COMP overexpression correlates with poorer DFS , OS , LRFS , and MFS . High COMP expression ties to worse overall survival (p = 0.0001), DSS (p < 0.0001), LRFS (p < 0.0001), and MFS (p < 0.0001). In conclusion, COMP overexpression links to poor prognosis and pathological features in iCCA, indicating its potential as a biomarker.Cartilage oligomeric matrix protein (COMP) interacts with various extracellular matrix proteins in tissues. Elevated COMP levels recently linked to worse overall survival in multiple cancer types. COMP's significance in intrahepatic cholangiocarcinoma (iCCA) remains uncertain. Here we report a retrospective study to explore COMP's impact on iCCA outcomes. We collected 182 patients' iCCA tumor tissues. COMP overexpression was associated with adverse factors like R1 resection ( High COMP levels have also been associated to breast cancer, hepatocellular carcinoma, prostate cancer, and colon cancer. In these cancers, COMP overexpression was associated with increased tumor growth, cancer metastasis, cancer recurrence, and overall shorter survival5. Against this backdrop, the prognostic significance of COMP in intrahepatic cholangiocarcinoma (iCCA) remains unexplored.Previous studies have elucidated the pivotal role of cartilage oligomeric matrix protein (COMP), also known as thrombospondin-5 (TSP-5), in various cancers. COMP's presence in multiple cell types and its contributions to maintaining extracellular matrix (ECM) integrity have been established. Notably, its functions encompass stabilizing ECM protein connections and enhancing tissue mechanical strength. COMP levels are elevated in a variety of musculoskeletal disorders7. CCA is a diverse collection of malignant cancers that arise in various parts of the biliary tree. CCAs are divided into three types: intrahepatic (iCCA), perihilar (pCCA), and distal CCA (dCCA), each with its own etiologies, risk factors, prognosis, and clinical and therapies. Together, iCCA and pCCA account for more than 90% of all CCAs in the world8. CCA is a rare cancer, but its incidence  and mortality  have been rising in recent decades, posing a global health problem11. Over the past few decades, the reported age-standardized incidence for iCCA has been steadily increasing in most locations worldwide, whereas the age-standardized incidence for dCCA has been decreasing12. Curative surgery remained the standard treatment for early CCA. Despite the improvement in CCA pathogenesis, diagnosis, and treatments over the last decade, patient prognosis has remained unchanged, with 5-year survival rates of 7–20 percent and tumor recurrence rates were as high as 48–56% after resection remaining unsatisfactory17. Several clinical markers, including as T stage, lymph node metastases, and histological grade, can be used to identify high-risk patients16. However, to establish further treatment plans after curative surgery, genomics-based prognostic biomarkers were warranted.After hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA) is the second most prevalent primary hepatic malignancy, accounting for around 15% of all primary liver tumors and 3% of all gastrointestinal cancersGiven the rising incidence and mortality of iCCA globally, coupled with the challenges in improving patient prognosis, an investigation into COMP's role in iCCA assumes importance. Therefore, the primary aim of this study is to assess COMP expression levels in iCCA patients and determine its potential as a prognostic marker. In summary, this study investigates the expression of COMP in intrahepatic cholangiocarcinoma (iCCA) and its implications for prognostic evaluation. By shedding light on COMP's potential as a prognostic marker, our work seeks to contribute valuable insights into enhancing the management and treatment outcomes of iCCA patients.p < 0.0001) in CCA, as shown in Table p < 0.0001). Thus, COMP was chosen for further analysis.For data mining, a published CCA transcriptome dataset (GSE26566) was used, which includes 104 patients who had radical surgery. We found 9 probes that covered 9 transcripts related to structural elements of the extracellular matrix  , advanced T stage (p < 0.001), large duct type (p = 0.004) and poor differentiated histology (p = 0.002).The association between COMP expression and various clinicopathological features was assessed using Pearson’s chi-square test. As determined using immunohistochemistry, poorly differentiated and higher T stage cholangiocarcinoma had greater COMP immunoreactivity than well differentiated and lower T stage cholangiocarcinoma Fig. . Table 2p = 0.001), poor OS , poor LRFS , and poor MFS  in multivariate analysis , DSS , LRFS  or negative connections (Supplementary Table 2) with COMP from the cholangiocarcinoma dataset . Later, employing the Gene Ontology (GO) classification system, these transcripts were utilized to prognosticate the functions of COMP. The results revealed that, in respect to biological processes , and the platelet-activating factor receptor (PTAFR) gene was identified. In view of molecular functions  that includes the platelet-derived growth factor subunit B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), collagen type I alpha 1 chain (COL1A1), COL1A2, COL3A1, and COL5A1 genes. Furthermore, with regard to cellular components , collagen type I trimer , and collagen sheet .To recognize the potential functions of ses Fig. A, the mo18. Several research have studied not only the morphological differences, but also the molecular distinctions between iCCA, pCCA, and dCCA21. Nakamura et al. demonstrated a variation in tumor anatomical location, indicating IDH, EPHA2 and BAP1 mutations and FGFR2 fusions in iCCA, whereas extrahepatic tumors preferentially show PRKACA and PRKACB fusions as well as mutations in ELF3 and ARID1B. Based on these findings, cholangiocarcinoma in different anatomical locations should be treated and examined differently20. Furthermore, cholangiocarcinoma's highly desmoplastic nature, substantial support from a rich tumor microenvironment, and significant genetic heterogeneity all contribute to its treatment resistance22.In contrast to HCCs, mass-forming iCCAs are characterized by a significant desmoplastic and hypovascularized tumor stroma, which is frequently the predominant histological characteristic of the tumor24. Although the function of COMP glycoprotein in a number of connective tissue illnesses has been well documented25, its function in carcinogenesis is still unknown. Evidence from recent studies has shown that COMP overexpression is associated with a poor prognosis and metastases in breast, prostate, thyroid, colon, and hepatocellular carcinoma28. In our previous study, we found COMP overexpression associated with poor survival and tumor invasiveness in urothelial carcinoma29.COMP is a large pentameric cartilage protein and is found in articular cartilage, ligaments, and tendons. The protein can also be found in the skin, breast tissues, and liver tissues2. Liver cirrhosis and viral hepatitis C and B have been recognized as risk factors for cholangiocarcinoma, especially intrahepatic disease33. The following associations were found in a meta-analysis34 of several case–control studies on the risk factors for intrahepatic cholangiocarcinoma: cirrhosis had a combined odds ratio (OR) of 22·92 (95% CI 18·24–28·79), hepatitis C of 4·84 (2·41–9·71), and hepatitis B of 5·10 (2·91–8·95). Based on this, we think that the COMP may also affect the prognosis of iCAA due to the shared etiology of HCC and iCCA. Moreover, cholangiocarcinoma is usually characterized by a prominent desmoplastic and hypovascularized stroma. There is now increasing evidence to suggest that the desmoplastic reaction, marked by a dramatic accumulation of α-smooth muscle actin positive cancer-associated fibroblasts (α-SMA + CAFs) with increased production of extracellular matrix proteins18. Based on these, we use the transcriptome dataset to assess the genes related to extracellular matrix structure constituent (GO:0005201). When compared to neighboring liver tissue, COMP gene was significantly elevated in CCA among these genes. Thus, COMP was chosen as a candidate for further survival analysis. To our knowledge, this is the first study to demonstrate COMP overexpression associated with poor survival in iCCA.As the extracellular matrix degrades with continued liver damage and fibrotic scar tissue builds up, the liver undergoes remodeling, which eventually results in fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC). Norman et el. demonstrated serum COMP could be an early marker of fibrosis, and that increased serum COMP levels could reflect the degree of cartilage breakdown during liver destruction and re- modeling, and also associated with HCC development36. Interestingly, Konstantinos et al. demonstrated that COMP regulates the cancer stem cell population through increasing the interaction between Notch3 and Jagged1, leading to increased activation of Notch3 signaling37.The Notch, WNT, and transforming growth factor (TGF) signaling pathways, among others, are highly active in iCCA. It is known that the Notch pathway has a role in biliary repair, growth, fibrosis, and stem cell niche maintenance. NOTCH3 overexpression was linked to the development and progression of iCCA, promoting cell survival through PI3K-AKT signaling38. It gives epithelial cancer cells the ability to develop mesenchymal characteristics with invasive capabilities, which promote colonization of metastatic sites39. The TGF-dependent pathway, whose signature has been discovered in iCCA stroma, is the prototypical inducer of EMT. In CCA, TGF either directly induces EMT or collaborates with other key EMT inducer pathways like EGFR42. Notably, transcription factors (EMT-TFs) that control the expression of epithelial and mesenchymal genes, including as the SNAIL, ZEB, and TWIST families, orchestrate EMT. Regardless of anatomical location, CCAs express EMT-TFs, which are linked to a poor prognosis44. Interestingly, COMP can stimulate tumor EMT, although the mechanism is unknown45. COMP glycoprotein has been demonstrated to be co-expressed with many EMT genes, and a clear association between high COMP expression and poor colon cancer survival has also been reported46. The evidences to support the link of NOTCH3 pathway, EMT pathway between COMP and iCCA are ambiguous initially but gradually apparent. However, the exact mechanism by which COMP acts as a poor prognostic factor in iCCA remains unclear and need further exploration.In the other way, the epithelial to mesenchymal transition (EMT) is a critical biological step in the migration and invasion of malignant tumor cells47. MiRs can be secreted extracellularly or encapsulated in