Panulirus argus) and native long-spined sea urchins (Diadema antillarum), indicating the potential for competition. We then used a laboratory experiment to compare activity and shelter use of each species when alone and when lionfish were paired with each native species. Spiny lobsters increased their activity but did not change their shelter use in the presence of a lionfish, whilst long-spined sea urchins changed neither their activity nor shelter use. However, lionfish reduced their shelter use in the presence of spiny lobsters and long-spined sea urchins. This study highlights the importance not only of testing for the non-consumptive effects of invasive species, but also exploring whether native species exert non-consumptive effects on the invasive.Indo-Pacific lionfish have become invasive throughout the western Atlantic. Their predatory effects have been the focus of much research and are suggested to cause declines in native fish abundance and diversity across the invaded range. However, little is known about their non-consumptive effects, or their effects on invertebrates. Lionfish use shelters on the reef, thus there is potential for competition with other shelter-dwelling organisms. We demonstrate similar habitat associations between invasive lionfish, native spiny lobsters ( Invasive predatory species are well known to cause consumptive effects on native species –3. Yet, Pterois volitans and Pterois miles [Coral reef ecosystems are in decline around the globe as a result of stressors including climate change , 12, unsis miles , hereaftis miles , with nuis miles , speciesis miles  and recris miles , particuis miles . Despiteis miles , 30.In addition to consumptive effects, lionfish are subject to and can exert non-consumptive effects, for example ‘fear effects’, whereby prey change their behaviour to reduce the risk of predation in the presence of a predator . In the Panulirus argus; hereafter referred to as lobsters) and long-spined sea urchins  are both native invertebrates that use reef shelters. Neither species has been the focus of research on lionfish impacts because spiny lobsters are rarely consumed by lionfish [Diadema being consumed by lionfish. Lobster is an economically important species exploited for food across much of its native range [Diadema is an ecologically important species for its keystone role in macroalgal grazing [Diadema use shelters on the reef to avoid predation [Diadema will recruit to artificial shelters that are placed on natural reefs [Studies investigating shelter use interactions between lionfish and native invertebrates are lacking. Lionfish show preference for broad-scale complexity such as caves and overhangs , which tlionfish  and therve range , whilst  grazing . Both loredation , 48 and redation –51. The redation , suggestal reefs , 54 furtDiadema, as seen in grouper-lionfish interactions [Diadema from shelters, thus exposing them to greater predation risk. Similar shelter preferences between lionfish, spiny lobsters and Diadema have been suggested [Diadema populations, which may affect economic benefits from the lobster fishery and ecological functions provided by Diadema.If lionfish show similar shelter preferences as native lobsters and ractions , 56, theuggested , and sheuggested  and the uggested  and conduggested , 61). CoDiadema for shelter on Caribbean coral reefs. We first tested whether lionfish, lobster and Diadema occupy similar habitats on the reef, by conducting in situ assessments of habitat complexity. We then tested whether the presence of lionfish influences the behaviour of lobsters and Diadema, using a controlled laboratory experiment with limited shelter availability. Our study clearly identifies the overlap in shelter preferences and illustrates the behavioural responses caused by interactions between native invertebrate species and invasive lionfish, highlighting the importance of non-consumptive impacts of invasive species on the ecology of native species.In this study we investigate whether invasive lionfish compete with native lobsters and Pterois volitans based on [Panulirus argus) and Diadema (Diadema antillarum) were studied in the shallow (3–12 m) near-shore reef system of La Ensenada in Tela Bay, Honduras, at the southern end of the Mesoamerican Reef (Lionfish (assumed to be based on ), lobstecan Reef  from Junn = 35), lobsters (n = 28) and Diadema (n = 22) were recorded during roving SCUBA dives at three sites on the La Ensenada reef system (n = 43) took place between 8:00 am and 2:00 pm across June, July and August. All three species are generally inactive and inside shelter during the day [Diadema. Details on the conversion from carapace length or test diameter to overall body size are provided in the experimental design section of the methods. These habitat associations were compared to randomly sampled background areas of reef (n = 36), obtained by laying three 50 m transects in random directions from the mooring line at approximately 5 m depth at each of the three collection sites  for six complexity categories were recorded on a scale from 1–5, with larger values indicating higher complexity, following . Maximumn = 35) were collected from the reef using hand-nets, whilst lobsters (n = 28) were caught by hand. Following capture, lionfish and lobsters were transferred to separate 57-litre drybags filled with seawater. Diadema (n = 22) were collected and transported in a large bucket of seawater. All specimens were slowly brought to the surface and then transported to the laboratory by 15-minute boat ride within the drybag or bucket they were collected in.Specimen collections were conducted by recreational SCUBA divers during roving dives in depths of 4.2–12.1 m. The individuals used in our laboratory experiment are a subset of those individuals that were collected during the habitat