patients, which was consistent with 97% CA lesion clearance, whereas C. nutans was able to reduce the HPV viral load for 42%, which was also consistent with 85% CA lesion clearance. These results were supported by our efficacy treatment analysis in that podophyllin cauterization was more effective than C. nutans. According to the C. nutans treatment, five of the six patients showed reduction in the HPV viral load in correlation with their CA lesion clearance. Only one patient (ID03) revealed high viral load even if the CA lesion was reduced, suggesting that C. nutans may not cure CA infected with HR-HPV.Podophyllin has been successfully utilized as a treatment for CA patients, and it has been found to be effective in reducing superficial CA . In randtivities . Evidencreatment . In Thaies daily . However studies . InteresC. nutans was found to reduce only LR-HPV viral load. Our results were consistent with other studies that podophyllin could effectively eliminate wart lesions from all HPV genotypes, resulting in no HPV detection [C. nutans, Sookmai et al. reported that C. nutans compounds could inhibit HPV infections in an in vitro study [C. nutans was less effective than podophyllin; hence, it is a suitable treatment for a single HPV infection and LR-HPV infection.Regardless of the efficacy treatment, there was a correlation between HPV genotyping and HPV viral load clearance. Podophyllin demonstrated significantly reduced viral load for both LR-HPV and HR-HPV, whereas etection . As to tro study . TherefoC. nutans treatment. To define the target genes, differentially expressed genes were screened by the volcano plot method. From total of 23 genes, two genes were upregulated and 21 were downregulated after podophyllin treatment. Only HLA-DPB1 was significantly upregulated, whereas OSM, IL8, CXCL1, CXCL2, and CXCR2 were the five most significantly downregulated genes. Interestingly, HLA-DPB1 was found to be upregulated after podophyllin treatment, which was a novel finding. HLA-DPB1 belongs to HLA (human leukocyte antigen) class II, which is associated with a major histocompatibility complex in viral pathogenesis [DRB1∗14, a polymorphism of HLA class II, confers a protection against cervical cancer [OSM gene was statistically significant observed. The OSM gene, Oncostatin M, an IL-6 family cytokine, helps to promote cell senescence and inhibits growth [OSM mRNA level was significantly upregulated in the HPV16-E6 but downregulated in the HPV negative group [OSM downregulation occurred with the podophyllin treatment. Therefore, podophyllin may inhibit or downregulate OSM gene due to the HPV infection clearance. Surprisingly, the expression levels of IL8 and CXCR2 genes were significantly downregulated. IL8 is an important regulator of the innate immunity; it is also known as a potent proinflammatory cytokine that exerts its effects through binding to its G-protein-coupled receptor CXCR1 and CXCR2 [IL8-CXCR1/CXCR2 has been shown to have therapeutic potential for a variety of solid tumors [IL8 and CXCR2 pathway may serve as a novel mechanism after podophyllin treatment. After C. nutans treatment, IFNL1 and IRF2 were found to be significantly upregulated, while none of other genes were found to be significantly downregulated. Interestingly, IFNL1 or Interferon Lambda 1 stimulates clearance of norovirus infection independently of adaptive immune system, suggesting that innate immune responses may have a role in determining viral persistence [IFNL1 treatment was reported to decrease HSV-2 shedding from the vaginal mucosa in in vivo study [IFNL1 by C. nutans treatment, which would lead to the activation of antiviral state in epithelial cell, was a critical first-line defense against pathogenic viruses. Interferon regulatory factor 2, IRF2, has emerged as a complex mediator of gene expression and cell cycle control [IRF2 promotor, displaying a complex negative feedback mechanism in response to signal transduction pathways [IRF2 gene would lead to elimination of the HPV infection. It is assumed that an increased immunosurveillance in CA lesions may allow the virus clearance with C. nutans treatment (The immune response in HPV-infected CA is considered to be the main determinant of CA disease and its associated immunomodulatory approaches, which have gained attention as promising strategies for CA treatment. Therefore, this is the first report on the mechanism of action associated with the immune response after podophyllin and ogenesis . It is es growth . Previound CXCR2 . Waugh ed tumors . We suggsistence . Furthervo study . Hence,  control . Lace etpathways . This coreatment .C. nutans treatment (CCL3, CXCL2, CXCL1, IL8, and STAT3 were found to be the five most significantly downregulated genes. A significant downregulation of the genes was shown in response to a decreased expression of CCL3, CXCL1, and CXCL2. It is suggested that podophyllin might induce a negative feedback on the proinflammatory pathway, leading to a decrease in the size of lesions. Downregulation of STAT3 after podophyllin treatment was also a novel finding. STAT3, a signal transducer and activator of transcription 3, modulates the transcription of the genes involved in the regulation of cell differentiation, proliferation, and immune responses. Frank et al. revealed that a selective inhibitor of STAT3 signaling pathway could be used as an anticancer drug [STAT3 after podophyllin treatment may be associated with the inhibition of HPV proliferation due to lesion and viral load clearance (Surprisingly, 108 genes associated with inflammatory process were significantly downregulated with the podophyllin treatment , whereasreatment . All of cer drug . We sugglearance .C. nutans-treated CA were compared. Some differences were found in immunomic profiling after podophyllin and C. nutans treatments. With podophyllin treatment, most genes involved in immune and inflammatory response were downregulated, whereas with the C. nutans treatment, those genes were upregulated. These results suggested that podophyllin and C. nutans may operate different mechanisms for HPV clearance. Downregulation of genes involving in immune and inflammatory response after podophyllin treatment suggested that its mechanism of action may not be correlated with the immune response; however, it may be related to previously described antiproliferative activity leading to CA clearance [C. nutans might not inhibit HPV replication directly. It is demonstrated that C. nutans could stimulate the production of cytokines; hence, increase the immune response, thereby diminishing CA and decreasing virus erosion [C. nutans. Alternatively, HPV clearance mechanism of C. nutans might not be involved in the inflammation signaling pathway. It is speculated that some other genes might play a crosstalk between the immune response and inflammation signaling pathways leading to HPV clearance by podophyllin or C. nutans. These shared molecular mechanisms may extend a potential target for a more effectively treatment of CA.In addition, the gene expression profiling associated with immune and inflammation panels of podophyllin and learance . It is s erosion . Further erosion . These fC. nutans as an alternative treatment for HPV-related CA with less long-term toxic effect.Nevertheless, there are some limitations of this study. Firstly, long-term follow-up more than 1 year was not completed. Secondly, a number of HPV-infected subjects was limited. Thirdly, heterogeneity leads to result uncertainty. Additionally, it is necessary to do further study on the molecular regulatory mechanism and other protein expression analysis of both drugs trials in order to identify therapeutic regimen. In turn, this may provide a guideline for the outcome on combination of these drugs for the CA treatment and indicate the underlying biological targets of CA in the future. However, this study might shed some light on applying C. nutans were, at least in part, due to reducing the inflammatory and modulating immune response signaling. Given the above findings, podophyllin possibly diminished HPV via both immune response and inflammatory signaling pathways, thereby allowing CA clearance. In contrast, C. nutans may promote the immune response while dampening inflammatory signaling, thereby given an overall tendency toward CA clearance.This study provides sufficient findings to demonstrate that the clinical efficacy and mechanism of action of podophyllin and


