To assess knowledge about human papillomavirus (HPV), vaccination, and HPV-related oropharyngeal cancer (OPC) and to evaluate HPV vaccine acceptability among a sample of undergraduate dental students. All third- and fourth-year dental students enrolled in any of the dental schools in Jeddah, Saudi Arabia, were invited to participate in this cross-sectional study. In total, 500 students completed a self-administered questionnaire on HPV, HPV vaccines, and HPV-related OPC knowledge. HPV, HPV vaccine and HPV-related OPC knowledge scores were calculated. Associations of participants characteristics with the knowledge scores and with willingness to take the HPV vaccine were assessed. Of the participants, 62% had heard of HPV infections, and 57% of HPV vaccines. Average knowledge scores were low: HPV knowledge score 3.8±4.5 out of 16, HPV vaccination knowledge score 0.9±1.6 out of 7, OPC knowledge score 0.9±1.2 out of 5. Clinical-year students had better knowledge and were more willing to take the HPV vaccine than were preclinical-year students, as were those vaccinated for hepatitis B versus those not vaccinated for hepatitis B. Students with higher HPV knowledge scores were more willing to take the HPV vaccine (66%) than were those with lower scores (43%) (p=0.018). Only 4% of males and 12% of females reported taking the HPV vaccine. Of those who refused it, 51% cited lack of knowledge as the primary reason. Knowledge about HPV, its vaccine, and HPV-related OPC is low among this sample of Saudi undergraduate dental students. Improving knowledge among them may increase their awareness, which could affect the care provided to patients. Human papillomaviruses (HPVs) are sexually transmitted, and can cause infections in various body parts, including the oral cavity and oropharynx. There are more than 40 subtypes of HPV. High-risk HPV types, such as HPV 16 and 18, are etiological agents for cervical and anogenital tract cancers, as well as oropharyngeal cancers (OPC), a subset of head and neck squamous cell carcinomas . A study in the United States estimated a 225% increase in the incidence of HPV-related OPC from 1988 to 2004; at that rate, the annual number of HPV-related OPCs would surpass cervical cancer by 2020 . OPC screening and HPV vaccination are important, as no specific diagnostic tool has been established for screening OPC that is equivalent to the Pap smear for cervical cancers . HPV vaccines have demonstrated high effectiveness in preventing cervical and anogenital tract cancers and have the potential to reduce the incidence of OPCs as well . The growing prevalence of HPV-related OPC is becoming a major concern, as it is increasing regardless of the presence or absence of other OPC risk factors  . Dentists can play a role in preventing HPV-related OPC by screening their patients for HPV-related OPCs  and by educating their patients about HPV and its risk factors, as well as encouraging them to get vaccinated. A study conducted among a sample of Florida dentists measured their willingness to discuss HPV vaccines with their patients and found that half of them (52%) fell into the precontemplation (not discussing and not intending) stage, followed by 40% of them in the contemplation (not discussing but willing to) stage, and only 9% in the maintenance  stage . Another study conducted in the United States found that dentists lacked the skills to address HPV-related issues with their patients, mainly because of the dentists’ young age, discomfort about discussing HPV topics, and lack of clarity about the role of dentists in discussing HPV knowledge and vaccines with patients . In addition, HPV knowledge among dental students was reported to be deficient by studies conducted in various countries, including the United States, Malaysia, and the Netherlands, and the need to implement educational material was highlighted . Studies in the Middle East on HPV and vaccine knowledge among dental students are scarce. Acceptance of the HPV vaccine might be influenced by a number of enabling factors or barriers . Several studies suggested that the cost of HPV vaccines is a major barrier, along with uncertainty about the side effects of the vaccine and the fear that daughters might engage in riskier sexual behavior after receiving it . In addition, the acceptability of the HPV vaccine was higher (7-fold increase) among female students than among non-students (such as employees), and the strongest predictor was a physician’s recommendation (93-fold increase) . Given the mounting evidence on the association between HPV and OPC, it is crucial to determine dental student’s knowledge regarding HPV and HPV-related OPCs. Dental students are future healthcare providers who can help increase public awareness and promote HPV vaccination among their patients. To our knowledge, few studies examined dental students’ knowledge about HPV and the acceptability of the vaccine. The objectives of this study were (i) to determine knowledge about HPV and HPV vaccination, (ii) to evaluate knowledge about HPV-related OPCs, and (iii) to assess the acceptability of the HPV vaccine, among a sample of dental students.Study Design and ParticipantsThis cross-sectional study was conducted in Jeddah, Saudi Arabia. The target population consisted of dental students enrolled during the academic year 2018-2019 in any dental school in Jeddah, Saudi Arabia. There are four dental schools in Jeddah; one is governmental, the Faculty of Dentistry at King Abdulaziz University (KAU), and three are private, Batterjee Medical College, Ibn Sina National College, and Alfarabi College. All dental students in these colleges in their third or fourth years were invited to participate in the study. Alfarabi College was excluded, as it did not have fourth-year students enrolled at that time. The reason for recruiting third- and fourth-year students is that they are still at an age when they can receive the HPV vaccine . Third-year students are not exposed to clinical practice, and so we referred to them as “preclinical students,” whereas fourth-year students were referred to as “clinical students.” Ethical approval was obtained from the Ethical Committee at the Faculty of Dentistry at KAU (043-0318). Written permissions were obtained from each dental school, and visits were arranged with school administrators to collect the data. Data CollectionA self-administered questionnaire was distributed to eligible participants between March and October 2018. Two trained medical students distributed the questionnaires to the students in their classrooms after explaining the study objectives. Implied consent was obtained, as students were informed that filling in the questionnaire indicated their agreement to participate in the study. Participants were also informed that their participation was completely voluntary and that the anonymity and confidentiality of their responses would be preserved.Survey InstrumentHave you ever heard of HPV infection?”, followed by a multiple-choice question about sources of HPV knowledge. Seven HPV vaccination knowledge questions were preceded by the question, “Have you ever heard of the HPV vaccine?”. The fourth section consisted of questions about HPV vaccine acceptability, including the following questions: “Have you ever received an HPV vaccine?”, “Would you be interested in taking the HPV vaccine?”, and “What is the main reason you would not take the vaccine?” . Participants were also asked if an awareness program could help them decide whether to take the vaccine or not, and if they were willing to discuss the HPV vaccine with the same and with the opposite gender patients. The fifth section included knowledge questions about HPV-related OPCs . The questionnaire was piloted on 20 fifth-year dental students to ensure clarity, practicability of the questions, and ease of completion. The questionnaire consisted of five main sections. The first section collected information about participants’ characteristics. The second and third sections included questions from a validated questionnaire that assessed general HPV and HPV vaccination knowledge . Sixteen general HPV knowledge questions were preceded by the question, “Statistical AnalysisI don’t know” or an incorrect answer. For each participant, the total general HPV knowledge score and the HPV vaccination knowledge score were calculated by summing the scores of the responses in each corresponding section, with a final score ranging from 0 to 16 for HPV knowledge and from 0 to 7 for HPV vaccination knowledge. In addition, a total HPV-related OPC score was calculated for each participant by summing the scores of the responses to the OPC knowledge questions, with a final score ranging from 0 to 5. Lower scores indicated worse knowledge. The main outcome variables in this study were (i) HPV knowledge, (ii) HPV vaccination knowledge, (iii) HPV-related OPC knowledge, and (iv) HPV vaccine acceptability. A total knowledge score for HPV and HPV vaccination was calculated. The response to each knowledge question was given a score of 1 for a correct answer and 0 for “Vaccine acceptability was categorized into a dichotomous variable, the categories being “Yes” and “No”/“I don’t know.” The associations between the following variables and vaccine acceptability were assessed: gender, academic year, cumulative grade point average (GPA), previous hepatitis B vaccination, previous history of sexually transmitted diseases (STDs), marital status, and the knowledge variables. Continuous variables were summarized by using means and standard deviations, and categorical variables were summarized by using frequencies and percentages. An independent sample t-test and a one-way analysis of variance were used to assess differences in mean values of the knowledge scores for categorical variables, as indicated. The chi-square test was used to assess the associations between categorical variables and vaccine acceptability. A p-value of ≤0.05 was considered statistically significant. SPSS Version 21  was used for data analysis.The questionnaire was completed by 500 out of 602 dental undergraduate students, yielding a response rate of 83%. The characteristics of the participants are summarized in Associations between participants’ characteristics and their willingness to take the HPV vaccine are shown in Around two-thirds of the dental students in our study were aware of HPV infection. HPV and HPV-related OPC knowledge was significantly higher in clinical students than in preclinical students. Although 57% of students had heard of the HPV vaccine, only 9% reported being vaccinated. Overall, the knowledge of dental students regarding HPV vaccination was poor. The baseline knowledge of undergraduate students is an important area of emphasis because it will increase their contribution to primary prevention of HPV-related OPCs . The proportion of students who know of HPV infection varies widely in the