Cytology-based screening has been for decades the conventional method of screening. Ancillary techniques have been added like immunocytochemistry with P16/Ki67 and L1-Capsid, but these methods require maintenance of complex infrastructure and highly trained personnel as well as relatively short screening intervals. HPV-based screening method to detect high-risk groups is a faster and automated method, which does not need morphologically highly qualified personal with high social costs. In the study, we have focused on the distribution of cervical lesions in the age groups with concordance of detection HPV high-risk subtypes (HPV-HR) and on the safety of the screening method. In the Institute for Pathology and Cytology-Schuettorf-Leer-Germany 146.800 samples of women from the age of and above 35 years were analyzed between the beginnings of 2020 until the beginning of 2021. 63.710 cases under 35 years old were analyzed. The samples were processed for both conventional cytological techniques and for molecular detection and subtyping of HPV-HR according to the advice and measurements of BD-manufacture. In this study, we have studied the histopathological results . Low grade cervical lesions (CIN I) were 0.13% (< 35 Ys), 0.35% (35–40 Ys), 0.36% (41–50 Ys), 0.25% (51–60 Ys) and 0.098% (> 60Y s), which was statistically significant . Without dysplasia 0.19% (< 35 Ys), 0.5% (35–40 Ys), 0.56% (41–50 Ys), 0.51 (51–60 Ys) and 0.26% (> 60 Ys). There is no significance between occurrence of cervical dysplasia and without dysplasia despite of detection of HPV-HR subtypes . The only use of HPV-subtyping is not a secure method and a protective way for women. There are worldwide many HPV-positive cases, which have been psychologically impaired with higher costs, although they have no cervical epithelial changes during the HPV-infection. There are many HPV-negative cases, in some studies up to 13% of cases, which develop cervical cancer. We have the opinion and are convinced that the screening should be both morphologically via cytological examination and may be with adding immunocytochemistry to detect the really dysplastic cervical lesions. HPV-subtyping may be added every three years to detect the concomitant subtype.The world health organization (WHO) called for coordinated global action in 2018 to eliminate cervical cancer, ensuring that every woman is screened and treated for precancerous lesions (World Health Organization. Cervical cancer: an NCD we can overcome. Geneva, 2018.  Cytology-based screening has been for decades the conventional method of screening. Ancillary techniques have been added like immunocytochemistry with P16/Ki67 and L1-Capsid, but these methods require maintenance of complex infrastructure and highly trained personnel as well as relatively short screening intervals. Immunocytochemistry has raised the sensitivity and specificity of the diagnosis but is still in many countries not well established2. The molecular detection of HPV high risk groups in women above 30–35 years has been proposed as a method of accuracy with higher sensitivity to detect women with risk to develop precancerous lesions. The advantage of this method is that it is a machinery automated work without the need to have highly trained and highly qualified personnel, but it is expensive. Due to lower specificity, HPV-based screening should not be done under the age of 35 years old3. In Germany at the beginning of 2020, a co-test-based screening program was applied for women at and above 35 years, which means performing conventional cytological analysis and HPV-HR-test for each woman above 35 years and in some cases under 35 years old. This is a retrospective study and experience after one year with the new system in Germany. This study will analyze, which of the methods give both clinicians and patients security to avoid progression of cervical dysplasia to cervical carcinoma for an early detection of cell changes under human papilloma infection. It will analyze, which HPV-subtypes are common and when they will appear and which age-groups are susceptible to produce dysplasia under HPV infection. It will show the distribution of these HPV-subtypes in the different age groups. Lastly, it will give us indirectly impression about the importance of detecting dysplastic cellular changes with conventional cervical cytology and the importance of morphological diagnosis.The world health organization (WHO) called for coordinated global action in 2018 to eliminate cervical cancer, ensuring that every woman is screened and treated for precancerous lesionsp value is < 0.05. The approval was granted by the ethics committee . The samples were anonymous with respect to measurements of data protection. All methods were performed in accordance with the relevant guidelines and regulations.In the Institute for Pathology and Cytology-Schuettorf-Leer-Germany 210.510 samples of women in the screening program were investigated between the beginning of January 2020 until the beginning of January 2021. 63.710 cases under 35 years old, 22.136 cases in the age group 35–40 years old, 34.667 cases in the age group 41–50 years old, 41.276 cases in the age group 51–60 years old and 48.721 cases in the age group older than 60 years old were investigated. The samples were processed for both conventional cytological techniques and for molecular detection and subtyping of HPV-HR according to the advice and measurements of BD-manufacture. We have divided the cases in this study in 5 groups . According to the BD manufacture, there were certain HPV-subtypes of high-risk groups studied. These are HPV 16, 18, 45, 31, 33, 52, 58, 35, 59, 56, 51, 39, 68, 73, 82, 53, 66, 70, 6, 40, 42, 43, 44/45, 33/58, 56/59/66, 35/39/68 . The samples were anonymous with respect to measurements of data protection. All methods were performed in accordance with the relevant guidelines and regulations. We are confirming that informed consent was obtained from all subjects and/or their legal guardian(s).Under the age of 35 years Fig. , there wIn the age-group 35–40 years old Fig. , there wIn the age-group 41–50 years old Fig. .In the age-group 51–60 years old Fig. .In the age-group over 60 years Fig. , there wUnder the age of 35 years, there were 3 cases with negativity for HPV analysis for high-risk subtypes but they were cytologically IIIg, IVb-g, and Vg and histopathologically were AIS, Sq.c.ca and endometrial carcinoma respectively Table .Between 35 and 40 years old: One case was negative for HPV analysis for high-risk-subtypes but was cytologically IIIg and histopathologically CIN II. Two cases were with HPV (18) but cytologically were IIg and IIIg then histopathologically CIN I and AIS. One case was with HPV (45) but cytologically IIg and histopathologically CIN II.Between 41 and 50 years old: One case was negative in HPV-HR-analysis but cytologically was IIIg and histopathologically AIS. A case was with HPV (52) and cytologically was IIIg and histopathologically CIN II. 4 cases were with HPV (16) and cytologically were IVa-g but histopathologically were AIS (2 cases), endocervical adenocarcinoma (1 case) and Sq.c.ca (1 case).Between 51and 60 years old: Three cases were negative in HPV-HR-analysis but cytologically were IIIg and histopathologically were CIN I and CIN II. Three cases were with HPV (18) and cytologically were IVa-g and IVb-g but histopathologically either CIN II or AIS or endometrial carcinoma.More than 60 years old: Two cases were with HPV 33/35 and cytologically were IVa-g but histopathologically was CIN I and CIN III.4. WHO had announced shocking deductions: one woman died of cervical cancer every 2 min. worldwide (WHO1). Currently studies point out that 20–25% of all human malignancies are related to micro-organism infections. Among these cancer-related pathogens, the human papillomavirus (HPV) has a prominent position, since the virus is responsible for about 30% of all infectious agent-related cancers as stated by Araldi RP and colleagues, 20185. Another work of de Martel C. and Colleagues, 20176, who have documented that 4.5% of all cancers worldwide ) new cancer cases per year worldwide are attributable to HPV. The association of infection with HPV16/18 and HPV 6/11/16/18/31/33/45/52/58 was also estimated. Other organs like vagina or anus or penis in men show also HPV-associated lesions. The most common types worldwide are HPV 16 and 18, which are the main types linked to carcinogenesis as reported by Palefsky JM, 20177, while HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82 are also oncogenic and responsible for little number of cases as reported by Schiffman and colleagues, 20098. It is well documented that nearly 90% of incident HPV infections are not detectable within a period of 2 years from the acquisition of infection and persist only in a small proportion as reported by Bosch and colleagues 20029. This is somehow compared with our study but not completely agree with the work of Palefsky JM, 20167, we have noticed among 210.510 cases about 1.1% with HPV-subtypes 56/59/66 in the age group 41–50 years old. These subtypes were detected with a rate of 0.93% in the age group 35–40 years old and with a rate of 0.99% in the age group 51–60 years old, which means that the peak of incidence was in the middle age group. This might explain the chronicity of this infection with these subtypes, that may lead to cervical dysplasia, as we have detected later in the histopathology results, that the peak of occurrence of CIN I (0.36%) was in the age group 41–50 years as well as AIS (0.01%) and endocervical adenocarcinoma of (0.014%). HPV-subtype 16 has an incidence in our work of 1.32% in the age-group 35–40 years and a little bit lower (1%) in the age group 41-50Ys, 0.7% in the age-group 51–60 years and 0.67% in the age-group of more than 60 years, which may explain the highest incidence of CIN I (0.35%), CIN II (0.42%) and CIN III (0.5%) in the age-group 35–40 years. The incidence of these lesions was small in the higher age groups. It should be noted that in the age-group 35–40 years, there was no dysplasia in 0.5%. This may explain that not every woman with HPV infection should develop dysplasia and this confirms the statement of Schiffmann and colleagues, 201810. They have also concluded that the true value of HPV primary screening is uncertain since a positive HPV test reflecting a developed cervical carcinoma may not lead to disease prevention. More confounding is the likelihood of HPV-negative, cytology-positive cervical cases, thus, the diagnostic value embedded in co-testing Pap-test models. In our work, there is no significance between occurrence of cervical dysplasia and without dysplasia despite of detection of HPV-HR subtypes . From Australia’s screening program with reflex cytology, there were high-grade lesions in 24.3% confirmed, which underscores the benefits through cell-morphology-based co-testing, and the potential deficits in HPV DNA screening. Related studies emphasize the need to minimize the false-positives arising from HPV DNA screening to avert harms to women, such as clinical overreaction, overtreatment and psychological distress, which are all associated with profound yet avertable social costs as stated by Schiffmann and colleagues, 201911. We showed in our study that the incidence of HPV-subtypes was low and matched with the occurrence of squamous cell carcinoma or endocervical adenocarcinoma. HPV 52, HPV 16, HPV 58, HPV 51, and HPV 39 were the most common genotypes accounting for 22.8%, 22.3%, 20.0%, 14.3%, and 13.6% of cases, respectively. The