To analyze knowledge, attitudes, and practices of adolescent students from public schools in the municipality of Teresina, state of Piauí, regarding human papillomavirus (HPV). Cross-sectional, analytical study carried out in 12 public schools in the municipality of Teresina, with a random sample of 472 15-year-old adolescents. All participants answered a validated questionnaire, which evaluated sociodemographic characteristics, level of knowledge about HPV, attitudes regarding vaccination and vaccination status. The levels of knowledge and attitude were classified by standardized scores and practice by the vaccination situation. The analyses were carried out using the SPSS software. In the bivariate analysis, simple logistic regression was used generating odds ratios to identify the associations of sociodemographic characteristics, knowledge, and attitude with HPV prevention practice. Variables that presented p-value ≤ 0.20 in the bivariate analysis were inserted in a multivariate logistic regression model. Statistical significance was set at p < 0.05. Among the participants, 27.3% had sufficient knowledge, 34.1% had positive attitudes, and 74.6% had adequate practice. In the multivariate analysis, we observed a statistically significant association among females , satisfactory knowledge , and positive attitudes  with proper practice. Being female, having a satisfactory level of knowledge about HPV and the vaccine, and having positive attitudes towards HPV vaccination reinforce the appropriate practice of vaccination. These findings demonstrate the need to expand the knowledge of adolescents, generating positive attitudes towards vaccination within an appropriate perspective. In Brazil, preliminary data from a 2017 population-based study conducted in 26 Brazilian capitals and the Federal District identified an HPV infection prevalence in 54.6% of the collected samples, of these, 38.3% had high-risk HPV. In the municipality of Teresina, state of Piauí, the detected prevalence was 53.3%2.Human papillomavirus (HPV) infection is extremely frequent worldwide and its transmission is mainly due to sexual contact, estimations point to 80% of sexually active individuals being infected with HPV at some point in their lives3. In Brazil, cervical cancer is the third most frequent type of cancer among women, with higher incidences recorded in states with lower socioeconomic development, such as those of the North and Northeast4.Although most times the infection is transient, its persistence has been directly associated with the development of cervical cancer, which represents an important health problem nowadays, due to its high incidence5. Immunization has shown important results in reducing the rates infection by the virus in countries where vaccination coverage is high and already has a proven impact on reducing the incidence of cervical cancer6. However, maintaining high vaccination coverage has been a challenge in Brazil5.Vaccination and cytological examination represent the main strategies for HPV prevention5. Also, note that currently adolescents constitute a group of high vulnerability to sexually transmitted infections (STIs) such as HPV, due to the early development of sexuality, the multiplicity of partners, greater sexual freedom, need for group affirmation, allied to resistance to condom use7.In this context, adolescents, who are the target audience of immunization, must have knowledge and awareness about the virus and recognize the importance of vaccinationThus, considering the importance of HPV vaccination and the high risk of exposure of adolescents to the virus, the need for investigation on the diagnostic conjuncture about what adolescents know, think, and practice regarding this health problem emerged. Thus, this study aimed to analyze the knowledge, attitudes, and practices of adolescent students from public schools in the municipality of Teresina, Piauí, regarding HPV.8, is 850,198 inhabitants.Cross-sectional, analytical study, developed by using a knowledge, attitude, and practice (KAP) survey, conducted in public high schools in the municipality of Teresina, capital of Piauí, whose population, according to the Brazilian Institute of Geography and Statistics (IBGE)The management of the state public schools of Teresina is organized in four Regional Teaching Management units (GRE). The inclusion criteria for the selection of schools in the study were: in the urban area of the capital and offering regular or comprehensive high school. Among 139 public schools, 91 met this inclusion criterion, distributed as follows: 26 in the 4th GRE; 25 on the 19th GRE; 18 on the 20th GRE; and 22 in the 21st GRE.For the study population, the inclusion criterion was to be a 15-year-old adolescent, enrolled in high school and regularly attending the selected schools at the time of collection. The choice for this age group occurred because by the age of 15, according to the National Immunization Program (PNI), the adolescent must have already received both doses of the HPV vaccine.To calculate the sample of students, a proportional stratified probabilistic sample was used, considering the population of 10,923 15-year-old adolescents of both sexes, enrolled in public high school, attending the 1st grade in the 2018 school year.9, which identified 28.9% of the population with adequate knowledge about HPV), accuracy of 5%, and significance level of 5%, obtaining a minimum sample of 386 adolescents. Applying a 20% rate for sample recomposition and assuming possible loss during the study, the planned sample size was at least 463 participants.The sample size was calculated by random sample for proportions, adopting a 95% confidence interval (95%CI), prevalence of 29% .’The variables of interest analyzed were: sociodemographic (gender and race/color) and economic characteristics and evaluation of knowledge, attitude, and practice. The economic characteristics of the families of adolescents were represented by an indicator constructed from information on quality of life, related to the health of the adolescentsƒi) bii. Where: i ranges from 1 to 7 goods; bi is equal to 1 or zero respectively, in the presence or absence of landline, cell phone, internet, automobile, motorcycle, computer , bathroom with shower. The weight attributed to the presence of each household good will be complementary to the relative frequency (ƒi) of each good in the total sample, that is, the rarer the good in the household, the greater the weight attributed to it.The ‘indicator of possessions (IP)’ was calculated by: IP = ∑ (1 − 12.The indicator was further refined by adding a weight that considers the amount of the “i” good found in the household (and not only whether it exists in the household). This variant is calculated by w = 0 to k, where “k” indicates the amount of the “i” good in the household. The dichotomized form of the variable was used, initially in quintiles, to separate the subgroup with the lowest possession of goods – supposedly the “least economically favored” – from the group with the highest possessionsThe evaluation of knowledge, attitude, and practice was consolidated into three categories: I disagree , I