association aspect of our study. Not all collected individuals were used because the cameras failed during some of the laboratory trials. Lionfish (Aquarium setup. Three tanks measuring 100 x 55 x 55 cm (length x width x depth) were used for acclimatisation and two tanks measuring 245 × 56 × 24 cm were used to run the trials. We used the largest tanks available to minimise the impact of confinement on interspecific competition [Diadema [petition . Trial tpetition , which apetition  and Diad[Diadema . Tanks w[Diadema . We aimeThe shelter was placed at one end of the tank, with the opening facing towards the centre of the tank. Gaps on either side of the shelter were blocked off using extra concrete bricks to prevent animals from sheltering between the edge of the tank and the shelter. Each acclimatisation tank was fitted with a Tetra whisper air pump  to maintain oxygenation of the water, however, air pumps were not used in the trial tanks because the much greater surface area would have allowed sufficient oxygen transfer and we did not want the air pumps to interfere with natural diffusion of chemical cues. At the end of every acclimatisation period and every trial, water quality  was tested using a saltwater master test kit and all values were found to be within acceptable water quality guidelines .Diadema [On arrival at the laboratory, each species was placed into a separate acclimatisation tank, with a maximum of two conspecific individuals per tank. Animals were collected between 8:00am and 2:00pm and tested the following morning, resulting in acclimatisation times of 16–24 hours. This was deemed to be enough time because at the beginning of each trial, all individuals were displaying normal day-time behaviour of resting inside shelter , 68, 69.Diadema , we chosDiadema , 71.n = 12, lobster alone n = 12, Diadema alone n = 12) to determine their activity and shelter use in the absence of other, potentially competing, species. Lobsters and Diadema were also tested in the presence of a lionfish  to determine whether behaviour differed between these ‘together’ trials and the ‘alone’ trials. Each animal was only used once. The individuals used in our laboratory experiment are a sub-set of those used in the habitat association study, thus we now report the range of sizes used for our laboratory experiment. Lionfish ranged in total length (TL) from 14.6–26.6 cm, with a mean (± standard error) of 21.2 (± 0.6) cm. Lionfish size at sexual maturity varies between the sexes and between locations but on average is 10 cm for males and 17.5 cm for females [Panulirus argus [Diadema ranged in test diameter from 4.0–5.6 cm, with a mean of 4.9 (± 0.1) cm, and were classified as adults because they had no banded spines [Diadema spp. [Diadema individuals used in our experiment ranged from 20–28 cm in total diameter.Single individuals of each species were tested alone  positioned 1 m from the end of the tank containing the shelter and angled down at 45 degrees. This positioning of the camera allowed us to see into the back of the shelter and 17 cm in front of the shelter. The camera filmed using infrared (850 nm wavelength) during both day and night because preliminary trials found that videos were often underexposed when filmed in daylight without the use of flash. Lionfish, lobsters and e 600 nm –79 so thDiadema were not marked because current tagging methods are invasive and can impact survival [Diadema were released by the mooring line at the collection site and subsequent individuals were collected a minimum of 20 m away. Average movement each night by Diadema is less than 5 m [Diadema were released back onto the reef within two hours of the trial ending. Immediately after the trial, lionfish were humanely culled using a two-step method for finfish euthanasia, whereby cervical transection was followed by pithing to destroy the brain tissue [Following the trials, we telson-clipped the lobsters to ensure that individuals were not re-used in subsequent experiments. survival . Insteadthan 5 m  and indithan 5 m , thus wen tissue . All accFor each 60 second video, the time spent: (i) active vs resting (activity), and (ii) inside vs outside the shelter (shelter use) were recorded. Individuals were recorded as inside the shelter if at least 25% of their body length was inside and were recorded as active if they were moving across the tank. Lionfish often moved their fins and lobsters moved their legs whilst stationary, but we categorised these behaviours as inactive because the individual remained in one location.Diadema together trials, the Diadema always started the trial inside the shelter, as previously explained.For our analyses, we calculated the time spent in each behaviour over the course of the day, but we removed the first hour of each trial to ensure that all animals had acclimatised to the trial tank for a minimum of one hour. Analysed trial data thus spanned 23 hours–comprised of 23 videos of 60 seconds. We calculated activity as the proportion of time spent active out of the time spent in view of the camera, since when animals were out of view we did not know whether they were active or inactive. We calculated shelter use as the proportion of time spent inside shelter out of the total trial time, since there was only one shelter in the tank so even when not in view of the camera we knew that animals were not inside the shelter . In addia priori significance level of 0.05 and were conducted in R version 3.6.2 [All tests were two-tailed with an on 3.6.2 . All graon 3.6.2  with theon 3.6.2 .in situ habitat associations, we performed non-metric multidimensional scaling (NMDS) with a Bray-Curtis dissimilarity matrix using the vegan package in R [Diadema).To visualise age in R , and ploin situ habitat associations for specific HAS categories, we conducted Kruskal-Wallis