highest infection rates were found in 20–30-year-old patients (35.1%). HPV 16 infection was the highest in the age-group 31–40 years, and HPV 52, HPV 58, and HPV 39 infections were highest in the age-group 20–30 years in the work of Shuang Lu and colleagues, 201512. The results showed that HPV 52 and HPV 16 were the most common subtypes found in 22.8% and 22.3% respectively of the study participants. HPV 58, HPV 51, and HPV 39 were also common and occurred in 20%, 14.3% and 13.6% of patients, respectively as reported by Shuang Lu and colleagues 201512. These results should be evaluated carefully as the incidence of HPV 52 was too small in all age groups with maximum of 0.4% in the age-group 41–50 years. The variability between nations may lead to variation in the results. The most common found subtypes were HPV 16 and HPV 18. It is found that people in Europe or South/Central America were more likely to be infected with HPV 18 than those in Asia. Other common subtypes were HPV 31 in Europe, HPV 33 in South/Central America, and several HPV genotypes  were in North America compared with other regions as stated by Bosch and colleagues, 199513. These results should be evaluated carefully, as HPV-18 was totally irrelevant in the age groups and has no association with the development of cervical carcinoma. Shuang Lu and colleagues 201512 have stated that the highest infection rates were found in patients 20–30 years old. At 31–40 years of age, the infection rates of HPV 16 were significantly higher than HPV 52 and HPV 58 infection rates. These differences may result from different clearance rates of HPV 52, HPV 58, and HPV 16. Low grade (I) cervical lesions, in the work of Shuang Lu and colleagues 201512, were most common in women 31–40 years old, and the prevalence of degree II + cervical lesions were highest in women 41–50 years old. In our study, we have detected the highest infection with HPV subtype 16 in age group 35–40 years old, followed by HPV 56/59/66 in age group 41–50 years old. We have also found that CIN I was high in the age group 41–50 years old (0.36%). CIN II and CIN III were highest in the age groups 35–40 and 41–50 years old. Under the age of 35 years old, we have also detected the same rates of CIN I, CIN II and CIN III of 0.13%, 0.23% and 0.31% respectively. Poljak14 has stated that HPV-based cervical cancer screening is more sensitive than cytology for detecting underlying CIN2+, CIN3 + and cervical cancer, is more accurate and objective, is less variable than cytology, requires less training, shows better reproducibility, offers a possibility of self-sampling for non-attenders, and provides safe extension of screening intervals in women with a negative screening result. Another work of Maver and Poljak15, that has proposed for HPV-based screening in some European countries and Australia. The work of Miller RA and colleagues16 has documented HPV-negative cervical cancers, which would be not detected, if we depend only on primary screening with HPV-test. Based on our work and on the work of Schiffmann and his colleagues, 201811 as well as the results of HPV-screening, we come to the conclusion that the only use of HPV-subtyping is not a secure method and a protective way for women. In the work of Baay M.F.D and his colleagues17, there were 13% HPV-negative cancers. There are too many HPV-positive cases, which have been psychologically impaired with higher costs, although they have no cervical epithelial changes during the HPV-infection. There are too many HPV-negative cases, which develop cervical cancer. We have the opinion and are convinced that the screening should be both morphologically via cytological examination and may be with adding immunocytochemistry as presented by Abbas and his colleagues, 20222 and the HPV-Genotyping as an automated fast method, but the results of both should be respectively analyzed and both clinicians and patients should be accordingly advised.Cervical cancer represents 570,000 new cases per year and accounts for the vast majority of all HPV-attributable cancer cases worldwide. 311,000 women died of the disease


HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) are sensitive to chemo-radiation therapy and have favorable survival outcomes compared with HPV-negative cancers. These tumors are usually not related to tobacco and alcohol exposure. Therefore, diagnosing HPV-positive OPSCCs for the appropriate disease management is crucial, and no suitable markers are available for detecting early malignancies in HPV-infected tissues. In this study, we attempt to find HPV-specific epigenetic biomarkers for OPSCCs.DAPK, FHIT, RASSF1A, TIMP3, AGTR1, CSGALNACT2, GULP1 and VGF) was analyzed by quantitative-methylation specific PCR (QMSP), and their associations with HPV positivity or RB/p16 expressions were evaluated.A total of 127 surgical samples were analyzed for HPV positivity and promoter methylation of a panel of genes. HPV detection was performed by PCR detection of HPV E6 and E7 viral oncoproteins. In addition, promoter methylation of a total of 8 genes (AGTR1 and FHIT were frequently methylated in HPV-positive OPSCC samples with a good area under the curve (AUC over 0.70). In addition, these genes' promoter methylation was significantly associated with p16 positive and RB negative cases, which were the characteristics of OPSCC cases with favorable survival outcomes. Either AGTR1 or FHIT methylated cases were significantly associated with HPV-positive cancers with 92.0% sensitivity (P < 0.001). Also, they had significantly better overall survival (P = 0.047) than both unmethylated cases.AGTR1 and FHIT methylation demonstrated a suitable detection marker of OPSCCs derived from the HPV-infected field, familiar with p16-positive and RB-negative phenotypes.A combination of The online version contains supplementary material available at 10.1186/s12885-022-10227-w. Oral Human papillomavirus (HPV) infection is strongly associated with oropharyngeal squamous cell carcinoma (OPSCC) , 2. AbouEpigenetic alterations of critical cancer-associated genes are considered as a hallmark of cancer. Van Kempen PM et al. reported a systematic review of differential methylation profiles between HPV-positive and HPV-negative OPSCC  in 2014.The primary purpose of this study was to identify differentially methylated candidate genes between HPV-positive OPSCCs and others. They could be used as markers for non-invasive early molecular detection approaches (screening) using bodily fluids  and non-invasive monitoring of recurrent disease. The secondary purpose was to find the correlation of methylation markers with representative HPV-related carcinogenesis pathway factors, p16 and RB.A total of 94 patients undergoing surgical resection of primary untreated OPSCCs at The Johns Hopkins Hospital from 1997 to 2008 were included. Written informed consent was obtained from all subjects and their legal guardians recruited under this protocol before participation in the study. All samples were obtained as anonymized materials following the Declaration of Helsinki. They were classified into 50 HPV-positive and 44 HPV-negative cases using the PCR detection method focusing on HPV E6 and E7 regions of HPV type 16 following the previous paper from our lab . Th. ThDAPK 3 [AGTR1 , CSGALNA2, GULP1 , 17 and LP1 [VGF , 19) werLP1 [VGF , 18, 20.Taq D (Invitrogen), dATP, dCTP, dGTP, and dTTP in a concentration of 200 μmol/l each and 6.7 mmol/l MgCl2. Amplification reactions were carried out in 384-well plates in a 7900HT Fast Real-Time PCR System  and were analyzed by the Sequence Detector System software . The relative level of methylated DNA for each gene in each sample was determined as a ratio of the QMSP value of the amplified gene to ACTB, multiplied by 1000 for easy tabulation, following the protocol we previously published [Bisulfite-modified genomic DNA samples served as templates for QMSP. Primers and probes were listed in Supplementary Table Sublished .IHC for RB was performed using the G3-245 mouse monoclonal antibody  at a dilution of 1:2000 after 20 min of steaming in 10 mM citrate buffer. Labeling was visualized using the DAKO LSAB kit . The percentage of neoplastic cell nuclei labeling in each case was assessed. Slides were considered RB negative when no neoplastic cell nucleus was stained. Detailed procedures and standardized IHC photos for RB staining were shown in a previous paper IHC for p16 was performed using the monoclonal antibody 16PO4 at a dilution of 1:100 . Positive p16 labeling was defined as the presence of nuclear and cytoplasmic reactivity. The percentage of cells showing nuclear and cytoplasmic labeling was recorded. The detailed procedure and standardized IHC photos for p16 staining were shown in the previous paper .For both antibodies, negative controls were performed by omitting the primary antibody incubation step. The staining intensity was evaluated by a senior pathologist and scored as 1 (Strong over 50%), 2 (Stained in 20–50%), 3 (Weak), or 4 (Negative). In addition, we defined p16 or RB expression level as follows: Positive (scores 1 and 2) and Negative (scores 3 and 4) because sample distributions were dramatically changed between HPV positive and negative samples by this threshold  curve analysis. All statistical analyses were performed using JMP 16 software . The level of statistical significance was set at A total of 127 surgical samples were included in this study. The incidence of HPV-positive and HPV-negative cases is significantly different considering gender, smoking history, and alcohol consumption  and FHIT  were frequently methylated in HPV-positive OPSCCs compared to others with fair AUC values (> 0.700). Interestingly, GULP1 and RASSF1A promoter methylation frequency was significantly higher in HPV-negative cases than in HPV-positive cases.We established an optimal cut-off value for each of the eight genes using 50 HPV-positive OPSCC cases and 77 others, including 44 HPV-negative OPSCCs and 33 controls by ROC curve analysis Fig. . All HPVAGTR1 and RASSF1A methylation were associated with advanced age, while CSGALNACT2, FHIT and GULP1 were associated with race. No drinking history affects DAPK methylation, and VGF methylation influences tumor grades 1–2.The association of promoter methylation of candidate genes with other clinicopathological parameters is shown in Supplementary Table SThe Cox proportional hazards model was used for univariate analysis to find prognostic factors of disease-specific survival of 94 OPSCCs. Age, race, smoking, alcohol consumption, HPV negative, p16 negative and RB positive were significant factors of disease-specific survival Table . Some ofAvailable immunohistochemical staining (IHC) data of p16 and RB were 89/94 (94.7%) and 54/94 cases (57.4%) respectively. The results of IHC were categorized into four groups. 