literature. A recent study in Jordan found that 80% of the clinical dental students had heard of HPV infection , whereas awareness of HPV was significantly lower among young female non-medical students targeted in Saudi (34.5%) and Nigeria (17.7%) . These findings highlight differences in the educational factor. In the current study, we noted that the lack of knowledge of HPV among dental students was high, which might be related to the low coverage that is given to HPV in educational programs in Saudi Arabia . Similar results have been reported among Indian dental students, where only 37% had heard of HPV infection .Our results are similar to those of other studies in which differences in academic years influenced students’ HPV knowledge . Poelman et al., (2017) reported a significant difference in HPV knowledge between masters students (75.0%) and bachelor students (54.3%). Doshi et al., (2015) found that the mean total HPV knowledge score was higher among interns than among undergraduate dental students, which could be explained by the interns having completed their clinical training. Knowledge of HPV is directly related to academic level, with clinical-year students being more aware of the infection, probably due to the incorporation of theoretical information about HPV in their curriculum. No significant association was observed between GPA and the HPV knowledge score. Those who were previously vaccinated against hepatitis B had higher HPV knowledge scores. We believe that one of the important determinants in receiving the vaccination is knowledge; people aware of the hepatitis B vaccine may have more information about other infectious diseases such as HPV. Although our current study found no statistical difference in knowledge between men and women, the majority of female students answered correctly in regard to HPV knowledge. This finding accords with previously reported results. Almutairi et al., (2019) found that female medical students were more knowledgeable about it than males were (p<0.001), and Rajiah et al., (2017) suggested that the knowledge of their female dental participants had a direct relationship with women’s health, since they were receiving sufficient information about cervical cancer, Pap smear tests, and HPV. Our finding that education was the participants’ main source of information is similar to that of other studies from Malaysia (38.7%) and Saudi Arabia (45.1%) among medical students . This similarity underlines the importance of a university-based curriculum to address HPV knowledge. However, another study found that the media was the students’ main source of HPV knowledge . In our study, the Internet represented 32% of the responses and the media 26% of responses. This result demonstrates the potential for social media for developing effective educational material and interventions tailored to Saudi adolescents. The low vaccination rate of  was similar to that in other studies, which reported a lower vaccination rate among medical Malaysian students (3.6%) and college students in Hong Kong (13.3%) . This rate could be related to the lack of educational programs in schools, the uncertainty over side effects, and cultural boundaries. A review reported the estimated HPV vaccination coverage globally: 1.1% in Asia, 1.2% in Africa, 31.1% in Europe, and 35.6% in North America . Many countries in these regions have added HPV vaccination to their national immunization schedules and raised public awareness about the usefulness of the vaccine in preventing some types of cancer.Similarly, the effect was also reflected in HPV vaccine knowledge scores. In our study, the mean HPV knowledge score was 0.9 on a 7-item scale. This figure is similar to the results of previous studies that reported a low level of knowledge about HPV vaccination among university students in Hong Kong  and Nigeria . This figure contrasts, however, to that in an international comparison study, which reported higher knowledge about HPV vaccine, as measured on a 7-item scale, among United Kingdom (mean 3.8), United States (mean 3.9), and Australian populations (mean 4.0) . In our study, 57% who were willing to take the vaccine had high HPV vaccine knowledge compared with 43% with high knowledge among those not willing to take the vaccine, although the difference was not statistically significant. It is striking that undergraduate dental students were insufficiently knowledgeable about HPV vaccination; this finding highlights the need for future efforts to raise awareness. As Chiang et al., (2016) reported, vaccination uptake is directly correlated with students’ baseline knowledge about HPV vaccination.Forty-five percent of the study participants intended to take the HPV vaccine, a relatively low rate compared with other studies among university and dental students, in which the rate ranged between 48% and 81.7% . Our participants’ low knowledge of HPV vaccination could explain the relatively low rate of intention to take the vaccine. In addition, Saudi society is religious and conservative, and sexually transmitted infections are less likely to occur in the absence of sexual behavior before marriage. Nonetheless, Rajiah et al. (2017) reported that 90% of their dental students would be vaccinated if they had the information. Therefore, knowledge about the vaccine is essential for students to decide about vaccination. In our study, 52% of the clinical students were willing to be vaccinated compared with 41% of the preclinical students. Sixty-six percent of those who scored high in HPV knowledge were willing to be vaccinated, whereas 57% who scored low were not willing. The current study also sheds light on the unwillingness of the participants  to receive the HPV vaccine. Because of the conservative culture of the Saudi population, the sexual nature of HPV infection might introduce barriers to HPV information seeking, vaccination education, and awareness, such as the fear of being labeled sexually active. Of our participants, 23% reported that HPV vaccination had not been discussed or recommended by a healthcare provider, and 20% worried about the safety of the vaccine. These findings are consistent with others: hence the need for strategies at a regional level, as well as community partnerships, constructed carefully so that women feel less stigmatized .Gender was a barrier to discussing HPV with patients. Participants expressed more hesitation in relation to discussing or recommending HPV vaccines with the opposite gender, yet the differences were not statistically significant. The low rate of willingness to address sex-related topics could be related to factors such as education, cultural barriers, religious factors, and individual beliefs . In a study conducted among Florida dentists, 44% of female dentists were in the action stage (recommending the vaccine) . A study of Dutch dental students found that they thought dentists should discuss HPV vaccination with their patients . Findings suggest that dentists have an important role in encouraging HPV vaccination . In our study, 65% of the male students and 68% of the female students thought that public vaccination awareness campaigns would help them to take the vaccine. The growing burden of HPV-related OPCs highlights the need for dental providers to play a role in this emerging public health problem. HPV-related OPC prevalence increases across calendar periods: hence the need to focus on prophylactic HPV vaccination . The incidence of OPC in Saudi Arabia is 2.4% and that of cervical cancer is 1.4% . A recent study that evaluated the prevalence of HPV with head and neck cancer showed an increase in OPCs over the years, 21% being associated with HPV infection . Macpherson et al., (2003) reported that dental practitioners were more confident in identifying potential precancerous lesions than were medical practitioners. Nonetheless, in our study, participants’ overall knowledge about OPC was inadequate (mean 0.9±1.2 out of 5). Only 29% affirmed that there is a link between HPV infection and OPCs (data not shown). Studies showed higher knowledge in linking HPV and OPCs in Dutch (64%) and Spanish (75%) dental students . A study conducted in Saudi Arabia by Kujan et al., (2014) among all dental students in their fourth to sixth years that assessed oral cancer knowledge showed that 90% of dental students identified smoking, 87% identified alcohol, and 84% identified HPV as risk factors. Kujan et al., (2014) reported higher scores for those with a higher level of university education who had some exposure to oral cancer during their clinical placement. In our results, 48% of clinical students linked HPV with OPCs, compared with only 10% of preclinical students (data not shown). This finding highlights the importance of knowledge in identifying HPV as a risk factor for OPC. Another factor could be that clinical students have higher confidence because they have more knowledge and experience . Female dental students demonstrated better HPV-related OPC knowledge than did their male counterparts, and the difference was significant. These findings are aligned with those of an earlier report , suggesting that female students may gain their knowledge from cervical cancer awareness programs. Despite the literature reporting that HPV-related OPCs develop more in males, they scored low in our study . The limitation of this study is that women were overrepresented among our participants, signaling the potential for possible selection bias. Another limitation is that the long-term effect of the students’ education could not be tested, as we targeted participants who were still young enough to receive HPV vaccination according to the CDC’s recommendation . Despite these limitations, our study had a high response rate. Given the sampling technique  and the high response rate, these results should be representative of the dental students of the specified age in the dental schools in Jeddah. However, caution should be practiced when generalizing the results to other dental schools. In addition, previous studies conducted in Saudi Arabia on HPV were within the context of cervical cancer and mostly among women. Our study targeted both genders and inquired about overall knowledge of HPV, vaccination, and its relation to OPC, due to the growing prevalence of HPV and HPV-related OPCs. In conclusion, overall knowledge about HPV, the HPV vaccine, and HPV-related OPCs is deficient among this sample of Saudi undergraduate dental students. Those in their clinical year were more knowledgeable about HPV. More than half of them reported willingness to take the vaccine. Targeting providers’ education programs and addressing the gaps in dental students’ knowledge may increase the students’ willingness to raise public health awareness and recommend HPV vaccination. Further assessment of student knowledge after implementation of an educational curriculum is warranted.