tests, which were chosen because the HAS data were ordinal, rather than continuous. We conducted one Kruskal-Wallis test for each of the six HAS categories: rugosity, variety of growth forms, substratum height, maximum refuge size, percentage live cover and percentage hard substratum. Each Kruskal-Wallis test compared HAS between background, lionfish, lobster and Diadema quadrats. When the results were significant- indicating a difference in HAS value between quadrat types- we ran post-hoc Dunn tests [Diadema, lionfish-lobster, lionfish-Diadema and lobster-Diadema. Our post-hoc Dunn tests were run using the Benjamini-Hochberg method to correct for multiple testing (by controlling the type I error rate) whilst retaining greater power than the Bonferroni method [To examine nn tests  to compai method .Our data for activity and shelter use were proportions. The denominator of the fractions was large (up to 1380), thus allowing the proportions to take a wide range of values. For this reason our data can be described as non-count-based proportions and thus were analysed using beta regression , which cEquation 1. This equation, modified from [ied from , convertDiadema shelter use) differed from the next best model by less than two AICc units, with the next best model containing one extra parameter. These extra parameters can be considered uninformative [Model selection followed the two-step process detailed in , wherebyormative , thus weormative .Diadema alone, lionfish-lobster together, lionfish-Diadema together) was always included in the mean models but we also tested whether to include trial tank  in the mean and precision models. We tested the difference in behaviour (activity and shelter use) between trial types in the 23-hour dataset. Trial tank was never included in the best fitting mean or precision models and thus will not be discussed further.Beta regression analyses (hereafter referred to as BR) were conducted using the betareg package in R  and AICcLionfish culling was carried out in accordance with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals . All expp = 0.001; p<0.05 for all; p<0.05 for all; p>0.05 for all; Diadema . The median refuge size HAS were: background (6–15 cm), Diadema (16–30 cm), lobsters (16–30 cm) and lionfish (31–50 cm). Despite these differences between species, there was still considerable overlap in HAS for maximum refuge size between species , in the presence of a lionfish during the 23-hour trial. The presence of lionfish had no effect on activity  of Diadema. Lionfish and lobsters shared the shelter in one out of twelve trials , whilst lionfish and Diadema shared the shelter in two out of seven trials . In ten of the twelve lionfish-lobster together trials , the species that entered the shelter first was also the species that spent the most time inside the shelter , even though Diadema always began the trial inside the shelter.Lobsters were 1.8 times more active .Lionfish used shelter 1.9 times more when alone than when in the presence of lobsters and 2.2 times more when alone than when in the presence of Diadema .Lionfish were 1.9 times more active in the presence of in situ habitat preferences as native lobsters and Diadema redation . Howeverredation , so in aredation . For foue region , reducinDiadema in Utila, Honduras, found that they preferred refuges of the size 5–15 cm [Diadema select shelters that scale with their test size to avoid predation [Diadema test diameter to total body size, we found that the Diadema were larger in overall size than the lobsters and lionfish. Although Diadema were larger, it is likely that they chose smaller shelters than lionfish or lobsters because they choose shelters that scale with their test diameter [Diadema may only be subject to shelter competition from small lionfish.We found significant differences in maximum refuge size between species. A previous study from Honduras found that lionfish preferred shelter sizes of 6–15 cm and 16–30 cm . However 5–15 cm , which iredation , therefodiameter , rather Homarus americanus) spend more time active in the presence of other lobsters than when alone and this activity is related to fighting and defending a shelter [Lobsters became more active in the presence of lionfish. This increase in activity may be related to aggression, as spiny lobsters display several aggressive behaviours . America shelter , so the  shelter , and so  shelter , suggest shelter , visual  shelter  and tact shelter  cues and shelter . Our expDespite the increase in activity, there was no change in shelter use in the presence of a lionfish, suggesting that the lobsters may be dominant and able to displace lionfish from the shelter, or prevent displacement by lionfish once they are inside the shelter. The lack of change in shelter use suggests that the increased activity observed in the presence of a lionfish is unlikely to be related to submissive behaviour and movement away from the shelter. Lionfish and lobsters shared the shelter in one of our trials, suggesting that even on reefs with limited, and already occupied, shelters, lobsters may still be able to access the shelters. However, we found that prior occupancy influenced shelter use, as in other fish and crustaceans , 109. WeDiadema. Several fish families and known predators of Diadema [Diadema are less likely to flee from a high-quality crevice [Diadema will avoid shelters containing conspecific odours, which is thought to be a method of avoiding shelter competition [Diadema shared the shelter in two of our seven trials, suggesting that lionfish are not considered a shelter competitor and thus the presence of a lionfish inside the shelter does not impact Diadema shelter use. However, we found that prior occupancy had no effect on which species used the shelter the most, suggesting that in some cases lionfish may be able