The progression rate from CIN1 to CIN3 is 9.0% and that for invasive cancer is 1.0%. The large majority of CIN1 lesions regress spontaneously, and the treatment of CIN1 is still controversial.The aim of this study is to investigate the responsible HPV genotype in the low‐grade SILs, then to predict the presence of high‐grade SILs, and determine whether further treatment is needed.We use the methods of manual microdissection with FFPE tissue specimens and the E6/E7 uniplex polymerase chain reaction (PCR) to detect HPV in the lesions.The HPV test was performed on 72 biopsy tissue specimens, and 55  of them were HPV positive. Nine  of them escalated to CIN2 after LEEP or cervical conization, and 46  were still CIN1. There were 17  cases with HPV‐negative results in cervical biopsy tissues.HPV test of cervical biopsy diagnosed with CIN1 has a positive predictive value of 16.4% in the presence of CIN2 or higher lesions, a negative predictive value of 94.1%, a specificity of 25.8%, and a sensitivity of 90.0%. HPV test of cervical biopsy tissues for the prediction of HPV infection in LEEP or cone surgery tissues had a positive predictive value of 80.0%, a negative predictive value of 82.3%, a specificity of 56.0%, and a sensitivity of 93.6%.It is the first time that we have detected HPV genotype in the low‐grade SILs by the methods of manual microdissection with FFPE tissue specimens and the E6/E7 uniplex PCR. Patients with cervical biopsy tissue diagnosed with CIN1 and with a negative or only low‐risk HPV type result can be considered for follow‐up. Conversely, in cases of cervical biopsy tissue diagnosed with CIN1 positive for high‐risk HPV, surgery or a close follow‐up program can be selected. Patients with cervical biopsy tissue diagnosed with CIN1 and with a negative or only low‐risk HPV type result can be considered only follow‐up. Conversely, in cases of cervical biopsy tissue diagnosed with CIN1 positive for high‐risk HPV, surgery or a close follow‐up program can be selected. However, about 90% of them are transient, and these viruses can be cleared from the patient within a few years.Epidemiological studies have shown that HPV in cervical squamous cell carcinoma (Cx‐SCC) has a prevalence of 98.7%,The progression rate from CIN1 to CIN3 is 9.0% and that to invasive cancer is only 1.0%. In fact, the large majority of CIN1 lesions (57%–90%) regress spontaneously,Therefore, the main target of a predictive test is to have an extremely high or even an absolute negative predictive value, which means that no cases were predicted to be low‐grade SILs. However, the finding of high‐grade SIL had an acceptable positive predictive value. With such a predictive test, it may be possible to reduce the number of patients overtreated for CIN1 lesions that may not progress or may even regress spontaneously.The aim of this study is to investigate the detection of HPV genotype responsible for the lesion by the methods of manual microdissection with FFPE tissue specimens and the E6/E7 uniplex polymerase chain reaction (PCR), then to predict the presence of higher‐grade SIL lesions, and determine whether further treatment is needed.22.1We selected patients who underwent cervical cancer screening at the Gynecological Clinic of China Medical University Affiliated Shengjing Hospital from January 2017 to January 2019. Due to abnormal results in the cytological test (Thinprep Cytologic Test) and HPV test using cervical canal cells, 5367 patients underwent further colposcopy or cervical multiple‐point biopsy in the Cervical Lesion Clinic. The endpoint diagnosis was based on the histopathological diagnosis, and the highest grade was chosen . In total, 1033 patients were diagnosed as CIN1, and only 76 of them underwent further treatment, including the Loop Electrosurgical Excision Procedure (LEEP) or cervical diagnostic conization, while the remaining 957 cases were simply monitored with a cytological test, HPV test, and colposcopy. Consent was obtained from all subjects before surgery for the use of their cells or histological specimens for this study, and the protocol was approved by the Ethics Committee of China Medical University Shengjing Hospital.The Thinprep Cytologic Test (TCT) was performed through a liquid‐based thin layer machine  and was then examined and diagnosed by an experienced cytopathologist. The criteria of diagnosis were based on The Bethesda System classification revised in 2001.The HPV test of cervical canal cells was performed using the HPV typing test kit (Kaipu Bio Ltd). The kit can identify 13 high‐risk types , two possible high‐risk types (HPV53 and HPV66), and six low‐risk types . A positive result for more than one type was considered a multi‐type infection. However, five possible high‐risk types, namely HPV26, 67, 70, 73, and 82, could not be detected. The TCT and HPV tests were performed within 1 week from the first visit.Colposcopy was performed to examine the presence of cervical lesions and then three or four punch biopsy specimens were obtained from abnormal lesions. Both the steps were performed by an experienced gynecologist.As for further treatment, LEEP surgery was performed in the Cervical Lesion Clinic, whereas cervical conization was completed in the operating room. All final histopathological diagnoses were revised in consensus by two experienced pathologists. If the diagnosis results were inconsistent, a third high‐level pathologist assisted the diagnosis.2.2With the assistance of a pathologist, we obtained all the 72 patients’ FFPE blocks, which contained biopsy tissue and LEEP or conization tissue.The FFPE blocks were sectioned according to the following procedure: 1 slide of 4 μm section was used for histopathological diagnosis, and 4–5 10 μm slides were used for DNA extraction and HPV genotyping, and the last 4 μm slide was used for histopathological evaluation to confirm target lesion remained. The microtome was cleaned with 70% alcohol, and the blade was replaced with each tissue block in order to avoid contamination. The tissue slides were stored in a refrigerator at −20°C until the next procedure.All the control slides with HE staining were reviewed by the same pathologist, and the final diagnoses were confirmed. The areas of abnormal lesions, and normal squamous or glandular epithelium were marked separately on each control slide. Viewing the marked areas, abnormal area, or normal epithelium on the tissue slides were independently dissected by hand using a sterilized needle under the inverted microscope. Each tissue fragment was placed into a 1.5 ml tube with 50 μl of alkaline lysis reagent . Next, the tube was incubated in a thermo‐shaker  at 300 rounds per min (rpm) for 15 min at 95°C, the tube was then spun down for 10 s, and the procedure was repeated at 300 rpm once, at 900 rpm twice. Subsequently, the same amount of acidic neutralizing solution  was added into the tube, mixed thoroughly, and centrifuged  for 1 min. Finally, 30–50 μl of supernatant was transferred into a new 1.5 ml tube and diluted 5 times with distilled water (DW), and 5 μl of the solution was used in the next procedure for HPV genotyping, and the remaining solution was stored in a freezer at −20°C.2.3The uniplex E6/E7 PCR test was designed to amplify E6 and E7 genes of 39 common HPV types using type‐specific primer pairs. This assay was able to detect 13 high‐risk types , 11 possible high‐risk types , 15 low‐risk types , and the beta‐globin gene as a positive control individually. The specific procedures have been described in previous papers.2.4U test to analyze continuous variables (patient’s age), and the Chi‐square test with Yates’ correction was used to compare categorical variables . Fisher’s exact test was adopted when the total sample size was less than 10. A cutoff value of p < 0.05 was considered statistically significant. All the statistical analyses were performed with SPSS version 19.0 (IBM).We used the Mann–Whitney 33.1A total of 72 patients were included with an average age of 41.8 (23–62 years) years. The prevalence of ASCUS+, LSIL+, and HSIL (or ASC‐H) among them was 100.0%, 85.3%, and 25.0%, respectively (Table There were 11 cases with HPV‐positive result in biopsy tissue but HPV‐negative result in postoperative tissue specimens (details in Table p = 0.005). The prevalence of HR‐HPV, pHR‐HPV, and LR‐HPV in the unescalated group was 30.6%, 3.2%, and 43.5%, respectively, and that for the escalated group was 100.0%, 0, and 0, respectively (Table p < 0.001), whereas LR‐HPV type was more often in the unescalated group (p < 0.022). Of the 72 LEEP (or conization) issue specimens, 14 were negative for HPV, and all of them were CIN1  patients with postoperative pathological escalation to CIN2, and 62  patients were still CIN1. No escalation to CIN3 or worse was found  in tissue specimens after cervical biopsy or LEEP (or conization), and 15 patients  were negative both in cervical biopsy and LEEP (or conization) tissue specimens.p = 0.49). At the same time, it can be concluded that the HPV test of cervical biopsy diagnosed with CIN1 has a positive predictive value of 16.4% for the presence of CIN2 or higher lesions, a negative predictive value of 94.1%, a specificity of 25.8%, and a sensitivity of 90.0%.Among the 72 cervical biopsy tissues, 55  cases were positive with HPV, 9  of them escalated to CIN2 after LEEP or cervical conization, and 46  were still CIN1  were still HPV positive in LEEP (or conization) tissue specimens, however, 11 cases were HPV negative  after LEEP (or conization) Table . Of the 3.3Among the 72 tissues, after cervical LEEP or conization, there were 10 cases of CIN2, and high‐risk HPV was detected in all of them; 9 cases were single‐type infections, of which HPV16 was detected in 4 cases, HPV52 in 2 cases, and HPV18/51/58 only in 1 case for each type  cases were negative. Among 48  cases that were positive for HPV, 6 were positive with HPV58, 3 were positive with HPV16/51/52/68, and 2 were positive with HPV35/39/45/53. The least frequent were HPV18, 31, 56, and 59, with only one case for each type, and there were 27 cases of low‐risk HPV infection . This is consistent with the conclusions from previous studies, and it also shows that the progression of CIN takes a long time.In our study, 10 patients were finally diagnosed as CIN2 after LEEP or cervical conization. This means that higher‐grade lesions were missing during cervical biopsy under colposcopy in 13.89% of the patients, which was lower than the result of other studies.A previous study from our group showed that at least one HR‐HPV or pHR‐HPV can be detected in CIN2 or CIN3 FFPE tissues,Among the 10 CIN2 cases, HPV16 was the most common type, followed by HPV51/52, and nine of them were single‐type infections. The exception was case no. 68, which was positive for both HPV42 and HPV51  tested negative in the HPV test as mentioned above. Therefore, the evidence for this is still insufficient, and the limitation of the present research is the small size of the samples. A total of 72 cases were eligible, and only 10 cases were pathological upgrade after LEEP or conization. Multicenter with large sample size study is needed for further research.For case no. 13, we found HPV16, HPV52 in biopsy tissue, while only HPV16 was found in cervical canal cells. This may be due to the uniplex E6/E7 PCR HPV detection method has higher sensitivity, and the HPV52 virus load in cervical canal cells is too low to be detected. For the other three cases, case nos. 24, 38, and 41, we think the HPV genotypes detected in cervical canal cells may not be the types that eventually lead to lesions, they may only be temporary infection, and the responsible HPV that really causes lesions in cervical canal cells may not be detected. This difference may be caused by different sensitivities between the two HPV detection methods, which is also one of the limitations of this study.In conclusion, the strength of this study is that we detected responsible HPV genotype in the low‐grade SIL lesions by the methods of manual microdissection with FFPE tissue specimens and the E6/E7 uniplex PCR. In the present study, we believe that patients with cervical biopsy tissue diagnosed with CIN1 and with a negative or only low‐risk HPV type result can be considered for follow‐up without further treatment such as LEEP or conization. Conversely, in cases of cervical biopsy tissue diagnosed with CIN1 positive for high‐risk HPV , surgery (LEEP or conization), or a close follow‐up program can be selected, though this warrants further investigation in multicenter trials with large samples.There is no conflict of interest in the paper.Conceptualization, H. Wang and S. Zhang; Methodology, H. Wang; Statistical analysis, X. Han; Pathological analysis, Z. He; Data curation, D. Zhang; Writing—original draft preparation, H. Wang; Writing—review and editing, S. Zhang; Visualization, S. Zhang; Supervision, S. Zhang; Project administration, S. Zhang. All authors have read and agreed to the published version of the manuscript.Consent was obtained from all subjects, and the protocol was approved by the Ethics Committee of China Medical University Shengjing Hospital.