1: Staining over 50% cells, 2: Staining in 20–50% cells, 3: Staining in 1–20% cells, and 4: Negative. Details of staining scores and the number of samples in each category are available in Supplementary Table SAGTR1, DAPK, FHIT and TIMP3 genes significantly associated with p16 positive cases . In contrast, CSGALNACT2, DAPK and FHIT promoter methylations were significantly associated with RB negative cases  were HPV-positive associated OPSCC markers. Among the novel 4 genes tested in this study, AGTR1 was strongly correlated with HPV-positive OPSCC characteristics.To determine whether there is any correlation of p16 or RB expression with candidate gene methylation, the distribution of QMSP values among IHC positive and IHC groups was compared by the Mann–Whitney U test. Promoter methylation of AGTR1 and FHIT methylation markers based on AUC over 0.7 of ROC analysis results . Also, they had significantly better overall survival  expression through p53 degradation (24), affecting the promoter methylation of relevant genes. Another group found that methylation arises as a host cell's defense mechanism to silence viral DNA . AlthougAGTR1, DAPK, FHIT, TIMP3 and VGF. Among the analyzed genes, promoter methylation of RASSF1A is less frequent in HPV-positive cases than HPV-negative cases, consistent with the previous report that promoter methylation of RASSF1A is inversely correlated with HPV infection in head and neck and cervical cancer [Several DNA methylation markers have been identified in HPV-positive cervical cancers , 33. Thel cancer , 34. Altp53 mutation is not common [n = 23) were compared with p16-/RB + cases (n = 24). Only negative smoking history  was a significant factor of p16 + /RB- phenotype. Disease-specific survival of p16 + /RB- cases was significantly favorable than p16-/RB + cases . The promoter methylations of AGTR1, CSGALNACT2, DAPK, FHIT and TIMP3 were significantly correlated with the p16 + /RB- phenotype , AGTR1 showed a significant correlation with cancer specificity, HPV infection specificity and p16 + /RB- phenotype  [AGTR1 methylation was found in 60% of NSCLC and 65% of colorectal cancers. Mitra et al. revealed that AGTR1 was a target of oncogenic miR-155, and was silenced via miR-155 up-regulation in head and neck cancer [AGTR1 expression was influenced by promoter hypermethylation.Among four novel methylation markers in OPSCC   and colo (NSCLC) . They shk cancer . They alA panel of OPSCC-specific and HPV-specific methylation markers can be available for early oropharyngeal cancer detection. One of the advantages of methylation marker detection is its high sensitivity and rapidity. We previously reported a rapid gene methylation detection procedure for head and neck cancers . It takeThere are some limitations of our study. First, since most cases were symptomatic and advanced and the samples came from surgically resected cases, the results may partially reflect the characteristics of advanced cancers. Second, an optimal cut-off of methylation level may be the cohort-specific value, which needs to be optimized by multiple data set testing. Third, we used a relatively small number of the single institute sample set, and data should be ascertained by more extensive multicenter cohort data. Finally, biological studies must be performed in contrast with HPV infection and promoter methylation.ATGR1 methylation and FHIT methylation could be a sensitive biomarker of HPV-positive OPSCCs, which has a characteristic of the p16 positive and RB negative phenotypes.In summary, we have detected a panel of novel HPV-positive and OPSCC-specific methylation markers. Especially, the combination of Additional file 1: Supplementary Figure S1. ROC Curves for determining optimal cut-off values between HPV-positive OPSCCs (n=50) and other samples including HPV-negative OPSCCs (n=44) and control samples (n=33). Supplementary Figure S2. Disease-specific survival curves of 94 OPSCCs depending on various clinicohistological factors, including HPV status.Additional file 2: Supplementary Table S1. Candidate promoter methylated genes. Supplementary Table S2. Primers and probes for QMSP. Supplementary Table S3. Association of methylation markers with clinicopathological data (n=94). Supplementary Table S4. Classification of cases based on p16 and RB Staining. Supplementary Table S5. Correlation of QMSP values with p16 and RB expression (Mann-Whitney U test).


Lactobacillus crispatus can restore eubiosis in women with HPV infections and hence achieve viral clearance.Recent studies have shown the importance of the microbiota in women's health. Indeed, the persistence of Human Papilloma Virus (HPV)-related lesions in patients with dysbiosis can be the antechamber to cervical cancer. The aim of this study was to evaluate whether long term administration of oral Lactobacillus crispatus M247  versus the control group, that hence only in follow-up .In total, 160 women affected by HPV infections were enrolled at the Department of Gynecological Obstetrics of “San Paolo” Hospital, Italy between February 2021 and February 2022. The women were randomly assigned to two groups, one in treatment with oral p = 0.05). Total HPV clearance was shown in 9.3% of patients undergoing only follow-up compared to 15.3% of patients in the group taking long term oral Lactobacillus crispatus M247 (p = 0.34). However, the percentage of HPV-negative patients, assessed with the HPV-DNA test, documented at the end of the study period was not significantly different from the control group.After a median follow-up of 12 months (range 10–30 months), the likelihood of resolving HPV-related cytological anomalies was higher in patients in treatment with the long term oral probiotic (group 1) versus the group that perfom only follow-up (group 2) (60.5% vs. 41.3%, Lactobacillus crispatus M247 than in the control group. Larger studies are warranted, but we believe that future research should be aimed in this direction.Despite the limitations of our analysis, we found a higher percentage of clearance of PAP-smear abnormalities in patients who took long term oral Trial registration This study is retrospectively registered. Lactobacillus crispatus M247 in patients with HPV infections could restore eubiosis and consequently achieve viral infection control [Lactobacillus crispatus M247 in order to modify the microbiota and increase HPV clearance, was recently evaluated [Lactobacillus crispatus M247 is a tested probiotic for oral administration shown to have fecal and vaginal colonizing properties [Lactobacillus crispatus M247 on cervical cytology and the persistence of HPV infection. According to our experience, albeit partial, oral administration of the Lactobacillus crispatus M247 strain is a safe treatment and has a real potential to achieve the regression of PAP-smear alterations.The vaginal microbiota in healthy women (eubiosis) contains a diversity of anaerobic and aerobic microorganisms. Lactobacilli are the most common, principal subpopulation. In selected situations, this balance can deteriorate (dysbiosis) and other pathogens may develop, decreasing the host anti-bacterial defenses . The los control , 11. Thevaluated . Lactobaoperties , 14. We Lactobacillus crispatus M247 is a well-documented probiotic for oral administration that demonstrates fecal and vaginal colonizing properties. The rationale for the use of Lactobacillus crispatus M247 in HPV-infected women are the evidences showing that that Lactobacillus crispatus is detected more recurrently in patients without human papillomavirus (HPV) infection [nfection –13.Lactobacillus crispatus promotes colonization in the intestine that, in a few days [Lactobacillus crispatus that feeds the vaginal compartment even when the probiotic is no longer taken for a few weeks/months, which would not be possible after only topical application of the product. Thanks to the adhesion properties of Lactobacillus crispatus M247, this probiotic can long remain within intestinal and vaginal cells [The administration of oral lactobacilli is based on the evidence that the intestinal and vaginal microbiota are linked, and that most vaginal dysbiosis may originate at the intestinal level –17. Partfew days  allows tal cells .Lactobacillus crispatus for 12 months (range 9–14 months). Group 2 (n = 80) constituted the control group that undergoing only routine follow-up every six months. The wet mount microscopy, PAP-smear and colposcopy were performed for every enrolled patient every 6 months, while the HPV-DNA test was repeated after 12 months. As literature reported, wet mount microscopy reflects the functional vaginal lactobacillary flora better than the Gram stain [This is a pilot study, performed between February 2021 and February 2022 at the Department of Gynecological Obstetrics “San Paolo “of Bari. The initial study group included 205 sexually active women, aged between 30 and 64 years old, with a Papanicolaou test reporting low-grade cervical cytology in the form of atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (L-SIL) and with positive HPV-DNA test. Exclusion criteria were pregnancy or breastfeeding, CIN2–3, immunological diseases or corticosteroid treatment, severe comorbidities. Patients were enrolled after a detailed explanation of the study aims, and underwent routine follow-up. Of the 205 patients selected according to the inclusion criteria, 160 women finally took part; features of the enrolled patients are summarized in Table am stain . Lactobaam stain . In caseThe Pap test was executed by collecting exo- and endocervical cells by means of an Ayre spatula and a cytobrush. The cells were successively streaked on a glass slide and spray fixed. Smears were classified according to the 2014 Bethesda System . The tesIn total, 160 women aged between 30 and 65 years, with low-grade cervical cytology in the form of atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (L-SIL), or with a negative PAP-smear but positive HPV-DNA test for HPV infection, were selected for this study. For each patient the following parameters were recorded.p ≤ 0.05. All statistical analyses were performed using the PlotIT program Ver. 3.2 .Bacteriological examination every 6 months, PAP-smear and colposcopy every 6 months, HPV-DNA-test every 12 months. The collected data were inserted into an Excel database  and subjected to Least Significant Difference Testing, set at Patients referred a greater than 98% adherence to therapy; they declared that the treatment was well tolerated, with no significant side effects. Only nausea was reported by 2 patients, that reported taking it in the morning, so we suggested taking it before meals and the disorder subsided.Lactobacillus crispatus M247 (lMG-P-23257) The product is manufactured by Alfa Omega  and traded by Pharmextracta SpA . The product was notified to the Italian Health Authorities in 2019 (March 1st) with notification number 115450.The probiotic product used in our clinical study was Crispact® sachets. Each sachet contains no fewer than 20 billion colony forming units of Lactobacillus crispatus M247 long-term oral users   versus the control group. . The most common HPV genotype was type 16, found in 27.6% of positives, followed by type 51, 45 and 35 (17.1%), type 18 and 52 (16.1%) and type 68 (14.0%). HPV genotype 66 was found in 12.3% and 58 (9.5%) of positives.In total, 160 women were included in the study. Each patient underwent fresh bacteriology  testing before the PAP test; signs of infection was elicited in 25% of group 1 patients  and 18.4% of Group 2 patients . Moreover, 95% of patients with vaginosis in group 1 and 98% in group 2 also referred intestinal disturbances . Vaginosis was identified following Amsel criteria  and by tp = 0.034) in patients with only follow-up (9.3%) as compared to patients who took long term oral Lactobacilli (15.3%).However, HPV-DNA negativity documented at the end of the study period, after a median follow-up of 12 months (range 9–14 months), was not significantly different (Cervical cancer is the most common neoplasia among patients in developing countries and the second most frequent female cancer worldwide . FrequenThe incidence of HPV could vary by country . ParticuIndeed, even if the effectiveness of vaginal lactobacilli appears limited, the concomitant use of oral and vaginal lactobacilli could be considered to potentially obtain HPV negativity in less time and probably also to reduce recurrences , 47. ConLactobacillus crispatus M247 could improve the microbiota and consequently the cytology, while the efficacy on definitive viral clearance, demonstrated with the HPV test, has still to be fully explored. Therefore, even if the data are encouraging, further studies of larger samples are warranted to verify the effectiveness of long term therapy with a probiotic, such as the analysis of microbiota community state types. Moreover, in cases of persistence, the viral strain could be evaluated by viral genotyping.The results of our study appear to encourage the use of probiotics in patients with low-grade HPV lesions. Therefore, our data must be carefully evaluated, since in the HPV natural history, 60–70% of patients undergo spontaneous regression within 12 months . ConsequLactobacillus crispatus M247 showed a potential effect on resolving cervical abnormalities, through restoring the physiological vaginal balance. Indeed, the high percentage of clearance of PAP-smear abnormalities obtained with oral Lactobacillus crispatus M247 long term administration offers encouraging evidence. For further confirmation, a metagenomics analysis of the vaginal, cervical, and intestinal microbiota in HPV infected women should be executed. Consequently, our experience could be a starting point for further discussions on this topic.In this study, long term administration of oral