EVs, thereby taking part in intercellular communication48. Transcytosis, a type of transcellular mechanism for crossing of EVs through the interior of a cell, has also been implicated in various solid tumors49. Interestingly, the results of our bioinformatic analysis revealed that, in terms of biological processes, the most significant term correlated with COMP upregulation was positive regulation of transcytosis /PTAFR pathways has also been suggested to cause hepatocellular carcinoma cell migration and invasion50. However, whether COMP may promote IHCC progression through transcytosis and PTAFR needs further investigation. Moreover, in terms of molecular functions, the most remarkable term correlated with COMP upregulation was platelet-derived growth factor binding  that play crucial roles in modulating cholangiocarcinoma development52. The COL1A1, COL1A2, COL3A1, and COL5A1 genes are also identified as CAF signature in cholangiocarcinoma54. Accordingly, the associations among the expression levels of COMP, platelet-derived growth factors, and CAF-derived collagen in IHCC development are quite interesting and deserve further examination.Recently, microRNAs (miRs) and extracellular vesicles (EVs) have gained attention as promising non-invasive biomarkers for diagnosis and prognosis in cholangiocarcinomaOur research has certain limitations. Firstly, it is a retrospective study conducted at a single institution and lack of experimental validation. Secondly, the exact molecular mechanism underlying disease progression and adverse outcomes in COMP-overexpressing IHCC remains unclear. Thirdly, there is currently no standardized immunostaining and scoring scheme for assessing COMP expression. Due to the lack of agreed staining standards, it is difficult to reach a consensus in this type of research. Fourthly, The GEO transcriptome dataset we use is from cholangiocarcinoma patients rather than iCCA patients, which may also cause some limitations in this study. Lastly, to validate our findings, prospective multicenter studies are required.In conclusion, COMP overexpression was associated with worsening clinical-pathological characteristics. COMP is also related with poorer survival in iCCA, supporting its function as a biomarker for iCCA prognosis. This is the first study to our knowledge that clarifies the role of COMP in iCCA. To completely comprehend the mechanism and apply these findings to clinical practice, additional research is required.A transcriptome dataset (GSE26566) containing 104 iCCA patients who received curative surgery was obtained from the NCBI Gene Expression Omnibus (GEO) database. Without pre-selection, all probe sets were used. The raw data was then transferred into the Nexus Expression 3 software, which was used to calculate gene expression levels. Comparative analyses were conducted to detect the significantly differently expressed genes connected to extracellular matrix structural constituents  by comparing CCA part vs. normal surrounding liver and CCA part vs. normal intrahepatic duct. After analysis, differentially regulated genes in CCA part vs. normal surrounding liver and CCA part vs. normal intrahepatic duct were discovered (P < 0.0001 and log ratio > 2)  of Chi Mei Medical Center with the approval number of 09,912,003. All research was performed in accordance with relevant guidelines/regulations. We gathered patients' retrospective demographic and clinical data, including pathological characteristics, oncological survival follow-up, and cause of mortality. Patients with acute blood diseases, abnormal bone marrow, concurrent cancer, or insufficient clinical data were excluded from the research. The tumor stage was assessed using the eighth edition of American Joint Committee on Cancer (AJCC) Tumor, Node, Metastasis (TNM) system developed in 2017. This study followed the REMARK (Reporting Recommendations for Tumor Marker Prognostic Studies) guidelines.55.Specimens were prepared according to protocol. The primary COMP antibody was incubated for 1 h on the sections . Then the primary antibody was diluted 1:200 in TBS with 1% BSA. Antibodies were then detected using a DAKO ChemMate EnVision Kit . Cell blocks from cell lines known to express COMP were used as positive controls. Sections were processed without the primary anti-COMP antibody as negative controls. Two pathologists used the following equation to calculate the H-score to estimate COMP immunoreactivity: H-score = SPi (i + 1), where Pi is the percentage of stained tumor cells in various intensities ranging from 0 to 100%, and I is the degree of staining (0 to 3 +). If there were any scoring disagreements, the two pathologists assessed the slides at the same time and agreed on an H-score. The immunostaining was categorized into low and high expression levels based on the median H-score as previously mentionedCOMP in intrahepatic cholangiocarcinoma, we appraised the relations between the mRNA expression levels of COMP and its coexpressed genes from the cholangiocarcinoma dataset  in The Cancer Genome Atlas (TCGA) database. Later, the top 200 differentially expressed transcripts showing positive connections or negative connections with COMP were downloaded. These transcripts were then annotated by the Gene Ontology (GO) classification system and ranked by fold enrichment.To recognize the potential functions of The connection between COMP expression and other clinicopathological characteristics was assessed using Pearson's chi-square test. We measured two outcomes: metastasis-free survival (MFS) and disease-specific survival (DSS). Using univariate and multivariate analysis, relevant COMP expression and clinicopathological variables were discovered as predictors of DSS  and MFS (measured from curative surgery to the first metastasis). The survival curves were created using the Kaplan–Meier method and a log-rank test. All relevant parameters from the univariate analysis were integrated into the multivariate Cox proportional hazards model to find the independent variables. IBM's SPSS Statistics V.17.0 software  was utilized for statistical analysis. The statistical significance criterion was chosen at P < 0.05.Supplementary Information.


Results: We included 28 articles with a total of 22,652 participants. The overall pooled prevalence of HR-HPV genotypes was 55.13%, albeit high heterogeneity between studies. The overall pooled prevalence of HR-HPV genotypes in HIV-positive individuals was 75.51%, compared to 52.97% in HIV-negatives (OR = 4.68 (0.71–30.76)). HPV 16 (18%), 35 (10.12%), 52 (9.98%), 18 (9.7%) and 45 (6.82%) genotypes were the most prevalent. Twelve studies identified the most frequently reported risk factors associated with HR-HPV, with HIV infection (66.66%), multiple sexual partners (41.66%) and young age (41.66%) being the most reported risk factors. Conclusions: The combined prevalence of HR-HPV genotypes among women in general and HIV-infected women in particular remains high in SSA. The presence of several genotypes not covered by the vaccine is remarkable and suggests the need for revision of current vaccination policies to prevent HR-HPV infections.Background: Sub-Saharan Africa (SSA) carries the highest burden of high-risk human papillomavirus (HR-HPV) in the world, driven by, and together with, HIV infection. This systematic review aimed to identify HR-HPV genotypes and their associated factors among women in SSA. Methods: A systematic review and meta-analysis of studies conducted in SSA on HR-HPV was conducted. Standard electronic databases were searched. R software version 3.6.0 was used for meta-analysis, with  Human papillomaviruses (HPVs) are viruses responsible for the occurrence of lesions in the anogenital and oropharyngeal regions, which can result in condyloma acuminata, warts that can develop into precancerous lesions and then some years later into cervical cancer ,2. Of noRegarding the pathophysiology, transformation of a cervical lesion into cervical cancer in HIV-negative women takes place over several years, generally induced by a group of so-called high-risk oncogenic viruses (HR-HPV), including the genotypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV42, HPV44, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV62, HPV66 and HPV68 .In case of co-infection with HIV, the burden of HPV is higher, with more rapid development of cancer from a cervical lesion, in the population of women living with HIV (50.8%) compared to their HIV-uninfected peers (22.6%). This therefore implies a higher vulnerability to HPV and cervical cancer in individuals co-infected with HIV ,12. ImpoChlamydia, Herpes simplex virus) and multiple sexual partners appear as the major drivers . The overall pooled prevalence of HR-HPV genotypes in HIV+ individuals was 75.51% [95% CI: 51.35–93.30] (2 = 0.1313 (NS = 8); p-value < 0.01 and I2 = 99%] and a significant difference in prevalence reported across included studies. The overall pooled prevalence of HR-HPV genotypes in HIV-negative individuals was 54.19% [95% CI: 17.33–88.58] (2 = 81.52 (NS = 6); p value < 0.01 and I2 = 99%], with a significant difference in prevalence reported across included studies   with hig5–93.30]  with hig3–88.58]  with hig studies .The pooled prevalence of HR-HPV genotypes was also estimated in the studies . The observed genotypes are classified as follows in order of prevalence: HPV16 (18%), HPV35 (10.12%), HPV52 (9.98%), HPV18 (9.7%), HPV45 (6.82%), HPV51 (6.60%), HPV58 (6.40%), HPV56 (6.20%), HPV33 (6.10%), HPV31 (5.90%), HPV39 (4.20%), HPV68 (3.96%), HPV59 (3.41%), HPV66 (2.20%), HPV62 (0.30%) and HPV61 (0.10%). Remarkably, most of these identified genotypes are not yet included in vaccines, particularly in those available in African countries. Most countries in sub-Saharan Africa have easy access to the bivalent and quadrivalent vaccines due to limited resources or lack of evidence for the 9-valent vaccines regularly used in developed countries . 