Papillomaviruses (PVs) are ubiquitous intracellular pathogens that have co-evolved with many different species. Regardless of species, infection with PVs typically induces benign hyperplastic growths of epithelial cells . In humaAccording to the most recent global statistics of new cancers, in 2018, HPV was estimated to be responsible for virtually all cases of cervical and anal cancers, a significant fraction of vaginal 78%), vulvar (25%), penile 53%), and an increasing number of head and neck cancers —inclusive of the oropharynx (30%), oral cavity (2%), and larynx subsites (2%) [%, and an8%, vulvaPioneering research conducted in the early 90s demonstrated the ability of recombinant L1, HPVs major capsid protein, to self-assemble into virus-like particles (VLPs). These VLPs were type-restrictive in their ability to induce neutralizing antibodies, and therefore multiple VLPs generated from the major wart-inducing and carcinogenic types were necessary to achieve broad protection . The ava−) counterparts that has implications for diagnosis, treatment, and outcomes. In recent years, many clinical research initiatives, specifically in the head and neck region, have profiled these differences at the epidemiological, molecular, immunological, and clinical levels. Indeed, in the context of HNCs, the HPV+ subtype has more immune cell infiltration into the local tumor microenvironment, greater incidence of T-cell activation, and higher levels of immunoregulatory stimuli compared to HPV− HNCs [+ and HPV− cervical cancers [− cervical cancer samples since virtually all cervical cancers are caused by this sexually transmitted virus.An exciting, and hot area, of clinical research on HPV-induced cancers is the revelation of distinct differences compared to anatomically-similar HPV-negative (HPVPV− HNCs ,11,12. I cancers ; however+ HNC may be due to the continuous expression of the viral E6 and E7 oncoproteins within the local tumor microenvironment that initiate an immune response. In addition, patients with HPV+ HNC also have greater sensitivity to chemotherapy and radiotherapy compared to those with the HPV− subtype, leading to a more favorable prognosis, with lower recurrence rate, and longer overall survival time [+ HNC, with the goal of sparing individuals from high-grade toxicities induced by aggressive radiation and chemotherapies that were originally developed for the treatment of individuals with the HPV− subtype of cancer [+ HNC could be due to the differences in the immune landscape of these anatomically identical carcinomas with very different etiologies. Intriguingly, prognosis for patients with HPV+ cervical cancer is also better than their HPV− counterparts, supporting the presence of parallel processes of treatment response in completely separate anatomical sites [These distinctive immunological features observed in HPVval time ,15. Thisf cancer ,17. The al sites ,19,20.+ HNC. Indeed, these tumor microenvironments have higher levels of LAG3, PD1, TIGIT, TIM3, and VISTA compared to their HPV− counterparts [+ HNC, with high levels of PD1 expression, displayed improved outcomes when treated with PD1 inhibitors compared to those patients with the HPV− subtype [+ HNCs may similarly respond to other immune checkpoint inhibitors, which has implications for de-intensification from the highly toxic traditional therapies and the possible reduction of treatment-induced sequelae.An abundance of clinical research has provided clear evidence that immune checkpoints, negative regulators of the immune response, are upregulated in HPVterparts ,21,22,23 subtype . This de− counterparts from an epidemiological, molecular, immunological, and clinical perspective. For this Special Issue, we invite authors working in the field to submit state-of-the-art original research and comprehensive reviews that encompass the current landscape of human papillomavirus clinical research, from infection to cancer. Although the primary focus is on infection and cancer of the head and neck region, we will consider HPV relevant manuscripts from other anatomical disease sites.From the explicit demonstration of the viral origins and contagious nature of warts in the 20s, to the ground-breaking discovery of select HPV types as the causative agents of cervical cancer and a subset of head and neck cancers in the ‘80s and early 2000s, respectively. Clinical research on this important human pathogen has continuously shaped the field and pushed the boundaries of our collective knowledge. Today, it is well-established that infection with certain types of HPVs can give rise to cervical cancers, other anogenital cancers, and HNCs—one of the fastest rising cancers in some countries despite the availability of effective vaccines. The culmination of nearly a century of clinical research initiatives have established certain HPV types as potent human biological carcinogens, collectively responsible for an estimated 4.1% of the global cancer burden that are distinct from their HPV


In settings with high HIV prevalence, cervical cancer incidence rates are up to six‐fold higher than the global average of 13.1 cases per 100,000 women‐years. To inform strategies for global cervical cancer elimination, we used a dynamic transmission model to evaluate scalable screening and treatment strategies, accounting for HIV‐associated cancer risks and weighing prevention gains against overtreatment.We developed a dynamic model of HIV‐HPV co‐infection and disease progression, which we calibrated to KwaZulu‐Natal, South Africa. Our baseline scenario reflects the current practice of HPV vaccination with a multi‐visit screening and treatment strategy involving cytology and colposcopy triage. We evaluated 13 comparator scenarios with increased vaccination coverage and one‐time, two‐time or repeat HIV‐targeted cervical cancer screening with the following single‐visit strategies: HPV DNA testing, HPV genotyping, automated visual evaluation (AVE) and HPV DNA with AVE triage. In all scenarios, HIV antiretroviral therapy, condom use and voluntary male medical circumcision continue at baseline levels. We simulated cancer incidence under each scenario from 2020 to 2120 using the 25 best‐fitting parameter sets. We present the median and range of model output from these simulations to account for parameter uncertainty.We estimate that cervical cancer incidence will decrease by 87% with the continuation of current cervical cancer and HIV prevention strategies, from an age‐standardized rate per 100,000 women of 80.4  in 2020 to 10.7  in 2120. Scenarios scaling up vaccination and single‐visit strategies resulted in near‐ and long‐term gains. With repeat HIV‐targeted screening, incidence rates were projected to be 29–34% lower in 2030 relative to the baseline scenario, and elimination  was achieved with HPV DNA testing in 2095 and with AVE in 2114. A strategy of HPV DNA with AVE triage optimized the tradeoff between cancer cases averted and overtreatment.Single‐visit screening strategies could avert a substantial burden of cervical cancer and accelerate progress towards elimination in settings with a high burden of HIV. Increasing the screening frequency among women with HIV and reducing loss‐to‐follow‐up for treatment will be key components of a successful elimination strategy. HoweverIn sub‐Saharan Africa, the region with the highest cervical cancer incidence rates , high HIIn this analysis, we project cervical cancer incidence over the coming century under a range of scenarios using a dynamic model fitted to KwaZulu‐Natal, South Africa, where HIV prevalence among reproductive‐aged women in 2016 was 37% . The sta2The Data‐driven Recommendations for Interventions against Viral InfEction (DRIVE) model is a compartmental model of HIV and HPV co‐infection and natural history, adapted from a previously published model . It simuHPV and HIV transmission is modelled through sexual contact in heterosexual partnerships. Rates of partnership formation depend on sex, age and sexual risk group, with assortative mixing by age and risk group. The model includes cofactors that modify HIV transmission: ART, voluntary male medical circumcision (VMMC) and condoms. We model the use of ART only for individuals with full HIV viral suppression. Condom use also reduces HPV transmission, and the risk of HPV acquisition increases with declining CD4 count among individuals with untreated HIV.2.1We reviewed the literature to obtain context‐specific estimates for model inputs related to demographic dynamics, sexual behaviour, use of HIV and cancer prevention interventions, and transitions between health states. After identifying parameters for which empirical evidence is limited or uncertain, we used a multi‐phased approach to calibrate our model to historical outcomes. In a preliminary step, we used hand‐calibration to assess the sensitivity of the model to specific inputs and refine prior ranges. We then used an Approximate Bayesian Computation‐Sequential Monte Carlo algorithm , 19, 20 2.2We assessed 14 primary scenarios with varying strategies and/or coverage of HPV vaccination, screening and precancer treatment Table . Key difThe next set of scenarios model four single‐visit screening and treatment strategies with 90% 9vHPV coverage beginning in 2021: (1) oncogenic HPV DNA testing; (2) HPV DNA testing with genotyping for HPV types 16, 18, 31, 33, 45, 52 or 58; (3) AVE; and (4) oncogenic HPV DNA testing with AVE triage. In these scenarios, 48% of individuals aged 35–39 are screened as in the baseline scenario, but the performance of the screening strategies differs Figure . SensitiThe final set of scenarios project the impact of repeat screening, including more frequent screening for women with HIV. Using the same four strategies outlined above, we model (1) twice‐lifetime screening for all women at ages 35–39 and 45–49 years, reflecting WHO targets [In all scenarios, we use published estimates for diagnostic performance and treatment efficacy by HIV status, with estimates reported for women with HIV conservatively applied to those without viral suppression. Because diagnostic and treatment performance are associated with CD4 cell count , 28, we To account for uncertainty in screening parameters, we conducted sensitivity analyses evaluating 14 additional scenarios. The first four isolate the effect of differing performance characteristics of single‐visit strategies by holding retention for treatment, screening frequency and coverage at baseline levels. We additionally evaluate moderate retention for treatment with HIV‐targeted screening using HPV DNA testing (70% retention for thermocoagulation and 50% for LLETZ). A sixth scenario increases screening coverage from 48% to 70% for the strategy of HIV‐targeted HPV DNA testing, aligning with WHO targets [2.3Using the 25 best‐fitting parameter sets from model calibration, we simulated HPV and HIV transmission, natural history and demographic dynamics from 2020 (the year before new interventions are introduced) to 2120. Outcomes include cervical cancer incidence rates over time, age‐standardized to the 2015 World Standard Population and the number of cervical cancer cases averted under each scenario relative to baseline. Time to elimination is defined by the years at which estimated cancer incidence crosses the threshold of 4 cases per 100,000 woman‐years  and a higher threshold of 10 cases per 100,000 . To acco3The baseline scenario of cytology with colposcopy triage and 57% HPV vaccination resulted in an 87% decrease in cervical cancer incidence from 80.4 cases per 100,000 in 2020 (range 58.2–112.1) to 10.7 cases per 100,000 in 2120  with HIV‐targeted screening.Median incidence crosses below the 10 per 100,000 threshold for all scenarios except the baseline  and the World Health Organization. Partial support for this research came from a Eunice Kennedy Shriver National Institute of Child Health and Human Development grant, P2C HD042828, to the Center for Studies in Demography & Ecology at the University of Washington.The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.Supplementary Appendix: Model inputs and methods.Click here for additional data file.