to exclude Diadema from their shelters.Lionfish had no effect on activity or shelter use of  Diadema  and pred Diadema , so the  crevice . In the petition . LionfisDiadema. Lionfish increase their activity in response to intraspecific competition [Diadema because they perceive them as a shelter competitor. Lionfish may not have increased their activity in response to lobsters because the lobsters may have been perceived as a dominant competitor, as is suggested by other studies on shelter use interactions between fish and crustaceans [Diadema indicates that lionfish are not simply responding to over-crowding . Instead, lionfish behavioural responses are determined by the identity of the competitor, as is observed in other fish species [Lionfish did not change their activity in the presence of lobsters but they became more active in the presence of petition , thus westaceans , 112. Th species .Increased activity in lionfish may lead to less energy being available for other functions such as growth. In areas where lionfish are culled by divers, increased activity of lionfish may make them more conspicuous and easier to capture, thus aiding culling efforts. But an increase in lionfish activity may not always be beneficial for the native community. Increased activity may increase the likelihood of interactions with native species, potentially leading to other non-consumptive effects, such as reduced grazing by herbivorous fish , 35.Diadema. Our findings lend support to anecdotal evidence that reefs with high urchin densities support few lionfish [In contrast to other invasive species, where shelter use of the invasive is unchanged in the presence of native species , 111, welionfish , potentilionfish , thus lelionfish , so any Studies on the commercial spiny lobster fishery have shown that lionfish are often caught as by-catch in traps  and the Although our results suggest that lionfish do not affect lobster shelter use, and therefore may not reduce lobster catches, this does not mean that lionfish have no effect on spiny lobster fisheries. The presence of lionfish in lobster condos increases handling time by fishers as they work more cautiously around lionfish to avoid envenomation and this may lead to reduced earnings as fishers are unable to visit as many condos each day . HoweverDiadema following the 1983–1984 mass mortality [Diadema shelter use. We initially hypothesised that lionfish would enter the shelter and exclude the Diadema, but our results showed that lionfish had no effect on Diadema activity or shelter use. This lack of response by Diadema suggests that lionfish may not interfere with restoration efforts and that the presence of lionfish on a reef may not a barrier to recovery of Diadema.A lack of structural complexity and the subsequent lack of predation refugia has been suggested as a barrier to recovery of ortality . One resortality . Lionfisortality , 119 andLobster traps may prove useful for lionfish management, as studies have shown catches of almost 3000 lionfish in a single lobster fishing season . TrappinDiadema activity or shelter use. Invasive species research often focuses on the impacts on the invaded community or on trophic interactions exerted by native species on invasive species, however, we have shown the importance of investigating non-consumptive effects of the native species on the invasive. These effects may prove detrimental to the invader or may lead to additional negative effects on native species. By studying the interactions between lionfish and two different invertebrates, we have demonstrated that non-consumptive effects of native species on invasive species may be widespread. Our research suggests that we should look beyond the consumptive effects of invasive predators and consider how native species may induce changes in invasive species behaviour.Our results have demonstrated the importance of testing for non-consumptive effects of invasive species. We found that lionfish are not detrimental to all native species, as there was no effect of lionfish on S1 Tablen = 35), lobsters (n = 28) and Diadema (n = 22) were collected from all three sites for use in the laboratory experiments. We recorded Habitat Assessment Scores (HAS) for 12 background quadrats at each site and for each individual that was collected. A total of 43 SCUBA dives took place between 8:00 am and 2:00 pm across June, July and August 2019. Coordinates are given in WGS84.Co-ordinates of the mooring lines at each of our three study sites in Tela Bay, Honduras. Lionfish ((CSV)Click here for additional data file.S2 Tablen = 35), lobsters (n = 28) and Diadema (n = 22) and for randomly sampled background quadrats (n = 36). HAS values range from 1 (low complexity) to 5 (high complexity). Date, site and animal size are also provided.Six HAS values were recorded for each quadrat containing lionfish ((CSV)Click here for additional data file.S3 TableDiadema test diameter are also provided .23 observations of 60 seconds were recorded during each 23-hour trial, thus each trial comprises 1380 seconds of observation. The time that each animal spent active in view of the camera, inactive in view of the camera and inside shelter is shown in seconds (columns G-O). Time spent active was divided by total time in view of the camera (time active + time inactive) to give proportion of time active . Time spent active or inactive was only recorded when the individual was in view of the camera, thus time active + time inactive is not necessarily equal to total trial time (1380 seconds).Time spent inside shelter was divided by total trial time (1380 seconds) to give proportion of time inside shelter . These proportions were than transformed using equation one to remove any values of 0 or 1 . Total length of lionfish, lobster carapace length and (CSV)Click here for additional data file.