L1 gene, nucleotide variability, and phylogeny were explored in relation to HIV in samples from Botswana and Kenya.The variation of human papillomavirus (HPV) genotypes shapes the risks of cervical cancer and these variations are not well defined in Africa. Nucleotide changes within the A total of 98 HPV-positive cervical samples were sequenced to identify different HPV variants. Phylogenetic inferences were used to determine HPV genotypes and investigate the clustering of sequences between women living with HIV (WLWHIV) and -women not living with HIV (WNLWHIV).Out of 98 generated sequences, 83.7% (82/98) participants had high-risk (HR) HPV genotypes while 16.3% (16/98) had low-risk (LR) HPV genotypes. Among participants with HR-HPV genotypes, 47.6% (39/82) were coinfected with HIV. The prevalence of HR-HPV genotypes was statistically higher in the Botswana population compared to Kenya . Multiple amino acid mutations were identified in both countries. Genetic diversity differed considerably among WLWHIV and WNLWHIV. The mean pairwise distances between HPV-16 between HIV and HIV/HPV as well as for HPV-18 were statistically significant. Six (6) new deleterious mutations were identified in the HPV genotypes based on the sequencing of the L1 region, HPV-16 , HPV-18 (S424P), HPV-45 , and HPV-84 (F458L). The majority of the patients with these mutations were co-infected with HIV.L1 gene were identified in both countries which can be further investigated using functional assays.Genomic diversity and different genomic variants of HPV sequences were demonstrated. Candidate novel mutations within the The online version contains supplementary material available at 10.1186/s12879-022-07081-3. Cervical cancer continues to be a major public health problem particularly in less-resourced countries with an estimated 570,000 cases diagnosed, and 311,000 death rates recorded in 2018 . To dateMolecular epidemiological studies assessing the phylogenetic association of HPVs based on oncogenic risk and supporting specific biological and pathological traits distinctive to HPV genera, species and types are still limited. Previous studies in Africa have explored the frequency and distribution of the HPV genotypes in HIV-infected women , with coL1 region of HPV genotypes were used to determine the mean pairwise distances and assessing for any signature mutations associated with cervical cancer among the HIV/HPV-coinfected patients. Phylogenetic analyses were used to (i) assign HPV genotypes, and (ii) determine clustering of sequences within and between the two countries.The objective of this study was to use Sanger genotyping protocol to detect and genotype HPV isolates in cervical cancer specimens of patients’ living- and not living- with HIV obtained from Botswana and Kenya. Apart from determining the prevalence and distribution of HPV genotypes (LR and HR) among the two groups (HPV versus HIV/HPV-coinfected), the mutation profiles (nucleotide) and amino acid changes) within Formalin-fixed paraffin-embedded (FFPE) tissue specimens from women living with HIV (WLWHIV) and -women not living with HIV (WNLWHIV), diagnosed with invasive cervical cancer previously typed using the Abbot Real-Time polymerase chain reaction (PCR) and Linear Array HPV Genotyping Test, LA-HPV  from prior retrospective cross-sectional studies , 21 fromWe proposed to describe the sequence variation of samples with single HPV infections for the two HR-HPV genotypes (HPV-16 and -18) which are responsible for the majority of cervical cancer cases reported in both countries within the ~ 450 base pairs (bp) region amplified by these HPV genotyping systems. Only samples harbouring HPV-16 or -18 were used in this study, samples with mixed HPV infection were excluded from this study. Demographic and clinical data including age, cancer stage, country of origin and HIV status were obtained for samples from Botswana. However, only HIV status was available for Kenya samples. Cervical cancer staging was performed as previously described .DNA extraction was performed from the tissue samples archived using a previously established protocol . The extL1 targeted by the LA-HPV and the Abbott Real-Time PCR method, conventional PCR for HPV DNA viral amplification of a ~ 450 bp HPV-specific segment from the L1 gene covering nucleotide positions (5722-6162) numbered according to NC001526 HPV-16 reference genome was performed using the forward primer MY09 5′-CGTCCMARRGGAWACTGATC-3′ and the reverse primer MY11 5′-GCMCAGGGWCATAAYAATGG-3′. Five microliters of DNA were added to 15 μL of reaction mix containing 1 × PCR buffer, 0.2 mM dNTPs, 4 mM MgCl2, 0.3 μΜ of each primer, and 2 U/μL of Platinum Taq DNA Polymerase, High fidelity .To isolate a region of The thermocycling conditions were denaturing at 96 °C for 10 s, annealing at 50 °C for 5 s, and final extension at 60 °C for 4 min for 25 cycles. PCR products were subjected to electrophoresis in 4% agarose (Applichem) using 1 × TBE buffer (Applichem) and visualized under UV light. PCR products were then purified using commercially available SureClean Plus (Bioline) and the purified products were sequenced directly via automated sequencing using two overlapping PCR primers (both forward and reverse). The BigDye Terminator v3.0 kit  was used for sequencing using the automated Sequencer .L1 Taxonomy Tool Analysis Results was utilized to check for similarity with the known HPV reference sequences. Additionally, the BLAST tool was used to determine the subset of sequences with high EO value that can be included in the phylogenetic tree. Finally, referenced alignments per each genotype were performed using AliView version 1.26 and were used for phylogenetic analyses while the translated alignment was used for mutation analysis. We developed a specific workflow to answer the study objectives  followed by generation of consensus sequences in FASTA file format, which were used, for downstream analysis. An online Phylogenetic inference was performed using the Bayesian Markov chain Monte Carlo (MCMC) approach implemented in the Bayesian Evolutionary Analysis with the Sampling Trees software (BEAST v1.10.2) with an uncorrelated log-normal relaxed molecular clock, General Time Reversible substitution model, and gamma site heterogeneity. The MCMC was set at a chain length of 500,000,000 with parameters logged every 10,000. The tree was visualized in FigTree v1.4.3 after a 10% burn-in using Tree Annotator v1.8.4. Posterior probabilities 0.90 and above were noted as statistically significant. The sequences generated in this study are available in GenBank under accession numbers (awaiting accession numbers).1 dataset). Here, the top 100 sequences after sorting by E value and covering the full L1 region were obtained from the NCBI blast search and compared to the N1 dataset. Comparisons were done at the aa level to exclude synonymous polymorphisms. Genetic diversity and any signature mutations associated with HIV/HPV co-infection were investigated by comparing the HPV sequences isolated from patients with HPV mono-infection versus those with without. Mutations that had not been noted previously in the literature were termed “candidate novel mutations”. The candidate novel mutations were further assessed using Protein Variation Effect Analyzer (PROVEAN) [To assess for any known mutations or signatures amino acids that may be associated with immune pressure among HPV patients in Botswana and Kenya, 95 aligned sequences with high coverage were included in the analysis (NPROVEAN)  and SNAPPROVEAN)  to deterRaw data were collected, processed and coded using Excel. Study demographics were presented as percentages for categorical variables and compared among participants with HR-HPV and LR-HPV HPV genotype using the Chi-square test. Wilcoxon rank-sum test was utilised to compare continuous variables. Differences among the prevalence of HPV genotypes in Botswana and Kenya was assessed using a comparison of proportion test. All the statistical analysis was done using Stata version 15  and p-value < 0.05 was considered as statistically significant.The present study analysed 98 samples positive for HPV-16 or -18 only from 2 previous studies , 21. AllL1 Taxonomy Tool Analysis. After quality control (QC), 3 sequences were excluded for mutation and phylogenetic analyses because sequences were too short (< 450 bp). In total, 19 HPV genotypes were determined, -6, -11, -16, -18, -35, -39, -42, -45, -53, -54, -55, -56, -58, -59, -73, -81, -89, -122, and -159 . All the studied HPV sequences clustered with reference and had posterior probabilities > 90%, and could be used to assign the genotypes with confidence. Phylogenetic inference with maximum likelihood and MCMC methods showed that there were no isolated clusters among HPV sequences from both countries. This was also true when trees for HPV-16 (n = 29) and HPV-18 (n = 12) sequences from this study and the respective GenBank references for the In total, 68 nucleotide base substitutions were detected using reference sequences for each genotype. Additional file L1 region, HPV-16 , HPV-18 (S424P), HPV-45 , belonging to the HPV HR-HPV group and HPV-84 (F458L) belonging to the LR-HPV group.The present study defined mutations as amino acid changes that differed from that of reference sequence and candidate escape mutations were those that have not yet been reported in 1000 most similar sequences. Six (6) new variants were identified based on the sequencing of the L1 region of HPV positive cervical samples, we were able to confirm the majority of the previously reported HPV genotypes [L1 were found. These observations, in light of the slow evolution of HPV at the population level, suggest that strong selection pressures are at play in each infection cycle. We analyzed the mutation frequency in the panel for HPV genotypes in WLWHIV and WNLWHIV and found mutations within HPV genotypes. We could not assess the clinical relevance  of the detected variants due to small sample size. However, it is