Digital technologies are being increasingly utilized in healthcare to provide pertinent and timely information for primary prevention, such as vaccination. This study aimed to conduct a systematic review to describe and assess current digital health interventions to promote HPV vaccination among adolescents and parents of adolescents, and to recommend directions for future interventions of this kind. Using appropriate medical subject headings and keywords, we searched multiple databases to identify relevant studies published in English between 1 January 2017 and 31 July 2022. We screened and selected eligible studies for inclusion in the final analysis. We reviewed a total of 24 studies, which included interventions using text messages (4), mobile apps (4), social media and websites (8), digital games (4), and videos (4). The interventions generally improved determinants of HPV vaccination, such as HPV-related knowledge, vaccine-related conversations, and vaccination intentions. In particular, text message and social media interventions targeted improved vaccine uptake behaviors, but little meaningful change was observed. In conclusion, digital health interventions can cost-effectively provide education about HPV vaccination, offer interactive environments to alleviate parental vaccine hesitancy, and ultimately help adolescents engage in HPV vaccine uptake. The burgeoning field of digital health, which includes categories such as mobile health  and telemedicine, is at the forefront of healthcare transformations and public health efficacy. The ubiquitous and evolving nature of digital tools, including mobile and web-enabled devices, has led to a proliferation of technology-mediated interventions across the healthcare continuum, from prevention to post-treatment surveillance . To imprDespite the growing number of digital health-based programs for child and adolescent vaccinations ,11,12,13A systematic review of the literature was conducted and reported using the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist . The detEligible articles about digital health interventions for HPV vaccination promotion were reviewed extensively. The literature search was performed from the three major bibliographic databases of Medline Ovid, Embase, and Cochrane. With the conjunction “AND” and the disjunction “OR,” the following Medical Subject Heading (MeSH) terms and relevant keywords were used in the search process: child, adolescent, teenager, teen, youth, parent, guardian, Human Papillomavirus, HPV vaccination, HPV vaccine, intervention, digital, e-health, smartphone, mobile health, mHealth, mobile app, game, social media, text message, and web-based. Reference lists of selected articles from multiple journals were manually checked for other potentially relevant articles and all “related to” or similar articles of the identified articles were followed. In addition, a hand search of the grey literature  was conducted. Titles, abstracts, and keywords were further screened for inclusion upon obtaining results that adhered to the search criteria.Peer-reviewed articles written in English were included in this systematic review. Given the speed at which technology develops, the review aimed to explore the most recent research trends in digital health interventions for HPV vaccination promotion. Accordingly, the inclusion time period included articles that were published within the last 5 years, between 1 January 2017 and 31 July 2022. The review included articles specifically about the design, implementation, and evaluation of interventions that deliver information, increase awareness, and promote the uptake of HPV vaccination using digital technology that encompasses electronic tools, mobile devices, and web-enabled devices. Furthermore, the review included articles about digital health interventions that targeted exclusively adolescents, parents of adolescents, and parent-adolescent dyads. Articles were excluded from the review if they were observational studies, systematic reviews, meta-analyses, or content and sentiment analyses.Study selection involved a four-step process. First, we systematically searched the databases using the MeSH terms and keywords. When selecting the studies, we independently reviewed the titles and abstracts of all records identified as relevant to this systematic review. Second, duplicate citations across the databases were identified using Endnote, manually verified, and subsequently removed. Third, the remaining abstracts were checked for eligibility. Fourth, the full-text articles of the included abstracts were screened according to the inclusion and exclusion criteria. This iterative selection and screening process was followed by a final synthesis of the eligible studies, which were qualitatively analyzed. This qualitative analysis entailed categorizing the interventions according to the technology used to deliver the interventions, the theoretical basis of the interventions, as well as the content, outcome measures, and results of the interventions. The same steps were taken with articles identified in the grey literature.Quality assessments were performed to assess the methodological quality of included studies. We independently used the Cochrane Collaboration tool , which in = 16), were not targeted to parents or adolescents (n = 11), were based on analyses of content or sentiment (n = 4), were not technology-based (n = 8), or had abstracts only (n = 2). We identified a total of 24 studies published between 1 January 2017 and 31 July 2022 as the final set of records for the review. No additional eligible articles were identified.The literature search and review process are shown in Among the 24 articles selected for analysis, four studies focused on text messaging ,31,32,33Four studies in this review were SMS text message interventions ,31,32,33Text messages with tailored content in theory-informed interventions did not yield drastic success as anticipated. While timely text message reminders helped shift parents from the pre-contemplation and contemplation stages to preparation and fully vaccinated stages, educational information in the messages tailored to their stage of decision-making had minimal impact on their adolescents’ vaccine series completion . In addiFour studies pertained to mobile apps developed to provide parents with information regarding adolescent HPV vaccination and promote uptake and completion of the HPV vaccine series ,35,36,37Two studies evaluated HPV vaccination intention and vaccine initiation behavior following the use of the apps ,37. HPV Eight interventions in this review were delivered via social media platforms and websites alike. In two interventions, which were a Facebook campaign to cultivate mother champions of HPV vaccination and a website providing mothers with tailored feedback from virtual assistants, both groups of mothers had significantly increased knowledge about HPV and HPV vaccine ,39. In tRegarding vaccination behavior, a Facebook campaign, Health Chat, delivered to mothers was effective, as mothers reported significantly higher initiation of their adolescents’ HPV vaccination at 12- (71.3%) and 18-months (73.3%) posttest, compared to baseline (63.4%). Completion of HPV vaccination was conveyed by 62.5% and 65.9% of mothers at 12 and 18 months, respectively, both of which represented a significant increase compared to the baseline rate of 50.2% . AnotherFour studies illuminated the feasibility of digital games to increase knowledge about HPV and HPV vaccination and vaccine uptake among parents and adolescents ,47,48,49Four video-based interventions for parents to improve HPV and HPV vaccine knowledge and intention to vaccinate their adolescents reported positive results ,51,52,53RCT studies included in this review were at relatively low risk for selection and reporting biases, whereas detection bias was the most frequently occurring bias. Many of the studies did not provide clear and thorough information regarding their risk for performance and reporting biases. Regarding the single-group nonrandomized studies included in this review, they fulfilled most of the items listed on the assessment tool. However, none of the studies had a sufficiently large sample size to provide confidence in their findings. Moreover, none of the studies reported the blinding of outcome assessment or included outcome measures that were collected multiple times before and after the intervention. A full report of the quality assessment for each study is included in the Over the last five years, HPV promotion interventions have been developed and delivered using various technologies: text messages, mobile apps, social media, digital games, and videos. Text messaging, as an early basic feature of mHealth , continuOur findings indicate that user perceptions and effects of the interventions were generally positive albeit with a few unexpected results. Contrary to the lack of theory-informed mHealth interventions reported by a previous review , almost While digital technologies are not supplanting traditional in-person medical consultations ,73, seveAnother interesting point raised by this review is that although the interventions, particularly those delivered via apps, social media, and digital games, were well-received and strengthened key determinants of HPV vaccination, little meaningful change was observed in the actual vaccine uptake or vaccination rates ,44,45,46Except for five studies that targeted parents of daughters ,43,44,51Finally, this review affirmed the recurrently mentioned challenge of population appropriateness for digital health interventions. Despite their cost-effectiveness and convenience, digital interventions for vaccine adherence may not produce positive outcomes if they are implemented for populations of low socioeconomic status . For exaThere are several limitations in this systematic review. A limited number of databases were searched, which may have led to selection and reporting biases, and included studies were limited to distinct geographical settings, mainly in the US and European countries, although unintended. Social media interventions were limited to Facebook campaigns. The evidence in this review may also not be robust enough to represent all the current digital interventions for HPV vaccination because we focused only on parents and adolescents as the intervention recipients, without targeting providers. The strengths of this study are its focus on an overarching collection of digital tools and technology-mediated HPV promotion interventions, beyond mHealth. We were able to verify the advantages of using digital interventions but also their overlooked weaknesses. This review can also serve as a useful guide for countries with high digital technology penetration but low awareness of HPV vaccination to develop successful digital interventions for HPV vaccination, which can pave the path to adopt gender-neutral vaccination in the near future.In this systematic review, we examined 24 digital health interventions incorporating text messages, mobile apps, social media and websites, games, and videos to promote adolescent HPV vaccination. Digital health interventions hold tremendous potential as a cost-effective and timely strategy to promote HPV vaccination; however, closing the intention-behavior gap remains a difficult task. While mobile apps and digital games facilitated education and active discussions around HPV thereby positively influencing knowledge and vaccination intentions, text message and social media interventions aimed to improve vaccine uptake behaviors, although the change was not always significant. Nonetheless, the interventions were found to support informed decision-making of adolescents and parents about HPV vaccination, expediting their communication with healthcare providers. Finally, continual revision of the interventions based on user feedback and considerations of parent characteristics is key to improving HPV vaccination outcomes among adolescents.