Background: HPV vaccine hesitancy is a key contributor to the sub-optimal HPV vaccination uptake in the United States. We aimed to determine the association between healthcare providers’ self-efficacy in HPV vaccination hesitancy counseling and HPV vaccination acceptance after initial and follow-up counseling sessions. Methods: Population-based cross-sectional study of healthcare providers (HCPs) practicing in Texas. Logistic regression analyses were used to determine the odds of HPV vaccination acceptance by vaccine-hesitant patients. Additionally, generalized estimating equations were used to compare HPV vaccination acceptance by hesitant patients after follow-up versus initial counseling sessions. Results: 1283 HCPs completed the survey with a mean (SD) age of 47.1 (11.3) years. HCPs who believed that they were very/completely confident in counseling HPV-vaccine-hesitant parents had higher odds of observing HPV vaccination acceptance very often/always after an initial counseling session (adjusted odds ratio (AOR): 3.50; 95% CI: 2.25–5.44) and after follow-up counseling sessions  compared to HCPs that perceived they were not at all/somewhat/moderately confident. The odds of HPV vaccination being accepted very often/always by vaccine-hesitant parents was 61%  higher after follow-up counseling sessions compared to an initial counseling session. The results were similar for the counseling of HPV-vaccine-hesitant adult patients. Conclusions: The confidence level of HCPs in counseling hesitant parents and adult patients impacts HPV vaccination acceptance. Importantly, acceptance was higher after follow-up counseling sessions than initial counseling sessions. HCPs should receive training in HPV vaccination counseling to enhance their confidence in counseling hesitant patients and should utilize every visit to counsel hesitant patients. HPV vaccination is safe and effective as a public health intervention to prevent HPV-associated cancers and minimize HPV-related morbidities and mortalities ,2,3,4,5.The rising HPV vaccine hesitancy among parents of adolescents has been reported as one of the reasons for the low HPV vaccination rate ,11. MoreIn addition to parental or patient factors, provider factors, such as their confidence level and knowledge of HPV vaccines, are notable modifiable factors impacting vaccination coverage rates ,22,23. TThis was a cross-sectional study from a population-based survey of HCPs in Texas conducted between January and April 2021. The survey was developed and distributed by the University of Texas MD Anderson Cancer Center after a series of testing and feedback from key stakeholders. A pilot test was conducted in December 2020 to ascertain questionnaire usability, comprehensibility, the questions flow, and integrity of skip logic. The target population included all HCPs currently practicing in Texas whose email addresses were available in the LexisNexis Master Provider Referential Database . HCPs weHCPs observed acceptance of HPV vaccination by hesitant parents of pediatric patients (9–18 years) were assessed separately following initial and subsequent (follow-up) counseling sessions based on two survey questions: After you have counseled HPV-vaccine-hesitant parents, please indicate the overall frequency at which they accept vaccination when their child is 9–18 years old: (1) After initial counseling session; and (2) Following subsequent counseling sessions. Possible responses to both questions were “Never”, “Rarely”, “Sometimes”, “Very often”, “Always”, and “Not sure”. Each dependent variable was recategorized as a binary variable, 0 = “Never/Rarely/Sometimes” and 1 = “Very Often/Always”. All “Not sure” responses were dropped.HCPs observed acceptance of HPV vaccination by vaccine-hesitant adult patients were assessed separately following initial and subsequent (follow-up) counseling sessions, as above, with identical possible responses and recategorization, with the only difference being a modification of the question to consider the older age group: After you have counseled HPV-vaccine-hesitant adult patients (>18 years old), please indicate the overall frequency at which they accept vaccination: (1) After initial counseling session; and (2) Following subsequent counseling sessions.The primary independent variable was HCP’s perceived self-efficacy or confidence in knowledge and ability to counsel hesitant parents of pediatric patients, as well as hesitant adult patients using the following two questions: (1) How confident are you in your knowledge and ability to counsel parents who are hesitant to vaccinate their child? (2) How confident are you in your knowledge and ability to counsel HPV-vaccine-hesitant adult patients (>18 years)? Possible responses to both questions were “Not at all”, “Somewhat”, “Moderate”, “Very”, or “Completely”. In order to compare HCPs with high self-efficacy to those with low/moderate self-efficacy, we recategorized our independent variables as binary, 0 = “Not at all/Somewhat/Moderate” and 1 = “Very/Completely”.We assessed the following HCP-related factors: age , sex , race/ethnicity , and region of practice . We used the zip codes where HCPs primarily worked to determine their region of practice based on the 2013 Rural-Urban Continuum Codes (RUCC) . RUCC cop-value < 0.05.Descriptive statistics were presented for the independent variables stratified by each dependent variable using proportions and Pearson’s chi-square test for comparison. We had separate logistic regression models for each of our four study outcomes. We predetermined variables for inclusion in our models based on the literature and relevance to our study aim. Multivariable logistic regression analyses were used to estimate the odds of HPV vaccination acceptance by both hesitant parents of pediatric patients, as well as hesitant adult patients after initial and follow-up counseling sessions while adjusting for all covariates included in the final models. Additionally, we examined the association between these covariates and HPV vaccination acceptance by hesitant patients. Furthermore, we estimated the odds of HPV vaccination acceptance by both hesitant parents and adult patients after follow-up counseling sessions compared to the initial counseling session using generalized estimating equations (GEE) with logit link and binomial family. In addition, for our GEE models, we utilized an exchangeable correlation structure with robust standard errors for computation. Multivariable analyses for logistic regression and GEE models were adjusted for HCPs’ age, sex, race/ethnicity, region of practice, years in practice, number of patients seen per week, role in practice, and facility type. All analyses were conducted using Stata/IC Version 15.1. Statistical significance was set as a two-sided A total of 1283 HCPs completed the survey representing a response rate of 7%. There was no significant difference between respondents and non-respondents with regard to provider type and sex for which data was available for non-respondents. The mean (SD) age of participants was 47.1 (11.3) years. Overall, 966 (77%) of the HCPs were females and 297 (23%) were males. Of the total HCPs, 614 (48%) provided vaccination services to both adult and pediatric patients, 404 (31%) provided vaccination services to only adult patients, and 265 (21%) provided vaccination services to only pediatric patients See .p-value: <0.001). Additionally, there was a difference in the distribution of age category , practice type , and years of practice experience  among HCPs that often/always versus never/rarely/sometimes observed HPV vaccination acceptance by hesitant parents after an initial counseling session.Of the 879 HCPs that provided pediatric services, 548 (62%) responded to the question on the frequency of HPV vaccination acceptance by hesitant parents of pediatric patients after an initial counseling session . Of thesAs seen in p-value: <0.001). Additionally, there was a significant difference in the frequency of years of practice experience among HCP that often/always compared to those that never/rarely/sometimes observed HPV vaccination acceptance by hesitant parents after follow-up counseling sessions .Based on the same 879 HCPs as above, 537 (61%) responded to the question on the frequency of HPV vaccination acceptance by hesitant parents of pediatric patients after follow-up counseling sessions . Of thesFollowing multivariable logistic regression analyses , HCPs whResults of the generalized estimating equation  revealedp-value: <0.001).Of the 1018 HCPs that provided adult services, 462 (45%) responded to the question on the frequency of HPV vaccination acceptance by hesitant adult patients following an initial counseling session . Of thesFollowing multivariable logistic regression analysis , HCPs whp-value: <0.001).Based on the same 1018 HCPs as above, 456 (45%) responded to the question on the frequency of HPV vaccination acceptance by hesitant adult patients after follow-up counseling sessions . Of thesResults of multivariable logistic regression analysis  revealedAdditionally, results of the generalized estimating equation analyses  showed tIn this study, using a sample of HCPs currently practicing in Texas, we found that those perceiving greater self-efficacy in counseling hesitant patients were more likely to observe HPV vaccination acceptance either after an initial counseling session or after follow-up counseling sessions. The latter finding corresponds with a previous study that found a two-time increase in odds of secondary acceptance after a follow-up counseling session from an HCP . These rNotably, the positive association between HCPs’ perceived self-efficacy and observed HPV vaccination acceptance after initial and follow-up counseling sessions held true for hesitant parents of pediatric patients, as well as for hesitant adult patients. Indeed, since HPV vaccination acceptance is higher among adolescents than young adults, our finding points to the far-reaching effect of providers’ self-efficacy in increasing HPV vaccination acceptance across different patient demography ,35. We aOur study also revealed that hesitant parents of pediatric patients and adult patients are more likely to accept HPV vaccination following repeated counseling sessions than an initial counseling session. This finding supports a previous study, which revealed that patients’ interaction with the healthcare system and regular visits to a provider are associated with HPV vaccination initiation . In addiFurthermore, our study has important implications for increasing HPV vaccination by targeting provider-level factors such as self-efficacy in counseling hesitant patients. HCPs would benefit from HPV vaccination training in line with the CDC ACIP guidelines for providers to counsel and recommend the HPV vaccines to their patients at every clinical encounter regardless of whether they were counseled but refused vaccination during their previous clinic visits . VariousOur study has some limitations. This was a cross-sectional study; as such, we are unable to infer causality. In addition, HPV vaccination acceptance was the perception of HCPs and may not reflect actual event rates. This study was designed from the perspective of HCPs who reported changes in HPV vaccination acceptance among pediatric and adult patients. Given that we did not directly sample patients, our study cannot provide insights into HCP’s self-efficacy in counseling hesitant pediatric patients transitioning into adulthood (≥18 years) between initial and follow-up counseling sessions. Additionally, there is the potential for recall bias as HCPs had to recollect their experiences in the past. Furthermore, this study may be prone to residual confounding from unmeasured covariates. For example, we did not account for time-changing variables, such as HCP training, that may have occurred between initial and follow-up counseling sessions. However, the generalizability of our results is increased by using a statewide survey of HCPs in Texas.In conclusion, we found that HCPs more confident in counseling hesitant parents and adult patients were more likely to observe HPV vaccination acceptance after an initial or follow-up counseling session. However, HPV vaccination acceptance was more frequent after follow-up counseling sessions compared to an initial counseling session. HCPs’ age and race/ethnicity were associated with HPV vaccination acceptance when counseling hesitant parents. HCPs should utilize every opportunity to counsel HPV-vaccine-hesitant patients. Additionally, HCPs will benefit from interventions to enhance their self-efficacy in counseling hesitant patients and their caregivers. To better understand the effect of multiple counseling sessions on HPV vaccination acceptance, future studies should evaluate the number of counseling sessions predictive of HPV vaccination acceptance by hesitant patients.


It is realized that the first intron plays a key role in regulating gene expression, and the interactions between the first introns and other introns must be related to the regulation of gene expression. In this paper, the sequences of mitochondrial ribosomal protein genes were selected as the samples, based on the Smith-Waterman method, the optimal matched segments between the first intron and the reverse complementary sequences of other introns of each gene were obtained, and the characteristics of the optimal matched segments were analyzed. The results showed that the lengths and the ranges of length distributions of the optimal matched segments are increased along with the evolution of eukaryotes. For the distributions of the optimal matched segments with different GC contents, the peak values are decreased along with the evolution of eukaryotes, but the corresponding GC content of the peak values are increased along with the evolution of eukaryotes, it means most introns of higher organisms interact with each other though weak bonds binding. By comparing the lengths and matching rates of optimal matched segments with those of siRNA and miRNA, it is found that some optimal matched segments may be related to non-coding RNA with special biological functions, just like siRNA and miRNA, they may play an important role in the process of gene expression and regulation. For the relative position of the optimal matched segments, the peaks of relative position distributions of optimal matched segments are increased during the evolution of eukaryotes, and the positions of the first two peaks exhibit significant conservatism. An intron sequence is regarded as a kind of non-coding sequence of interrupted gene, and its functions are being discovered. A large number of studies have shown that intron can regulate gene expression as a kind of regulatory element , for exaThe most basic and important interaction among bases is base matching, for example, the formation of a correct codon-anticodon pair is essential to ensure efficiency and fidelity during translation, and circRNA formed by exon cyclization or intron cyclization contains long flanking introns with complementary repeats . BesidesThe first introns have gained increasing attentions in recent years because of their unique features that are located in close proximity to the transcription, and the distinct deposition of epigenetic marks and nucleosome density on the first intronic DNA sequence , and it The genome consists of an extremely complex network of interactions among functional elements, and its functions are achieved primarily through these interactions. We have known that a complete match between siRNA and targeted genes can lead to targeted genes silencing, and high but incomplete matching between miRNA and targeted genes can suppress gene expression. It means base matching is an important way for non-coding RNA to interact with targeted genes, intron as a kind of non-coding DNA is rich in eukaryote genomes, introns must interact with each other, and the interactions can be embodied by the modes of base matching. Based on this, in this work, the mitochondrial ribosomal protein gene sequences were selected as samples, the characteristics of the optimal matched segments between the first intron and the corresponding reverse complementary sequences of other introns were analyzed, and the variations of the characteristics along with the evolution of eukaryotes were studied.Homo sapiens, Mus musculus, Fugu rubripes, Drosophila melanogaster and Caenorhabditis elegans. Considering that the mitochondrial ribosomal protein gene has many advantages in biological research as a kind of housekeeping gene, they are involved in the key process of all protein translation and have very good evolutionary conservatism  were selected as our samples, they were from ervatism , thus foThe intron sequences were obtained from the above gene sequences, then, they were transformed into their reverse complementary sequences except the first introns. Next, similar alignments were done by the local similarity matching software called Smith-Waterman.FLm, FGCm, and Fmat respectively, they were defined as follows,We calculated the length, GC content, matching rate of each optimal matched segment considering that they must provide the basic characteristics of the optimal matched segments, then divided the optimal matching segments into several groups, respectively, according to their lengths, GC contents or matching rates. And then calculated the frequencies of the optimal matched segments with different ranges of lengths, GC contents and matching rates, marked with FLmi is the frequency of the optimal matched segments whose length are within the ith group, NLmi is the amount of the optimal matched segments in the ith group, and nL is the amount of the groups divided according to their lengths. FjGCm is the frequency of the optimal matched segments whose GC contents are within the jth group, NjGCm is the amount of the optimal matched segments in the jth group, and nGC is the amount of the groups divided according to their GC contents. Fkmat is the frequency of the optimal matched segments whose matching rate are within the kth group, Nkmat is the amount of the optimal matched segments in the kth group, and nmat is the amount of the groups divided according to their matching rates.Where, The lengths of the first introns in different gene sequences are different, we standardized the first introns as sequences with 100 bp length in order to conveniently compare the relative position distributions of the optimal matched segments. The method of length standardization as follows ,iL is the length of the ith first intron, ijN is the jth base site of the ith first intron, and ijn is its relative position corresponding to the ith standardized first intron. In this way, the first introns are all transformed into 100 bp long sequences.Where, According to the base site of each optimal matching sequence on the first intron, each base site of the first intron is scored, if in the optimal matching region, base site is scored 1, but if not, it is scored 0, and the definition of matching score as follows ,ijf is the score of the jth base site on the standardized ith first intron, ian and ibn are the initiation base relative site and the termination base relative site of the optimal matched segments on the standardized ith first intron. Thus, for each optimal matching sequence, the first intron is transformed into a sequence consisted of 0 and 1. if there are m optimal matching sequences in a gene, we can obtain m sequences consisted of 0 and 1. On this basis, we divided the 100 sites of each number sequences into 10 regions on average, the relative position frequency of the optimal matched segments on each site and in each region are defined as follows,Where, Fjr is the relative position frequency of the optimal matched segments on the jth base site of the standardized first intron, Fkr is the relative position frequency of the optimal matched segments in the kth region, ijf is the score of jth base site on the standardized first intron, kap and kbp are the initiation base site and the termination base site of the kth region, iaN and ibN are the initiation base site and the termination base site of the optimal matched segments, and m is the total number of the optimal matched segments in the gene.Where, The optimal matched segments between the first introns and the reverse complementary sequences of other introns in each mitochondrial ribonucleo protein gene of five species were counted, and the dataset of the optimal matched segments was established. Then, the lengths of the optimal matched segments of each species were counted, and the frequencies of the optimal matched segments with different ranges of lengths were calculated by Homo sapiens, Mus musculus and Fugu rubripes are up to 100 bp in length, and the ratio of the optimal matched segments of Homo sapiens concentrating at 90–100 bp is up to 24.6 percent. In addition, the length distribution of the optimal matched segments of Homo sapiens is similar to that of Mus musculus. The results showed that the optimal matched segments of high eukaryotes have a longer length and a wider length distribution than that of the low eukaryotes, and it means the length and the ranges of length distribution of the optimal matched segments are increased along with the evolution of eukaryotes.The lengths of the optimal matched segments of five species are mainly concentrated at 10–50 bp, while some optimal matched segments of FGCm are decreased along with the evolution of eukaryotes, but the corresponding GC