2 = 0.3109 (NS = 04); p-value < 0.01 and I2 = 100%). The pooled prevalence of HR-HPV in South Africa was 59.42% [95% CI: 41.26–76.36] with a heterogeneity of (x2 = 0.1025 (NS = 12); p-value = 0 and I2 = 100%). In Uganda, the pooled prevalence of HR-HPV was 33.02% [95% CI: 12.27–58.05] with a heterogeneity of (x2 = 0.0499 (NS = 03); p-value < 0.01 and I2 = 99%). In Togo, the pooled prevalence of HR-HPV was 40.14% [95% CI: 22.05–59.74] with a heterogeneity of (x2 = 0.0290 (NS = 03); p-value < 0.01 and I2 = 96%). Data from Burkina Faso and South Africa revealed a pooled prevalence of 43.04% [95% CI: 39.20–46.92] with a heterogeneity of (x2 = 0.0006 (NS = 02); p-value = 0.06 and I2 = 72%); while four countries had no applicable heterogeneity  , Linear Array HPV Genotyping Test , HPV care, genotyping by PCR restriction fragment length polymorphism analysis , slide-based reverse hybridisation, multiplex RT-PCR, Sacace Biotechnologies, Anyplex II HPV28 real-time PCR , The Hybrid Capture DNA 2 (HC2) test (97% sensitivity and 97% specificity), INNO-LiPA HPV genotyping Extras delay) . The varHIV infection, a history of having sex with three or more sexual partners, young age, condom use, sexually transmitted infection, single marital status, precarious employment, use of hormonal contraceptives, alcohol consumption and tobacco use were the most frequently reported risk factors associated with HR-HPV infection .Five studies reported the rate of HPV positivity in HIV-positive and HIV-negative women respectively. In HIV-negative women, the rate of HPV positivity was 29.97% , while the rate of HR-HPV positivity was 61.72% (1751/2837), with HIV-infection increasing by more than four times the risk of being infected by HR-HPV (OR = 4.68 [0.71–30.76]) .Nine studies reported data on the cervical lesion status of participants in their studies; in total, 3814 tests were performed, and 459 participants had low grade squamous intraepithelial lesion (LSIL) compared to 534 who had high grade squamous intraepithelial lesion (HSIL). Two studies reported that the HR-HPV genotypes most commonly found in cervical lesions in the selected studies were 16 and 35 .The objective of this study was to conduct a systematic review with meta-analysis to identify the different HR-HPV genotypes and their relationship with HIV, to verify their association with high-grade cervical dysplasia in sub-Saharan Africa, and to verify their adequacy with available vaccines. Thus, the synthesis of studies with detailed data on HIV-infected women showed that the prevalence of HR-HPV is high but similar to the prevalence of HR-HPV in HIV-uninfected women; this observed difference seems surprising. However, this result may also reflect the success of antiretroviral treatment (ART) among people living with HIV(PLHIV) due to scale-up of ART, which in turn ensures immune reconstitution to normalcy at level similar to that of HIV-negative individuals .We decided to include studies that used a complete diagnostic kit capable of demonstrating at least eight HPV-HR genotypes. Although these kits are very costly for resource-limited countries and require state-of-the-art technical facilities with qualified technicians, they have the advantage of demonstrating the HPV-HR genotypes that are prevalent in a region, and this information could have an impact on the appropriate vaccine for sub-Saharan Africa to eradicate this virus responsible for several public health problems .HIV infection, a history of sex with three or more partners, young age of the woman, smoking, use of hormonal contraceptives were found to be the most frequently associated risk factors in this study. Smoking is known to act as an immunosuppressant, which decreases the host immune response to invasion of an HR-HPV genotype. With regard to sexuality, early sexual debut is an important risk factor, partly because of the immaturity of the cervix during adolescence, which makes it more susceptible to infection and damage by HR-HPV, and several authors have already shown that having sex with multiple partners significantly increases the possibility of exposure to HR-HPV ,27.With a positivity rate of 55.13% across all included studies, several high-risk oncogenic genotypes were revealed , with the most prevalent being HPV16 , followed by HPV35 (10.12%), HPV52 (9.98%) and HPV18 (9.87%). With the variability of genotypes identified from country to country, according to data generated by researchers in sub-Saharan Africa, it is essential to have data from each region in order to develop a good policy for the prevention of genital HPV infections which are significantly prevalent in the general population and in PLHIV in particular, who remain a vulnerable population when not put on ART . The perThe association between HIV and HPV is well documented, with HIV increasing the risk of HPV infection and persistence . FurtherAll of the genotypes identified, especially those not yet included in the literature review, were made possible by the use of comprehensive molecular diagnostic techniques, which are increasingly available to laboratories in sub-Saharan Africa, thanks to collaboration between researchers, institutions and organisations in the North and South . AuthorsVery few studies have reported data on the status of cervical lesions during infection and other coinfections. The available data nonetheless confirm that HIV is a major risk factor for the development of LSILs and HSILs, although the relative impact on HSILs is smaller . This isIn brief, it appears from our findings that accessibility to HPV screening tools needs to be expanded both within health facilities and at the community level. An optimal diagnostic algorithm, adapted to the context also needs to be developed and validated in low-income settings like sub-Saharan Africa, accompanied with training of health professionals, timely diagnosis and appropriate monitoring of women living with HIV/HPV co-infection as a vulnerable population. We must also enhance the sensitisation of stakeholders and community engagement for adherence to HPV screening and prevention strategies.This systematic review indicates that the rate of HR-HPV genotype positivity among women in general and in HIV-infected women in particular remains high in sub-Saharan Africa, with no significant disparity compared to their HIV-negative peers, probably due to widespread access to antiretroviral treatment (“test and treat” strategy) aimed at eradicating this pandemic. The study also showed that an alarming number of HR-HPV genotypes are circulating in sub-Saharan Africa, with a considerable presence of genotypes not covered by the vaccination policy implemented by the health systems of certain countries, calling into question existing public health policies against cervical cancer. Cervical cancer diagnosis and treatment programs therefore need to be strengthened, while vaccination policies against new emerging HR-HPV genotypes need to be implemented in these contexts.


Penile cancer (PC) is a rare male malignant tumor, with early lymph node metastasis and poor prognosis. Human papillomavirus (HPV) plays a key role in the carcinogenesis of PC. This review aims to summarize the association between HPV infection and PC in terms of virus–host genome integration patterns (the disrupted regions in the HPV and PC genome), genetic alterations, and epigenetic regulation (methylation and microRNA modification) occurring in HPV and PC DNA, as well as tumor immune microenvironment reprogramming. In addition, the potential of HPV vaccination strategies for PC prevention and treatment is discussed. Understanding of the HPV-related multidimensional mechanisms and the application of HPV vaccines will promote rational and novel management of PC. Penile cancer (PC) is a rare malignant tumor in the male genitourinary system, with an estimated 36,068 new cases and 13,211 deaths worldwide in 2020 . Due to So far, two major carcinogenic pathways have been identified as underlying causes of PC: one related to inflammation, phimosis, and lichen sclerosis, and the other to human papillomavirus (HPV) infection . The latThis review emphasizes the virus–host genome integration patterns, DNA alterations, epigenetic regulation, and tumor microenvironment reprogramming caused by HPV infection in PC, as well as prophylactic/therapeutic vaccines targeting HPV.Eligible studies in the English language were searched in Web of Science, Pub Med, Embase, and Cochrane from the date of inception up to 1 September 2023. Our search strategy included the following terms in the title/abstract: (“HPV” OR “human papillomavirus” OR “human papilloma virus”) AND (“penile” OR “penis”) AND (“cancer” OR “tumor” OR “carcinoma”) AND . In addition, we reviewed the references of the acquired literature to obtain more appropriate studies.HPV is a spherical, epithelia-philic virus with circular double-stranded DNA embedded in a protein shell. Currently, more than one hundred HPV subtypes have been detected , in whicEarly studies of HPV genome integration with PC cellular DNA mainly focused on virus genotyping via Southern blot assays and found HPV16 to be the most common subtype of HPV integrated in the PC genome ,20,21,22The HPV E2 protein has been reported to participate in the negative control of E6 and E7, which cause the inactivation of two key tumor suppressors (p53 and pRb) and tumorigenesis in the cervical epithelium ,25,26,27In HPV-induced cancers, the inactivation of p53 and pRb is usually attributed to the presence of HPV E2 disruption. However, the results of some other PC studies are inconsistent with this assumption ,33,34. TAlthough HPVs have been classified into hundreds of “subtypes”, more specific classifications within a certain subtype are established based on differences in genome sequence, referred to as “HPV variants” . These vThe alterations in the HPV16 DNA sequence have been implicated in the malignant transformation of squamous epithelium and in the progression of cancer. In cervical cancer, the frequency of AA and E variants was found to be significantly higher compared to normal cervical samples (50.3% vs. 1.1%) . SpecifiSeveral studies have focused on HPV variants in PC. Tornesello et al. identified European and non-European variants in 44.4% and 55.6% of 18 HPV16 PC samples from the Italian population, with the AA variant achieving the highest frequency . This reIt is worth noting that specific sequence mutations might occur in a particular variant of the virus, resulting in an increased risk of oncogenesis. Tornesello et al. revealed LCR, E6, and E7 mutations in a certain HPV16 variant, Af-1. These mutations were identified at nucleotide positions 7714 (T→A), 7461 (C→G), 7489 (G→A), 7521 (G→A), 7764 (C→T), 7786 (C→T), and 7833 (G→T) in LCR, positions 132 (G→C), 143 (C→G), 145 (G→T), 286 (T→A), 289 (A→G), and 335 (C→T) in E6, as well as positions 789 (T→C) and 795 (T→G) in E7. Moreover, the LCR in mutated Af-1 had more promoter activity and mutated LCR-driven E6/E7 showed higher transforming activity in vitro than those in classical Af-1, highlighting the critical role of sequence alterations in PC pathogenesis . SimilarThe accumulation of spontaneous somatic mutations in normal cells can lead to the development of corresponding cancers . SpecifiHaras missense mutation at codon 61 in an advanced lymph node metastasis of HPV18+ PC, occurring 7 years after the initial lesion. This finding suggests that this specific genetic alteration may occur during long-term