Lactobacillus decreased. Compared with the HPV group, the prevalence of HPV E6/E7 and oncogene expression level were significantly upregulated in cervical intraepithelial neoplasia (CIN) and cervical cancer (CC) groups. Additionally, a positive correlation between the expression of the HPV oncogene and the genera Sneathia, Salmonella, Leptotrichia, Pseudomonas, and Roseovarius in the HPV group was observed. In the CIN group, the enrichment of the genera Sneathia and Megasphaera was weakly linked with HPV oncogene overexpression. In the CC group, a strong association between the overabundance of the genera Peptostreptococcus and Enterococcus and the high expression of HPV oncogene was also found. The increased diversity of the vaginal microbiota and the decreased Lactobacillus abundance were significantly associated with the severity of cervical disease, and the expression of the HPV oncogene could also be regulated by certain pathogens in different stages of cervical lesions.As the fourth most common gynecological cancer, cervical cancer has resulted in more than 300,000 deaths worldwide in 2020. The expression of the human papillomavirus (HPV) oncogenes E6 and E7 is significantly involved in the initiation and progression of cervical neoplasia. Additionally, the composition of the vaginal microbiome was also closely associated with the ability of HPV to induce cervical cancer. However, the relationship between the expression of HPV E6/E7 oncogene and the composition of the vaginal microbiome has not been clearly explored. In our present study, to investigate the relationship between the HPV E6/E7 oncogene and vaginal microbiome, cervical swabs from 115 patients were collected, and their vaginal microbiomes were analyzed by using metagenomics sequencing. Along with the progression of cervical lesions, the diversity of cervical flora increased gradually, and the abundance of IMPORTANCE The main findings of this study were that we clarified the associations of the different bacterial species with the expression of human papillomavirus (HPV) oncogenes at different stages of cervical cancer. Along with the severity of cervical lesions, the abundance of the genus and species of Lactobacillus obviously declined, while the aerobic and anaerobic bacteria, as well as the prevalence and expression of HPV E6/E7 oncogene, were increased dramatically. Based on the statistics from the Global Cancer Statistics 2020 report, cervical cancer (CC) was considered the fourth female malignant tumor in both morbidity and mortality . The resLactobacillus in the vaginal microbiome is highly correlated with the elimination of HPV infection and significantly inhibited the development of cervical cancer. Furthermore, the vaginal microbiome dominated by a variety of Lactobacillus is considered healthy and positively correlated with microecological functions. Studies showed that the metabolites produced by Lactobacillus may participate in local cellular innate immunity, thus inhibiting the infection and replication of the HPV virus , precancerous lesions with hrHPV category (n = 40), and invasive cervical cancer category (n = 41). In addition, all patients were divided into HPV oncogene-negative (n = 29) and -positive (n = 86) groups  for further analysis, ignoring the severity of cervical lesions. After quality control, 8.34 billion 150-bp paired-end clean reads were obtained, and the average reads for each sample were 7.25 ± 0.91 billion. After removal of human DNA contaminants, 11.3 million microbiome reads were generated, with a median of 117.87 (47.08 to 598.24) thousand reads per sample .The samples collected from a total of 115 patients were sequenced, and then these patients were classified into following three categories: hrHPV positive without cervical lesion category (P < 0.001). Moreover, we also observed significantly low Chao1 indexes in the patients of the HPV infection group compared with those of the CIN and CC groups (P < 0.001), whereas no significant difference was found between the patients of the CIN and CC group   . After aLactobacillus genus , while the average enrichments of the Gardnerella genus , Prevotella , and many other pathogenic bacteria were increased dramatically along with the progression of cervical lesions  and Lactobacillus crispatus , but the mean abundance of Gardnerella vaginalis , Prevotella bivia , and various aerobic and anaerobic bacteria was increased dramatically with the severity of the precancerous lesions  .Compared with the patients of the CIN and CC groups, the patients of the HPV group exhibited a significantly high average enrichment of the  lesions  and B. C lesions  and D. V, and CC . In the  < 0.05) . SignifiP = 0.003) (P < 0.001) (As shown in = 0.003) . Meanwhi< 0.001) . Additio< 0.001) , E and G< 0.001) . HoweverSneathia , Salmonella , Leptotrichia , Pseudomonas , and Roseovarius , while a weakly positive correlation of the HPV oncogene expression with the genus Ureaplasma  and Klebsiella  was also observed in the HPV group. Additionally, a strong positive association of Sneathia amnii  and Salmonella enterica  with the HPV oncogene expression were found at the species level, while we also observed a weakly positive association of Ureaplasma parvum  and Klebsiella pneumoniae  with the presence of HPV oncogenes. In the CIN group, the high abundance of the genus Sneathia  and Megasphaera  were weakly associated with the HPV oncogene expression. At the species level, we observed strong associations of Sneathia sanguinegens , Megasphaera sp. UPII_135-E , Megasphaera genomosp. type_1 , and Megasphaera sp. UPII_199-6  with the HPV oncogene expression. Additionally, we also observed the strong correlations of the overabundance of genera Peptostreptococcus  and Enterococcus  with the high expression of the HPV oncogene, as well as the weak association between the Parabacteroides  and the overexpression of the HPV oncogene, Meanwhile, the associations of Peptostreptococcus anaerobius , Enterococcus faecalis , Bacteroides thetaiotaomicron , and Porphyromonas uenonis  with the HPV oncogene expression were also discovered at the species level.Next, we conducted the Spearman correlation analysis on these three groups to explore the correlation between the vaginal microbiome and HPV oncogene expression. The results showed that the HPV oncogene expression was strongly correlated with the presence of the genus Lactobacillus genus among the three groups , but weakly associated with the Neobacillus  in the CIN group and Mycoplasma  in the CC group . In line with the results of the genus level, significantly positive associations of the serum blood estrogen with the high abundance species of Lactobacillus iners  among the three groups, Lactobacillus farciminis  in the HPV group, and Lactobacillus crispatus  in the CIN group were also found. Additionally, in the patients of the HPV group, significantly positive correlations of the WBC count with the genera Enterococcus, Anaerococcus, Gemella, Staphylococcus, Peptostreptococcus, Bacillus_f__Bacillaceae, unclassified_p__Candidatus_Saccharibacteria, and Clostridium were discovered. Furthermore, in the CC group, we simultaneously observed a positive association of the WBC and neutrophil with the genus Bifidobacterium and a negative association with the genus Veillonella. As shown in Streptococcus pneumoniae, Streptococcus mitis, Prevotella sp. S7-1-8, Mageeibacillus indolicus, Streptococcus pseudopneumoniae, Streptococcus anginosus, Fusobacterium gonidiaformans, Peptostreptococcus anaerobius, and Megasphaera sp.UPII_135-E in the HPV group; Atopobium vaginae, Mycoplasma hominis, and Anaerococcus lactolyticus in the CIN group; and Prevotella timonensis, Anaerococcus tetradius, Porphyromonas asaccharolytica, Streptococcus anginosus, and Dialister micraerophilus in the CC group . Interestingly, when we conducted a similar correlation analysis in the HPV oncogene-positive group, a positive association of the HPV oncogene expression and Peptostreptococcus anaerobius and Bacteroides thetaiotaomicron was observed again, suggesting the close relation of these bacteria to the progression of cervical cancer. Furthermore, we also found a significantly negative correlation between the abundance of L. crispatusand L. iners and the alpha diversity in HPV oncogene-positive and -negative groups .We further explored the correlations between the vaginal microbiome and blood indexes. The results showed that the serum estradiol level was positively associated with the cervical enrichment of the Lactobacillus iners, Lactobacillus crispatus, Escherichia coli, and Lactobacillus gasseri were considered potential biomarkers for identifying HPV infection to any cervical lesions. Additionally, the Bifidobacterium genus was treated as an excellent microbiota biomarker for precancerous patients. Also, Prevotella, Porphyromonas, and Bacteroides genera were considered great microbial markers of the cervical cancer group. After performing 10-fold cross-validation on random forest models between every two groups as a previous study described .Linear discriminant analysis (LDA) effect size (LEfSe) modeling was conducted to distinguish differences in bacterial abundance that might be linked to the progression of cervical lesions  could effectively maintain a healthy vaginal environment, which could exert a vital role in the elimination of HPV infection , colposcope, and biopsy were generally employed in the screening and diagnosis of the patients with CIN and CC in the clinic. Recently, noninvasive microbiome models of the intestinal tract for the diagnosis and prediction of many types of cancer, such as colorectal, breast, and liver cancer, have been established . Similar lesions , 40. Int lesions . Thus, c lesions , 43. TheWe conducted this comprehensive study to reveal the potential relationship between cervical lesions, cervical microbiota, and HPV oncogene expression using metagenomics sequencing. The current study also has several shortcomings that limit the conclusions that can be drawn. First, the study size number was small and did not have a validation cohort. A large and multicenter verification is required for confirming the conclusion of the current study. On the other hand, due to the contamination of human host DNA, effective sequencing data of vaginal microorganisms are limited, and more depth-sequencing methods that remove the human host genome are required to validate our results. Furthermore, the genotype of hrHPV was not equivalent in each group, and since, here, we conducted a cross-sectional study, a longitudinal cohort is needed to verify our result. Furthermore, in our present study, several important indicators of vaginal inflammation were not included, and the variables, such as vaginal pH and Nugent score, should be included in future studies. Lastly, our study demonstrated the potential relationship between the HPV oncogene, the cervical microbiota, and the development of cervical cancer, but the causality and underlying molecular mechanisms of these three aspects still need to be further investigated.Lactobacillus and species was reduced and the abundance of anaerobic and aerobic bacteria was increased, while the prevalence of HPV E6/E7 and the expression of oncogenes were elevated. The HPV oncogene overexpression was associated with distinct bacterial species during different stages of cervical cancer. More studies are needed to explore the underlying molecular mechanisms of different pathogens that can inhibit or advance the formation of cervical lesions.Along with the severity of cervical lesions, the abundance of the genus All nonduplicated patients were recruited from June 2020 to March 2021 at Renmin Hospital of Wuhan University and had a Han Chinese background. These patients were pathologically diagnosed with precancerous lesions or invasive cervical cancer for the first time. Additionally, patients met the following criteria: (i) not received cervical and vaginal surgery or chemoradiation, (ii) not consumed oral contraceptives and/or antibiotics in 3 months, (iii) not received hormone replacement therapy in 1 year, (iv) absence of vaginal lavage or sexual intercourse within 24 h before sample collection, and (v) must not have been diagnosed with any other gynecological tumors and immunosuppressive diseases. Furthermore, patients with hrHPV infection without cervical lesions were used as the HPV infectious group. All negative cervical lesions of the control populations were confirmed by TCT and colposcopy.Cervical samples were collected according to the standard procedures . All samThe Qiagen DNeasy PowerSoil pro kit  was used to extract DNA from cytobrush samples with several modifications. Briefly, the cervical brush was vigorously vortexed with cell preservation fluid for 5 min, and then 1 mL of the mixed liquid was pipetted into a new tube and centrifuged for 15 min at 15,000 rpm. Nucleic acids were extracted according to the kit instructions. The RNA was extracted by adopting the magnetic total RNA extraction kit . The optical density of DNA and RNA 260/280 was detected using a Nanodrop 2000c spectrophotometer .