interesting that the new variations in high-risk genotypes were only identified in samples from HIV patients. The most striking finding in this report is the high proportion of mutations in the HPV‐genotypes (including both those arising among WLWHIV and those arising among WNLWHIV). Amino acid variations were found in the L1 region of the HPV genotypes , HPV-18 (S424P), HPV-45 , belonged to the HPV HR-HPV group and HPV-84 (F458L) for the LR-HPV group. These alterations are described for the first time, and functional implications resulting from this variation need further analyses. Changes in the L1 region of the HPV genome may be important for discriminating the infectious potential of different variants, as well as in defining epitopes relevant to vaccine design. Some previous studies investigating HPV-16 full-length sequences in cervical specimens have shown that the contiguous deletions identified to be highly associated with cancer are suggestive of a pattern of HPV integration [L2, LCR, E6 and E7.Given the uniquely large and diverse collection of HPV-genotyped cervical samples worldwide, we were able to evaluate the genetic diversity within HPV genotypes and report on the geographic distribution of HPV variants, as well as measure their association with cervical cancer in Botswana and Kenya. Several studies have investigated HPV prevalence and type-specific distribution in cervical cancer patients from Botswana and Kenya, and found that HPV-16 or -18 (HPV-16/18) were identified in 93.8% from Kenya, and 61.8% from Botswana. Non-HPV-6/18 genotypes were detected in 17.2% from Kenya, and 47.8% from Botswana . Again senotypes , 24–26. egration –37. The L1, making it essential to take into account the HPV variants lineages or population stratification when developing vaccines. The limitation of this study was the small sample size for samples from Botswana and Kenya; we could not determine whether the synonymous mutations that are described have any impact on the protein expression. We attempted to investigate HPV full-length sequences in FFPE cervical cancer specimens, but were unable to do so because of highly fragmented samples. Further study is required to determine whether variants represent a higher risk for cervical cancer. Additionally, functional studies regarding HPV polymorphisms across the HPV genome variants should be conducted to explore the biological evidence of carcinogenicity.Our work showed genetic variability of L1 single amino acids changes were identified. New variations in high-risk genotypes were only identified in samples from HIV patients. However, it was not possible to correlate disease severity with any particular variant. The results illustrated that multiple HPV variants are available in both countries. Further studies with complete sequencing of HPV genomes from large population-based and case–control studies of cervical pre-cancer and cancer are required to understand viral carcinogenesis and possibly to improve preventive and therapeutic strategies in the future.We demonstrated the genomic diversity of HPV sequences and phylogenesis of HPV genotypes in Botswana and Kenya samples giving important information as six new Additional file 1: Table S1. Amino acid sequence variation in sequences of L1 gene of HPV genotypes amongst cancers from Kenya and Botswana.


E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein.A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes  and four T cell epitopes . Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs.The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia. Human papillomavirus (HPV) causes the most common viral infection in the human reproductive tract system; it consists of small double-stranded DNA pathogens that infect the epithelium. Recently, more than 200 HPVs have been identified and differentiated by their genomic sequences. Furthermore, around 40 types of those HPV’s variants infect mucosal epithelium, and epidemiology studies found its strong association with cervical cancer. The HPV type-16 causes approximately 50% of cervical cancers globally, while its combination with type-18 increases the cases up to 66%. Additionally, the five-high risk types  are responsible for 15% cervical cancers, and 11% of all cases associated with HPV infection .in vitro method in acidic condition (pH 5.4), generating homogenous L1-VLP 30-40 nm size in the prokaryotic system of Escherichia coli BL21 DE3. Therefore, it is suggested to be promising and suitable for the development of L1 HPV type 52 vaccine.In conclusion, truncated gene encoding L1 HPV type 52 was successfully constructed, expressed, and assembled by


Participants completed a self-administered questionnaire on self-sampling experiences and preferences. Both samples were tested for 14 high-risk (hr) HPV types and E6/E7 mRNA using the Abbott RealTime HR-HPV and Aptima HR-HPV mRNA assays, respectively. Results: The overall agreement for hr-HPV typing between 527 paired samples was good  with high sensitivity (86.2%) and specificity (88.0%). HPV-16  had higher agreement rate than HPV-18  and the other 12 hr-HPVs . Two hundred and eighty-five (285) sample pairs tested for E6/E7 mRNA showed fair agreement . Furthermore, self-sampling was reported as comfortable (90.5%) and painless (86.7%), with 88.4% of women preferring self-collection. Conclusions: Self-collected samples had good agreement with the healthcare professional-collected samples for the detection of hr-HPV DNA and the procedure was highly preferred by women. Self-sampling using SelfCerv can be used as an alternative to healthcare professional sampling in clinic-based routine cervical cancer screening.Background: The SelfCerv Self-Collection Cervical Health Screening Kit  is an applicator tampon designed for self-collection of vaginal samples for the detection of human papillomavirus (HPV) deoxyribonucleic acid (DNA) and E6/E7 messenger ribonucleic acid (mRNA). The study aimed to evaluate the performance of the SelfCerv applicator tampon for the detection of hr-HPV for cervical cancer screening, and further to investigate women’s experiences and preferences regarding self-sampling. Methods: Vaginal samples were collected from 527 gynecology clinic attendees aged ≥18 years at a tertiary hospital in Gauteng Province, South Africa. Self-samples were collected using the SelfCerv kit, followed by endocervical samples collected by a healthcare professional using Cervex-Brush Cervical cancer is one of the most preventable and treatable diseases. However, there were an estimated 604,127 new cervical cancer cases and 341,831 deaths in 2020 worldwide [Persistent infection with one of the oncogenic types of human papillomavirus (HPV), primarily types 16 and 18, is a necessary but not sufficient cause of cervical cancer . The rolA self-collected sample is an easy to perform, preferred, and comfortable sampling method that is increasingly adopted for the detection of HPV ,9. SeverThis study aimed to evaluate the performance of the SelfCerv Self-Collection Cervical Health Screening applicator tampon compared to health professional-collected samples for the detection of hr-HPV for cervical cancer screening, and further, to investigate women’s experiences and preferences regarding self-sampling versus healthcare professional sampling.This was a cross-sectional study conducted at a tertiary hospital in Gauteng Province, South Africa, between 2016 and 2018. Paired samples were collected from 527 women who were enrolled in the study following obtaining written informed consent. Samples were sent to the HPV and STIs Training Centre for Africa at the Department of Virology laboratory for testing. Women aged 18 years and older were included in the study, and those who had undergone a complete hysterectomy or were going through their monthly menstrual cycle were excluded.® Combi  with the assistance of a speculum. The collection brush containing endocervical cells was immediately rinsed into a ThinPrep PreservCyt solution vial  and then discarded. Participants were then requested to complete the self-sampling part of the questionnaire. Both samples were placed in a medical box and transported to the laboratory for processing. Upon arrival, 20 mL of ThinPrep PreservCyt solution was added to the specimen bottle containing the tampon. The bottle was swirled and then squeezed to remove the tampon from the bottle. The solution from the specimen bottle was transferred into an empty ThinPrep vial and the vial was labeled properly.Participants received verbal instructions on how to collect the self-sample using a SelfCerv Self Collection Cervical Health Screening Kit . The SelfCerv package included pictorial diagrams and instructions for use. The SelfCerv Kit includes an applicator tampon, a patient information sticker, and a specimen bottle containing 4 mL of saline buffer. While waiting to be attended to at the clinic, consenting participants were individually taken to a private room and asked to insert the applicator tampon into the vagina until it met with resistance, and then remove the applicator and return to the waiting room. Participants then completed part 1 of the questionnaire. After tampon insertion for two hours, as specified by the manufacturer, the participants were requested by the healthcare professional to remove the tampon, taking care not to touch the tampon with their hands. The tampon has a cord/string that was gently pulled to remove it from the vagina and place it in a specimen bottle containing the saline buffer, making sure that the cord/string remained outside the bottle. To avoid contamination, there was no direct contact of the tampon with the hands during insertion and removal of the tampon. The healthcare professional then examined the participants and obtained an endocervical sample using a Cervex-Brush“Were you comfortable using the self-collection device?”, “Did you experience any pain when self-collecting the sample?”, “Would you prefer to self-collect the sample for cervical cancer screening in the future?”, “Would you prefer to use the self-collection device to collect a sample for cervical cancer screening in the future?”, “Would you prefer the healthcare professional to take the sample for cervical cancer screening”?, “Are you worried that you have not collected the sample properly?”