HPV infection [+ PC, showing that high-risk type HPVs and mutated p53 may coexist in these tumors [+ PC cases, and revealed 38 altered cytobands with 2314 CNAs, 89.5% of which were located at the HPV integrated positions in the host genome and found that the gain on 14q12 was correlated with HPV16 and multiple infections [+ samples, respectively, but not in HPV16/18- tumors. Moreover, 30.8% of HPV16/18+ tissues were classified as having a high tumor mutational burden (≥10 mutations/Mb) [+ PC patients, including heterozygous deletions and single nucleotide change in STK11 [For PC, some works have also been conducted to analyze the role of viral infection in host genomic mutations. A case report first reported a c-nfection . Furthere tumors . Silva efections . Anotherfections . Nazha eions/Mb) . Other sin STK11 , as wellin STK11 . These a+ PC [+ PC ,65,66,67DNA methylation, which has been recognized as a hallmark of cancer, is a non-mutational epigenetic reprogramming mechanism . This moMethylation events also occur in HPV DNA. In cervical cancer, a higher incidence of HPV16 DNA methylation (47%) was detected at E2-binding sites  and the Sp1-binding site (Sp1BS) of the LCR region (regions involved in regulating E6/E7), compared to in normal tissues and cervical intraepithelial neoplasia (CIN) . Another+ PC cellular genome in addition to in HPV DNA. Generally, p16ink4a (a cyclin-dependent kinase inhibitor) is inhibited by Rb protein [ink4a expression in PC via a feed-back control [ink4a expression has been observed during HPV infection, several studies have demonstrated that the p16ink4a gene can be methylated. The proportion of p16ink4a methylation events in HPV+ PC samples ranged from 10% to 46.8% [ink4a may be involved in tumorigenesis and progression in PC. Since the p16ink4a protein induces cell cycle arrest [ink4a gene counteracts the proliferation inhibition caused by elevated p16ink4a expression due to the HPV E7/Rb axis, thereby promoting PC progression.Methylation events occur in HPV protein ,81. Thus control ,83,84. Eto 46.8% ,86,87, we arrest , presume+ (7 samples) and HPV− (18 samples) groups. Specifically, 65 genes were found to be hypermethylated in HPV+ cases, including CD70, HN1, FZD5, and FSCN1, which are involved in tumor migration, invasion, immunomodulation and other processes [+ PC tissues, respectively, and established a correlation between these two hypermethylation events and pathological variables [+ PC progression through similar pathways. Moreover, epigenetic silencing of FHIT via methylation has been observed in 100% of PC samples with viral infection [+ PC.Other studies have provided additional evidence regarding the methylation of host DNA in PC patients with HPV infection. One of these studies reported that 88.0% of PC cases infected with high-risk HPV had elevated global 5-methylcytosine levels (>60%), while 30.4% had increased global 5-hydroxymethylcytosine levels (>60%), suggesting that 5-methylcytosine, rather than 5-hydroxymethylcytosine, may serve as a stable epigenetic marker . The finrocesses ,94,95,96ariables . It has ariables ,99. Thernfection . These a+ PC DNA [+ PC DNA ,97,100.MicroRNAs (miRNAs), a class of small endogenous non-coding RNAs, promote cancer development by negatively regulating gene expression . Studies+ PC cases compared to those in HPV− PC cases, and to correlate with an expected elevation of EGFR expression. Further in vitro experiments verified a negative relationship between the levels of HPV16 E6 and miR-146a, which affects the proliferation of keratinocytes [+ PC tissues, 11 of which could target the TP53 and RB1 genes, suggesting an epigenetic mechanism of HPV infection for TP53/RB1 suppression [+ PC samples, with 5.5% of the alterations found within the UTRs. These alterations included miRNA targets in the UTRs of four cancer-associated genes . It was speculated that miRNA-directed down-regulated expression of these genes might be impaired due to UTR variants during HPV infection [Several studies have revealed changes in miRNA levels in PC patients infected with HPV. MiR-146a, known for its tumor suppressor function through binding to the 3’ UTRs of EGFR and NOTCH1 mRNA in breast cancer and glioma ,108, wasinocytes . TP53 anpression . In addipression . Canto enfection . Thus, e+ PC [+ PC ,109,110.+ and PD1+ tumor infiltrating lymphocytes, which conferred a favorable prognosis for anal and oropharyngeal cancer [It has been reported that HPV infection affects the immune microenvironment in cervical cancer through HPV-stimulated immune evasion via the reduction in immunostimulatory IFN-α of natural killer cells and the elevation of M1 to M2 transition in macrophages ,112. Addl cancer ,114. Rec+/CD8+ ratio in HPV-positive lymph nodes in PC patients, exhibiting an obvious expansion of CD8+ cytotoxic T lymphocytes [+, CD8+, or FOXP3+) and macrophages (CD68+ or CD206+) and found that PC samples with high-risk subtypes of HPV harbored a higher density of stromal Granzyme B+CD8+ T cells compared to HPV− tumors, indicating a highly immunogenic property of HPV [+ PC tissues rather than in HPV− PC samples, further confirming that HPV can induce immune activation. Intriguingly, a type of immune-suppressing T cell, CD4+Foxp3+ Treg cells, was detected more frequently in virus-infected tissues, indicating the complexity of HPV-induced immune regulation [+ and HPV− cases, which could be attributed to patient heterogeneity, sample size, testing methods, and other factors [Tumor infiltrating immune cells are the key component of the immune microenvironment. An early study analyzed the influence of HPV on the amount of Langerhans cells, a kind of immature dendritic cell that functions as immune surveillance, in penile epithelial cells. The results showed a significant reduction in Langerhans cells occurring in HPV-infected genital tracts, while local treatment with 5-fluorouracil restored a normal density of Langerhans cells, suggesting that HPV may promote immune evasion . Anotherphocytes . Chu et y of HPV . Similargulation . However factors ,120.+ PC cells than in HPV− PC cells. Specifically, the rate of PD-L1 positivity was 32.7–53.8% in HPV+ PC samples, whereas it was 49.4–64.4% in those without HPV infection [+ PC patients generally have a better prognosis than HPV− PC patients [+ PC exhibited a higher density of intratumor PD-1-positive T cells than the HPV− tumors, indicating immune evasion and higher immunotherapeutic potential for HPV+ PC [+ PC patients due to activation of PD1+ cytotoxic T lymphocytes. Certainly, this assumption needs further validation in large-scale clinical trials.Immunotherapy, including programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) inhibitors, has become a preferred option in combination with chemotherapy or targeted treatments . PD-L1 enfection ,120,123.patients . Additio HPV+ PC . In fact HPV+ PC . The afoConsidering the nature of virus-induced carcinogenesis in HPV-associated malignancies, vaccines targeting HPV may be an effective strategy for the prophylaxis of these cancers. HPV vaccination has been recommended worldwide for females and in certain countries for males to prevent HPV infection and the development of HPV-related neoplasms. Numerous studies have confirmed a significant reduction in the incidence of HPV infection and HPV-related carcinomas in the cervix, anus, and oropharynx in vaccinated individuals compared to those in the unvaccinated population ,126,127.Routine HPV vaccination is currently recommended for boys and men by the U.S. Advisory Committee on Immunization Practices . Recentl+ PC patients. Strong activation of CD4+ Th and CD8+ T lymphocytes against the HPV vaccine was detected, and dose-dependent responses with HLA-DR, a marker of T cell activation, were observed against gardasil at 0.1, 0.3, and 1.0 μg/mL, supporting T cell stimulation depending on the HPV vaccine [In addition to its prophylactic effects, HPV vaccination may confer therapeutic action against HPV-related cancers. Therapeutic vaccinations targeting HPV 16/18 E6 and E7 proteins have showed clinical responses in anal and cervical intraepithelial neoplasia ,134. For vaccine . In addi vaccine . However+ PC genome, such as CNAs and point mutations , are potentially affected by viral infection. Epigenetic changes are also significant in PC development. CpG methylation has been observed within the regions of HPV L1 and LCR, including the E2BS- and Sp1-binding sites. Specific methylation events and up- or down-regulation of miRNAs in HPV+ PC often induce tumor migration and therapy resistance via regulating the expression of corresponding genes. From the perspective of the PC immune microenvironment, anti-tumor immune cell types  increase with the up-regulation of PD1 expression on T cells. This enhancement potentially facilitates the efficacy of immunotherapy in PC patients. Finally, HPV vaccination may serve as an effective strategy for preventing HPV infection and PC development, and as a treatment option for HPV+ PC patients.HPV, particularly genotype 16, is involved in the malignant transformation of normal penile epithelium and the progression of PC in multiple ways. Regarding virus–host genome integration, the E1 region is disrupted more frequently in PC, and the integration positions in the PC genome tend to indicate the corresponding regulation of various tumor suppressors or oncogenes. In terms of DNA alterations, HPV DNA and the PC genome exhibit distinct characteristics. Non-European variants are more common in PC, sometimes with specific point mutations in the Af-1 region. DNA alterations in the HPVSurely, some limitations exist in this review. First, the number of eligible studies in some sections (HPV DNA methylations and HPV DNA mutations) is relatively inadequate due to the low incidence of PC, leading to potentially insufficient conclusions. Second, PC populations and the detecting technologies of HPV–PC genome integration vary from study to study, which may bias the results in disagreement. Third, the epigenetic effects from HPV infection have only been detected in some studies, which is not enough to clarify and validate the impact of methylation and miRNA/mRNA regulation on PC progression.In future studies, a more detailed investigation of the integration patterns of the HPV–PC genome, PC gene methylation, and miRNA/mRNA regulation, and consequently the expression regulation of host genes and downstream molecular changes during HPV infection, is warranted to gain a deeper insight into the pathogenesis of HPV-related PC. Moreover, the research focusing on the development of therapeutic vaccines targeting specific HPV DNA regions is indispensable for novel treatment options of PC patients.