−ΔΔCT) method. Thin-prep cytology test (TCT) samples were prepared by the AZR-D and ATP-E liquid-based cytology machine  according to the manufacturer’s instructions.HPV genotypes were detected according to the manufacturer’s handbook (Tellgenplex HPV27 genotyping test). The hrHPV infection was defined as any infectious HPV genotype of 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 82. The expression of the HPV E6/E7 oncogene mRNA was determined using the high-resolution melt curve method . The copies of HPV E6/E7 per 1,000 cells were measured using a single reference gene marker, HPRT, and the relative expression of the HPV E6/E7 gene was calculated using the threshold cycle , and paired-end reads were generated with 150 bp. The Fastp software (version 0.20.0) was applied to trim the 3′ end and 5′ end of the barcode sequence and to remove sequences with a length less than 50 bp and an average quality value lower than 20, the unidentified bases after mass shearing, the reserve high-quality pair-end reads, and the single-end sequences. Before the data analysis, sequence reads were compared with the human host DNA sequence using the software BWA (version 0.7.9a), and the host genome-contaminated reads with high similarity were removed. The remaining high-quality reads were assembled using Multiple Megahit (version 1.1.2), and the shortest contigs were over 300 bp. The MetaGene was used to predict open reading frames (100 bp) from previously assembled contigs. The gene sequences predicted by the sample were clustered by CD-HIT software to constitute a nonredundant gene catalog and to obtain the base sequence of the gene of the nonredundant gene catalog . To align the high-quality reads of each sample with the nonredundant gene database  and to calculate the abundance information of the genes in the matching sample, the SOAPaligner software was applied.−5), and the species annotation result was obtained using the taxonomic information database matching the NR library. Then the counts of each species were calculated using the total gene number corresponding to the species, and the counts of each species were calculated as the domain, kingdom, phylum, class, order, family, genus, and species at each taxonomic level to count the abundance of species in each sample to construct the corresponding classification abundance profile at the taxonomic level. To avoid interference from the interkingdom species, the genes annotated to nonbacteria, such as fungi, viruses, and parasites, were excluded from the final nonredundant gene set. As described previously  database  . Serum bThe samples and clinical data collected in the present study were approved by the Medical Ethics Review Committee of Renmin Hospital, Wuhan University, China (ethics record number WDRY2021-K042). All patients signed informed consent. We confirmed that all methods were carried out in accordance with the Declaration of Helsinki and relevant local and national guidelines and regulations.post hoc test and a two-way P value of <0.05 was considered statistically significant.The types of vaginal microbiome were classified as described previously . The bioPRJNA771720).All raw data from the metagenomics sequence have been uploaded to the NCBI Sequence Read Archive database (accession number:


Identification of HPV infection is usually performed on cytological specimens, despite the often transient virus types. HPV profile analysis of pathologically confirmed lesions can also be performed on formalin-fixed paraffin-embedded (FFPE) cone samples and should be taken as standard during follow-up. We compared HPV profiles of cytological and FFPE specimens of women diagnosed with HSIL. Archived PAP smears and FFPE cones from 49 patients were processed. For genotyping, the HPV Direct Flow CHIP test was used. All samples were positive. HPV profile agreement of the two sample types was 84.16–100%. Mono-infections occurred in 12.24% and 61.22% in PAP smears and FFPE specimens, respectively; while multi-infections were detected in 87.76% and 38.78%, respectively. The most abundant genotypes were HPVs 16, 31, and 51/33. Of all infections, 56.25% and 64.93% were caused by nonavalent vaccinated type (VT) HPVs; while 50.69% and 38.96% belonged to non-nonavalent VT HPVs, in PAP smears and FFPE specimens, respectively. Our results confirmed the importance of HPV genotyping of FFPE cone samples. We also confirmed a remarkable presence of non-vaccinated HPV types in HSIL cases indicating the importance of vaccine development. There are over 207 different human Papillomavirus (HPV) genotypes classified to date, by varying distribution and prevalence across different populations and geographical regions ,7,8,9,10Although it is generally accepted that both HSIL and invasive cancers are mostly driven by a single HPV type, in about 60% of the cases multiple HPV genotypes are detected ,20,21. TData comparing HPV genotype profile of cytological and cervical FFPE cone samples at the time of diagnosis, from the same patient, are limited and incomplete in the literature and completely absent in Central-Eastern Europe.Therefore, in a retrospective study, we aimed to determine the occurrence and agreement of HPV genotypes in women diagnosed with HSIL, by comparing the HPV genotype profile of cytological samples and cervical cone samples, applying a method which is sensitive to 35 different genotypes of HPV (18HR and 17LR). We think that our data could present additional evidence on the role of persistent HR-HPV infection in the development of HSIL or invasive cervical cancer. Moreover, by detecting a high number of HPV genotypes, our study allows us to draw attention to the relevance of HPV vaccines, especially to the widely used nonavalent vaccine , and to the occurrence of non-nonavalent vaccinated type (VT) HPVs, providing important information for vaccine development.Archived conventional PAP smears and FFPE cone samples of 49 women with a diagnosis of HSIL were included in our retrospective study. The study was based on women that attended the Department of Obstetrics and Gynaecology and other outpatient clinics in Pécs, between 2019 and 2020, due to routine gynecological screening and diagnostic follow-up. Conization was performed within a 6 month period after cytological sampling in all cases and the genotyping was accomplished during the spring of 2021.Cytological conventional PAP smears of each case were evaluated by three independent cytopathologists based on the Bethesda 2014 system. In the histological specimens, evaluation of the individual areas that mostly represent HSIL was made according to standard criteria, 2020 WHO classification, on hematoxylin and eosin-stained sections, observed by experienced histopathologists. For further processing, 3 series of 10 µm-thick slices of archived FFPE cervix cone samples were cut. Besides, exfoliated cervical cells from archived PAP smears were scraped by sterile scalpel and were suspended in 500 µL sterile gynecological (GYN) specimen preservation solution (CelltraZone) and were used for HPV genotype analysis.HPV genotype determination was done by the HPV Direct Flow CHIP system  at both sample materials. The test is able to differentiate between 18 high-risk and 17 low-risk HPV genotypes as, HR HPVs 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 and LR HPVs 6, 11, 40, 42, 43, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, 81, and 84. The system includes GP5+/GP6+-based PCR, reverse dot blot hybridization, and automatic readout. An additional fragment (268 bp) of the human beta-globin gene is co-amplified during the PCR to assure the quality of the input starting material. Samples were processed based on the manufacturer’s instruction following the protocol for cytology in GYN transport medium and paraffin-embedded tissue sections for direct PCR.Processing of cytological samples: 500 µL of cytological samples were centrifuged for 2 min at 2000 rpm at room temperature. The pellets were dissolved in 400 µL PCR sterile water and the centrifugation step was repeated. The pellets were dissolved in 300 µL sterile water; 30 µL of the solution was used for PCR amplification.Processing of FFPE tissue samples: Three 10 μm thick sections were dissolved in 400 μL mineral oil and then incubated at 95 °C for 2 min. After removing the mineral oil, 60 μL lysis buffer with 1.5 μL DNA release  was added to the samples and incubated at 60 °C for 30 min, followed by inactivation at 98 °C for 10 min. Furthermore, 3 μL of crude cell extract supplemented with 27 μL DNase/RNase free water was used for PCR amplification. The lyophilized PCR HPV mix contained PCR buffer, dNTP(U/T), DNase/RNase free water, biotinylated primers, DNA polymerase, and UNG. The primers used are specific for the amplification of a fragment of the L1 region of the HPV genome. Besides, primers for the amplification of the human beta-globin gene are included and used as an internal control for the PCR reaction. The amplification cycling conditions in IANLONG PCR Thermal Cycler (Genesy 96T) were the following: pre-incubation at 25 °C for 10 min then 94 °C for 3 min; 15 cycles of denaturation at 94 °C for 30 s, annealing at 47 °C for 30 s and elongation at 72 °C for 30 s; 35 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s and elongation at 72 °C for 30 s and final elongation at 72 °C for 5 min; followed by cooling at 8 °C. Amplicons were denatured at 95 °C for 10 min then cooled on ice for 5 min.The hybridization process was performed semi-automatically in hybriSpot (HS12) following the manufacturer’s instructions. The management of the samples, the images, the analysis, and the report of the results was performed by hybriSoft software.p values < 0.05 were considered to be statistically significant.To compare PAP smears and FFPE cones of the patients we applied McNemar and McNemar–Bowker tests as appropriate. To observe agreement between the sample types  we used weighted kappa statistics with a 95% confidence interval. In the case of genotype analysis, we calculated the ratios’ exact confidence interval, using the Poisson distribution and Poisson test. Only Overall, 49 women with a median age of 39 years (ranging from 17 to 64 years of age) were included in our retrospective study. The patients mostly resided in the southwest part of Hungary. All patients had high-grade cytology and histopathology. The HPV test was performed on conventional PAP smears and FFPE cone samples. The test, provided positive β-globin results in all cases, suggesting that the samples’ DNA quality and quantity were suitable for the analysis. All patients proved to be HPV positive.HPV genotype classification was based on the HPV Direct Flow Chip Test’s  description. HPV DNA was detected, in all of the 49 cases in both sample types. The PAP smears and FFPE samples showed identical genotype (HPV genotypes are the same in both sample types) in 15 (30.61%), compatible genotype  in 34 (69.38%) specimens, while discrepant genotype (present only in FFPE sample) was not found. It means that if we considered FFPE to reference genotype profile, the identity is 100% with the appropriate PAP smears. By examination of the agreement between the cytological and histological profiles, we found a moderate correlation , which is due to the fact that the sampling conditions are completely different. p < 0.001.Of the 49 PAP smears, 6 (12.24%) appeared with mono-infection, while 43 (87.76%) occurred with multi-infections. In contrast, 30 (61.22%) of the 49 FFPE samples showed mono-infection, while 19 (38.78%) had a multi-HPV infection. By comparing the two categories we found that