do not agree nor disagree, or I agree (I agree and I strongly agree); and it was conducted as follows: to classify the degree of knowledge of adolescents regarding HPV, the answers of the questionnaire on the subject were evaluated with a numerical value assigned for each answer and the correct answers totaling six points, corresponding to 100%.14, in which the degree of knowledge was categorized as unsatisfactory knowledge (0 to 74%) and satisfactory knowledge (75 to 100%). The answers were considered adequately correct when: the adolescent disagreed that men do not get HPV, agreed that women vaccinated against HPV need to undergo Pap tests, agreed that HPV can be contracted through sexual activity, disagreed that HPV is very rare, agreed that HPV can cause cervical cancer, and disagreed that the HPV vaccine protects against all types of cancer.Subsequently, scores adapted from the study by Almeida et al.Regarding the determination of attitudes, the instrument was composed of positive and negative statements about the aspects involving vaccination and each alternative was assigned a numerical value, with the most positive attitude totaling eight points, which corresponds to 100%. Subsequently, the attitude was classified according to the percentage as follows: from 0 to 74% negative attitude, from 75 to 100% positive attitude.The attitude was considered positive when the adolescent agreed that he strongly values his health, agreed that the prevention of diseases and infections is important to him, disagreed that a vaccine shot can be a bother, disagreed that he fears that HPV vaccines are very painful, agreed that he is not afraid to receive vaccines, disagreed that he is concerned about the side effects of the vaccine, disagreed that he feels tense when he hears other boys talking about HPV vaccination, agreed that he is concerned that he may develop HPV cancer in the future.The practice was evaluated using the vaccination schedule. The adolescent responding that he has received both doses of the HPV vaccine was considered an appropriate practice.All variables of the instrument for data collection were organized and coded in a dictionary called codebook. Then, this data was entered in a Microsoft Office Excel 2016 spreadsheet, and after double typing, the data was exported to the Statistical Package for the Social Sciences (SPSS), version 21, to perform the statistical analyses. Adolescents whose questionnaires were incomplete were excluded.Sociodemographic data and the classification of knowledge, attitude, and practice (vaccination situation) were initially analyzed by descriptive statistics. The bivariate test of association between qualitative variables  used was the simple logistic regression, referred to here as unadjusted odds, to select the possible variables that could explain the appropriate practice.15. However, the significance criterion of the variables in the model was the p < 0.05 level association.To explain the joint effect of the variables on the appropriate practical outcome (yes/no) multiple logistic regression was used using the adjusted Odds Ratio (ORa). The inclusion criterion of variables in the logistic model was the p < 0.20 level association in the bivariate analysisThis study was approved by the Research Ethics Committee of the Universidade Federal do Piauí under Opinion No. 2,868,990, in accordance with Resolution No. 466/2012. The adolescents signed an informed assent form, and the parents or guardians signed an informed consent form as a form of common agreement to participate in the research.A total of 472 students participated in the study since, at the time of data collection, all adolescents aged 15 years in one of the selected classrooms expressed the desire to participate. Of the total, 60.8% were female, 39.8% were male and most considered themselves as mixed race (60.8%). The evaluation of the economic factor resulted in dichotomous variables, having as cut-off point the quintiles, in which the group with the lowest possession of goods scored up to the 1st quintile and the group with the highest possession of goods scored above the 1st quintile. Most adolescents (79.7%) belonged to the group with the highest possession of goods .The percentage of adolescents with insufficient knowledge about HPV was 72.7%, and 65.9% had negative attitudes towards infection prevention. We identified the appropriate practice (being vaccinated) in 74.6% of adolescents .Females are more vaccinated (92.7%) than males (46.5%). The race/color variable showed no significant differences between the categories regarding the practice. The evaluation of the variable indicator of possessions identified a slightly higher percentage of vaccinated adolescents (77.1%) in the group with the lowest possession of goods .When assessing the association between sociodemographic variables, knowledge, and attitude with practice, we observed that the female gender  and knowledge  were statistically associated .In the multivariate model, the variables that explained the proper practice were sex, knowledge, and attitude. Female adolescents are 15.62 times more likely to be vaccinated , sufficient knowledge increases this chance by 2.09 times  and positive attitude increases vaccination by 1.89 times  .16. A study conducted in Rio de Janeiro, municipalities of Niterói and São Gonçalo, in 2018, with 807 adolescents, also included most participants (80.3%) in the group with the highest possession of goods, despite covering students from public and private schools12.Most adolescents participating in this study are female, declare themselves as mixed race and fall into the group with the highest possession of property. The profile data of this sample regarding gender and race/color are close to the analysis of the National School Health Survey 2015, conducted with a national sample of 102,301 9th graders, in which most, 51.3%, are female, and mixed race, 43.1%17, the results of this research showed that most adolescents had insufficient knowledge about this condition. This corroborates other publications that point to insufficient knowledge about HPV among adolescent18 and in different population groups19. In Brazil, the study with national sampling5 conducted with young people aged 16 to 25 years identified an average percentage of 51.79% of correct answers regarding HPV and vaccination. In Mexico17, a survey with 242 students between 14 and 18 years old reported that 80% of them had a low level of knowledge about HPV.Although knowledge about HPV plays a crucial role in how people assume their sexuality, protection, and prevention20. However, when considering the level of knowledge only among the female public, we found a high percentage (70.7%) with insufficient knowledge, as in another national study19, which, despite the larger percentage of women than that of men, observed a low proportion of well-informed individuals.We observed that the female public presented a higher level of knowledge than the male, similar to the findings of other studies21.Most adolescents presented negative attitudes towards HPV prevention and vaccination. We evaluated aspects related to the perception of the importance of prevention and the fear and discomfort when receiving the vaccine. The perception of the severity and susceptibility of the individual to HPV infection, and their beliefs about the benefits and barriers to vaccination can predict the intention to receive the vaccine, and this intention predicts behavior22.The attitudes and beliefs of parents and adolescents regarding the safety and efficacy of the vaccine, concerns about side effects, lack of conviction that the vaccine is essential, especially among males, and the lack of awareness about HPV infection and its associated risks, are factors that hinder the maintenance of