The aim was to estimate the risk of subsequent extra-cervical Human Papillomavirus (HPV) related cancer in patients surgically treated for high grade cervical intraepithelial neoplasia (CIN 2–3). This is the first study in Italy investigating the occurrence of extra-cervical tumors in this cohort of patients.3184 patients surgically treated for CIN2–3 since 1992 at the Department of Surgical Sciences of University of Torino were considered. The risk of HPV-related cancer was calculated as Standardized Incidence Ratio (SIR), using as expected values tumour age specific incidence of resident population.173 second primary cancer (SCPs) were identified. SIR to develop cancer after treatment for CIN2–3 was 2.2 (CI 95% 1.89–2.50). Among these occurrences, 10 are in HPV related sites: 1 anus , 3 vagina , 1 vulva , 5 oropharynx .Significant risk has been also recorded for pulmonary  and bladder , with smoking as possible cofactor. We also found increased risk for breast  and ovarian cancers , probably due to an higher adherence to spontaneous and programmed screening programs.Our study supports the hypothesis of an increased risk of HPV-related tumours for CIN treated patients, mostly for CIN3. It is conceivable the need of early diagnosis for these cancers in this higher-risk populations. High-risk human papillomaviruses (hrHPV) are estimated to cause 5.2% of all cancers worldwide, with HPV16 alone responsible for approximately 70% of cervical cancer, that is the first most common HPV-related cancer in women  by age class (five-year groups: 0–85+), period  and cancer site. Person-years in the cohort were calculated with month approximation at follow-up date and time of surgery (as starting observation time).Expected cases by type of cancer were calculated in the reduced cohort applying corresponding specific rates , even for head and neck cancers the opportunity of a screening program is affected by the low incidence in the general population of the same, especially in women. Moreover, unlike tumours in the anogenital area, there is no pre-tumour lesion that anticipates oral and oropharyngeal cancers, this further compromises the usefulness of a screening strategy , 46.It remains to assess the impact of adjuvant HPV vaccination in treated patients: it has already demonstrated a protective effect in reducing recurrent intraepithelial cervical lesion but further studies are needed to evaluate the impact on HPV-related SPCs .There are some limitations in our study. First the type of treatment of cervical lesions, LLETZ, could have influenced the study outcome, since it removes less tissue than cold knife conization. If this approach is of utmost clinical benefit for reduction of adverse obstetrics outcomes, it can leave in situ more HPV infected tissue. Also positive margin status and glandular involvement from a less invasive treatment could have influenced, as a result of incomplete excision or a lesion hidden in the glandular crypts. Secondly, our database is not representative of the entire Italian population but based on a regional cancer registry. Furthermore, we assumed that SPCs can be attributable to HPV infection persistence but HPV testing was not available in our retrospective database.The increased risk of cancer in extra-cervical sites after CIN 2–3 treatment makes appropriate to introduce personalized follow-up and screening programs, while HPV vaccination needs further studies to assess its impact. The best strategy should be defined according to the economic budget of public health and to the workload of the hospital units in charge of their implementation.


Human papillomavirus (HPV) gained momentum as a potential etiological factor for many types of cancers. Therefore, the aim of this study was to assess the prevalence of HPV-16 infection among Sudanese patients diagnosed with Squamous Cell Carcinoma (SCC) and Salivary Gland Carcinoma. A descriptive, hospital-based study was conducted. 150 formalin-fixed paraffin-embedded blocks were collected.The study population included a total of 150 patients aged between 18 to 87 years with a mean age of 48.8 ± 11.9 years. Based on gender, females constituted 46.7% while males constituted 53.3%. The 150 patients were classified into 40 (26.0%) esophageal, 30 (20.0%) nasopharyngeal, 18 (12.0%) conjunctival, 18 (12.0%) tongue 12 (8.0%) laryngeal, 8 (5.3%) lip, 6 (4.0%) oropharyngeal, 6 (4.0%) mucoepidermoid, and 6 (4.0%) adenoid cystic, and 6 (4.0%) myoepithelial carcinomas. Odds ratio for male and female diagnosed with carcinoma was 1.025 . Molecular detection of HPV-16 revealed a prevalence of 26 (17.3%) patients were positive for HPV-16. According to cancer diagnosis, esophageal SCC patients showed a high proportion of HPV-16; 14/40 (35.0%). A statistically significant difference was seen for the distribution of HPV-16 positive patients based on cancer diagnosis, P value 0.001. Human papilloma virus (HPV) encompass a group of double-stranded DNA icosahedral viruses that belongs to the family