FFPE samples harbored a significantly higher number of mono-infection than PAP smears, p = 0.073), we found that the PAP smears harbored a higher number of HPV infections per sample than the FFPE cones, 3.21 (CI: 2.69–3.79) compared to 2.47 (CI: 1.82–3.29), respectively.When the infection numbers in multi-infected PAP smears and FFPE cone samples were compared, although the results were not significant due to the small sample size  compared to 17 cases (34.69% CI: 20.21–55.55%)  .From the 35 tested (18 HR and 17 LR), 16 high-risk and 12 low-risk HPV genotypes were detected in PAP smears, yielding a total number of 144 infections within the 49 samples.In the FFPE cone samples, 16 high-risk and 6 low-risk HPV genotypes were found resulting in a total number of 77 infections. p = 0.031) suggests that the number of HPV mono-infections in FFPE samples is higher than in PAP smears.In the PAP smears, three of the 35 HPV genotypes, namely, the nonavalent VT 16, 52, and 45 appeared as mono-infections. In contrast, in the FFPE cone samples, nine of the 35 HPV genotypes, the nonavalent VT 16, 31, 33, 45, 52, and 58 and the non-nonavalent VT 35, 68, and 82 appeared as mono-infections . The sigRegarding the occurrence of the nonavalent and non-nonavalent VT HPVs in PAP smears, 71 (49.30%) of the 144 infections were caused by the former types, while 73 (50.69%) were due to the latter ones. In addition, of the 73 non-vaccinated HPV infections, 52 (71.23%) were caused by high-risk HPVs, such as: HPV 51, 56, 35, 66, 53, 39, 73, 82, and 68. In the case of the FFPE samples, we found that 47 of all 77 infections (61.03%) were caused by nonavalent VT HPVs, while 30 (38.96%) belonged to the non-nonavalent VT HPVs; of which 26 (86.66%) were high-risk HPVs, such as HPV 51, 35, 53, 56, 82, 68, 73, 39, and 66. We also analyzed the agreements between the two samples from the same individual which are summarized in p = 0.002.The most abundant genotype, the HPV 16, was found in 46.93% of the FFPE samples and 55.10% of PAP smears. In the case of PAP smears, the HPV 16 appeared significantly more frequently in the form of a multi-infection than in FFPE samples, where it mostly appeared as mono-infection, Cervical conization is an effective treatment for HSIL, however, this treatment cannot eradicate high-risk HPV; a wide range of recurrence rates of HSIL after conization (0.35–69%) was reported, hence close follow-up of the patients is crucial ,28,29. UIn our retrospective study, we compared and analyzed the agreement of the HPV genotype profile of PAP smears and FFPE cone samples, originated within six months, from the same 49 women diagnosed with HSIL. The sensitivity and specificity of the applied method (HPV Direct Flow Chip) had been proved in independent studies ,33,34,35The overall agreement between PAP smears and FFPE cone samples was very good (100%), suggesting that cytological samples are reliable sources for verifying the presence of certain HPV genotypes.Either in the form of mono- or multi-infection, our results underlined the importance of high-risk HPV genotypes in the formation of precancerous HSIL, as all of the 49 PAP and FFPE samples harbored high-risk HPV genotypes.p < 0.001) proportion of multiple infections detected in the 49 PAP smears (87.76%) compared to the FFPE cone samples (38.78%).Cervical coinfection with more than one HPV genotype is common; women infected with one HPV type are more likely to harbor additional genotypes ,37. IndeHPV genotypes integrate into the human cell/genome, achieving increased viral oncoprotein expression and increased mRNA stability, which generates the imbalance of cellular genes that leads to carcinogenesis. In rare cases, multiple viral genomes could integrate, however, most of these become epigenetically silenced ,43,44,45Apart from the sample material, the most abundant genotype was the nonavalent VT HPV 16, with 46.93% and 55.10% of FFPE and PAP samples, respectively, followed by the nonavalent VT HPV 31, and non-nonavalent 51, together with the nonavalent VT 33 in the FFPE cone samples. These findings, together with the relatively low prevalence of nonavalent VT HPV 18, are consistent with a previous report in Hungary, although that study only examined cervical swab samples [Indeed, using the same method on two types of samples that originated from the same patients, we were able to analyze the distribution of 35 different HPV types giving reliable information about their infection forms (multi- or mono-infection), and thereby their oncogenic potential in cervical cancerogenesis. In FFPE cone samples the nonavalent VT HPVs 16, 31, 33, 45, and 52 and non-nonavalent VT HPVs 35, 68, and 82 appeared as mono-infections.Today the most effective weapons against HPV infection are vaccines. Currently, three effective HPV prophylactic vaccines are available, all of which contain HPV L1 proteins that are capable of forming virus-like particles (VLP). Of these, the nonavalent Gardasil 9 (Merck) covers the most HPV types, such as HPVs 6, 11, 16, 18, 31, 33, 45, 52, and 59. This vaccine was approved by the FDA in late 2014; in Hungary, it is available since 2015 ,47. NumeAn essential finding of our study was the high rate of non-nonavalent VT HPV prevalence in both sample types—50.69% in PAP smears and 38.96% in FFPE cone specimens from all infections . The results of this study suggest that either single or multiple nonavalent VT, as well as non-nonavalent VT HPVs, are associated with HSIL.Reports on HPV genotype profile and agreement of cytological and cervical FFPE cone samples with HSIL are rare and up to now, not available from Central-Eastern Europe. The ultimate goal of our study was to draw attention to the importance of HPV genotype profiling of cone samples in precancerous HSILs. Cone samples are the references regarding the diagnosis and the assessment of therapy efficiency during post-conization follow-up. Our results confirmed strong concordance between HPV genotypes found in cytological and tissue samples, suggesting that cytological samples are reliable sources for verifying the prevalence of certain, oncogenic HPV genotypes present in cone specimens.By applying detailed HPV genotype analysis, we pointed out the presence of non-vaccinated HPVs as well in HSIL cases; in addition, 71.23% and 86.66% of such infections were caused by high-risk non-vaccinated HPV genotypes in PAP smears and FFPE cone samples, respectively. Together with possible interactions between different HPV types, these findings should be considered in the design of vaccination strategies and highlight the need for the development of wider-spectrum vaccines against HPV.


Astrocytes have many important functions in the brain, but their roles in psychiatric disorders and their responses to psychotropic medications are still being elucidated. Here, we used gene enrichment analysis to assess the relationships between different astrocyte subtypes, psychiatric diseases, and psychotropic medications . We also carried out qPCR analyses and “look-up” studies to assess the chronic effects of these drugs on astrocyte marker gene expression. Our bioinformatic analysis identified gene enrichment of different astrocyte subtypes in psychiatric disorders. The highest level of enrichment was found in schizophrenia, supporting a role for astrocytes in this disorder. We also found differential enrichment of astrocyte subtypes associated with specific biological processes, highlighting the complex responses of astrocytes under pathological conditions. Enrichment of protein phosphorylation in astrocytes and disease was confirmed by biochemical analysis. Analysis of LINCS chemical perturbagen gene signatures also found that kinase inhibitors were highly discordant with astrocyte-SCZ associated gene signatures. However, we found that common gene enrichment of different psychotropic medications and astrocyte subtypes was limited. These results were confirmed by “look-up” studies and qPCR analysis, which also reported little effect of psychotropic medications on common astrocyte marker gene expression, suggesting that astrocytes are not a primary target of these medications. Conversely, antipsychotic medication does affect astrocyte gene marker expression in postmortem schizophrenia brain tissue, supporting specific astrocyte responses in different pathological conditions. Overall, this study provides a unique view of astrocyte subtypes and the effect of medications on astrocytes in disease, which will contribute to our understanding of their role in psychiatric disorders and offers insights into targeting astrocytes therapeutically. Astrocytes play many important roles in the brain, from facilitating metabolism and cell signaling ,2, to reReactive astrogliosis describes a spectrum of heterogeneous changes in gene expression ,47,48, cIn addition to disease heterogeneity and methodological differences, psychotropic medications are hypothesized to normalize disease-associated changes in astrocyte expression ,66,68, aIn this study we apply bioinformatic analysis of transcriptomic datasets to assess the relationship between different astrocyte subtypes  and psychiatric disorders, and between astrocytes and psychotropic medications. Our goal is first to explore how different subtypes of astrocytes are implicated in psychiatric disorders and second, to determine whether psychotropic medications are associated with molecular changes in astrocytes in disease.The gene-sets for each transcriptomically distinct subtype of mouse astrocyte were downloaded from the supplementary data of Batiuk et al. single-cell RNA sequencing astrocyte study . Full geAstrocytes gene-sets (GSE 35338) from a mouse model of stroke , 1 day timepoint) and a mouse model of neuroinflammation (lipopolysaccharide (LPS) 1 day timepoint) as described in , were obwww.brainrnaseq.org, access on 3 October 2022) from enriched human mature astrocytes and whole cortex homogenate samples [FPKM gene expression values were obtained from BrainRNAseq ( samples . Genes wp < 0.1) from subtype (SUB) compared to classical (CLA) hiPSC astrocytes (AST_hiPSC_SUBvCLA). Subtypes astrocytes are defined as GFAP+ and S100B+ cells with a distinct, non-bushy morphology. The second gene-set consists of 504 differentially expressed genes (adj p < 0.1) from schizophrenia (SCZ) compared to healthy control (HC) hiPSC astrocytes (AST_hiPSC_SCZvHC). The third gene-set consists of 430 differentially expressed genes (p < 0.05) from schizophrenia clozapine responders compared to clozapine non-responders (AST_hiPSC_SCZ-RvSCZ-NR). Gene-sets are listed in Three gene-sets of human induced pluripotent stem cells (hiPSC) derived into astrocytes were obtained from . The firhttp://shenlab-sinai.github.io/shenlab-sinai/, accessed on 14 September 2022). Disease-specific curated gene-lists were downloaded from DisGenNET , and see Curated disease-associated gene-sets for major psychiatric disorders major depression (MDD), bipolar disorder (BPD), and SCZ were downloaded from DisGeNET . To avoihttp://download.baderlab.org/EM_Genesets/, accessed on 16 April 2021). To compare enrichment of pathways across different astrocyte subtypes and diseases, the -log10 was used to generate the heatmap. To better identify the character of biological changes in the overlap results, a focused analysis of forty a priori functional themes was performed. As described in our previous study [Pathways affected in different astrocyte subtypes and diseases were determined using hypergeometric overlap analysis (HGA) with a background of 21,196 genes . The astrocyte-associated gene-sets and disease-associated gene-sets were tested against GO pathways associated with Biological Process (GOBP), Molecular Function (GOMF), and Cellular Component (GOCC). Updated lists of GO-pathways were obtained from the Bader lab , a density-score is obtained as:r and c represent the number of pathways in a theme and number of astrocyte subtypes and diseases, respectively. A zero element represents a non-significant disease-pathway relationship. For density associated with individual pathways and drug signatures, the density-index representing the fraction of non-zero elements in the number of astrocyte subtypes and diseases was used. In such an instance, the numerator of the above density formula represents count of zero elements in a vector. Whereas r and c in the denominator are