content of the peak values are increased along with the evolution of eukaryotes.The distributions of GC content of the optimal matched segments of five species ranged from 0 to 0.9. And comparing the results of the five species, it is found that the peak values of As seen from the matching rate distributions of the optimal matched segments, most of the matching rates are distributed between 60% and 80%. Interestingly, studies showed that the matching rates between miRNA and target mRNA are distributed between 65% and 95% . CompariFr values bigger than 10% are analyzed, the results showed that for Homo sapiens, there are 3 peaks with Fr values bigger than 10%, which are at 20–30 bp, 50–60 bp and 90–100 bp, and there is two peaks with Fr values bigger than 10%, which are at 20–30 bp and 50–60 bp for Mus musculus, 30–40 bp and 60–70 bp for Fugu rubripes and Drosophila melanogaster, but for Caenorhabditis elegans, there is only one peak with Fr value bigger than 10%, which is at 70–80 bp. This also indicates that the relative position frequencies of the optimal matched segments are distinctively differences among the five different species, and the peaks of relative position distributions of optimal matched segments are increased along with the evolution of eukaryotes, but the positions of the first two peaks exhibit significant conservatism.It can be seen from In order to further confirm the conservatism of the relative position distributions of optimal matched segments, As we can see from p-values between any two species are >0.05, it indicates that the differences between any two species were not statistically significant, means, in terms of the distributions on each site of the standardized first intron, the optimal matched segments exhibited high conservatism during species evolution. These results further confirmed the conservatism of the relative position distributions of optimal matched segments, which let us be sure that optimal matched segments are organized and functional sequences for each species.It can be seen from Homo sapiens, Mus musculus, Fugu rubripes, Drosophila melanogaster and Caenorhabditis elegans, respectively. Interestingly, we know that the siRNA, whose length is from 21 to 25 bp, guiding mRNA to silent by perfect complementarity with target mRNA  and the National Natural Science Foundation of China (31860304).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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This finding emphasizes the importance of concomitant screening of the anal region in case of cervical HR-HPV.High-risk HPV (HR-HPV) infection is an established risk factor of cervical cancer. Cases of anal cancer are increasingly observed in women. Since anal infection by HR-HPV is also a risk factor for anal cancer, the aim of this study was to investigate the relation between cervical and anal HPV and dysplasia, as their interaction remains unclear. The present study identified anal HR-HPV as an independent risk factor for patients with cervical HR-HPV , had earlier sexual intercourse , had more sexual partners , more dysplastic cervical cytology findings  and higher prevalence of anal HR-HPV . Furthermore, the HR-HPV group reported more anal intercourse . Multivariate analysis retained anal HR-HPV as independent risk factor for cervical HR-HPV . The results of this study emphasize that it is of upmost importance to screen women for anal HR-HPV when diagnosing cervical HR-HPV.Anogenital human papillomaviruses (HPV) are highly prevalent in sexually active populations, with HR-HPV being associated with dysplasia and cancers. The consequences of cervical HPV infection are well-known, whereas those of the anus are less clear. The correlation of cervical and anal HPVs with the increasing number of anal cancers in women has not been studied yet. The objective of our prospective study was to determine whether cervical and anal HPV correlated in a cohort of women recruited in a university hospital in Switzerland. Recruitment was conducted in the gynecology clinic, the colposcopy clinic, and the HIV clinic. Cervical and anal HPV genotyping and cytology were performed. Overall, 275 patients were included , and among them, 102 (37%) had cervical HR-HPV. Patients with cervical HR-HPV compared to patients without cervical HR-HPV were significantly younger (39 vs. 44 yrs,  Human papillomaviruses (HPV) are naked, double-stranded DNA viruses that rely on epithelium differentiation for productive infections. More than 200 different genotypes infect humans, having a tropism for specific epithelia such as skin, oral, and anogenital mucosa. Upon infection of the basal cells, viral DNA replication is tightly controlled by the host cell as well as viral factors. Viral production is intimately linked to terminal differentiation of the infected epithelial cell so that virions are produced only in the upper layers of the infected epithelium. Most infections clear spontaneously under immunological host control. However, long-term persistence over years can be observed. In those cases, cell host alteration and viral oncogene expression can lead to cellular abnormalities, and potentially to cancer .Anogenital HPVs are mainly sexually transmitted. They can infect various epithelia such as the vulva, vagina, cervix, penis, oropharynx, and anus. Specific HPV genotypes considered as high-risk (HR-HPV) are a necessary cause of most of cervical cancers . The samHIV is a major risk factor of recurrent HPV infection. Other risks such as tobacco use, men having sex with men (MSM), early onset of sexual activity, or multiple sexual partners have also been identified . CervicaThe aim of the present study was to determine if patients with cervical HR-HPV infection had concomitant infection of the anus with HR-HPV as well.ANGY for AnusGynecology. This prospective, cross-sectional, single-center study included women recruited at the Lausanne University Hospital (CHUV), a tertiary university hospital in Switzerland. Patients to be classified either in the cervical HR-HPV-positive group or cervical HR-HPV-negative group were recruited in three outpatient clinics: (1) gynecology clinic (patients with regular problems and follow-up); (2) specialized colposcopy clinic; and (3) HIV clinic. Total number of patients to include was 360, 120 from each of the three outpatient clinics. Based on the prevalence of anal dysplasia in each of the three groups , a sample size of 330 patients was calculated. Considering 10% margin error, the final sample size was set at 360 patients.The study was named Cervical and anal screening of HPV genotypes and cytology were performed by board-certified gynecologists and colorectal surgeons. The operation was completed during a single appointment. A cervical cytology smear (for PAP-test and HPV test), with biopsies if needed , was conducted after vaginal examination and colposcopy on a gynecological chair. The proctology team conducted a proctology examination with high-resolution anoscopy (HRA), along with cytological smears of the anal canal . Using a dedicated brush, ectocervical, endocervical, and anal samples were performed. HPV tests were performed with the MagNaPure 96 Total Nucleic Acids kit , with genotyping of 28 HPV genotypes  by the Anyplex™ II HPV28 kit .Furthermore, all patients were screened for HIV. A self-assessed questionnaire exploring sexual habits and HPV immunization status was submitted to patients as well . FollowiThe study was approved by the Institutional Review Board of “Commission Cantonale d’éthique Vaud” . Informed consent was obtained from all subjects involved in the study.Recruitment was conducted between September 2016 and October 2020, with premature study discontinuation of the clinical trial due to difficulties in recruiting patients and related to COVID-19 epidemic-related restrictions. Statistical power for the main outcome, cervical HR-HPV, was not impacted in the present statistics due to its high prevalence.p values lower than 0.05 were considered as significant. Variables yielding p values lower than 0.05 by univariate analysis were entered into a multivariate logistic regression model to determine variables independently associated with the presence of cervical HR-HPV. All statistical analyses were carried out using XLstat Biomed software .Patients were divided into two groups according to their cervical HR-HPV: positive or negative. The chi-squared test was applied to compare qualitative variables that were expressed as n (percent). The ANOVA test was used to compare quantitative variables that were reported as mean ± standard deviation (SD). Overall, 275 participants were enrolled until premature study discontinuation. Patient demographics are presented in Univariate analysis of cytopathologic factors associated with cervical HR-HPV is shown in p-value = 0.02), smoking , cervical LR-HPV , and anal HR-HPV  as factors independently associated with cervical HR-HPV  is debated, as the prevalence of cancer is low . The appYounger age was identified as an independent risk factor for cervical HR-HPV. As we know to date, there is a clearing of HPV in human cells over time, with some genotypes being more carcinogenic. Young patients tend to have a higher viral load but also different concomitant subtypes of genotypes .https://monographs.iarc.who.int/wp-content/uploads/2018/06/mono90.pdf (accessed on 17 October 2022)), stating that HR-HPVs, when they are found, are more often present with LR-HPVs. These findings support the hypothesis of coinfection/cohabitation models [Low-risk HPV was retained as risk factor for cervical HR-HPV. This is in line with the literature, as stated by Cogliano et al., along with the IARC classification of HPV genotypes , we should expect to see lower rates of HPV in women and decreasing cancer cases ,17,18. AIt could be very interesting to follow up with our participants, in order to observe the evolution of HPV prevalence over time, and the eventual development of cancers. On the opposite, one should not forget the natural course of HPV infection, with most of the viral load being cleared from cervical and anal cells as time passes . Several limitations of the present study need to be addressed. There is an inherent weakness due to the cross-sectional design of the study. For instance, the difficulty to reliably assess both HPV and dysplasia impedes the identification of a causal inference. However, as highlighted by Wang et al., the design is useful to establish preliminary evidence before planning future large-scale studies . The ratIn our experience, anal screening during a gynecologic exam is not acceptable for many women. The results of this study may help to increase the acceptance of anal screening.The strength of the present study is the prospective collection of concomitant samples from both the anus and the cervix, not only in patients with known cervical dysplasia, but also in a comparative group without any lesions. This study supports the assumption, as suggested by Acevedo-Fontánez et al., that women with gynecological HPV-related malignancies are more likely to develop secondary anal cancer than women in the general population .In conclusion, genital HPV is highly prevalent in women, and high-risk genotypes should be closely monitored in order to prevent cervical and anal dysplasia as well as cancer development. Most importantly, when cervical HR- HPV is diagnosed, anal screening for concomitant HPV or dysplasia should be proposed, as there can be dramatic consequences for the health of our patients if neglected.