high vaccination coverage23that resulted in only 60.15% of vaccinated adolescents24. This reduction is assumed to be a result of the first dose being administered in public and private schools, while the second dose was available in the Basic Health Units, like the current doses are, and few health professionals went to the target audience to administer the second dose in schools23. In the year following the vaccine implementation coverage decreased substantially25. This picture shows that the initial vaccination strategy in schools adopted in Brazil showed more success in the adhesion rates.At the beginning of the quadrivalent HPV vaccine implementation in the Unified Health System (SUS), which occurred in 2014, the country achieved a high vaccination coverage in the first phase of the campaign; however, the second dose showed a significant and progressive reduction26.The results showed that females had much higher chances of getting vaccinated when compared with males. The vaccine being initially made available by the SUS only for females and the dissemination of information regarding the consequences of HPV in women, such as cervical cancer, possibly contributes to the ignorance of the availability of this vaccination for both sexes and for the lower vaccination coverage among male adolescents27. In the United States, a study showed that Black women were less likely to have knowledge about the HPV vaccine compared to white women28. This scenario is a likely result of the greater access to the vaccine and to knowledge on the subject among white people, influenced by socioeconomic factors29.In this study, no sociodemographic characteristic was associated with knowledge and attitude about HPV. However, in another study conducted in Brazil, the Black race was associated with low levels of knowledge about HPV among women30, such as a study with high school students in Malaysia30, which identified that the intent to get a vaccination was significantly associated with the level of knowledge about cervical cancer. This scenario highlights the need for educational interventions aimed at raising the awareness of the population about the problem of the infection and the need for preventive measures.Females, sufficient knowledge, and positive attitudes about the vaccine and HPV were associated with adequate prevention practice by vaccination. However, the low rate of adolescents with sufficient knowledge and positive attitudes is a worrying scenario since it negatively influences acceptability and vaccine adhesion, as demonstrated in our study and other publicationsThe main strength of our study was to investigate with a sample that covered all public schools in the urban area of the municipality of Teresina, which provided an overview of the knowledge and attitudes of these students regarding the subject and allowed an evaluation of the rates of HPV vaccine adhesion. Another strong point of this study was to cover adolescents who recently became part of the target audience of HPV immunization in the public health network.Among the limitations is the lack of a standardized scale for the evaluation of knowledge and attitudes about the subject, we made the choice for the scoring procedure due to finding it more appropriate than evaluating with isolated questions. However, we consider that this study could provide important contributions to health strategies and policies aimed at the intensification of HPV vaccine coverage. The occurrence of information bias is also possible considering the self-declared answers, especially those related to sexual questions, which could lead adolescents to change their answers for fear of moral judgments. We used instruments with self-response to minimize those.The findings of this study demonstrated significant associations between sufficient knowledge and positive attitudes with the practice of vaccination, which shows that expanding the knowledge of adolescents, generating favorable attitudes, can be a valuable tool for HPV vaccine adhesion.The results also showed a low rate of vaccinated male adolescents and an association between females and HPV vaccination. This scenario indicates the need to include the male population in themes inherent to HPV prevention since infection is often associated only with cervical cancer, which underestimates its consequences among men, contributing to low vaccination coverage in this public. 1. No Brasil, dados preliminares de um estudo de base populacional, de 2017, realizado em 26 capitais brasileiras e no Distrito Federal, identificaram uma prevalência de infecção pelo HPV em 54,6% das amostras coletadas, dessas amostras, 38,3% apresentavam o HPV de alto risco. No município de Teresina-PI foi detectada uma prevalência de 53,3%2.A infecção pelo papilomavírus humano (HPV) é extremamente frequente em todo o mundo e sua transmissão se dá principalmente pelo contato sexual, estima-se que 80% dos indivíduos sexualmente ativos serão infectados pelo HPV em algum momento da vida3. No Brasil, o câncer do colo do útero é o terceiro tipo de câncer mais frequente entre mulheres, com maiores incidências registradas em estados com menor nível de desenvolvimento socioeconômico, como o Norte e o Nordeste4.Embora na maioria das vezes a infecção seja transitória, sua persistência tem sido diretamente associada ao desenvolvimento de câncer do colo do útero, que representa um importante problema de saúde na atualidade, devido sua elevada incidência5. A imunização mostrou resultados importantes na redução das taxas de infecções provocadas pelo vírus em países onde a cobertura vacinal é alta e já possui impacto comprovado na redução da incidência de câncer do colo do útero6. No entanto, a manutenção de elevadas coberturas vacinais tem sido um desafio no Brasil5.A vacinação e o exame citológico representam as principais estratégias de prevenção contra o HPV5. Além disso, destaca-se que atualmente os adolescentes constituem um grupo de alta vulnerabilidade às infecções sexualmente transmissíveis (IST) como HPV, devido ao desenvolvimento precoce da sexualidade, a multiplicidade de parceiros, maior liberdade sexual, necessidade de afirmação grupal, aliado à resistência ao uso de preservativo7.Nesse contexto, é essencial que os adolescentes, que representam o público-alvo da imunização, tenham conhecimento e consciência sobre o vírus e reconheçam a importância da vacinaçãoAssim, considerando a importância da vacinação contra o HPV e o elevado risco de exposição dos adolescentes ao vírus, emergiu a necessidade de investigação a respeito da conjuntura diagnóstica sobre o que os adolescentes sabem, pensam e praticam frente a esse agravo à saúde. Dessa forma, o objetivo deste estudo foi analisar os conhecimentos, as atitudes e as práticas de adolescentes estudantes de escolas públicas do município de Teresina-PI sobre o HPV.8, é de 850.198 habitantes.Estudo transversal, analítico, desenvolvido por meio de um inquérito de conhecimento, atitude e prática (CAP), realizado em escolas da rede pública que oferecem ensino médio no município de Teresina, capital do Piauí, cuja população, de acordo com o Instituto Brasileiro de Geografia e Estatística (IBGE)A gestão das escolas públicas estaduais de Teresina está organizada em quatro Gerências Regionais de Ensino (GRE). Os critérios de inclusão utilizados para a seleção das escolas no estudo foram: estar localizada na zona urbana da capital e oferecer ensino médio regular ou integral. Dentre as 139 escolas públicas existentes, 91 atenderam a esse critério de inclusão, distribuídas da seguinte forma: 26 na 4ª GRE; 25 na 19ª; 18 na 20ª e 22 na 21ª GRE.Para a população do estudo, adotou-se como critério de inclusão ser adolescente de 15 anos de idade, matriculado no ensino médio e frequentando regularmente as escolas selecionadas para o