papillomaviridae with a tendency to infect mucosal and cutaneous epithelia . More thPrevalence of infection by HPV-16 varies across populations, genders and habit attributes with incident infection as high as 60% been detected . The immA descriptive, cross-sectional study was conducted from January 2018 till June 2019. Formalin-fixed paraffin-embedded (FFPE) blocks of cases diagnosed with a variety of carcinomas were retrieved from the archive of the Radiation and Isotopes Centre Khartoum regardless of their sex and ages. Sections from the cases that were stained by hematoxylin and eosin were then reviewed to confirm the diagnosis.The study ethics approval and consent to participate were obtained by the ethics review board of the Faculty of Medical Laboratory Sciences, University of Khartoum. Informed consent was obtained from each participant prior to enrollment using writing informed consent.Since HPV-16 cannot easily be cultured, nowadays the most reliable method used in HPV-16 detection is detection of its DNA by Polymerase Chain Reaction (PCR) . SectionMolecular detection of HPV-16 was made using the previously published primers; forward primer 5-TTT TGG GTT ACA CAT TTA CAA G-3 and the reverse primer 5-TGT CTG CTT TTA TAC TAA CCG-3 for the PCR amplification . PCR reaPCR amplicons were visualized using UV-transilluminator , after loaded into 2.5% agarose gel  and electrophoresed at 100 V for 60 min . To avoid false negative and false positive results, PCR results were recorded as positive results in comparison to a 100 bp molecular marker  based on the presence of 121 bp band size in all the three DNA samples for each FFPE block. A double distilled water was used as negative control in each PCR run instead of adding DNA.P value < 0.05 was considered statistically significant.Data were analysed using the Statistical Package for Social Sciences software . Chi-Squared test was used to compare the status of HPV-16 infection among the study variables. Odds ratio was calculated with 95% Confidence Interval (CI). A The study population included a total of 150 participants aged between 18 to 87 years with a mean age of 48.8 ± 11.9 years. Based on gender, females constituted 46.7% while males were constituted 53.3% of our cohort. Odds ratio for male and female diagnosed previously with cancer was 1.025 . The cohort included 40 patients diagnosed with esophageal squamous cell carcinoma (SCC), 30 patients with nasopharyngeal carcinoma, 12 patients with laryngeal carcinoma and 6 patients with oropharyngeal carcinoma. Eighteen cases in our cohort had SCC of the conjunctiva, 18 patients had tongue SCC and 8 patients had SCC of the lip. Interestingly, 18 of the patients had carcinomas of the salivary gland carcinoma split equally between mucoepidermoid carcinoma, adenoid cystic carcinoma and myoepithelial carcinoma.Based on age group, the age group of 31–60 years constituted the majority of the study population 80.0% (120/150), of them 54/120 (45.0%) were females and 66/120 (55.0%) were males. A statistically significant difference was noted for the distribution of gender and cancer diagnosis based on age groups, P values 0.01 and 0.004, respectively  were in the age group of 31–60 years, followed by 6 25.0%) and 4 (66.7%) of the age groups 61–90 years and 1–30 years, respectively. The prevalence of HPV-16 among the study population was statistically significant based on age group, P value 0.002. According to cancer diagnosis, those were diagnosed as esophageal SCC showed a high proportion of HPV-16 positive patients; 14/40 (35.0%), followed by 6/12 (50.0%), 4/18 (22.2%), and 2/6 (33.3%) of those diagnosed with larynx cancer, tongue cancer, and adenoid cystic adenocarcinoma, respectively. A statistically significant difference was seen for the distribution of HPV-16 positive patients based on cancer diagnosis, P value 0.001. The distribution of HPV-16 positive patients based on gender, age groups, and cancer diagnosis is illustrated in Table 5.0% and The involvement of HPV-16 in various human cancers has led to significant advances in prevention and management of these cancers. For instance, the identification of the subset of oropharyngeal carcinoma that is HPV-16 positive showed unique impacts in the treatment and the prognosis of cancer , 13. TheThis study concludes that those who were diagnosed as esophageal SCC show a high proportion of HPV-16 and is worth mentioning that none of the cases diagnosed with oropharyngeal carcinoma were positive for HPV-16 which indicate a negative role for HPV-16 for oropharyngeal carcinoma initiation. Also, this study highlights the need for a routine testing of HPV-16.Samples investigated in this study were limited, therefore a larger sample size and incorporate different types of head and neck cancers could provide further insight.Studying the relation between the presence of HPV-16 and prognosis of the cancer to correlate the disease progression with the presence and absence of HPV-16 were not applicable to be done.