The Human Papillomavirus (HPV) vaccine has offered a great promise to reduce the cervical cancer burden; its utilization (uptake) however has been lagging. However, the levels and factors associated with the uptake of the vaccine have not been well investigated, especially in the local context.To assess the uptake of human papillomavirus vaccination and its associated factors among adolescent school girls in ambo town, Oromia, Ethiopia, 2020.An institution-based cross-sectional quantitative study design supplemented with the qualitative inquiry was employed to assess Human Papillomavirus vaccination uptake and its associated factors among 422 adolescent school girls in Ambo town, central Ethiopia from December 1–30, 2020. The collected data were coded, entered, and cleaned by using Epi info 7.2.3 and exported to SPSS version 25 for analysis. Descriptive statistics were used to compute summary statistics and proportions. Both bivariate and multivariable logistic regression was employed to identify factors associated with HPV vaccine uptake. Adjusted odds ratio and 95% confidence interval were used for the strength and directions of association. A P-value of < 0.05 was used to declare statistical significance. Qualitative findings have been analyzed with manual thematic analysis.The proportion of HPV vaccination uptake among school girls in this study was 44.4%. Hearing about HPV vaccine , availability of awareness creation , and favorable attitude  were the key identified factors associated with vaccination uptake. In addition, poor perception, fear of side effects, and misunderstanding were among the major factors identified by qualitative findings.There was low uptake of HPV vaccination among the school Adolescents in the study area. Availability of awareness creation programs, favorable attitude towards HPV vaccine, and hearing about HPV vaccine was significantly associated with the uptake of the HPV vaccination. Therefore, awareness creation and behavior change education are mandatory to scale up the vaccination. Cervical cancer is the fourth most common cancer affecting women and accounts for about 7.5% of cancer-related deaths in women globally . More thHowever, it’s a preventable form of cancer through the prevention of human papillomavirus (HPV) infection . This caSince its introduction, many countries have tried to implement school-based immunization programs which were remarkably successful in increasing the adolescents’ vaccination rate. Studies showed that HPV-vaccine uptake levels (at least one dose) varied significantly among countries as low as 1.1% to 94.4%. The review study shows that high-income countries such as Scotland and Taiwan that have successful programs have reached >80% . HoweverHowever, its acceptance by the public has been lagging for several reasons . To explWHO set strategy design and coverage improvement plans to reduce the burden of the disease and to reach the 90% goal of the global cervical cancer elimination program. To address such a strategy, different studies need to be conducted pre-and-post the deliverance of the vaccine expecting a high contribution to the success of the program. Therefore, an evidence-based study on the HPV vaccine and associated factors is a cornerstone for tracking the progress of the program to eliminate diseases caused by HPV and achieve the stated goal. However, in Ethiopia, the vaccine is newly introduced and studies conducted to analyze the vaccine uptake level and its associated factors are very limited. Therefore, this study might be used to identify barriers and factors affecting the utilization of the HPV vaccine which might be used as basic information for further interventions. Additionally, this study aimed at providing important information for policymakers and program implementers at national as well as regional levels, and the information derived from the study considered it would benefit the adolescent by providing directions on service provision. Therefore, this study assessed the uptake of HPV vaccination and its associated factors among adolescent school girls in Ambo town, Oromia, Ethiopia.An institution-based cross-sectional quantitative study design supplemented with the qualitative inquiry was employed to assess HPV vaccination uptake and its associated factors among Adolescent school girls in Ambo town, central Ethiopia from December 1–30, 2020. The qualitative inquiry was included to describe various aspects of behaviors, feelings, perceptions, and experiences related to the HPV vaccination program. Ambo town has sixteen primary schools of which nine are governmental and seven private schools and a total of five secondary schools of which four are governmental and one private school. The school-based registered number of adolescent girls aged 10–19 years among all government and private schools was 10,091. Based on the Ambo town health office report, the first cycle of HPV vaccination has been initiated with a campaign in all primary schools for only 14-year-old girls since 2018.The study was complemented by qualitative inquiry to explore in detail cultural, behavioral, and social factors that might affect HPV vaccine uptake. Accordingly, health professionals working on the HPV vaccination program at Ambo town health offices and health facilities, school teachers who were involved in HPV vaccination program in a town, and adolescent girls aged 14–18 years in schools who have participated in different health clubs  and purposively selected community leaders were interviewed during the qualitative inquiry.The sample size for the quantitative study was determined by using single population proportion formula with a prevalence (p) estimate of HPV-vaccine 50%, 5% of marginal error (d), and 95% confidence level. Finally, by considering 10% as a non-response rate the calculated final sample size was 422.. For the qualitative data, purposively selected key informants consisting of health professionals, community leaders, community volunteer workers, and teachers were organized where the study subjects gave their views on the questions in the key informant interview guide. In addition, some adolescent girls were selected for an in-depth interview.To select the study participants in the quantitative approach, six out of 16 primary schools and two out of five secondary schools in Ambo town were selected by simple random sampling using a lottery method. For each selected school sample size was determined proportionally according to the number of adolescent girls within the school. The study participant was selected from each school using simple random sampling by lottery method Structured questionnaires were prepared in English, translated into Afan Oromo, and then translated back into English to check for consistency. The questionnaires were adapted through reviewing different literature and previous similar studies.The questionnaire tool has eight sections that were developed to assess socio-demographic characteristics, knowledge about cervical cancer, HPV infection and vaccination, perceptions, attitudes towards HPV vaccination, presence of promotion, and sources of information about HPV vaccination and HPV vaccine uptake. Data was collected using self -administered Afan Oromo version questionnaires. To administer the structured questionnaire eight teachers as data facilitators- one for each selected School (experienced data collector) and three Nurses as supervisors were recruited. The training was given for one day on the objective, relevance of the study, confidentiality of information, respondent rights, informed consent, and techniques of interview.For the qualitative data, open-ended questions were prepared by focusing on the types of information required from the participant. Data were collected through an in-depth interview. One interviewer for each in-depth interview was recruited to conduct the interview. An in-depth interview was conducted for each category of adolescents who had not received, received one, and received two HPV vaccinations respectively, and a key informant interview (KII) was conducted on those who know the issue of the concern, an expert opinion, and can give a variety of perspectives about HPV vaccination. The interview had been recorded in tapes which were later transcribed.For the quantitative approach, to maintain the quality of the data, data collectors and supervisors were trained in data collection procedures. Before actual data collection time, the questionnaire (tool) was pretested for validity and reliability on 5% of the sample in schools that were not selected for the actual data collection to assess instrument simplicity, flow, and consistency; thereby possible adjustment or modification was considered/ on a time of deliverance and little correction on some questions had been incorporated. The principal investigator and supervisors were made spot-checking and reviewing all the completed questionnaires to ensure completeness and consistency of the information collected. The data collectors were supervised by three supervisors. Data entry has been done by the principal investigator to keep the accuracy of the data.For the qualitative approach, data quality was ensured by enhancing the trustworthiness and rigor of the study findings. The in-depth interview record and note-taking were transcribed immediately after the interview was conducted. The recorded tapes were listened and transcriptions were read and re-read several times carefully. From the potentially important and provisional notes, the researcher noted the interrelationships, connections, and patterns that emerged in the data. These were developed into themes.The quantitative data were checked for completeness, and consistency of the information obtained from the respondent and entered into Epi info version 7.2.2.6. Then data were exported to SPSS version 25 for cleaning, editing, and analysis. The data were checked for missed values and outliers. Descriptive analysis  was done to describe variables. Both bivariate and multivariable logistic regressions were performed to identify significant factors associated with HPV vaccine uptake. Those variables with p<0.25 in bivariate analysis were considered for multivariable logistic regression analysis. Multivariable logistic regression at the 95% confidence level was used to identify the predictors. A significance level of 0.05 was taken as a cutoff value for all statistical significance tests. Multi-collinearity was detected between predictors when checked using the variance inflation factor for each variable. Accordingly, two variables that yield 9.95 and 11.5 were removed on Multivariable logistic analyses (VIF = 9.95 & 11.5).Data from an in-depth interview were audiotaped and recorded then transcribed verbatim, then coded, arranged by themes, and analyzed thematically. Tape-recorded and note obtained