estudo no momento da coleta. A opção por esta faixa etária ocorreu porque ao completar 15 anos de idade, de acordo com o Programa Nacional de Imunizações (PNI), o adolescente já deve ter recebido as duas doses da vacina contra o HPV.Para o cálculo da amostra de estudantes utilizou-se amostragem do tipo probabilística estratificada proporcional, considerando-se a população de 10.923 adolescentes de 15 anos, de ambos os sexos, matriculados no ensino médio público, cursando a 1ª série, no ano letivo de 2018.9, que identificou 28,9% da população com conhecimento adequado sobre o HPV), precisão de 5% e nível de significância de 5%, obtendo-se amostra mínima exigida de 386 adolescentes. Aplicando-se uma taxa de 20% para recomposição da amostra e presumindo-se eventual perda durante a pesquisa, o tamanho amostral planejado foi de no mínimo 463 participantes.O tamanho amostral foi calculado por meio de amostra aleatória para proporções, adotando-se intervalo de 95% de confiança (IC95%), prevalência de 29% ’.As variáveis de interesse analisadas foram: características sociodemográficas (sexo e raça/cor) e econômicas e avaliação do conhecimento, atitude e prática. As características econômicas das famílias dos adolescentes foram representadas por meio de um indicador construído a partir de informações sobre qualidade de vida, relacionada à saúde de adolescentes escolaresbi é igual a 1 ou zero respectivamente, na presença ou ausência de telefone fixo, telefone celular, internet, automóvel, moto, computador , banheiro com chuveiro. O peso atribuído à presença de cada bem do domicílio será complementar da frequência relativa (ƒi) de cada bem na amostra total, isto é, tanto mais rara a presença do bem no domicílio, tanto maior o peso atribuído ao bem.O ‘Indicador de Bens (IB)’ foi calculado por: IB= ∑ (1-ƒi) bii. Onde: i varia de 1 até 7 bens; 12.O indicador foi ainda refinado adicionando um ponderador que considera a quantidade do bem “i” encontrada no domicílio (e não somente se este existe ou não no domicílio). Essa variante é calculada por w = 0 a k, em que “k” indica a quantidade do bem “i” no domicílio. Foi utilizada a forma dicotomizada da variável, inicialmente em quintis, para separar o subgrupo de menor posse de bens – pressupostamente o “menos favorecido economicamente” – do grupo com maior posseA avaliação do conhecimento, atitude e prática foi consolidada em três categorias: discordo , nem discordo nem concordo e concordo (concordo e concordo fortemente); e foi conduzida da seguinte forma: para classificar o grau de conhecimento dos adolescentes em relação ao HPV foram avaliadas as respostas do questionário sobre o tema, em que foi atribuído um valor numérico para cada resposta, sendo que as respostas corretas totalizam seis pontos, correspondendo a 100%.14, em que o grau de conhecimento foi categorizado em conhecimento insatisfatório (0 a 74%) e conhecimento satisfatório (75 a 100%). As respostas foram consideradas adequadamente corretas quando: o adolescente discordou que os homens não pegam HPV, concordou que as mulheres vacinadas contra o HPV precisam fazer exames de Papanicolau, concordou que o HPV pode ser contraído por meio de atividade sexual, discordou que o HPV é muito raro, concordou que o HPV pode causar câncer cervical e discordou que a vacina contra o HPV protege contra todos os tipos de câncer.Posteriormente, foram aplicados escores adaptados do estudo de Almeida et al.Quanto à determinação de atitudes, o instrumento foi composto de declarações positivas e negativas sobre os aspectos que envolvem a vacinação e, a cada uma dessas alternativas, atribuiu-se um valor numérico, sendo que a atitude mais positiva totaliza oito pontos, o que corresponde a 100%. Posteriormente, a atitude foi classificada de acordo com o percentual da seguinte forma: de 0 a 74% atitude negativa, de 75 a 100% atitude positiva.A atitude foi considerada positiva quando o adolescente concordou que valoriza fortemente sua saúde, concordou que a prevenção de doenças e infecções é importante para ele, discordou que ser furado por uma agulha pode incomodar, discordou que teme que as vacinas contra o HPV sejam muito dolorosas, concordou que não tem medo de receber vacinas, discordou que está preocupado com os efeitos colaterais da vacina, discordou que se sente tenso quando ouve outros garotos falando sobre a vacinação contra o HPV, concordou que está preocupado que possa ter câncer provocado pelo HPV no futuro.A prática foi avaliada por meio do esquema vacinal. Foi considerada prática adequada quando o adolescente respondeu ter recebido as duas doses da vacina contra o HPV.codebook. Em seguida, esses dados foram digitados em planilha do Microsoft Office Excel 2016, e após dupla digitação, os dados foram exportados para o Statistical Package for the Social Sciences (SPSS), versão 21, para realização das análises estatísticas. Excluíram-se os adolescentes cujos questionários apresentavam incompletude.Todas as variáveis do instrumento para coleta de dados foram organizadas e codificadas em um dicionário denominado de odds não ajustado, com objetivo de selecionar as possíveis variáveis que poderiam explicar a prática adequada.Os dados sociodemográficos e a classificação do conhecimento, atitude e prática  foram analisadas inicialmente, por meio de estatística descritiva. O teste bivariado de associação entre as variáveis qualitativas  utilizado foi a Regressão Logística Simples, referido aqui como Odds Ratio ajustada (ORa). O critério para inclusão de variáveis no modelo logístico foi a associação ao nível de p ˂ 0,20 na análise bivariada15. Todavia, o critério de significância das variáveis no modelo foi a associação ao nível de p ˂ 0,05.Para explicar o efeito conjunto das variáveis sobre o desfecho prática adequada (sim/não) utilizou-se a regressão logística múltipla, por meio da Este estudo foi aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal do Piauí, sob o parecer nº 2.868.990, em conformidade com a Resolução nº 466/2012. Os adolescentes assinaram um termo de assentimento livre e esclarecido e os pais ou responsáveis um termo de consentimento livre e esclarecido como forma de comum acordo em participar da pesquisa.Participaram da pesquisa 472 estudantes, uma vez que, por ocasião da coleta de dados, em uma das salas de aula selecionadas, todos os adolescentes com 15 anos manifestaram o desejo de participar. Do total de adolescentes, 60,8% eram do sexo feminino, 39,8% do sexo masculino e a maioria se definiu como pardos . A avaliação do fator econômico resultou em variáveis dicotômicas, tendo como ponto de corte os quintis, em que o grupo com menor posse de bens pontuou até o 1º quintil e o grupo com maior posse de bens pontuou acima do 1º quintil. A maioria dos adolescentes  foi categorizada como pertencente ao grupo com maior posse de bens .O percentual de adolescentes com conhecimento insuficiente sobre o HPV foi de 72,7%, e 65,9% apresentaram atitudes negativas em relação à prevenção contra a infecção. A prática adequada (estar vacinado) foi identificada em 74,6% dos adolescentes .O sexo feminino encontra-se mais vacinado  do que o masculino . A variável raça/cor não apresentou diferenças significativas entre as categorias em relação à prática. A avaliação da variável indicador de bens identificou um percentual um pouco mais elevado de adolescentes vacinados  no grupo com menor posse de bens .Ao testar a associação entre as variáveis sociodemográficas, o conhecimento e a atitude com a prática, observou-se que o sexo feminino  e o conhecimento  foram estatisticamente associados .No modelo multivariado, as variáveis que explicaram a prática adequada foram o sexo, o conhecimento e a atitude. Adolescentes do sexo feminino têm 15,62 vezes mais chances de serem vacinados , o conhecimento suficiente aumenta 2,09 vezes essa chance  