The Papillomavirus Rapid Interface for Modelling and Economics (PRIME) has been used around the world to assess the health impact and cost-effectiveness of human papillomavirus (HPV) vaccination in girls. We updated PRIME with new data and methods for demography, disability weights, and cervical cancer burden, and generated revised estimates of the health impact of HPV vaccination at the global, regional, and national levels for 177 countries.PRIME was updated with population demography of the UN World Population Prospects (UNWPP) 2019 revision, disability weights of the Global Burden of Disease (GBD) 2017 study, and cervical cancer burden from the Global Cancer Incidence, Mortality and Prevalence (GLOBOCAN) 2018 database. We estimated the lifetime health benefits for bivalent or quadrivalent and nonavalent vaccination of 9-year-old and 12-year-old girls at 90% coverage during 2020–29 in 177 countries. Health impact was presented in terms of cervical cancer cases, deaths, or disability-adjusted life-years (DALYs) averted per 1000 vaccinated girls in comparison with the counterfactual scenario of no vaccination, and the number of girls needed to be vaccinated to prevent a single case, death, or DALY.In estimating the health impact of HPV vaccination of 9-year-old girls, the combined updates to demography, disability weights, cervical cancer burden estimates resulted in a 26% increase in the estimated number of cases averted, a 51% increase in deaths averted, and a 72% increase in DALYs averted per 1000 vaccinated girls for both the bivalent or quadrivalent and nonavalent vaccines, compared with previous estimates. With the updated model, the bivalent or quadrivalent HPV vaccine was estimated to avert 15 cases, 12 deaths, and 243 DALYs per 1000 vaccinated girls, and the nonavalent HPV vaccine was estimated to avert 19 cases, 14 deaths, and 306 DALYs per 1000 vaccinated girls. The health benefits of vaccination of 12-year-old girls were estimated to be similar but slightly decreased in comparison with vaccination of 9-year-old girls.HPV vaccination provides greater health benefits and is more cost-effective than was previously estimated. The demography update, which incorporates population aging, has the largest effect on the health impact estimates. The WHO African region is expected to gain the greatest health benefits and should be prioritised for HPV vaccination.Gavi, the Vaccine Alliance; Bill & Melinda Gates Foundation. Cervical cancer has the fourth greatest global burden of cancer among women for both incidence and mortality, and is the leading cause of cancer death among women in 42 countries.3Cervical cancer is caused by infection with high-risk genotypes of human papillomavirus (HPV). Three HPV vaccines  are widely available in the global market, and are highly efficacious in preventing persistent infection and associated disease from the high-risk HPV 16/18 genotypes, which cause 70% of all cervical cancers.WHO recommends that all countries introduce HPV vaccination for primary prevention of cervical cancer, prioritising the primary target group of young adolescent girls, aged 9–14 years.In May, 2018, the WHO Director-General called for global action to eliminate cervical cancer as a public health problem through improved coverage for HPV vaccination, high-precision screening tests, and treatment and care.Evidence before this studyThe Papillomavirus Rapid Interface for Modelling and Economics (PRIME) was developed in 2014 in collaboration with WHO to assess the health impact and cost-effectiveness of human papillomavirus (HPV) vaccination in girls around the world. It was used to show that vaccinating 12-year-old girls against HPV was cost-effective in almost every country for the bivalent and quadrivalent vaccines. Since its development and the introduction of a nonavalent vaccine in 2014, new data and methods for demography from the UN World Population Prospects 2019 revision, disability weights from the Global Burden of Disease 2017 study, and cervical cancer burden from the Global Cancer Incidence, Mortality and Prevalence 2018 database have become available, and were used to update the model.Added value of this studyThe combined PRIME updates for demography, disability weights, and cervical cancer burden improve the HPV vaccination impact estimates in comparison with previous forecasts and suggest greater health benefits in preventing cases, deaths, and disability-adjusted life-years (DALYs) due to cervical cancer at the global, regional, and national levels. The demography update has the largest effect on the health impact estimates; population aging leads to increasing life expectancy among women and an increase in lifetime risk of cervical cancer incidence and mortality without vaccination. HPV vaccination can avert this potential increase in new cases and deaths, and because the number of girls needed to be vaccinated to prevent a single case, death, or DALY is lower than previous forecasts, HPV vaccination both provides greater health benefits and is more cost-effective than was previously estimated. Because of a higher burden of cervical cancer before vaccination in the WHO African region compared with other regions, HPV vaccination will provide the greatest health benefits in this region.Implications of all the available evidenceReaching the WHO goal of global elimination of cervical cancer as a public health problem will take decades. Therefore, HPV vaccine impact models, such as PRIME, need to regularly integrate new knowledge and updated data sources. With such updates, HPV vaccination remains highly cost-effective globally, and efforts to increase coverage and equity should be continued. Countries in the WHO African region should be prioritised for HPV vaccine introduction and scale-up.PRIME) is a model to assess the health impact and cost-effectiveness of HPV vaccination of girls for prevention of cervical cancer.14The Papillomavirus Rapid Interface for Modelling and Economics .