constant holding a fixed value of 1 and 27, respectively.To quantitatively summarize how common (close to 1) or unique (close to 0) a theme is across different astrocyte subtypes and diseases, we deployed a previously developed density-index . For a gEnrichment of astrocyte subtypes and drug-induced molecular signatures in the disease-associated gene-sets was calculated using HGA. Drug-specific gene markers were downloaded from the DSigDB library of gene-sets . In orde−1 haloperidol-decanoate (n = 10) via intramuscular injection every 3 weeks for 9 months or vehicle-treated with sesame oil (n = 10) under the same conditions. At the end of the 9 months, brains were removed and stored at −80 °C.All animal studies were carried out in accordance with the IACUC guidelines at the University of Alabama at Birmingham. Male Sprague Dawley rats were housed with a 12-h cycle of light and darkness. They were pair-housed and given access to food and water ad libitum. Rats were administered with either 28.5 mg kgt-test. α = 0.05 for statistical tests. Data were analyzed with GraphPad Prism v7.04 .RNA was extracted  from 14 µm thick fresh frozen sections of rat frontal cortex as previously described ,87. RNA https://kalganem.shinyapps.io/BrainDatabases/, accessed on 1 June 2022) [p-value of transcript expression in patients on/off medication is reported. No data is available from the SMRI for MDD patients on or off antidepressant medications.The R shiny application “Kaleidoscope” (ne 2022) , was usene 2022) ,90. The The DrugFindR R Shiny application was run as described  to identg for 10 min at 4 °C. The supernatant was layered onto 7.8 mL each of 2%, 6%, 10%, and 20% v/v Percoll in SEDH buffer  and centrifuged at 33,500× g for 5 min at 4 °C. The turbid layer between the 2% and 6% Percoll layers was collected, resuspended in SEDH buffer, and centrifuged at 1000× g for 20 min at 4 °C. The supernatant-containing gliosome fraction was centrifuged at 33,500× g for 40 min at 4 °C. The pellet was resuspended and washed ×2 using the same spin conditions. The pellet was resuspended in MPERS buffer. Two micrograms protein from the enriched gliosome fraction and total homogenate was loaded on a Pamgene chip and analyzed using the Pamgene12  as previously described [Brain tissue from a non-psychiatrically ill control subject  was obtained from the Alabama Brain Collection with consent from next of kin with IRB approval protocols. Approximately 500 mg of brain tissue was homogenized in 5 mL buffer  and centrifuged at 1000× escribed ,93,94. To determine whether different astrocyte subtypes are enriched in psychiatric disorders, we assessed the enrichment of (1) genes representing six mouse astrocyte subtypes (AST1-5 and “common”) , (2) genThe set of genes representing the “common”  mouse astrocyte subtype were the most frequently enriched, in line with findings that astrocytes play important roles in the pathophysiology of these disorders . The ASTBiological pathway enrichment  of astrocyte datasets and psychiatric disorders is shown in Signaling cluster pathways, including “modulation of chemical synaptic transmission”, “regulation of membrane potential” and “regulation of neurotransmitter levels” are commonly enriched in AST_hiPSC datasets and across psychiatric disorder datasets but have limited enrichment in mouse subtype or human astrocyte datasets, suggesting species and disease specific alteration in astrocyte-related signaling processes. High levels of gene enrichment in the “Synapse” subcluster were largely driven by the hiPSC- derived astrocyte gene-sets and support changes in synaptic transmission and astrocyte sensitivity to synaptic signaling in different human astrocyte subtypes (hIPSC_SUBvCLA), in disease (hIPSC_SCZvHC) and in response to medication (hIPSC_SCZ-NRvSCZ-R) . These fNext, we assessed the gene enrichment of astrocyte subtypes, psychiatric disorders, and the gene-sets representing different types of commonly prescribed psychotropic medications  from the DSigDB library . OverallThe DSigDB is a collection of drug datasets from different sources and include data generated from varied cell types and tissues that use a range of drug concentrations and dosage time points that may contribute to heterogeneous results. This is reflected in the gene enrichment heatmaps presented here, where common enrichment of astrocyte is highly variable across different drug datasets but is relatively consistent across disease datasets, suggesting robust gene enrichment in disease but not astrocytes.p > 0.05; To further explore the relationship between psychotropic medications and their effects on astrocytes, we carried out a “look-up” study of antipsychotics, antidepressants and mood stabilizers on an extensive range of astrocyte marker genes in rodent frontal cortex. We used those genes defined in the “mouse_common” gene list, which is comprised of genes found in at least 60% of astrocytes analyzed by . Figure Protein kinase activity, determined by an increase in phosphorylation signal intensity, is higher in a gliosome preparation from postmortem frontal cortex tissue relative to total homogenate from the same sample A,B. ThisUsing DrugFindR, we identified the FDA approved drugs with the greatest discordance (≤−0.60) with the hiPSC_SCZvHC gene signature . This huAstrocytes play an important, albeit still poorly understood role in many neurological and psychiatric diseases ,2,3,66. To improve our understanding of how astrocytes are altered in the brain in disease, and the effects of medications on astrocytes, we carried out bioinformatic analysis, taking advantage of emerging single-cell RNAseq datasets that define mouse astrocyte subtypes. As unique molecular signatures for different subtypes of human astrocytes have yet to be elucidated ,67, we iGene enrichment analysis  found thThere is extensive conservation of gene expression between human and mouse astrocytes . HoweverThis is further supported by the diverse patterns of gene enrichment of the mouse disease model astrocyte datasets compared to the astrocyte subtypes from healthy mice. However, differences in astrocyte gene-set enrichment in different disease datasets may also indicate that specific astrocyte subtypes are affected in particular disorders, as seen in mental depression, where mouse AST1, a subtype found in the hippocampus, was uniquely enriched. Overall, our data support a role for astrocytes in psychiatric disorders, but it will be necessary to characterize and study human astrocyte subtypes and their unique responses to different pathological states to fully understand their role in the brain and disease.Next, we looked at how astrocyte function relates to psychiatric disorders and saw that many of the same biological pathways were enriched in mouse astrocyte subtypes, in the mouse disease model astrocytes, the human astrocytes, and in psychiatric disorders. Pathway analysis identified common enrichment in the “metabolic”, “plasticity/structure” and “signaling” pathway clusters, highlighting the role of astrocytes in these processes and the dysregulation of these processes in schizophrenia and depression ,104,105.The “carbohydrate metabolism” pathway cluster is enriched in astrocyte subtypes and psychiatric disorders but has limited enrichment in mouse disease models or AST_hiPSC schizophrenia datasets. Astrocytes play an essential role in ensuring the bioenergetic requirements of neurons are met in the brain . We haveAdditionally, “regulation of protein phosphorylation” was enriched across astrocyte and psychiatric disorder datasets, implicating these processes in astrocytes under physiological and pathological conditions. We confirmed enrichment of protein kinase activity in a gliosome preparation relative to total homogenate in human postmortem brain tissue, and found that changes in activity in a subset of these kinases are also implicated in schizophrenia ,98. ThisA potentially confounding variable in understanding the role of astrocytes in disease is the effect of psychotropic medications that may be normalizing disease-related changes in astrocytes, or inducing structural, molecular, or functional changes in these cells ,108,109.While the general lack of consistent enrichment of drug-induced genes in astrocyte datasets is somewhat surprising ,115, ourOverall, these bioinformatic findings suggest that astrocytes are not significant primary targets of psychotropic medications used in the treatment of psychiatric disorders but different subtypes of astrocyte may be targets under specific conditions. In support of this, we found that chronic antipsychotic administration did not significantly affect astrocyte marker  transcript expression in the frontal cortex in rodent brain. Future studies will be required to examine marker expression in other brain regions. “Look-up” studies of rodent transcriptomic brain datasets also found few reports of significantly altered astrocyte marker transcript expression changes in response to different psychotropic medications, suggesting that chronic medication administration has a limited effect on transcript expression of established glial markers in rodent models. Importantly, “look-up” studies did find significant increases in expression of astrocyte gene markers in postmortem brain of schizophrenia subjects who were “on” antipsychotics compared to those who were “off” antipsychotics but not in bipolar disorder subjects who were “on” mood stabilizers compared to those who were “off,” highlighting the importance of considering specific disease- and drug-interaction effects on astrocytes. Future studies should consider a broader range of astrocyte genes to better capture understand the diversity of astrocyte subtypes and their responses in pathological states.Finally, we mined the LINCS chemical perturbagen gene signature repository to identify FDA-approved small molecules that could potentially reverse the gene expression changes associated with astrocytes in schizophrenia (AST_hiPSC_SCZvHC). Kinase inhibitors were among the top drug hits identified by this analysis and typically had stronger discordance scores than antipsychotics, suggesting that modulating astrocytic perturbation of kinases may have therapeutic potential in schizophrenia. A necessary limitation of this study is the use of astrocyte subtype molecular signatures derived from healthy mice for bioinformatic analysis, which may not reflect the subtypes of astrocytes found in human brain ,102. AddIn summary, this study provides a unique analysis of archived transcriptomic data and emerging molecular characterization of astrocyte subtypes to illuminate the relationship between psychotropic medications and astrocytes in the context of psychiatric disorders. We identified gene enrichment of distinct subtypes of astrocytes in psychiatric disorders but surprisingly, minimal enrichment of psychotropic medication-induced gene expression in astrocyte subtypes. This bioinformatic analysis was supported  by “lookup” studies and qPCR in rodents, suggesting that these medications have limited effects on inducing gene expression changes in astrocytes. However, our results highlight the importance of conducting truly translational studies, including postmortem brain studies that account for the effects of medication, due to the differences in astrocyte gene-sets in different species, cell subtypes and disease states. Interestingly, when we identified the biological processes associated with different astrocyte subtypes, regulation of protein phosphorylation was implicated across astrocyte and disease datasets. In combination with our exploratory studies of gliosome protein kinase activity, and in silico analysis of chemical perturbagens that reverse astrocyte-disease related gene signatures, our findings suggest that dysregulated astrocyte kinase activity is potentially a novel therapeutic target in schizophrenia. However, further work is required to elucidate the role of altered regulation of protein phosphorylation in astrocytes in the brain in psychiatric disorders. These findings serve as a starting point to better understand the complex relationship between psychotropic medications and astrocytes in psychiatric disorders, which could have significant consequences on our understanding of the pathophysiology of disease, and potentially offer insight into targeting astrocytes therapeutically.