Human papillomavirus (HPV)-based screen-and-treat (SAT) is recommended but implementation presents operational challenges. We implemented HPV-SAT at a research site in Khayelitsha, South Africa, screening 3062 women aged 30–65 years (44% women living with HIV [WHIV]). All were screened using point-of-care Xpert HPV and almost all received their HPV results on the same day. HPV-positivity occurred in 41.5% of WHIV and 17.4% of women without HIV (WNoH) reducing to 26.2% in WHIV and 10.4% in WNoH applying treatment eligibility criteria based on high viral load in the channels detecting HPV16, 18, 45, 16, 18, 31, 33, 35, 52, 58. Among those eligible for treatment, 91.3% were considered suitable for ablative therapy, and 94.6% underwent thermal ablation on the same day, with no serious adverse events. Twelve months later, 39.0% of WHIV and 65.2% of WNoH treated with ablative therapy were clear of HPV. In women who were HPV-positive but ineligible for treatment, 19.1% and 12.9% had histologically-confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) at 12 months. SAT programs need to weigh trade-offs between overtreatment versus delayed or no treatment for women who test positive for HPV. Treatment modalities for precancerous lesions need to be improved. Strengthening cervical cancer screening programs in Low and Middle Income Countries (LMICs) is essential to address global inequity2. Weak linkage between the screening test and follow-up treatment threatens the goals of the World Health Organization (WHO) elimination of cervical cancer as a public health problem agenda. Recognizing this critical gap in program efficiencies, WHO endorsed a simplified approach to screening called screen-and-treat (SAT). In SAT, a positive screening test result, a human papillomavirus (HPV) test, or visual inspection with acetic acid (VIA), identifies those at highest risk of precursor lesions that can be treated immediately—reducing attrition introduced by referral to colposcopy and histological confirmation. Although known to be safe and effective, including in women living with HIV (WHIV)6, much remains unknown about the implementation and scale-up HPV-SAT in real-world primary health care settings.Cervical cancer is one of the most preventable cancers due to the possibility of detecting and treating during the precancerous phase before the development of invasive disease. Yet, global inequality in access to quality health care has resulted in a highly-skewed prevalence, incidence, and mortality from cervical cancer8. This is done by restricting the definition of a positive test to a subset of HPV genotypes at higher viral load thresholds8. In addition, Xpert HPV can be used at the point-of-care (POC). POC testing is desirable because women can receive their screening results and be treated at a single visit. No additional reflex testing, triage tests, or new sampling is required.Some have questioned the desirability of HPV-SAT for populations with high HIV prevalence. WHIV have high rates of HPV co-infection, but not all will progress to cancer. Treating all WHIV who are HPV co-infected burdens the health care system and imposes unnecessary discomfort on women. Recently, our group proposed a modification to the interpretation of the Xpert HPV assay that reduces the number of women defined as “positive”, and thus reducing over-treatment, without substantial compromise in sensitivityHere we implemented this single-visit, HPV-SAT approach at a clinical research site in South Africa utilizing Xpert HPV, a POC assay, modifying the interpretation of this assay to define eligibility for treatment, and providing same-day ablative treatment for women who screened positive. Our goal was to evaluate real-life operational challenges in implementing this approach with task shifting to nurses and community health workers (CHW) to assess the feasibility of a broader roll-out.The study was implemented at a clinical research site co-located with a primary health care facility in Khayelitsha, outside Cape Town, South Africa. From May 2017 to September 2018, 3062 women, aged 30–65 years, residing in Khayelitsha were recruited into the study: 1346 (44.0%) were WHIV and 1716 (56.0%) were WNoH. Table Figure Next, a nurse-collected cervical sample was tested on-site with Xpert HPV. Overall, 857 (28.0%) women tested HPV-positive in one or more of the five channels at standard cut-offs (41.5% of WHIV and 17.4% of WNoH). Using the modified criteria, which involved restricting to three channels that detect HPV types 16, 18, 45, 31, 33, 35, 52, 58 and limiting further to more stringent cut-offs related to higher viral loads or multiple types, this number was reduced to 530 (17.3%) (26.2% of WHIV and 10.4% of WNoH).Almost all (99.8%) women received their HPV results on the same day. Four of 6 women who did not receive their results on the same day, received the results the following day. Most (98.1%) assays yielded a valid result on the first run. The median waiting time from sample collection to receiving results was 1.52 h (IQR 1.35–1.80 h). Exactly, 473 (15.6%) and 44 (1.5%) women waited for more than 2 h and 3 h, respectively. Longer wait times were associated with not getting a valid result on the first run  had conditions of concern on initial nurse examination and were reviewed by the on-site study doctor. Another 51/530 (9.6%) were considered unsuitable for immediate ablative therapy or the nurse was uncertain and were reviewed by the on-site doctor. On doctor review, 4/13  and 14/51  were referred back to the study nurse as suitable for ablative therapy. After this combined review, 484/530 (91.3%) were considered suitable for ablative treatment.Nearly 95% (458/484) of women referred for thermal ablation of the transformation zone were treated on the same day by the study nurse. The remainder (n = 26) received ablative treatment later date . Only 4 complications from thermal ablation were noted during the procedure—3 women complained of moderate or excessive pain and one had mild vaginal wall burn.At 6 and 12 months follow-up visits, 49 (11.6%) women reported that they had had complications following the procedure. Of these, 21 women had an unscheduled visit at the research site within 1–5 weeks post-treatment and an additional three women presented to a local clinic. All complications were minor and most were treated with antibiotics. No patients required up-referral, none were hospitalised, and none were severe enough to be reported at serious adverse events Table .Table 3SOf the 46/530 (8.7%) women who met modified HPV criteria for treatment but were unsuitable for immediate ablative treatment, 16 were treated with Premarin or antibiotics and scheduled for ablative therapy on-site after 6 weeks. Eleven were managed on-site, inclusive of 5 women who had Large Loop Excision of the Transformation Zone (LLETZ), 4 cone biopsies, and 2 cervical biopsies. Sixteen women were referred to a higher level of care  not visible, 6 the whole cervix was not visualized, 3 for technical reasons and 1 woman with a bleeding disorder). Three women in this group who had also been identified as being suspicious for cancer on initial review were referred to a higher level of care. All three had confirmed histological diagnoses of squamous cell carcinoma (SCC).In addition to the 13/55 women who had been flagged as having conditions of concern by the initial nurse evaluation and met modified HPV eligibility criteria (inclusive of the 3 cancers described above), 10/327 women who tested HPV-positive but did not meet eligibility criteria and 32/2205 women who tested HPV-negative were evaluated by the on-site study doctor. Of 42 in the latter two groups, one woman had histologically-confirmed SCC. This woman was classified as suspicious for cancer by the nurse on the initial exam and was in the HPV-negative group. She had an advanced cancer and was referred for chemoradiation but died from a pulmonary embolus before completing her treatment. She was retested using Xpert HPV and Roche Linear Array and both tests were negative for HPV. Of the 42 women, 5 had polyps removed, 24 were referred for a variety of gynecologic conditions and 12 were considered not to require further referral.All women who tested HPV-positive, whether or not they met modified treatment criteria, and a 10% random sample of women who tested HPV-negative were asked to return at 6 and 12 months  were seen at 6 months and 371 (76.7%) at 12 months.At 6 months, 29.2% of WHIV and 55.3% of WNoH and at 12 months, 39.0% of WHIV and 65.2% of WNoH had cleared all HPV. Clearance rates of the channels present at baseline were higher. At 12 months, 58.1% of WHIV and 68.9% of WNoH were negative for the channel they had tested positive for at baseline. Positivity for channels that were negative at baseline occurred in 19.5% of WHIV and 5.4% of WNoH. In terms of individual channel clearance rates, there were no differences between the channels  and P5  positive. A biopsy at 6 months yielded a diagnosis of cervical intraepithelial neoplasia grade 2 (CIN2) which prompted a LLETZ at 12 months, which diagnosed CIN3 and a microinvasive focus of 0.9 × 0.4 mm. The other two cancers were detected in WNoH. One woman was diagnosed on a biopsy taken at 6 months which showed a small focus of microinvasion with CIN3. She was persistently HPV 16 positive at baseline and month 6. The second WNoH had CIN2 diagnosed on a biopsy at 6 months and underwent a LLETZ at 12 months which diagnosed CIN3 with a focus of microinvasion measured at 0.2 × 0.2 mm. She was persistently P3  positive.At 6 months, 17.6% of WHIV and 12.9% of WNoH had CIN2 or worse (CIN2+) and at 12 months, 23.2% of WHIV and 14.4% of WNoH  were seen at 6 months and 245 (74.9%) at 12 months. At 6 months, 31.0% of WHIV had cleared all HPV and 46.7% of WNoH. At 12 months, 38.4% of WHIV and 58.6% of WNoH had cleared all HPV. The prevalence of CIN2+ at 6 months was 19.4% and 10.0% and at 12 months 19.1% and 12.9% in WHIV and WNoH, respectively  were seen at 6 months and 165 (74.0%) at 12 months. At 6 months, 14.9% of WHIV and 8.7% of WNoH tested HPV-positive. There were no cases of CIN2 or CIN3 among WHIV and 1 case of CIN3 in WNoH. At 12 months, 5.5% of WHIV and 6.4% of WNoH tested HPV-positive. No additional CIN2+ cases were identified.Operational fidelity to the HPV-SAT program was outstanding at our clinical research site. Recruitment was robust and steady. Almost all women had valid HPV test results on the first run. Almost all women stayed to receive their results. Greater than 90% of those eligible for treatment underwent thermal ablation on the same day. Task-shifting of HPV testing to a community health worker (CHW) with no prior laboratory training was successful. The program was nurse-led, with successful task-shifting of initial safety screening, sample collection, visual assessment for suitability for ablative therapy and, if suitable, performance of thermal ablation on the same day by the trained study nurse. Thermal ablation resulted in only minor complications in a small proportion of those treated. In sum, HPV-SAT successfully managed a round of screening at a single-visit, dramatically reducing the numbers of women who needed to be up-referred to specialist colposcopy services which are over-burdened in this setting.10. Failure of either ablative or excisional treatment of cervical cancer precursors has been well-documented in WHIV, due to decreased clearance of HPV infection, increased progressive disease and decreased regression in WHIV11. The proportion of WHIV in our study (44%) nearly doubles the 21% expected HIV prevalence in adults living in Khayelitsha12. This overrepresentation of WHIV in our study may reflect that people living with HIV access health services more often than women who are uninfected. Paradoxically, women without HIV infection are an under-screened population in our setting and special efforts must be made to ensure that they are adequately brought into services.South Africa has the highest rate of people living with HIV globally, with approximately 8.2 million in 2021, and cervical cancer screening services need to take into account the special needs of this group. The geography and variation in HIV prevalence influences screening outcomes, due to the different natural history of HPV infection in people living with HIV13. In our study, using a CHW to perform Xpert HPV testing on-site avoided logistical challenges with transporting samples to a central laboratory, loss of time, and the costs of transportation and laboratory-trained technicians. Most importantly, however, on-site testing enabled women to receive their results at the same visit, avoiding inevitable delays and attrition with results given at a subsequent visit. POC testing enabled the research clinical team to provide post-test counselling to HPV-positive women, an important intervention for comprehensive screening and treatment. A recent study from Peru reported in-depth interviews with women with a positive HPV test. Most women reported experiencing fear after receipt of a positive HPV test, particularly regarding how they became infected and by associating HPV infection with cancer, and death. This was followed by relief knowing that simple and fast treatments could be performed as an outpatient at primary care level. Counselling about the virus and expectations of treatment significantly reduced anxiety and fear among the participants14.The potential of POC testing to improve health care in LMICs has been widely discussedPOC testing will, however, require significant investment including, capital outlay for equipment as well as establishing reliable supply-chain management systems, commodities security and data management systems that may be less robust at the outset than utilizing established centralized laboratory systems. Reliable electricity supply is also needed which is a challenge in many LMICs, especially in South Africa. Our study was based at a clinical research site with research staff dedicated to screening, a GeneXpert instrument dedicated to HPV testing that did not need to be shared with other services, and adequate infrastructure and facilities. These aspects of HPV-SAT will require careful planning for sustainable integration within primary care facilities where patients with a range of health conditions are seen.15.Most women were informed of their test result within one and a half hours from sample collection. For most patients, the waiting time cannot be shortened further—Xpert HPV requires 60 min run time in the machine and the additional 30 min accounted for moving the samples from the exam room to the onsite laboratory, sample processing and loading, and results tabulation