. The data were double entered into Microsoft Excel.The questionnaire consisted of four main parts. The first part of the questionnaire collected demographic data including age, marital and employment status, place of residence, race, sexual practices, and reproductive history. The second and third parts included questions on HPV and cervical cancer. The fourth part included six closed-ended (Yes/No) questions to evaluate the participant’s experiences and preferences using the self-sampling device. Participants were asked to fill in part 4 of the questionnaire, which was on self-sampling experiences and preferences after they had self-collected and a healthcare professional had taken the sample. The questions to assess self-sampling experiences and preferences were: HPV genotyping was performed using Abbott RealTime High-Risk (HR) HPV assay  according to the manufacturer’s instructions. A volume of 0.8 mL of self-collected and healthcare professional-collected samples from each ThinPrep PreservCyt vial was transferred into Abbott Master Mix tubes and labeled properly. This assay detects 14 high-risk HPV types  and only genotypes HPV-16 and HPV-18 from the other 12 hr-HPV types. Probes for HPV-16 and HPV-18, “other hr-HPV” types  and IC were labeled with four fluorophores allowing their signals to be distinguishable in a single reaction to differentiate HPV-16 and HPV-18 from other 12 hr-HPVs. The HPV target cutoff (32.00 cycle threshold (Ct)) as well as the internal control target cutoff (35.00 Ct) is already established by the manufacturer. Samples with insufficient content of cervical samples are automatically invalidated. The endogenous human beta-globin sequence was detected as sample validity control for cell adequacyThe Aptima HPV mRNA assay  was used to detect E6/E7 viral mRNA from 14 hr-HPV types . Briefly, an aliquot of 1 mL was transferred into the Aptima Specimen Transfer Tube containing specimen transport media that lyses the cells and releases the mRNA and protects it from degradation. During testing, the mRNA was isolated from the specimen using capture oligomers that are linked to magnetic microparticles. Sequences complementary to specific regions of the HPV mRNA target molecules and a string of deoxyadenosine residues were contained in the capture oligomers. Sequence-specific regions of the capture oligomers bind to specific regions of the HPV mRNA target molecule during the hybridization step. HPV mRNA was amplified using transcription-based nucleic acid amplification and detection was achieved by Hybridization Protection Assay. Assay results were interpreted on the basis of the signal/cutoff ratio for the analyte, and specimens with signal/cutoff ratios of ≥0.5 were considered positive.Data analysis was performed using STATA version 14.1 . Results of categorical data were presented as frequencies and percentages. For continuous data (age), mean and standard deviation (SD) were calculated. The overall agreement  was determined using the kappa (κ) statistic. Kappa values were considered as poor (≤0.20), fair (0.21–0.4), moderate (0.41–0.60), good (0.61–0.80), and very good (0.81–1.00). Healthcare professional-collected test results were used as a reference standard to estimate the sensitivity and specificity of the applicator-tampon collection method.The mean (±standard deviation (SD)) age of participants was 36.8  years. Most (32.6%) of the participants were between the ages of 30 and 39, followed by those aged less than 30 years (28.7%). The majority (75.9%) of the participants were either single and/or divorced/widowed/separated, 54.8% were unemployed, and 85.0% were from semi-urban areas. Most (99.6%) of the participants were black Africans and 52.8% reported never being screened for cervical cancer before the study. The majority (44.2%) of the participants attended the clinic for routine Pap smear with the least (5.7%) of the participants seeking family planning services. The study population included 66.4% of women who were asymptomatic .DNA extraction and amplification was successfully achieved on all paired samples, as indicated with a positive internal control. For self-collected samples, the overall HPV prevalence was 47.6% (251/527); for healthcare professional-collected samples, 48.0% (253/527). HPV-16 in self-collected samples and healthcare professional-collected samples were detected in 63 (12.0%) and 62 (11.7%), respectively. Self-collection detected 6.5% (34/527) and 42.1% (222/527) for HPV-18 and other 12 hr-HPVs compared to 5.9% (31/527) and 41.9% (221/527) by healthcare professional sampling. The results for hr-HPV DNA and HPV E6/E7 mRNA testing were obtained for 527 and 285 participants, respectively . OverallThere were 19 disagreements between self and healthcare professional HPV tests for HPV-16, 17 disagreements for HPV-18, and 71 for other 12 hr-HPVs. Of all disagreements, 56/107 (52.3%) results were positive for self-collected samples and negative for the healthcare professional-collected samples. No follow-up is available for these women. Discrepant self-collected test results were positive at high cycle threshold values .A total of 526 out of 527 participants answered the questionnaire. Self-sampling using the SelfCerv was recorded as comfortable by 90.5% while 13.3% recorded experiencing pain. Most participants reported that they would prefer to use the SelfCerv self-sampling device (88.4%) or self-collect the sample (87.1%) regardless of the device. A total of 402 (76.4%) participants recorded that they would also prefer the healthcare professional to collect the sample. Very few participants (24.3%) were worried that they did not collect the sample properly .p = 0.007). The results further show that women aged 40–49 years were more likely to prefer the healthcare professional to collect the sample. Women in the age group 60 years and older were less worried that they had not collected the sample properly but more likely to have experienced pain when self-collecting the sample. A low proportion (12.6%) of participants who have never been screened before the study recorded not being comfortable using the self-collection device. Most unscreened participants (86.0%) recorded not having experienced pain during self-collection. The majority (89.9%) of participants who had never been screened recorded that they would prefer to use the self-collection device (SelfCerv) for future cervical cancer screening, as opposed to 28 (10.1%) who answered “no” to the question.As shown in Interventions to increase cervical cancer screening coverage are important. This is supported by the data from the current study, which show that the majority of women in the current study had never been screened for cervical cancer using the Pap test at the time of the study. The reasons why these women had never been screened were not explored. In HPV self-sampling, sample validity depends on the participants; hence, it is important to compare HPV test results from self-collected samples with those collected by a healthcare professional, which are considered the gold standard.The current study revealed 87.1% concordance between self-and healthcare professional-collected samples in detecting overall hr-HPV with κ 0.74, which showed good agreement. The level of agreement in our study was not as high as that in a study in Ghana among 194 women using the careHPV brush coupled with the careHPV assay , κ: 0.74For overall hr-HPV, the sensitivity and specificity of self-collected samples compared with healthcare professional-collected samples were 86.2% and 88.0%, respectively. Similar findings were reported in a study conducted among women with reproductive tract infections using Roche Reverse Line Blot Assay . In MalaThere are limited data on the performance of self-collected samples for hr-HPV mRNA detection. In the current study, the SelfCerv applicator tampon coupled with Aptima HR-HPV mRNA assay demonstrated a fair level of agreement . In South Africa and Kenya, Adamson et al.  and SenkIn the current study, the majority of participants were comfortable self-collecting the sample using the SelfCerv applicator tampon. Consistent with previous studies, participants reported self-sampling as being painless when using nylon-tipped flocked swabs and flocked swabs ,33 and cAlthough a high proportion of participants preferred the self-sampling method and were willing to self-sample again, some participants were still worried about having correctly collected the sample, which is a common concern that has previously been reported ,34. DespSince only 285-paired samples were tested, this could have affected the results. The questionnaire employed in the study did not include a question assessing the educational level of participants, nor did it assess any difficulties experienced during the insertion and/or removal of the tampon.The study shows that self-sampling using an applicator tampon was comparable to healthcare professional-collected samples in detecting hr-HPV DNA using the Abbott hr-HPV assay. However, the self-collected samples demonstrated a fair level of agreement for HPV E6 and E7 mRNA. Furthermore, we report a positive experience and a high preference for clinic-based self-sampling using the applicator tampon, which could be a way to increase primary screening coverage among women with limited access to screening services. The findings of the study must be interpreted with caution, as the SelfCerv might not be transferable to “harder-to-reach” populations who are often underscreened or not screened at all. Further research is needed to evaluate the effectiveness of applicator tampon-based self-collection and HPV testing for true cervical disease.