e a atitude positiva aumenta 1,89 vezes as chances de vacinação  .16. Estudo realizado no Rio de Janeiro, Niterói e São Gonçalo, em 2018, com 807 adolescentes escolares também enquadrou a maioria dos participantes  no grupo com maior posse de bens, embora esse estudo abrangesse alunos de escolas públicas e privadas12.A maioria dos adolescentes participantes deste estudo é do sexo feminino, se autodeclara como pardos e se enquadra no grupo com maior posse de bens. Os dados do perfil dessa amostra referentes a sexo e raça/cor se aproximam da análise da Pesquisa Nacional de Saúde Escolar 2015, realizada com uma amostragem nacional de 102.301 alunos do 9º ano, em que a maioria, 51,3%, pertence ao sexo feminino, e se caracterizou como pardos 43,1%17, os resultados desta pesquisa evidenciaram que a maioria dos adolescentes apresentou conhecimento insuficiente a respeito desse agravo. Corroborando outras publicações que apontam para um conhecimento insuficiente sobre o HPV entre o público adolescente18 e em diferentes grupos populacionais19. No Brasil, o estudo com amostragem nacional5 realizado com jovens de 16 a 25 anos, identificou uma porcentagem média de 51,79% de respostas corretas sobre o HPV e a vacinação. No México17, realizou-se uma pesquisa com 242 estudantes com idade entre 14 e 18 anos em que 80% apresentaram baixo nível de conhecimento sobre o HPV.Apesar de o conhecimento sobre o HPV desempenhar um papel muito importante na forma como as pessoas assumem sua sexualidade, proteção e prevenção20. No entanto, ao considerar o nível de conhecimento apenas entre o público feminino, foi evidenciado um elevado percentual  com conhecimento insuficiente, como em outro estudo nacional19, em que apesar de o grupo de mulheres ter apresentado um percentual superior ao de homens, ainda se percebe uma baixa proporção de indivíduos bem informados.Observa-se que o público feminino apresentou um nível de conhecimento superior ao masculino, resultado semelhante ao encontrado em outras pesquisas21.A maioria dos adolescentes apresentou atitudes negativas em relação à prevenção do HPV e à vacinação. Foram avaliados aspectos relacionados à percepção da importância da prevenção e ao medo e desconforto ao receber a vacina. A percepção da gravidade e da suscetibilidade do indivíduo em relação à infecção pelo HPV, bem como suas crenças sobre os benefícios e barreiras à vacinação podem prever a intenção em receber a vacina, e essa intenção prediz o comportamento22.As atitudes e crenças dos pais e adolescentes em relação à segurança e eficácia da vacina, preocupações acerca dos efeitos colaterais, falta de convicção de que a vacina é essencial, especialmente entre o sexo masculino, além da falta de conscientização sobre a infecção pelo HPV e seus riscos associados, podem ser caracterizados como fatores que dificultam a manutenção de coberturas vacinais elevadas23que resultou em um percentual de apenas 60,15% de adolescentes vacinadas24. Supõe-se que essa redução tenha ocorrido porque a primeira dose foi administrada nas escolas públicas e privadas, ao passo que a segunda dose ficou disponível nas Unidades Básicas de Saúde, como ocorre atualmente, e poucos profissionais de saúde se dirigiram até o público-alvo para realizar a segunda dose nas escolas23. No ano subsequente à disponibilização da vacina houve uma diminuição substancial na cobertura25. Esse quadro mostra que a estratégia inicial de vacinação nas escolas adotada no Brasil apresentou mais sucesso nas taxas de adesão.No início da implantação da vacina quadrivalente contra o HPV no Sistema Único de Saúde (SUS), que ocorreu em 2014, o país atingiu uma elevada cobertura vacinal na primeira fase da campanha, no entanto, houve uma redução significativa e progressiva para a segunda dose26.Os resultados demonstraram que o sexo feminino apresentou chances muito superiores de se vacinar quando comparado ao sexo masculino. O fato de a vacina ter sido disponibilizada pelo SUS inicialmente apenas para o sexo feminino e a difusão de informações relacionadas às consequências do HPV nas mulheres, como o câncer cervical, possivelmente contribui para o desconhecimento da disponibilidade dessa vacinação para ambos os sexos e para as menores coberturas vacinais entre os adolescentes masculinos27. Nos Estados Unidos, um estudo evidenciou que as mulheres de raça negra possuíam menor probabilidade de ter conhecimento sobre a vacina do HPV em comparação com as brancas28. É provável que esse cenário ocorra devido a um maior acesso à vacina e ao conhecimento sobre o tema entre as pessoas da raça branca, influenciado por fatores socioeconômicos29.Neste estudo, nenhuma característica sociodemográfica foi associada ao conhecimento e à atitude sobre o HPV. Contudo, em outra pesquisa realizada no Brasil, a raça negra foi associada a baixos níveis de conhecimento sobre o HPV entre as mulheres30, a título de exemplo, a realizada com estudantes do ensino médio na Malásia30, a qual identificou que a intenção de se vacinar foi significativamente associada ao nível de conhecimento sobre o câncer do colo do útero. Tal cenário evidencia a necessidade de intervenções educativas voltadas para a conscientização da população a respeito da problemática da infecção e da necessidade de adoção de medidas preventivas.O sexo feminino, o conhecimento suficiente e as atitudes positivas sobre a vacina e o HPV foram associados à prática adequada de prevenção por meio da vacinação. Todavia, o baixo índice de adolescentes com conhecimento suficiente e com atitudes positivas representa um cenário preocupante, pois influencia negativamente na aceitabilidade e adesão à vacina, como demonstrado neste estudo e como citado em outras publicaçõesA principal força deste estudo foi realizar uma investigação com uma amostragem que abrangeu todas as escolas públicas da zona urbana do município de Teresina, o que forneceu um panorama sobre os conhecimentos e atitudes desses estudantes em relação ao tema e permitiu uma avaliação das taxas de adesão à vacina contra o HPV. Outro ponto forte desta pesquisa foi abranger adolescentes que recentemente fizeram parte do público-alvo da imunização contra o HPV na rede pública de saúde.Dentre as limitações, pode ser citada a inexistência de uma escala padronizada para avaliação de conhecimentos e atitudes a respeito do tema, a opção pelo procedimento de pontuação ocorreu por acharmos mais adequado do que avaliar com perguntas isoladas. No entanto, considera-se que esta investigação poderá fornecer importantes contribuições para as estratégias e políticas de saúde voltadas para a intensificação da cobertura vacinal contra o HPV. A ocorrência de vieses de informação, também é possível considerando-se as respostas autodeclaradas, sobretudo aquelas relacionadas às questões sexuais, que poderiam levar os adolescentes a alterarem as suas respostas por receio de julgamentos morais. Para minimizá-los foram utilizados instrumentos com autorresposta.Os achados deste estudo demonstraram associações significativas entre o conhecimento suficiente e atitudes positivas com a prática da vacinação, o que evidencia que ampliar o conhecimento dos adolescentes, gerando atitudes favoráveis, pode ser uma importante ferramenta para adesão à vacina contra o HPV.Os resultados também evidenciaram um baixo índice de adolescentes masculinos vacinados e uma associação entre o sexo feminino e a vacinação contra o HPV. Esse cenário indica a necessidade de incluir a população masculina em temas inerentes à prevenção ao HPV, pois frequentemente a infecção é associada apenas ao câncer do colo do útero, subestimando-se as suas consequências entre os homens, o que contribui para as baixas coberturas vacinais nesse público.