before returning results to the patient. Given available staff on site, one four module GeneXpert instrument was adequate for the number of women who could be seen daily. Instruments with larger capacities would allow for a higher testing volume with increased clinical staff capacity. The observation that most patients waited for return of their results and treatment, if needed, can be interpreted that the same-day, HPV-SAT is acceptable to patients. However, we did not directly inquire if the waiting time was acceptable. In addition, this wait time does not include time spent prior to sample collection or the time required for counselling and treatment, if necessary, after the HPV result was conveyed. We made provision for a waiting area with refreshments and made sure, through counselling and education, that women understood what was involved in a round of screening. In routine services it may be difficult to accommodate such long visit durations and to provide waiting area space. A possible solution could be to conduct informed consent, counselling and education at one visit, and then have a second visit for HPV testing and treatment. However, such an approach would add another layer of complexity and increase the likelihood of attrition. Adequate linkage to care is crucial when testing and treatment occur in different locationsAn important outcome of using our modified definition of a positive HPV test  to define eligibility for treatment was that it reduced by almost 40% the numbers of women who needed treatment. This benefit, however, came at the cost of relatively high rates of HPV persistence and pre-cancerous disease in those who were HPV-positive without meeting our modified criteria . While our approach reduces overtreatment, in settings with such high rates of HIV-positivity, HPV prevalence and cervical disease, restricting treatment to only those with a high viral load, results in women at lower but still high risk of disease being left untreated. In a situation where treatment resources are scarce, our approach is justified as it targets scarce treatment resources to the highest risk women. However, where treatment resources permit, widening the net may be more justified. Rationing treatment should be driven by resource availability and the ability to provide high levels of care to screened women. The specific cut-offs selected for programs should be based on a pragmatic approach which takes the context in which the program is offered and the resources available into account.Our data support the very high negative predictive value of a negative HPV test, in women both with and without HIV infection and support the notion of a wider interval for rescreening of at least 5 to 10 years, which in itself will result in reduced burden on screening resources.The initial nurse pre-screen examination detected four cancers. One of these four was HPV-negative and would have been missed by an HPV screening program that did not include this safety net. Although the purpose of screening is to detect pre-cancerous disease, in under-screened populations like the one studied here, programs need to be cognizant of the importance of detecting existing cancers and facilitating pathways into care. We recommend that screen-and-treat programs retain and use the clinical skills of the health care providers including visual inspection to detect clinically visible cancers and appropriate history taking to elicit symptoms related to genital tract disease.18, no serious adverse events or hospitalisations were recorded following thermal ablation supporting the safety of this procedure by a trained nurse.Consistent with prior studies19. A study performed in Honduras, not included in the meta-analysis, reported a higher cure rate (94%) based on being free of CIN2+ on histology20. Cure rates for women living with HIV were not reported out separately in the meta-analysis19.Rates of histologically confirmed disease post-treatment were relatively high (23% CIN2+ in WHIV and 14% in WNoH) indicating that thermal ablation was only partially successfully in treating precursor lesions. As we did not perform histological assessment of women at baseline, it is not possible to calculate the cure rate. Nevertheless the rate of 86% of women without HIV being free of histologically confirmed disease is identical to the cure rate reported in a meta-analysis for studies conducted in low and middle income studies although lower than the 94% cure rate they report for studies from higher income countries21. In a multisite study in rural China, 86% of treated women had cleared all high risk HPV types after thermal ablation, although rates were lower for clearance of HPV16/18 (68%)22. A randomized trial of thermal ablation vs. cryotherapy also conducted in China reported rates of HPV clearance exceeding 80% in both groups23.Our protocol defined clearance of HPV as the primary marker of treatment effectiveness and at the 12 month visit, only 39% of WHIV and 65% of WNoH were clear of all HPV. Even among women without HIV, these clearance rates are lower than have been reported from elsewhere. In a study from Cameroon, 84% of women treated with thermal ablation had cleared their HPV infection by 12 months24. All the women recruited to our study were South African women living in the peri-urban settlement of Khayelitsha where women have a high rate of exposure to these modifiable risk factors. Whether exposure to these risk factors explains the high rates of persistence of infection and disease post thermal ablation in this study will require further research.Reasons for the low clearance rates in our study are unknown but it is interesting that if clearance is defined strictly as clearance of a channel present at baseline, clearance rates are higher. At 12 months, 59% of WHIV and 69% of WNoH were clear of the infection they had at baseline compared to 39% of WHIV and 65% of WNoH defining clearance as being free of all high-risk HPV. The low rates of HPV clearance in our study, particularly in WHIV, may in part be explained by newly acquired infections or activation of latent infections. Our study recruited a largely unscreened or under-screened population and the prevalence of HPV, even among women without HIV, was high. We further restricted ablative therapy to those with higher viral load on the most oncogenic HPV genotypes mostly likely selecting those most challenging to treat. Larger lesions, poorer immunity due to co-morbid infections such as tuberculosis, HIV, high levels of stress and poor-quality nutrition related to poverty and low socio-economic status are associated with increased risk of pre-cancerous lesions25, 647 women tested positive for HPV using Xpert HPV, of whom 603 were considered eligible for same day ablation (94%) and 602 underwent treatment, a similar proportion as in our study. It may be that over 90% suitability for ablative treatment in both our and the PNG study is too high, particularly among unscreened populations of women, with high levels of preinvasive and invasive cervical disease. However, similar proportions of screened women were suitable for same day thermal ablation in studies performed in Malawi, 93.3%26 and Cameroon, 90.9%27, both low resource settings where the majority of women are unscreened.Our study classified more than 90% of those meeting HPV eligibility criteria for treatment as being suitable for ablative therapy. In a study of 4285 women in Papua New Guinea (PNG)28. This study recruited 231,741 women in three countries with an overall HPV-positivity rate of 13.6% and the majority tested using self-sampling. There was a delay of one month in receiving careHPV results which contributed to loss to follow up. However, 84.7% of women in Guatemala, 67.1% in Nicaragua and 58.8% in Honduras were suitable for and underwent ablative therapy, initially using cryotherapy and later using thermal ablation28.The Scale-Up Project conducted in Guatemala, Nicaragua and Honduras used careHPV testing with triage either by Pap or VIA, and, if triage-positive, women were referred for ablative therapyThis study has several strengths: Our clinical research site, co-located in a real-world primary health care setting, serves a largely unscreened population of women in the targeted age group of 30–65 years. All women were recruited from Khayelitsha, an area typical of low resource settings with high levels of co-morbid conditions and inadequate health care services. Study procedures were nurse-driven and performed on-site but with back-up of a study medical officer when required. HPV testing was performed by a CHW using a validated POC, HPV DNA PCR assay. The CHW was quickly up-skilled and proved capable of performing the test and undertaking the interpretation for eligibility for treatment. The ability to perform on-site HPV testing enabled over 90% of women eligible and suitable for ablation to receive treatment at the same visit. Linking screening to treatment is a major advance in cervical cancer prevention and will lead to greater coverage, efficiency and effectiveness of screening programs.Limitations of the study include the lack of histological sampling at baseline to determine prevalence of CIN2+ and hence our inability to estimate cure rates. We did not perform an in-depth acceptability evaluation, other than documentation of adherence to protocol. Future studies of screen-and- treat warrant a deeper assessment of women’s experiences and whether these pose a barrier to establishing services.Our study provides strong evidence that screening with a point-of-care HPV test and treating women using thermal ablation at the same visit has potential to be safe and feasible in a low resource primary care setting but further implementation studies are required.We have identified limitations to the approach of restricting treatment to only those with high viral load on certain HPV genotypes. While this approach has the advantage of reducing the number of women requiring treatment as well as the rate of overtreatment, rates of CIN2+ in women with HPV at lower viral loads was still quite high and these women are at risk of developing cervical cancer. The decision to use such modified cut-offs, and thereby restrict treatment, should be based on available resources and the context in which screening is implemented, balancing the trade-offs.The relatively high persistence of HPV infection and CIN2+ at 6 and 12 months post treatment in women in our study highlights the need for more research to improve treatment options. Currently available options  have major limitations and constraints. Other treatment options and/or adjunctive therapies urgently need to be investigated, including therapeutic vaccination.This demonstration study supports the feasibility and safety of HPV-SAT for cervical cancer prevention. HPV-SAT will add value to other efforts to eliminate cervical cancer as a public health problem. Implementing HPV-SAT should be incremental, with careful attention to the infrastructure and ecosystem required for successful and efficient outcomes and full integration into routine primary health care services.8. A positive HPV screen using the modified definition, was followed by a trained nurse visually assessing the woman for suitability for ablative therapy and, if suitable, thermal ablation was performed on the same day. The study was approved by the Human Research Ethics Committee of the University of Cape Town and the Institutional Review Board of Columbia University. All research was performed in accordance with relevant guidelines and regulations of these committees and the Declaration of Helsinki. All participants provided written informed consent.The study was implemented at a clinical research site co-located with a primary health care facility in Khayelitsha, outside Cape Town, South Africa. All clinical staff were employed by the research study. A single-visit, HPV-based screen-and-treat program was implemented which recruited women of known HIV status aged 30–65 years from the surrounding community. Point-of-care Xpert HPV was the primary screening test, with a positive result defined according to a modification based on HPV genotype and viral load restriction, previously validatedFigure ®ThinPrep®PapTest solution  for HPV testing.A trained study nurse performed a gynecologic examination, visually inspecting the lower anogenital tract and cervix without acetic acid for clinically detectable cancers and other markers suspicious for cancer. This examination was designed as a safety net for possible HPV-negative cancers. All women flagged as suspicious for cancer or with other concerning conditions, such as polyps, infection, atrophy or poor visibility of the cervix, were reviewed by an onsite study doctor regardless of HPV results. Next, the nurse collected a sample from the cervix using a plastic spatula and a cervibrush into 20mls of PreservcytThe nurse-collected specimen was HPV tested using Xpert HPV  on-site. Xpert HPV is a cartridge-based real-time PCR test run on the GeneXpert platform with results obtained in 1 h. A 4-module GeneXpert instrument was dedicated to the study. The system requires consistent electricity supply and linkage to a laptop computer, and printer. An inverter was installed to maintain power to the instrument given frequent interruptions to the electricity supply. Xpert HPV detects 14 high-risk HPV types with results reported in 5 separate channels: HPV16 (P1); HPV18,45 (P2); HPV31,33,35,52,58 (P3); HPV51,59 (P4); and HPV39,56,66,68 (P5). Xpert HPV is CE- marked since 2014 and received WHO prequalification in 2017. The test was validated by the National Health Laboratory Service (NHLS) of South Africa in 2016. The tests were run by a trained Community Health Worker (CHW) with no previous laboratory training and women were asked to wait for their results. The CHW received 3 days of training in running the assay, maintaining quality control and classifying the women into eligible or not eligible for treatment. Other members of the study team were also trained to perform these steps in circumstances when the designated CHW was absent. The time when the sample was collected was recorded on the participants’ record as well as the time when the result was returned to them to calculate the amount of time that was required to wait for the HPV result.8. Previously, we found that sensitivity of HPV testing to detect precursor lesions was minimally affected and specificity substantially improved if the definition of a positive result was restricted only to channels P1, P2 and P3 on Xpert HPV detecting HPV genotypes 16, 18, 45, 31, 33, 35, 52, 588. We demonstrated further that with minor trade-offs in sensitivity, specificity could be