Time trends prevalence of human papillomavirus (HPV) genotypes including negative and untypable infections were analyzed during a 15-year period (2005–2019) among 5807 subjects with abnormal pap-smears and/or cervical intraepithelial neoplasia (CIN). The rates of HPV16 dropped by 13% every 3 years  in the CIN1 biopsy, while HPV16 status was unchanged over time in the CIN2+ biopsy. In CIN1 lesions, there was a corresponding increase of HR-HPV types unrelated to nonavalent vaccine. The rates of HPV 18, 31, and 52, decreased by 35% , 19% , and 21%  every 3-year interval in CIN2+, respectively. Overall, the prevalence of negative/untypable HPV specimens in the entire database increased from 9.6% (129/1349) in the period 2011–2013 to 17.6% (161/913) and 28.4% (224/790) in the 2014–2016 period and in the 2017–2019 period, respectively . HPV 16 prevalence decreased significantly among subjects with low-grade cervical squamous lesions. A significant increase of both HPV types unrelated to nonavalent vaccination and negative/untypable HPV infections was reported. The prevalence of HPV types among subjects with abnormal pap smears in Northern Italy is changing. Many variables including demographic factors and possibly vaccination could be responsible for this modification. Persistent high-risk human papillomavirus (HPV) infection, mostly caused by HPV 16 and HPV 18, is the leading cause of invasive cervical cancer .Before the introduction of the vaccination program in Italy, the distribution of HPV types in CIN lesions over the period 1985–2007 in Northern Italy changed significantly as a consequence of the introduction of new HPV types from other geographical areas and/or modifications of social and behavioral variables . In partAlthough currently available HPV genotyping assays are very accurate in the identification of HPV types with proven and probable carcinogenicity, 1–2% of cervical samples shows a positive signal in HPV lines but fails to show a positive signal in subsequent HPV typing ,8,9. TheData for this study were extracted from a database containing prospectively collected clinical, colposcopic, and virological information from subjects aged 21 to 65 years of age attending the Colposcopic Service of our Department due to an abnormal screening pap smear. For the purpose of the study, we evaluated 5 consecutive 3-year periods from 2005 to 2019.Subjects were referred by the cytological screening service of our department and external institutions and from private practice. Exclusion criteria included age <21 years, ongoing pregnancy, history of positive HPV test or treatment for CIN or total hysterectomy before enrolment, and non-squamous lesions on pap smear or with preinvasive or invasive lesions. Institutional Review Board (IRB) approval by our institution was obtained .The database was composed by a series of anamnestic items compiled after structured interviews at entry and by clinical, colposcopic, and virological items compiled at entry and during the follow-up. All patients were treated according to an established protocol including HPV DNA detection and genotyping and colposcopy with targeted biopsies. Cervical samples for HPV genotyping were obtained immediately before colposcopy. After speculum examination, scrapes were taken with a cervix brush, suspended in ThinPrep-PreservCyt Solution , and stored at 4 °C. DNA extraction was performed by lysis and digestion with proteinase K. HPV sequences from the L1 region were amplified by means of the polymerase chain reaction (PCR) using SPF10 primers in a 50 μL final reaction volume for 40 cycles. Appropriate positive and negative controls were introduced for each set of reactions. Concurrent amplification of beta globin sequences was used as a control for DNA adequacy. HPV type-specific sequences were detected by the line probe assay INNO-LiPA HPV genotyping assay version V2 up to 2009 and version EXTRA subsequently , according to the manufacturer’s instructions. Hybridization patterns were automatically analyzed by the LiRAS system and checked by two independent readers. The risk of the HPV type associated with the development of cancer is based on the data of the International Agency for Research on Cancer (IARC).For the purpose of this study, we classified HPV types into four categories: high risk with proven carcinogenicity targeted with novalent HPV vaccines , high risk HPVs not targeted with nonavalent vaccines , Low risk HPVs , and HPV untypable  .A standardized colposcopic examination was performed immediately after cervical brushing for HPV genotyping by two different gynecologists  certified by the Italian Society of Colposcopy. Multiple targeted cervical biopsies were obtained in all cases where CIN2+ was suspected on colposcopy and in all cases of high-grade squamous cervical lesions (HSIL) irrespective of colposcopic impression. Endocervical curettage was performed, according to the clinician’s judgment, when the extent of the lesion or the squamocolumnar junction was not entirely visible (NTZ Type 3) or in the case of atypical glandular cells (AGC) on Pap smear. Histological diagnoses were based on consensus decision of two expert gynaecological pathologists . In the analysis of the data, we either used the histological diagnosis of punch biopsy or, in more severe cases, the diagnosis was determined after cone biopsy obtained by the loop electro-excision procedure (LEEP) or by cold knife excision.Univariate statistical analysis was carried-out with Kruskal–Wallis analysis of variance and chi-square test to compare continuous and categorical variables, respectively. The Bonferroni method for multiple comparisons was used to evaluate partitioned chi-square tests in multiway contingency tables. Chi-square for trend was used to test for univariate linear trend. Adjusted prevalences and prevalence ratios (PR) were obtained by using the Poisson regression model including the different types of HPV infections as outcomes and age upon examination, HIV status , nulliparity , ancestry , and multiple HPV infections  as confounding variables. All the analyses were carried out with STATA 13.0 .Out of 5864 subjects between 21 and 65 years of age attending the colposcopy service of our department in the 2005–2019 period and potentially eligible for this study, we excluded 57 (1%) subjects because colposcopy was impossible and/or unsatisfactory, leaving 5807 women for the final analysis. Unfortunately, full data about HPV vaccination in naïve subjects was available only in a small number of subjects referred to our department, so inferences about the role of vaccination upon HPV type distribution cannot be made. p < 0.001 and chi-square = 88.8, p < 0.001, respectively). Regarding the types of HPV detected over time, although the frequencies of the different categories were heterogeneous , there was a significant reduction in the rates of LR-HPV and an increase in HR-HPV unrelated to nonavalent vaccine genotypes in the last 3–6 years compared to previous periods. Overall, the tests for trends also confirmed an increase in the rates of HR-HPVs unrelated to vaccination, negative and untypable HPVs. After adjustment for potential confounders, the increase of the three-year prevalence ratio of HR-HPV unrelated to nonavalent vaccination was homogeneous across ages at examination strata being 1.21 (95% CI = 1.1–1.33), 1.16 (95% CI = 1.1–1.26), and 1.17 (1.05–1.31) among women of 21–29, 30–45 and 45–65 years, respectively. In the 2011–2019 period, after adjustment for potential confounders the prevalence ratios of negative and untypable HPVs increased by 1.53 (95% CI = 1.36–1.72) and 2 (1.78–2.36) times every three-years, respectively.In To evaluate the potential displacement of HPV types, we studied the distribution over time of the 7 HR-HPVs  targeted by nonavalent vaccines both among cytological  and histWhen the analysis was restricted to the 3475 women who had a positive colposcopically-directed biopsy , the ratThe overall prevalence of the seven HR-HPVs targeted by nonavalent HPV vaccines were substantially unchanged during the period of the study  irrespective of the severity of cervical lesions and age category A. The rates of HR-HPVs unrelated to nonavalent vaccines increased every three-year period among subjects with ASCUS/LSIL , HSIL , CIN1 lesions on biopsy  but not among subjects with CIN2+ histological diagnosis  B.The rates of LR-HPVs C decreasOverall, the prevalence of negative/untypable HPV specimens in the entire database increased from 9.6% (129/1349) in the 2011–2013 period to 17.6% (161/913) and 28.4% (224/790) in the 2014–2016 period and 2017–2019, respectively  D. Among The corresponding rates of negative/untypable HPV in the same periods among CIN2+ subjects were 3.7% (8/214), 4.1% (6/141) and 14.6% (22/151) .Finally, in the 2011–2019 period in the overall database, the increase in the prevalence ratio of negative/untypable HPV was homogeneous across age at examination strata being 1.9 (95% CI = 1.6–2.3), 1.76 (95% CI = 1.54–2) and 1.6 (95% CI = 1.36–1.87) at 21–29, 30–45 and 45–65 years, respectively.The distribution of HPV types involved in cervical infection depends on the characteristics of the population, including geographical area, presence and severity of lesions and age. In the present study, during 15 years, the rates of HPV 16 and other HR-HPVs such as HPV-18, 31, 52, 58 dropped significantly among subjects with low grade lesions. On the other hand, among subjects with CIN2+ on biopsy the prevalence of HPV 16 and other HR-HPV related to nonavalent vaccines remained stable during the period of observation. The behavior of HR-HPV unrelated to nonavalent vaccines increased correspondingly in low risk cervical lesions mirroring the behavior of HPV16 infection suggesting a potential replacement of HPV16 by other HR-HPVs. Finally, the prevalence of the specimens negative for HPV or with untypable HPV infections increased consistently in the 2011–2019 period, irrespective of the severity of cervical lesions and age category.The variation in the rates of single HPVs associated with cervical lesions during different periods of time could be influenced by several factors including age, a proxy for sexual activity and HPV exposure, parity and race, prevalence of other sexually transmitted diseases and severity of cervical lesions in the population studied . In