from the in-depth interview were transcribed carefully by investigators into Afan Oromo language word by word and arranged with the written notes taken at the time of the interview. The information was translated into English. Thematic content analysis was employed to describe the ideas obtained from the in-depth interview. Through this process, a verbatim quotation was used to illustrate responses on relevant issues and themes. Finally, it was incorporated with the quantitative findings to provide comprehensive and complete information.In this study, uptake of the HPV vaccine was defined as the proportion of those Adolescents girls who had received at least one dose of the HPV vaccination . A good Ethical clearance was obtained from Ambo University, College of Medicine and Health Science Research review and ethics committee with the reference number AU/PGC/29/2020. Informed oral consent was obtained from each respondent after explaining the purpose and procedure of the study. Written informed consent was obtained separately from parents to ensure their legal preparation on behalf of their children for adolescents aged 18 and below. To keep the confidentiality of any information provided by study subjects, the data collection procedure was anonymous. Participation was voluntary and withdrawal from the study at any time of data collection was considered. No name or other identifying information was included in the questionnaire..A total of 414 respondents participated in the study with a response rate of 98.1%. The age of respondents ranged from 14 to 18 years with a mean age of 15.69 and standard deviations of 1.34 years. Regarding parental educational status, more than a third 155 (37.5%) and 183 (44.1%) of the respondent’s fathers and mothers had no formal education respectively . On the other hand, unavailability of pre-information 70(30.4%) fear of side effects 34(14.78%), and being absent during vaccination day 29(12.61%) were some of the reasons for hesitating to get vaccinated among unvaccinated school girls .More than half 230 (55.6%) had not received any dose of the HPV vaccine, 97 (23.4%) had received one dose and 87 (21%) had received two doses. The proportion of the HPV vaccine uptake in the study area was 44.4%. The main reasons mentioned by the respondents for being vaccinated were having pre-information 41(22.28%), believing that it has benefits 36(19.57%), and encouragement from health workers 34(18.48%) However, the interview identified some thought-provoking factors related to HPV vaccination that were left unidentified by the quantitative data. There was weak integration while the HPV vaccination program/campaign/ and the absence of a standard program was the main challenging factor for its implementation. There was no regular, coherent, and stable program/schedule. This leads uncomfortably among stakeholders which even affects their attitude. This was assured by some of the health extension workers:Some stakeholders’ unfavorable attitude was another challenge that affects the vaccination program. They considered the duty as only given for health care workers, valuing (considering) the benefit, inability to cooperate.”“Most of the adolescent school girls, 367 (88.6%) had good overall knowledge about Cervical cancer, HPV infection, and the HPV vaccine knowledge. More than half of the adolescent school girls 230 (55.6%) had a favorable attitude toward the HPV vaccine. The qualitative inquiry investigated high perceived severity among adolescents. Health workers explained that illness and deaths associated with cervical cancer witnessed in the communities were an eye-opener to parents that cervical cancer is fatal and its prevention is critical. Having high perceived severity of the disease and any prior experience related to its consequence could increase fear of the disease which had a determinant effect on views of the vaccine. Such exposures ultimately encourage communities to vaccinate their daughters.On the other hand, the most common individual barriers to vaccination were concerns about side effects. In addition to this, pain associated with vaccination was another concern. Participants with a higher awareness of their low tolerance for pain felt greater anxiety about the intramuscular injection. Moreover, the vaccine was highly misunderstood as some believed that the vaccine was a hoax by the government, meant to reduce population growth. This was supported by the word of a participant who hasn’t been vaccinated:“If it is not aimed to reduce the population growth why it has been given for females only?” asks the participant of the interview. Many of them associate it with reproductive issues considering rumors circulated in society. In some places, peer influences were highly seen. Especially, from boys who repetitively influence girls by seeing the drug description/leaflet and pressure the girls not to take the vaccine. Even some adolescents had a concern as much as: “I do not trust it since it has been widely spoken that it [HPV vaccine] is to reduce the number of communities, especially the number of Oromos and hence I couldn’t be sure on it”.. Regarding the potential source of information about the HPV vaccine, health workers 170 (41.06%)and mass media 160 (38.64%)were the main sources of information .More than half 213 (51.4%) of the adolescent school girls reported the unavailability of promotion and health education regarding the HPV vaccine at school and 333 (80%) of the girls responded that promotion regarding HPV vaccine by health workers through community outreach is lacking. However, 238 (57.5%) of the respondents retorted to the availability of providing full information about HPV vaccines before vaccination We don’t know when we get the vaccine because they gave us leaflet only and told us as we were recommended to be vaccinated without full information” (in-depth interview of schoolgirls).In both in-depth interviews and KII, there was a strong expression of social connectedness as a community-level facilitator to promote vaccination. In addition to this, adolescent participants described school-based promotion and vaccination as very important, however, there would less emphasis on promotion and health education. “The study also additionally identified the association between uptake of HPV vaccination and some factors or characteristics of the respondent. On bivariate logistic analysis fathers’ level of education, mothers’ occupation, hearing about the vaccine, attitude towards HPV vaccine, availability of promotion in the area, availability of awareness creation about cervical cancer or HPV vaccine at school, and provision full information about HPV vaccine before vaccination were found to be associated with the HPV vaccine uptake.Multivariable logistic regression analysis revealed that fathers’ level of education, mothers’ occupation, hearing about the vaccine, attitude towards HPV vaccine, and availability of awareness creation about cervical cancer or HPV vaccine at school were significantly associated with uptake of HPV vaccination..Accordingly, adolescents who have heard about the HPV vaccine before vaccination had the odds of receiving the vaccine 2.5 times higher than those who haven’t heard about it . The odds of HPV vaccine uptake were two times higher among adolescents who had a positive attitude toward the vaccine than those who have a negative attitude towards the vaccine . Also, the odds of HPV vaccine uptake were 2.5 times higher among adolescent school girls who reported the presence of awareness creation about cervical cancer or HPV vaccine is available at school  Striving for the program coverage and access to the HPV vaccine is very important to decrease the burden of cervical cancer. In this study, the uptake level among the study group was 44.4%. The finding was consistent with a finding from a similar study in the Minakulu sub-county Oyam district, Uganda, in which the uptake was 42.4% . HoweverAdditionally, the study also tried to investigate factors associated with HPV vaccine uptake. Accordingly, hearing about the vaccine/having pre-information about the vaccine before vaccination was significantly associated with vaccine uptake. The result was in line with the qualitative result from a focused group discussion of a study from the Ibanda District of Uganda in which being exposed to information was associated with HPV vaccination . This miOn the other hand, the finding of the study indicated that having a positive attitude is associated with higher odds of HPV vaccine uptake. This is supported by the finding in another study conducted in the Liara district of Uganda; in which the uptake of HPV vaccine among adolescents with a positive attitude is three times higher than those with a negative attitude . As wellMoreover, the availability of awareness creation about cervical cancer or HPV vaccine specifically at school was associated with the vaccine uptake. This was also supported by qualitative findings; were the respondents described as availability of awareness creation in their school (even if for an irregular/short period) help them to gain more information about the benefits of the vaccine which in turn influenced them to take the vaccine. The result of this study was also consistent with findings from Uganda where they revealed the prevalence of HPV vaccine uptake was higher among adolescents who had been encouraged by a health worker or a village health team member to go for HPV vaccination . This maThe study employed a mixed study design to gain detailed data from the research participants. Therefore, the problem has been addressed by both quantitative and qualitative research approaches. On the other hand, the study didn’t address all components of perception which might well be addressed by the health belief model. Also, the study was limited to school facilities, it was difficult to generalize to all adolescents of similar age groups living in the same town.In this study finding, having pre-information about the vaccine before vaccination, attitude, and the existence of awareness creation about cervical cancer or HPV vaccine specifically at school were found to be statistically significant with HPV vaccine uptake. The main barriers to HPV vaccination were lack of pre-information, fear of side effects, poor perception, and no regular program or schedule for the immunization. Therefore, regular HPV vaccination schedules and sensitizations should be done to build positive attitudes towards the vaccine which have paramount importance to increasing the uptake. The finding also indicated the need to improve health education programs for adolescent vaccination. Also, to minimize the effect of the limitations mentioned above scholars with similar interests are recommended to conduct a community-based study.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.