Global trend is moving towards the use of natural phytochemicals to fight against pathogens. Human cervical cancer is directly associated with onco-potent type of Human Papilloma Virus (HPV). There is no known medicine for clearance of HPV type whose persistence is the cause of occurrence and re-occurrence of cervical cancer. The different species of fig fruit and their latex are reported to have HPV associated genital warts clearance capability.In the current investigation, the effect of the methanol extract of Ficus benghalensis L. fruits on HPV type18 viral load in HeLa cell line was tested by doing PCR using HPV L1 primers (MY09/My011) and the cytotoxicity was also analysed by MTT assay. The induction of apoptotic activity in terms of DNA fragmentation and hyper-chromic effects of DNA was analysed. The PCR results showed a reduction in the HPV18 DNA and also the treatment exhibited a promising cytotoxicity with IC50 value at 211.86 μg/ml. The DNA samples from treated HeLa cells showed DNA shearing and laddering as a mark of apoptotic DNA fragmentation  and the The Ficus benghalensis L. fruit extract reduced the HPV viral load in HPV18 containing HeLa cells and showed an effective cytotoxicity on HeLa cell line. It also could induce the apoptotic activity in HeLa cell line and this study results suggest that the Ficus benghalensis L. fruits can be used to fight against cervical carcinoma, acting on HPV load. Cancer is a life threatening disease, which is not having any particular cure at present. Cervical cancer is the second most common types of cancer in women worldwide. In the current scenario, the incidence and mortality of cervical cancer is less due to the implementation of advanced prevention and screening techniques. Human Papilloma Virus (HPV) is the major causative agent for the development of cervical cancer as it is principally transmitted by sexual mode. The HPV is a group of over 200 different related types, in which about 40 HPV serotypes infecting genital tract cause various lesions . Among the HPV types, few are identified as onco-potent types, which cause approximately 5% cancer burden of all cancers worldwide and are categorized as high risk types . The high and low risk HPV types are mainly transmitted sexually before the symptoms appear on the infected person and it also can be transmitted non-sexually by birth or via mothers milk or by close contact as per the data of last 2 decades . More than 90% of cervical cancer and few non cervical cancers like anal, vulvar and oesophageal are caused by high risk type HPV 16/18 . According to the IARC report, the cause of several cancers of oropharynx, base of the tongue, tonsils, cervix uteri, penis, vulva, vagina and anus origin have shown a strong relationship with HPV infection .® HPV HR) and nucleic acid amplification methods , PapilloCheck®) have been approved by FDP for screening high risk type HPV infection in addition to the cytological Pap test. The detection of HPV by polymerase chain reaction (PCR) is the main FDA approved method for screening high risk type HPV in clinical samples due to its high sensitivity and reliability . All the diagnostic methods have reported a high prevalence of high risk type HPV16/18 in cervical cancer. We have also reported 95% invasive cervical cancer cases infected with onco-potent HPV types HPV16/18 using PCR based detection method . As the persistence of onco-potent HPV in epithelial cells is responsible for the development and recurrence of cervical cancer, the viral load needs to be eliminated in all the HPV infected cells. The HeLa cell line is isolated from adenocarcinoma of uterine cervix and it contains HPV type 18 as viral particles as well as an integrated form as per many published research articles . The L1 capsid protein having conserved sequence is responsible for HPV-host cell interaction for the entry of HPV, accounting to persistent infection in cancer cells and has shown binding affinity in HeLa cell line . The hyper-methylation of L1 gene was highly correlated with the severity of neoplasia in HPV 18 positive clinical samples  and it affected the transcriptional expression of other genes in HeLa cell line . The aim of this study is to determine the viral load in HeLa cell line after treatment with the extract of fig fruit to find its anti-viral effect. The viral load is determined widely, by testing the presence of viral capsid proteins and is responsible for virion maturation, assembly, package and release. Many studies have tested the gene for capsid protein L1 to determine the viral load of HPV  and the L1 ORF has type-specific conserved domain of its protein. The consensus of PCR primers for the highly conserved region in L1 ORF was used for HPV identification . Hence, we studied the viral load by following the conventional method of detecting the L1 gene in HeLa cell line. In this study, we have utilized the widely used and clinically evaluated MY09/MY11 consensus primers , which amplify the L1 region of HPV in HeLa cells .HPV is a non-enveloped, epitheliotrophic virus with double stranded circular DNA of 7900 bp with eight overlapping open reading frames (ORF). The HPV 18 genome is of 7837 bp with 6 early (E) genes , which code for non-structural regulatory proteins for the replication and other transformation related functions of HPV . The late (L) genes, L1 and L2 encode the major and minor capsid proteins (the capsid contains 72 pentamers of L1 gene and 12 molecules of L2 gene), which are responsible in viral particle formation for subsequent infection to other cells. The early genes regulate viral replication whereas; a few of them have the transformation potential, which makes the treatment difficult . The HPV type, viral load and integration status of HPV predict the patient’s prognosis . Several molecular testing devices such as; signal amplification methods . Among these, Ficus is one of the plant species found to be used as both food and medicine. The aqueous extract of the bark of Ficus religiosa has been reported for its apoptosis induction in HeLa cell lines and it has reduced the HPV 18 onco-proteins E6 and E7 . In Iranian medicine, the latex of fig fruit is used to treat papillomatosis, hypoglycemic induction, cancer suppression and anti-helminthic effect . The ‘Indian Fig’ fruits  belonging to the Ficus family are widely available. Ficus benghalensis L. species have antioxidant property with singlet oxygen quencher molecule. The methanol extract of the bark of Ficus benghalensis L. possess hepato-protective activity in rats . An effective cytotoxic agent namely 6-O-acyl-d-glucosyl-sitosterols has been isolated from fig fruit latex and found to show in vitro inhibitory effects on proliferation of various cancer cell lines . It has been found that Ficus benghalensis L. fruit extract has various effects on the cancer cell line, which includes cytotoxic activity and apoptosis . Hence, we have evaluated the anti-cancer effect of Ficus benghalensis L. fruit extract on HPV 18 containing cervical cancer cell line (HeLa) in vitro. Furthermore, its antiviral effect on HPV 18 gene upon treatment was analysed in this study by assessing the viral load of HPV 18 in treated cell lines.Sample Extraction The Ficus benghalensis L. fruits were collected and authenticated by the Botanical Survey of India (BSI). The authentication number is BSL/SRC/5/23/2017/Tech./2923 and the letter is kept for future reference. The fruits were washed and dried at room temperature and by using methanol as solvent, the majority of the compounds were extracted by using soxhlet apparatus. The obtained extract appeared as semi-solid brown paste and stored in a refrigerator until use.HPV18 containing cervical cancer cells2 at 37°C. HeLa cells were purchased from NCCS Pune, India and were grown by following the instructions using RPMI1640  as medium. Each 500 ml of the medium consisted of 5.2 g of RPMI powder, 1 g