addiA significant reduction in the prevalence of HPV-16 related cervical lesions in the last 10 years, especially among low grade lesions and severe lesions among younger women, has been already reported by other authors ,14. SimiThe biological reasons for the reduction in the rates of HPV 16 infection and the corresponding increase in the rates of HR-HPVs are various. Although in stratified analysis, the modifications in the rates of HR-HPVs were confirmed only in the groups of young women of Italian ancestry, the increase in the rates of foreign women attending our colposcopic service during the 15 years studied suggests that immigration could play a significant role in the relative prevalence of HR-HPVs by introducing new HPV types in the population. Several studies support this view suggesting that ‘new’ HPV types are very common among immigrants in Italy [Multiple HR-HPV infections are rather common, involving from 10–40% of HPV infections ,18. SeveHowever, most of our data suggest that a reduction of the rates of HPV 16 and other HR-HPVs related to vaccination among women with abnormal pap smears, and conversely the increase in HR-HPVs unrelated to vaccination, was mainly found among young women with low grade lesions. This is in agreement with a population study of HPV infection in the general population carried out in self-collected cervico-vaginal samples ; in thisThe increase in the rates of HPV negative and HPV untypable specimens during the period of 2010–2019 in our study is puzzling. As reported by several studies, the prevalence of HPV negative specimens among women with abnormal pap smears, CIN or invasive cervical cancer is about 12–14% ,25, a raFrom a methodological viewpoint, the INNO-Lipa extra system is considered a reliable method for genotyping HPVs when compared to hybrid capture methods , althougUntypable HPV infection is defined as having positive PCR results using consensus primers (SPF10) but a failed positive signal on genotyping assays ,8. This In our study, the increased prevalence of HPV negative or HPV untypable specimens was confirmed in CIN 1 and CIN2+ histological lesions and was independent of age suggesting that the relative modifications of the frequencies of HPV types in cervical samples recorded in recent years involve not only HPV unrelated to vaccines but also to uncommon HPV types, or, possibly, to HPV quasispecies .A definite approach including virological, cytological and histological homogeneous data during a 15-year period is the main strength of the study. On the other hand, data were limited only to abnormal pap smears and were collected in a single center; for these reasons, our results cannot be applied to general population. In conclusion, during a 15-year period there were significant modifications in the prevalence of different types of HPVs among women with abnormal pap smears. The reduction in the rates of HPV 16, 18 and other HR-HPVs was mirrored by an increase in the rates of HPVs unrelated to vaccines and by HPV negative/untypable specimens.


Data about the genotype-specific human papillomavirus (HPV) prevalence in the Czech Republic is limited. We aimed to evaluate the prevalence and concordance of genotype-specific HPV infection detected in semen samples, penile swabs and cervical swabs from non-vaccinated heterosexual couples without HPV-associated disease.® HPV Test and PapilloCheck®. Only the PapilloCheck® test was used to detect HPV in penile swabs. The genotype-specific prevalence and concordance of HPV infection not targeted by vaccine were evaluated using Fisher exact test.Semen samples and penile swabs were collected from male partners and cervical swabs were collected from female partners of heterosexual couples treated for infertility (n = 195). Presence of HPV DNA in semen samples and cervical swabs was analyzed using the cobasP < 0.001). The most prevalent hrHPV genotype were HPV53  in semen samples, HPV16  in penile swabs and HPV39  in cervical swabs. Low-risk (lrHPV) genotypes were detected in 5.13% (10/195) of semen samples, 15.9% (31/195) of penile swabs, and 4.10% (8/195) of cervical swabs (P < 0.001). Male sexual partners of HPV-positive women were more likely to be infected with at least one of the same HPV types than female sexual partners of HPV-positive men .Both partners were infected with any HPV type in 13.8% (27/195) of couples and, of these couples, 55.6% (15/27) harbored at least one mutual genotype. High-risk HPV (hrHPV) genotypes were detected in 12.3% (24/195) of semen samples, 31.3% (61/195) of penile swabs, and 19.5% (38/195) of cervical swabs (This study showed that the detection of HPV infection differ by anatomic site and gender. Regardless the anatomic site, high prevalence of HPV genital infection was found in both Czech men and women.The online version contains supplementary material available at 10.1186/s12985-021-01551-x. Human papillomaviruses (HPVs) are agents of common sexually-transmitted diseases that affect sexually-active human populations . AlthougIn the Czech Republic, data on the genotype-specific prevalence of HPV in women is limited –8. Even Seven hundred and twenty-eight (728) couples treated for infertility in two Czech fertility centers, Fertimed Ltd., Olomouc and Arleta IVF Ltd., Kostelec nad Orlici, were enrolled into the study from July 2013 to November 2016. These two Czech fertility centers operate in the same geographical region. Long-term monogamous couples who engaged in regular sexual activity with no barrier contraception, demonstrated no symptoms of HPV-associated disease, and received no prior vaccinations against HPV were included. Only 195 couples provided both semen samples and penile swabs from male partners as well as cervical swabs from female partners that were suitable for evaluation . Semen samples were obtained at the same time by masturbation after 3–5 days of sexual abstinence. After liquefaction of the ejaculate at room temperature, at least 0.1 mL of ejaculate was put into 20 mL of cobas® PCR Cell Collection media. Clinician-collected cervical brushes were rinsed in 20 mL of cobas® PCR Cell Collection Media. All samples in cobas® PCR Cell Collection Media were transported and stored at room temperature until testing according to the manufacturer’s recommendations for gynecologic samples.Male study participants self-collected penile swab samples by wiping a dry cotton swab at least three times around the coronal sulcus and the top of the glans. Each swab was then rinsed in 0.5 mL of cobas® HPV Test  according to the manufacturer’s recommendations for cervical swabs management , respectively, P = 0.669; Wilcoxon exact test]. Similarly, the median age of participating men presenting hrHPV-positive penile swabs was no different from that of men presenting hrHPV-negative penile swabs . Likewise, for participating women, no difference in median age was observed between those presenting hrHPV-positive cervical swabs and those presenting hrHPV-negative cervical swabs .The median ages of male partners and female partners from couples (n = 195) were 35 years  and 32 years  of these couples harbored at least one mutual genotype. Contrary to our findings meta-analysis conducted by Reiter et al. .P = 0.055; Pearson chi-squared test). This finding aligns with studies suggesting more likely genital transmission of α-HPV from women to men [We found that male sexual partners of HPV-positive women are more likely to be infected with at least one same HPV genotype than female sexual partners of HPV-positive men  and cervical swabs . This difference could be caused by regional and age variabilities in studied populations.We found that HPV infection in penile swabs was detected two times more frequently than in cervical swabs (41.0% vs. 22.1%), and the frequency of detection in semen samples was even lower (15.9%). Similar to our study, Giuliano et al.  reportedThe global HPV prevalence in women with normal cytology findings is estimated at 11.7% with considerable regional differences. Regardless of the region, the highest HPV prevalence is observed in women under 25 years of age 24.0%) and declines with age with up to 7.5% HPV prevalence observed in women older than 55 years .0% and d, 27. In Contrary to observations in women, HPV prevalence data in the asymptomatic male population are very limited and no published study reports on HPV prevalence in the Czech male population is available to date. HPV is rarely tested in men, with testing usually occurring only because of HPV-associated disease, or due to research mainly on the male partners of women with HPV-associated disease.The largest study focused on genital HPV prevalence in healthy men was HIM study  which waIn recent meta-analysis, semen samples of men from infertile couples demonstrated significantly higher HPV prevalence than semen samples from men of the general population . HPV preIn the meta-analysis, hrHPV/lrHPV infection was detected in 10.0%/8.3% of men from general population and 15.5%/10.3% of male fertility clinic attendees . In our The current study draws attention to the high HPV prevalence not only among Czech women but also in Czech men and shows the differences in HPV prevalence and genotype-specific concordance by anatomic sampling sites and gender in Czech heterosexual couples.Additional file 1. Prevalence, single-type infection and coinfection of hrHPV and lrHPV genotypes detected in participating couples. Statistically significant data (P ≤ 0.05) are shown in bold. HrHPV includes HPV31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82 genotypes. LrHPV includes HPV6, 11, 40, 42, 43, 44/55. The HPV result for a given sample was obtained when at least two detection methods were concordant. The presence of HPV53, 70, 73, 82 and lrHPV genotypes  was evaluated using PapilloCheck® HPV-Screening only. The presence of HPV in penile swabs was evaluated using PapilloCheck® HPV-Screening only.Additional file 2. HPV concordance in paired samples taken from male and female partners of participating couples. CS cervical swab, PS penile swab, SS semen sample, NA not applicable. Statistically significant data  are shown in bold. *Concordance in any type of sample from male partner with sample from female partner any-type concordance-both partners are HPV positive same-type concordance-both partners are HPV positive for 1 or more HPV types in common.