The Lancet reports a substantial reduction in cervical cancer incidence after the introduction of the HPV immunisation programme in England.HPV infection causes the majority of cases of cervical cancer but can be prevented via vaccination. A new study in  Cervical cancer is one of the most common cancers in women and most cases are caused by human papillomavirus (HPV) infection, particularly high-risk HPV types 16 and 18. HPV vaccination has been shown to protect against HPV infection and cervical intraepithelial neoplasia (CIN)—a precursor to cervical cancer—in clinical trials, but there is little real-world evidence of the effectiveness of HPV vaccines in reducing cancer rates.Falcaro and colleagues report population-based cancer registry data to estimate the effect of Cervarix-based HPV vaccination on the incidence of cervical cancer and cervical carcinoma in situ, or grade 3 CIN (CIN3), in women in England1.The UK began to offer HPV vaccines to school-age girls in 2008, with a bivalent vaccine targeting HPV 16 and 18 called Cervarix. Now, The authors compare cervical cancer and CIN3 incidence rates in three cohorts who were offered the vaccine at different ages with earlier cohorts who were not offered the vaccine, using several statistical models to account for various confounding factors. They report relative reductions in cancer cases of 34% in those offered the vaccine at age 16–18, 62% for age 14–16, and 87% for age 12–13. Reductions in CIN3 incidence were even greater, reaching 97% in the group aged 12–13.Although data on vaccine status for individuals within each cohort was not available, these striking results provide strong evidence that HPV vaccination protects against cervical cancer and provide hope that it could be eliminated in the near future. Continued effort will be required to promote vaccine uptake and ensure vaccines reach low- and middle-income countries where access to cervical cancer screening and treatment is limited. Further research is also needed to determine the extent to which vaccination protects against other HPV-associated cancers, such as head and neck cancer, and to determine the impact of the more recently used quadrivalent vaccine Gardasil.


The COVID-19 pandemic requires thinking about alternatives to establish ECMO when often-limited hardware resources are exhausted. Heart-lung-machines may potentially be used for ECMO but contain roller pumps as compared to centrifugal pumps in ECMO-circuits. We here tested roller pumps as rescue pump for ECMO-establishment.We set up in vitro circuits on roller pumps from C5 heart-lung-machine with 5 l/minutes flow. In two series, we placed either PVC or silicon tubing for an ECMO circuit into the roller pump. We assessed the mechanical stress on the tubing (aiming to run the pump for at least 1 week), measured the temperature increase generated by the friction and assessed flow characteristics and its measurement in simulated situations resembling tube kinking and suction.The roller pumps led to expected and unexpected adverse events. PVC tubing burst between 36 and 78 hours, while silicon tubing lasted for at least 7 days. At 7 days, the silicone tubing showed significant signs of roller pump wear visible on the outside. The inside, however, was free of surface irregularities. Using these tubings in a roller pump led to a remarkable increase in circuit temperature . Kinking or suction on the device caused the expected dramatic flow reduction (as assessed by direct measurement) while the roller pump display continued to show the preset flow. The roller pump is therefore not able to reliably determine the true flow rate.Roller pumps with silicone tubing but not PVC tubing may be used for running ECMO circuits. Silicone tubing may endure the roller pump shear forces for up to 1 week. Thus, repeated tubing repositioning may be a solution. Circuit heating and substantial limitations in flow detection should increase attention if clinical use in situations of crisis is considered. Thus, a center’s therapeutic potential may not fully be exploited mainly due to the limitation in available ECMO pumps. However, most centers that are heavily affected by the pandemic also experience a substantial decline in standard cardiac surgery procedures, freeing up perfusion resources by availability of the heart-lung-machines. In addition, heart-lung-machines (HLM) contain several pumps per unit and ECMO units only need one pump. Unfortunately, heart lung machines primarily use roller pumps, while for ECMO centrifugal pumps are used. Using roller pumps for longer time frames may result in tubing failure.The growing experience shows that individual centers or areas may be affected so severely and rapidly, that the often only small number of ECMO units available is quickly exhausted.We here tested the possibility to use a roller pump as rescue pump for the establishment of ECMO circuits. We specifically wanted to test the durability of standard ECMO perfusion sets in a roller pump by using either classic PVC or silicone tubing and assessed heat development and hemodynamic characteristics.An in vitro perfusion circuit was established on a C5 heart-lung-machine (LivaNova) using the large roller pump that normally generates cardiopulmonary bypass flow.® PVC blood supply tubing.Set-up 1: pump tube 1/2″×3/32″ RAUMEDIC ECC noDOPSet-up 2: 1/2″×3/32″ RAUMEDIC ECC-SIK, silicon tubing.The roller pump occlusion was set to fully occlusive. The level sensor was installed at the hard-shell reservoir and the level control was connected to the roller pump. Running times of the pumps were recorded using the Data Management System of the heart-lung-machine . In case of pump tubing rupture, the volume loss decreased the level in the reservoir, the level sensor stopped the pump and the online DMS system recorded the event. From these data we concluded the running time of the pump tubes. Temperature measurement was established at the arterial temperature port of the oxygenator. The oxygenator (Terumo Capiox RX25) also served as system resistance and the heat exchanger of the oxygenator was used to heat the system to the working temperature.Once operational, we first assessed heat generation through friction of the tubing. The pump was started at room temperature and run for 3.5 hours. Temperature increase was measured. After that, an ECMO heating device (HU 35) was connected to establish perfusate temperature of 36.5°C. Then, durability of tubing roller pump was assessed. We performed three experiments per group and intended to reach 7 days of perfusion. In two cases with silicone tubing perfusion was run for up to 10 days. While running the experimental set-ups for durability, we performed hydrodynamic testing, such as kinking of the tubes or simulated different situations of poor venous drainage. Tubing occlusion was performed either by manual kinking the tubing for “full occlusion” or by using a screw adjustable clamp and compressing the diameter by 50% for “half occlusion.”We demonstrate in this in vitro analysis that Roller pumps with silicone tubing but not PVC tubing may be used for running ECMO circuits. Silicone tubing may endure the roller pump shear forces for up to 1 week. Repeated tubing repositioning may be a solution. Circuit heating and substantial limitations in flow detection should be taken into account if clinical use in situations of crisis is considered.Under normal conditions, centrifugal blood pumps are used to run ECMO circuits. The reason is based on the physical principles that apply to their action. Centrifugal force is used to set the blood into rotation with help of impellers. The rotational speed transfers the energy of the pump to the blood. The generated flow depends on centrifugal head size and is dependent on in- and outflow pressure. Flow is generated with the energy that exceeds the amount needed to generate the tubing pressures. Thus, if pressure increases, flow decreases and if the pump stops, blood is free to flow back according to the prevailing pressure gradients.In contrast, a roller pump is a robust and efficient pump, where pump flow depends on tube size and rotation speed. There is no backflow while the pump is stopping, for instance in an emergency situation. There is no control or compensation for inflow occlusion or negative pressures. Obstructions in the outflow tubing result in significant pressure increases without limitations in flow over a wide range, because the pump can work against the resistance until its pressure limits will cause it to stop. These differences in design, and especially the lack of mechanical shear forces on the equipment, have made the centrifugal pump the worldwide standard for ECMO circuits.79 The technical issues such as suction and kinking of tubing are important, have to be recognized by the team but should not limit its use. However, increased attention appears required to run such a system. Including negative pressure measurements for limiting pump speed in case of suction may also be helpful. The connection of the patient to a much larger device (heart-lung-machine) is also likely to limit maneuverability of the patient. In addition, the mechanical shear forces on the tubing appears to be a limiting factor. While PVC tubing may not be used at all because it may not even last for 2 days, silicone tubing appears to be a possible alternative, at least for up to a week. Our findings are also consistent with Peek et al. who assessed durability of three different types of tubing in a roller pump setting and demonstrated tubing rupture between on averaged 7 and 244 hours. Our conclusions are also supported by our finding that use of PVC resulted in a significantly higher degree of circuit heating under our experimental conditions, which we attribute to the greater degree of friction with the PVC tubing. The silicone tubing did not break and did not show any significant signs of erosions on the inside of the tubing for at least 1 week. However, the outside showed signs of mechanical stress that may raise concern for longer use. This concern is supported by the one breakage on the ninth day of roller pumping. Thus, in case such a set-up is planned to be used in the real practice setting, it may be wise to move the tubing piece subjected to the roller pump every week in order to distribute the shear forces to different parts of the circuit. This suggestion is also consistent with current practice in the pediatric setting.79 In addition, it is known that centrifugal pumps work more gently on the blood than roller pumps over longer operating times and that there may be an increase in hemolysis, microbubble transmission with longer use of roller pumps.1014Our work here suggests that a roller pump may under emergency conditions be used in vivo. This suggestion is supported by the fact that roller pumps are routinely used in pediatric ECMO circuits, although the hemodynamic conditions in children require much less flow.The differences in measured and displayed flow in A final consideration is the durability of roller pump function itself. Build for providing cardiopulmonary bypass support, which is usually limited to several hours, roller pumps may run into problems such as overheating or pump failure when run for days or weeks. We did not observe any problems in the operation of our roller pumps and the whole heart and lung machine during the time frame of our experiments. The manufacturer does not suggest using roller pumps under these conditions. Thus, if this set-up is required in practice is appears wise to have a second roller pump present as backup.We demonstrate in this in vitro analysis that roller pumps with silicone tubing but not PVC tubing may be used for running ECMO circuits. Silicone tubing may endure the roller pump shear forces for up to 1 week. Repeated tubing repositioning may be a solution. Circuit heating and substantial limitations in flow detection should be taken into account if clinical use in situations of crisis is considered. Importantly, this last resort-type ECMO circuit appears to require more attention and limits maneuverability of the patient.