of sodium bicarbonate, 5 ml of sodium pyruvate (1 mM), 5 ml of L- glutamine (2 mM) and 50 mL of heat inactivated foetal bovine serum (FBS) along with Streptomycin  in deionised water. The medium was then sterilised by filtering through 0.22 μM pore filter. The pH of the medium was adjusted to 7.4 using concentrated HCL or NaOH and kept at 4°C before use. Cell lines were maintained in a humidified atmosphere of 5% COMTT Assay5 cells. After 24 hours of incubation, the fruit extract was added at different concentration  and the DMSO was added for control. Then, 20 μL of MTT (5 mg/mL) was added to all the wells after 24 hours. The reduction of yellow MTT into insoluble dark blue coloured formazan product was measured. The insoluble dark formazan products were then solubilized with an organic solvent (DMSO) and the solubilized formazan were then measured spectrophotometrically at 570 nm using a micro ELISA plate reader and the percentage of cell viability was calculated using the following formula:The inhibitory activity of the methanolic extract of Ficus benghalensis L. towards HeLa cell line was determined by using the colorimetric method of tetrazolium salt; 3- 2,5-diphenyltetrazolium bromide (MTT) reduction . Briefly, HeLa cells suspension with 80-90% confluence was added in triplicates into 96 well cultured plates at concentrations of 1x10Percentage cell viability = Absorbance of control - Absorbance of test X 100Absorbance of controlDNA Fragmentation Assessment5 cells of the cell suspension for both treated (211.86 μg/ml (IC50)) and untreated cells. After treatment, cells were centrifuged at 3,000 rpm for five min and the pellet was suspended in 300 μL of cell lysis solution, containing SDS 0.5% and proteinase K to a final concentration of 0.05 ng/mL and incubated at 56°C, overnight. After incubation, equal volume of phenol: chloroform: isoamyl alcohol was added and the aqueous layer was separated. The DNA was then precipitated by adding ammonium acetate (1.4 mM) and an equal volume of ice cold isopropanol. It was then centrifuged at 10,000 rpm for 15 min at 4°C and the obtained DNA pellet was washed in ice-cold 95% ethanol, and again centrifuged at 14,000 rpm for 10 min at 4°C. The pellet was dried and suspended in 100 μL of TAE buffer. This DNA sample was loaded in 0.8% agarose gel, and the DNA bands were observed and analysed using gel documentation system .DNA extraction was performed from 1x10UV Spectrophotometric AnalysisIn order to check the hyper chromic effect of the DNA sample as an indication of DNA damage, the absorbance of the treated (211.86 µg/ml) and untreated DNA sample at 260 nm were noted. Milli Q water was used as blank . PCR Analysis for HPV 18 Gene:The isolated DNA was analysed for the concentration, purity and integrity by measuring them at wavelength A260 and A280 as well as by performing 0.8% agarose gel electrophoresis. The DNA samples having the ratio of A260/A280 as 1.8-2 and a thick band near the well in agarose gel were quantified and subjected to PCR. The DNA of control cell lines was not intact in agarose gel. The isolated DNA samples of both treated and untreated cells were amplified through PCR amplification, which target a portion of the HPV LI region (approximately 450 bp). The amplification of ß-globin fragment (268 bp) was carried out as an internal control to indicate that the sample was adequate for HPV analysis and that no inhibitor was present. Each 25 μL reaction mix contained 12.5 μl Taq DNA polymerase 2x Master mix, 1 μl of forward and reverse HPV LI consensus primers  (MY09 and MY11) , and 1 μCytotoxicity by MTT Assay50) was found to be 211.86 μg/ml in the given graph . Our present study on Ficus benghalensis L. showed a higher cytotoxic activity against HeLa cell line, with an IC50 value of 211.86 μg/ml at 24 hrs of incubation. Cervical cancer is the second leading cancer among women with an estimation of 445,000 new cases in 2012 (84% of the new cases worldwide). According to WHO , more than 85% of deaths occurs in low and middle income countries. It is stated that about 70% of cervical cancers occurs in developing countries. HPV infection is the major cause of cervical cancer, but finding the HPV treatment effect at molecular level is a difficult task. The HPV18 inclusions and L1 capsid proteins were observed in HeLa cell line which suggest the presence of L1 gene in HeLa cells and its ability to express L1 proteins . In order to develop a novel approach in the treatment of cervical cancer to target the HPV, we have used the Ficus benghalensis L. plant material as the plant materials have been used traditionally for HPV treatment as therapeutic agent . The current study was thus, focused on finding the effect of Ficus benghalensis L. fruit extract against the HPV generated from the integrated genome in the cervical cancer cells. The cytotoxic activity of Ficus benghalensis L. fruit extract against HeLa cell line was analysed in this study by MTT assay. Ficus ariculata was earlier shown to possess anticancer activity against HeLa cell line, indicating an ICThe DNA fragmentation analysis is done for understanding the apoptotic activity and deactivation pattern of the gene codes, for the enzymes involved in cancer. The DNA of apoptotic cells undergoes fragmentation as shown in many studies . The Apoptosis (cell death) and its signalling pathways have a remarkable impact on the progression of cancer  and initiation of apoptosis is a highly desirable goal for controlling the cancer and its pathways . In our study, the DNA fragmentation was observed in HeLa cells treated with Ficus benghalensis L. fruit extract at a concentration of about 211.86 μg/ml. The control cancer cells did not show any fragmentation as shown in 50). Majority of HPV studies have used the MY09/11 primers that are potentially capable of detecting many mucosal HPV types in a single PCR reaction  and are also specific to a highly conserved HPV L1 gene region . Analysing the presence of Human Papilloma Virus (HPV), its types and its viral load is the basic step in evaluating the risk of cervical cancer and most of the available data indicate that the cervical lesions are related to HPV-16 and HPV-18 . The HeLa cell line was used as positive control for the presence of HPV 18 in many studies . The latex of fig fruit and other parts were reported to be traditionally used for HPV treatment. In this study, we intend to analyse the effect of Ficus benghalensis L. fruit extract on the HPV viral load in HeLa cell lines by PCR method. In the current investigation, the difference in HPV 18 viral load between the treated and untreated HeLa cells was evaluated using PCR method by treating the cells with 211.86 μg/ml  indicated that the sample was adequate for HPV analysis and that no inhibitor was present. Our results have shown a significant reduction in the level of HPV DNA in terms of the band width in agarose gel and the density in treated sample when compared to the control sample as shown in In conclusion, this study illustrates the decrease in HPV 18 viral load upon treatment with methanolic Ficus benghalensis L. fruit extract as compared with the untreated HeLa cell line. The cytotoxic activity of fig fruit extract on Hela cells also showed a high percentage inhibition of about 87.92% for 350 μg/ml for an incubation period of 24 hrs. The enhanced DNA fragmentation observed in treated cell lines in terms of DNA laddering and hyper chromic effect implies that the apoptotic activity was induced by the effect of Fig fruit extract treatment.Conceptualization: V. M. Berlin Grace. Investigation and Data curation: B. Lydia. Methodology: V. M. Berlin Grace. Project administration: D. David Wilson. Writing – original draft: V. M. Berlin Grace and D. David Wilson. Review & editing of manuscript: D. David Wilson