To evaluate the feasibility of delivering the Live Long Walk Strong (LLWS) rehabilitation program among community dwelling, mobility limited older Veterans in the VA Boston Healthcare System. Community dwelling Veterans 50 years and older identified as being at high risk for mobility decline based on self-report task modification and AM-PAC mobility questions. All Veterans received 10 sessions over 8 weeks of LLWS Physical Therapy care focusing on novel impairments related to mobility decline and behavioral change strategies. Sessions were delivered 1:1 with a Physical Therapist over 45 minutes. To assess feasibility, we tracked recruitment and retention metrics. We assessed length of each session, number of sessions attended, and any reason for withdrawal. To examine technological feasibility, we recorded number and type of issue along with resolution of the issue. A total of 178 Veterans were contacted to participate. Twenty Veterans were enrolled into the LLWS virtual pilot between October 2020 – May 2021. Among our 20 enrolled, 5 did not complete the program. Reasons for not completing included: being enrolled in another exercise study simultaneously and non-related medical complications. Among those completing, an average of 9.7 out of 10 intervention sessions were completed. An average of 1.8 technology difficulties per Veteran was experienced within the intervention. The most frequent technology issues experienced were related to camera positioning and Wi-Fi bandwidth resulting in delayed video and audio. LLWS is feasible to deliver as a virtual mode of care in middle and older aged Veterans at high risk for mobility decline.


Approximately 43% of males over the age of 65 years are Veterans. Veterans may be at elevated risk for functional declines due to barriers to health care access leading to accelerated loss of independence. This compared the prevalence of functional limitations in Veterans and non-Veterans. Data from two National Health and Examination Survey collection periods, administered 2013-2014 and 2015-2016, were used to compare physical functioning data between male Veterans (N=369) and non-Veterans (N=738) matched 1:2 for sex, race, and BMI. Individuals were considered a Veteran if they self-reported having “served in active duty in the U.S. Armed forces.” Pearson’s chi-square tests were used to assess differences in the prevalence of various self-reported functional limitations between groups. Veterans  were disproportionately affected by self-reported functional limitations caused by long-term physical, mental, or emotional illnesses . Twenty-five % of Veterans reported that these limitations kept them from working compared to 18% of non-Veterans (p<0.003). Veterans (38%) were also more likely to report being limited in the amount of work they could perform compared to non-Veterans (27%) (p<0.01). Additionally, Veterans (20%) were more likely to report the use of special healthcare equipment  than non-Veterans (12%) (p<0.001). These data suggest that Veterans are at greater risk for functional limitations caused by self-reported long-term physical, mental or emotional illness. Therefore, further research is needed to determine if home- and community-based services could prevent further functional decline, ultimately allowing Veterans to maintain independence.


The last decade has seen a continued escalation in rates of human papillomavirus related oropharyngeal malignancy (HPV-OPC). This has occurred despite established national vaccination programs. In contrast, HPV associated cervical cancer incidence rates have declined, due in part to effective cervical cancer screening programs, many of which have moved towards the detection of high-risk HPV (hrHPV) as an early marker of malignant potential. This raises questions as to whether similar hrHPV screening methods could be used for early detection of HPV-OPC. Persistent oral hrHPV is a prerequisite for the development of HPV-OPC and can be accurately detected in saliva. Despite this, single point saliva testing for hrHPV lacks sufficient sensitivity and specificity to allow for effective population screening. Recent published literature suggests the use of serial saliva testing in targeted high-risk individuals, with an emphasis on biomarker persistence and intensity patterns, as a potential means of detecting even subclinical microscopic disease. When coupled with serological testing, this has the potential to provide an accurate test for screening at risk individuals. Despite these promising developments, several significant barriers to an effective targeted screening program remain. Rates of human papillomavirus (HPV) related oropharyngeal malignancy (HPV-OPC) are increasing rapidly throughout high-income countries. HPV-OPC numbers have now surpassed those of cervical cancer, making it the most common HPV related malignancy in many nations. Risk factors for the disease include male sex, current smoking status, and a greater number of lifetime sexual partners. Vaccination programs aimed at eradicating HPV related malignancy have had variable uptake and even where implementation has been successful the impact on HPV-OPC is not expected to be seen for decades.Persistent oral high-risk HPV (hrHPV) is a risk factor for the development of HPV-OPC. The most common hrHPV types found in the western world are -16,-18,-31,-33 and -35  demonstrOne of the principal barriers to developing a strategy for HPV-OPC screening is our lack of understanding of the natural history of the disease and in particular the risk and time course of progression from persistent hrHPV infection to malignancy. Unlike the cervix, HPV-OPC does not seem to follow a stepwise pattern of dysplasia, intraepithelial neoplasia, and invasive malignancy. The tonsillar crypts lack a basement membrane and an insitu form of the cancer is purported not to exist. Additionally, the oropharynx is an anatomically extensive region, making assessment and directed tissue sampling very difficult. This raises concerns regarding our ability to identify those in whom the disease is progressing from persistent hrHPV infection to HPV-OPC and by doing so, limiting further intervention to these specific individuals. Without this discrimination, screening will either lack sufficient sensitivity, or alternatively sufficient specificity, and we will risk over-treatment for those in whom the infection would have naturally cleared without progression, or fail to detect significant disease frequently leaving patients unassured. One emerging possibility to bypass this difficulty may be analysis of the temporal pattern of HPV expression and in particular detection of a rapid increase in the levels of HPV DNA over time.One limitation to our current understanding of the potential for screening for OPC has been the lack of prospective studies of HPV biomarkers that demonstrate a link between persistent hrHPV and incident malignancy. Last year we reported the first case of an individual diagnosed with HPV-OPC following serial saliva testing for HPV-16 DNA as part of a prospective, longitudinal study. We detected occult, p16 positive OPC in an asymptomatic individual where MRI gave a negative result , 4. ThisIn our study we demonstrated that oral HPV-16 DNA was able to detect a 2 mm malignancy. The lesion was undetectable both clinically and radiologically. Despite the relatively high association of oral HPV-16 DNA with HPV-OPC positive tumours [For screening to be beneficial, earlier diagnosis must provide patients with better clinical outcomes. HPV-OPC tends to present as locally advanced disease and despite having a favourable prognosis (10–20% 5-year recurrence rates), morbidity from treatment of advanced disease can be considerable, leaving some with lifelong pain, dysphagia, and speech issues. Those in whom the disease is found early can receive single modality treatment either through surgery or radiotherapy with excellent cure rates and lower side effects with virtually all patients achieving a normal diet and minimal long-term morbidity. Diagnosing the disease at an earlier stage would therefore appear desirable. However, given the low mortality of disease overall, this assertion remains controversial. Additionally, the difficulty in clinically diagnosing the disease at very early stages calls the feasibility of such an approach entirely into question, and it remains unclear as to how biomarker-positive patients should be clinically assessed, investigated and treated.The apparent link between oral HPV copy number and progression from infection to microscopic disease is exciting and needs to be replicated on a larger scale. More accurate means for assessment of persistently HPV positive patients need to be established and new treatment options such as immunotherapeutic vaccines for clinically occult, very small volume disease need to be investigated. Recent findings help to inform us about both the natural history of this disease and the sensitivity and specificity of oral HPV DNA in detecting the disease. Rates of HPV-OPC are increasing rapidly. Further large-scale studies are the requirement for progressing our understanding of the feasibility of personalised screening in high-risk populations.