further improved by restricting to lower cycle threshold (CT) values  on these three channels, with slightly different CT cut-offs identified for WHIV and for women not living with HIV (WNOH)8. For this demonstration study, we developed a manual look-up table for the CHW running the HPV test to classify women into one of three categories: (1) HPV-positive according to our modified interpretation and therefore in need of treatment, (2) HPV-positive but not meeting our modified criteria and (3) HPV-negative. For women who were positive on only one of the three channels of interest, we used the CT cut-off identified in our validation study that yielded a sensitivity of ~ 84% and a specificity for WHIV of 78% and WNOH of 92%. These were for WHIV 34.5, 29.2 and 35.1, and for WNOH 39.2, 28.1 and 34.9 for P1 (HPV16), P2  and P3  respectively. In addition, we included all women positive on more than one of the three channels of interest regardless of CT value  the cervix was accessible (2) there was no evidence or suspicion of invasive cancer (3) the squamocolumnar junction was visible (4) there was no marked inflammation, infection or severe atrophy and (5) any visible aceto-white lesion covered less than 75% of the cervix and did not extend into the endocervical canal. Nurses could refer women to the on-site doctor if they were uncertain about their interpretation of the findings or if they considered the woman needing a higher level of care. The on-site doctor had the option of referring women back to the nurse if women were deemed suitable for same-day ablative therapy. Women were offered same-day ablative treatment unless a scheduled appointment was required.Study nurses used thermal ablation to perform ablative therapy. Thermal ablation uses a heated probe to destroy cells and tissues on the surface of the cervix, at a temperature of 100–120 °C and applied for 20 s, over the acetowhite lesion. Repeated applications of the probe are permitted. No local anaesthetic is required. The device is battery-driven . Any immediate complications were noted, and women were advised to return to the site or to their nearest primary care clinic if they developed any untoward symptoms such as pain, bleeding or an offensive discharge. While women were encouraged to return to the site for any complications, they were also given a letter that included relevant details about the screening and treatment procedure to present to other facilities. Women were encouraged to report to the nearest health facility emergency service in case of complications, especially bleeding, occurring afterhours, nights, weekends or public holidays. All women were also counselled to abstain from sexual intercourse or to use a condom if abstaining was not possible for 4 weeks after the procedure.Women who met the modified criteria for a positive HPV screen, but were not suitable for ablative therapy, were referred to the on-site doctor for assessment. If Large Loop Excision of the Transformation Zone (LLETZ) was indicated, it was usually performed by an on-site study doctor. If the on-site doctor determined that further investigations or other interventions were needed, the woman was referred off-site to secondary or tertiary-level services.The primary outcome measured operational fidelity to the screening algorithm and quantification of the proportions of women proceeding through the steps in the screening cascade Fig. .Figure Women who had undergone ablative therapy were asked at the 6- and 12-month visits about any complications they had experienced following treatment. The protocol defined the primary marker of ablative treatment effectiveness as the repeat HPV testing with Xpert HPV at 6 and 12 months post-treatment. Histological sampling was not performed before treatment precluding evaluation of the effectiveness of treatment based on this marker. Confirmed CIN2+ at 6 and 12 months were used as secondary markers of treatment failure.In women who tested HPV-positive on conventional cut-offs but did not meet modified criteria and in the random sample of women who tested HPV-negative, confirmed CIN2+ was used a marker of the magnitude of disease missed by the screening algorithm.We aimed to recruit 3000 women over 18 months, 1000 of whom were estimated to be WHIV. We estimated that ~ 480 women would meet the treatment HPV criteria, ~ 300 women would test HPV-positive but would not meet our criteria and that ~ 250 HPV-negative women would be randomly-selected for follow-up, leading to 1030 women scheduled for follow-up visits. The sample size was determined based on logistical, human and resource constraints with the number of women we could enrol, screen and follow-up.Descriptive statistics for categorical  and continuous variables were calculated as appropriate . Statistical analysis was done in SAS 9.4 .Supplementary Information.


Participants’ post-intervention vs. pre-intervention scores for attitude (mean = 35.46 ± SD = 5.46 vs. mean = 23.42 ± SD = 8.63), knowledge (M = 28.48 ± SD = 5.14 vs. M = 16.56 ± SD = 7.19), confidence (M = 8.96 ± SD = 3.43 vs. M = 6.17 ± SD = 2.84), and intention for vaccine acceptance (M = 4.73 ± SD = 1.78 vs. M = 3.29 ± SD = 1.87) increased significantly (p < 0.001). The intervention showed that for every one-unit increase in the participants’ self-confidence and attitude scores, the odds of the HPV vaccination acceptability increased by 22% (95% CI: 10–36) and 6% (95% CI: 0.1–12), respectively. Intention for vaccine acceptance, F (1167) = 6.89, and attitude toward vaccination, F (1167) = 19.87, were significantly higher among parents than adolescents (p < 0.001), after controlling for the baseline scores. These findings suggest that the intervention targeting parents’ and adolescents’ attitudes and knowledge has the potential to increase HPV vaccination acceptance in Ghana.The study examined whether the 3R  communication model intervention can impact parents’ and adolescents’ HPV vaccination acceptability. We used face-to-face methods to recruit participants from three local churches in the Ashanti Region of Ghana. Participants completed pre- and post-intervention assessments based on the validated Theory of Planned Behavior survey. We organized two face-to-face presentations for parents and adolescents separately for parents ( Human papillomavirus (HPV) is the most common sexually transmitted disease in the world ,2. Inces®, Gardasil®, and Gardasil 9®) have contributed to the decline in HPV-related cancer cases and deaths . For every one unit increase in the intervention score of parents’ positive attitudes, the odds of a higher (vs. lower) likelihood of vaccine acceptance for their adolescent increased by 21% while holding baseline scores constant [AOR = 1.21 ]. For every one-unit increase in the intervention score of parents’ negative attitudes, the odds of a lower (vs. high) likelihood of vaccine acceptance for adolescents increased by 15% while holding baseline scores constant [AOR = 1.15 ].Adolescents only: For every one-unit increase in the intervention score of adolescents’ confidence, the odds of a higher (vs. lower) likelihood of vaccine acceptance increased by 18% while holding baseline scores constant [AOR = 1.18 ]. For every one-unit increase in the intervention scores of adolescents’ positive attitudes, the odds of a higher (vs. lower) likelihood of vaccine acceptance increased by 7% while holding baseline scores constant [AOR = 1.07 ]. This community-based intervention assessed whether the 3R communication model was an effective model in changing Ghanaian parents and their unvaccinated adolescents’ attitudes, knowledge, and intention for HPV vaccination acceptance. The first main takeaway of the intervention is that parents with varied educational backgrounds, income status, and insurance status benefited from the intervention. Parents with a less educational background gained significant knowledge about HPV vaccination, and they also developed positive attitudes toward HPV vaccination. Parents with a low income had a significant improvement in their intention to accept HPV vaccination and their attitude toward vaccination. Our findings support a previous study which concluded that structure–educational interventions have the potential to improve parental awareness, knowledge, and perceptions toward HPV and the acceptability of the vaccine .A second major finding is the overall impact of the intervention on both parents and adolescents. The intervention showed significant effects on the adolescent and parent attitude toward HPV vaccination. Individuals’ positive attitudes toward a given behavior are associated with performing that behavior, whereas negative attitudes deter the performance of that behavior . The posIndividuals’ behavioral intention is proximal to performing a behavior, and participants’ scores for intention for getting vaccination increased. Overall, the 3R communication model was effective in influencing the participants’ intentions for HPV vaccination acceptance. We found that participants’ likelihood of HPV vaccination acceptance was associated with their self-confidence. Our findings showed that, for every one-unit increase in the self-confidence scores, the participants’ likelihood of getting the HPV vaccination increased by 19%. The participants’ likelihood of HPV vaccination acceptance was associated with their positive attitude scores. For every one-unit increase in the post-intervention positive attitude scores, the participants’ likelihood of getting the HPV vaccination increased by 9%. Our findings concur with other studies suggesting that attitudes about the vaccine are the most proximal determinants of vaccine uptake and likely mediate some of the effects observed by the other variables ,57. Our The subgroup findings showed that there was a significant effect on parents’ confidence and attitude toward vaccination. For every one-unit increase in the scores for confidence and attitudes, the likelihood that parents would allow their adolescents to vaccinate increased by 28% and 21%, respectively. The 3R communication model significantly reduced the parents’ negative attitude toward vaccination acceptance. For every one-unit decrease in the negative attitudes score, the likelihood that the parents would allow their adolescent to vaccinate increased by 15%. The adolescents’ confidence and attitude scores toward vaccination significantly increased. The one-unit increase in the scores for confidence and attitude the likelihood that the adolescent would get vaccinated increased by 18% and 7%, respectively. However, the percentage increases in parents’ confidence and attitude scores were greater than those in adolescents. Previous studies found similar results regarding parents’ attitudes and knowledge about cervical cancer and HPV, as well as the subsequent intention to receive the vaccination. Perceptions from parents about the HPV vaccine correlated with adolescent uptake of vaccination . In relaOur community-based educational intervention among parents and their adolescents was created utilizing the 3R  communication model and was shown to significantly improve attitude, knowledge, confidence, and intention regarding HPV vaccination acceptance . The sigAmong other demographics, previous studies with HPV-focused interventions delivered through various media forms have shown the effectiveness of educating parents ,71,72,73This study had some limitations. The quasi-experimental design nature of this study did not include a control group to compare results and confirm any causal relationships between the intervention and results. Additionally, since we used a single group pre- and post-intervention design, there may have been confounding factors influencing the final results; therefore, the findings of the study should be interpreted with caution. Moreover, without long-term follow-up, this study could only determine the intent of HPV vaccination and not the actual uptake of vaccination. In future research, a randomized control trial with a longer follow-up time is necessary to analyze the results of actual uptake of vaccination and the long-term effectiveness of improving parents’ knowledge and attitudes toward the HPV vaccination. Secondly, this study design utilized a convenience sample of adults and children willing to participate with interest in the study topic. This limitation could be addressed in the future with additional resources by enlisting a comparison group of parents and adolescents who opted not to participate in the study. Additionally, this study did not evaluate actual HPV vaccination behavior as we had limited resources to assess whether the intention for HPV vaccination acceptance among parents and their unvaccinated adolescents resulted in actual vaccination. In future interventions, an arrangement could be made to have a mobile vaccination clinic available so that participants who are willing and able to afford it can have access to vaccination right away. Although the cost of a vaccine can be a barrier to vaccination, after the intervention, the participants did not seem to worry about their ability to pay. One of our survey questions asked participants the following: “If my adolescent gets the vaccination, I will not be able to pay the vaccination bill”; this item did not seem to have any impact on the participants’ self-confidence for HPV vaccination acceptance. However, future studies to access actual vaccination will help determine participants’ ability to afford it. The strengths of this study include being one of the first to test the effect of intervention on Ghanaian parents’ attitudes and behaviors toward HPV vaccination acceptance. Additionally, this study assessed the efficacy of the 3R communication model in increasing the HPV vaccination intent of Ghanaian parents and adolescents. This information has important implications for the importance of education and addressing attitudes and perceptions when informing parents and adolescents of HPV vaccination. According to previous studies, framing the HPV vaccine message within the context of sexual behavior is ineffective because it solidifies the stigma and religious objections associated with vaccination . Our 3R An intervention utilizing the 3R communication model was effective in increasing parental and adolescent knowledge, attitudes, and confidence toward the HPV vaccination, as well as intention of HPV vaccination acceptance. These findings suggest that the 3R communication model, when used to guide intervention, has the potential to increase HPV vaccination uptake in Ghana. This information has important implications for the importance of education and addressing attitudes and perceptions when informing parents of HPV vaccination.


Background: Dental infections, which are the main cause of tooth loss, are known to be caused by bacteria. However, recent research suggests that other organisms, such as viruses, may also play a role. In this study, we aim to detect the presence of human papillomavirus (HPV)-16 and assess its prevalence in tissues infected with various dental infections, including aggressive and chronic periodontitis, pericoronitis, and periapical infection, as well as healthy gingival tissues, saliva, and gingival crevicular fluid, for comparison.Methods: A cross-sectional study including 124 adult healthy patients presenting with dental infections requiring dental extractions were conducted to assess the prevalence of HPV-16 in saliva, infected, and healthy tissues using quantitative polymerase chain reaction (PCR) tests. Samples were collected and a categorical scale was used for the prevalence. Statistical analyses were performed using Chi-square for the prevalence of HPV-16.Results: Among the HPV-16-positive PCR cases, the prevalence of HPV-16 was highest in periapical infection tissues as compared to chronic periodontitis, aggressive periodontitis, pericoronitis, and control tissues.Conclusion: The prevalence of HPV-16 in periapical infection samples was the highest among the studied dental infection samples. Thus, a primary conclusion can be drawn about the presence of an association between HPV-16 and the occurrence of periapical infection. Dental infections, or infections of dental origin, belong to the most commonly encountered infections in the oral cavity. They are considered the main cause of tooth loss as they affect the tooth-supporting tissues such as in periodontitis, or the periapical alveolar bone when it comes to periapical infection or abscess, or the hard and soft tissues surrounding an erupting third molar causing pericoronitis . It has Study designThis study was designed as a cross-sectional study that included 124 adult healthy patients. The patients were recruited from a private clinic in Beirut, Lebanon, between January 2020 and January 2022 using a non-probability convenient sampling technique. The tissue or saliva specimens were collected by one clinician and all samples were evaluated by one examiner who was blinded to the disease conditions of the patients.Ethics statementThe study was designed and conducted based on the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) and a flow diagram was prepared Figure . The stuInclusion criteriaAdult healthy patients, American Society of Anesthesiologists (ASA) I, who presented with dental infections that included AP, CP, PI or PC infections and requiring dental extractions were included in this study.Exclusion criteriaExclusion criteria included children and medically compromised patients (ASA II and III), including non-consenting patients with a history of cognitive dysfunction, pregnant or lactating women, as well as patients undergoing chemotherapy or radiotherapy.Sample size determinationThe sample size was determined based on the primary outcome, prevalence, with a 95% confidence interval assuming an alpha of 5%, an estimated prevalence of 50%, and a margin of error of 10%. Sample collectionInfected gingival tissues were collected from extraction sockets of teeth with either one of the studied pathologies, while healthy gingival tissue samples were collected from extraction sockets of teeth that were extracted for orthodontic or prosthetic reasons and were confirmed to be healthy.Saliva and GCF samples were collected from some of the participants and analyzed for the presence of HPV-16. Patients were provided with a sterile container in which to spit. GCF samples were collected by inserting paper points into the gingival sulcus. Infected and healthy tissues were collected from both the socket and/or the periodontal ligament of the extracted teeth. Sample tissues were then rinsed with a sterile saline solution, and all samples were stored at -80°C for future analysis.Outcome measuresThe primary outcome was the prevalence of HPV-16, which was evaluated and reported as a ratio in saliva, GCF, healthy and infected samples.Sampling quantification of HPV-16DNA extraction and isolation were performed using the QIAamp® DNA mini kit , as per manufacturers' instructions, through three main steps: lysis, followed by DNA precipitation, and DNA elution. DNA purity and yield were evaluated using a nanodrop spectrophotometer  in addition to q-PCR analysis .Quantitative polymerase chain reaction analyses (q-PCR)The q-PCR technique was used to determine the presence of HPV-16 in fluid and tissue samples. The q-PCR technique was conducted using a SYBR green mix in a CFX96 system . Products were amplified using primers detailed in Table Seven hundred twenty-four patients were screened, of which 570 patients were excluded, and 154 patients included. Among the recruited patients, 124 finally participated in the study. Tissue samples were obtained from 32 CP, 38 PI, 27 PC, and 17 AP  tissues as compared to CP (15.62%), AP (7.14%), PC (5.88%), and control (9.75%) tissues. HPV-16 was detected in 3.79% of the saliva samples and no positive cases of HPV-16 were detected in any of the GCF samples  as compared to healthy tissues from each participant in the Lebanese population. Among the HPV-16-positive PCR cases, the prevalence of HPV-16 was highest in PI tissues (23.68%) as compared to other investigated infection tissues, healthy tissues, saliva and GCF samples with a confidence interval of this prevalence ranging between 11.44 and 40.24. As the confidence interval reflects the prevalence of this virus from our samples in the Lebanese population with a 5% error, the high value of the lower limit of this interval (11.44 significantly superior to zero) indicates that this virus has a significantly high prevalence in the Lebanese population. As for CP, the prevalence of HPV-16 was 15.7% with confidence interval ranging between 5.28 and 32.79, so this suggests a possible association between HPV-16 and CP in the Lebanese population. In the control samples, the prevalence of HPV-16 detected was 9.75% with confidence interval ranging between 4.31 and 18.32, which suggests that HPV-16 may be present in healthy tissues similarly like other viruses. On the other hand, the low prevalence of HPV-16 in AP, PC, and saliva , and the approximately zero lower limit of confidence interval  suggests that no association is found between AP and PC and HPV-16, and that this virus is not found in saliva. In addition, it should be noted that the values of prevalence obtained in AP and PC were close to those obtained for control tissues, which supports our conclusion of the absence of association between HPV-16 and AP and PC.On the other hand, our results were non-conclusive concerning the association between HPV-16 and periodontitis, while some previously conducted studies did not find an association ,11. On tBesides, there are several factors that could contribute to the variability in prevalence and controversies between studies, such as demographic factors, systemic and oral health status, clinical tissue sampling, and laboratory techniques. The analysis was performed using a real-time PCR technique for assessing the presence of virus in each sample category and to compare the results between different sample categories. According to the available information, no previous studies have assessed and compared the viral presence of HPV-16 in all these dental infections. In the oral cavity, the detection rate of HPV varies considerably in published data, ranging from 0-100%, depending on the status of the disease, tissue origin, detection techniques, and other environmental risk factors such as geographic regions [Although the presence of HPV-16 has been explored in AP, CP, and PI, the literature is scarce, particularly in PI studies -18. AmonIt is important to point out that the quantity of HPV-16 in both fluids, saliva, and GCF, was significantly lower than all other healthy and infected tissue samples (3.79 and 5% respectively). This could be plausibly attributed to the fact that HPV-16 is an intracellular micro-organism  and has Moreover, multiple HPV genotypes may be present in dental infections. The most commonly identified genotypes are HPV-16 and HPV-18, with HPV-16 being more prevalent in the normal oral cavity than HPV-18 . As viraIn summary, our study found that the prevalence of HPV-16 was higher in tissue samples than in fluid samples such as saliva and GCF. Furthermore, infected tissue samples, and more specifically PI samples, showed a significantly higher prevalence of HPV-16. Therefore, a primary conclusion can be drawn about the presence of an association between HPV-16 and PI. This association may suggest that patients with dental infections may require special care regarding cross-contamination and their high risk of oral carcinoma due to their elevated chance of having an underlying HPV-16 infection. However, further studies and investigations are necessary to confirm this hypothesis.