(GLOBOCAN) database.PRIME data inputs include country and age-specific cervical cancer incidence, prevalence, and mortality among females from the Global Cancer Incidence, Mortality and Prevalence PRIME uses country-specific life tables to model the annual population size of longitudinal birth cohorts. The life tables were previously generated from population size of birth cohorts and all-cause female mortality estimates by age from the WHO 2009 Global Health Observatory, which remained static over time.Disability weights were updated from estimates of the Global Burden of Disease (GBD) 2001 studyCervical cancer burden was updated from the International Agency for Research on Cancer estimates for GLOBOCAN 2012v be the age at vaccination and i any given age, where v is less than or equal to i; then cervical cancer burden averted at age i is the product of five estimates: cervical cancer burden caused by all HPV genotypes at age i before vaccination, country-specific proportion of cervical cancer caused by high-risk HPV types, two-dose vaccine coverage at age v, vaccine efficacy against high-risk HPV types, and proportion of female population that has not experienced sexual debut by age v.HPV vaccine impact on cervical cancer burden  averted is calculated as the proportional reduction in age-specific incidence, prevalence, and mortality in each country at single-year age intervals from 0–100 years. Cervical cancer burden was calculated as follows: let We analysed five scenarios to assess the health impact of HPV vaccination using PRIME . The basWe present vaccine impact metrics in terms of cervical cancer burden averted per 1000 vaccinated girls and number of girls needed to be vaccinated to prevent a single cervical cancer case, death, YLD, year of life lost (YLL), or disability-adjusted life-year (DALY). These two vaccine impact metrics are not affected by vaccination coverage, with the limitation that PRIME includes only direct effects of HPV vaccination and excludes indirect herd effects.The funders of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. All authors had full access to data in the study, and final responsibility for the decision to submit for publication.PRIME was updated with population demography from the UNWPP 2019 revision, disability weights from the GBD 2017 study, and cervical cancer burden from the GLOBOCAN 2018 database. In the base scenario (S1) with the bivalent or quadrivalent vaccine, the numbers of 9-year-old girls that were needed to be vaccinated were 82 to prevent one case of cervical cancer, 132 to prevent one death, and seven to prevent one DALY; with the nonavalent vaccine, the numbers were 65 to prevent one case, 105 to prevent one death, and six to prevent one DALY . In the The health impact of bivalent or quadrivalent and nonavalent HPV vaccination of 12-year-old girls at 90% coverage during 2020–29 on the averted lifetime burden of cervical cancer in 177 countries for the five comparative scenarios is presented in The lifetime health impact of HPV vaccination of 9-year-old girls during 2020–29 on disease burden averted in the six WHO regions is presented in In comparison with the counterfactual scenario of no vaccination, routine vaccination of 9-year-old girls during 2020–29 would have the greatest health benefits in eSwatini, Zambia, Malawi, Tanzania, Zimbabwe, and Burundi. 72, 63, 56, 54, and 53 cases of cervical cancer would be averted for the bivalent or quadrivalent vaccine and 88, 78, 69, 67, and 66 cases averted for the nonavalent vaccine per 1000 vaccinated girls in eSwatini, Zambia, Malawi, Tanzania, and Zimbabwe, respectively. 58, 47, 45, 44, and 43 deaths would be averted for the bivalent or quadrivalent vaccine and 72, 59, 55, 54, and 54 deaths averted for the nonavalent vaccine per 1000 vaccinated girls in eSwatini, Zambia, Malawi, Burundi, and Zimbabwe, respectively. 1228, 966, 947, 903, and 897 DALYs would be averted for the bivalent or quadrivalent vaccine and 1517, 1194, 1171, 1117, and 1108 DALYs averted for the nonavalent vaccine per 1000 vaccinated girls in Malawi, Burundi, eSwatini, Zambia, and Tanzania, respectively. The countries to potentially gain the largest HPV vaccination impact are the same for bivalent or quadrivalent and nonavalent vaccination of 9-year-old and 12-year-old girls.The countries that will experience the largest health benefits through HPV vaccination are all in the WHO African region. Within the other regions, the countries that will experience the greatest number of DALYs averted through HPV vaccination are the Maldives, Indonesia, and Nepal in the South-East Asia region; Bolivia, Jamaica, and Belize in the region of the Americas; Fiji, the Solomon Islands, and Papua New Guinea in the Western Pacific region; Kyrgyzstan, Romania, and Lithuania in the European region; and Morocco, Somalia, and Djibouti in the Eastern Mediterranean region.In comparison with previous estimates, the updated estimates resulted in increases in the numbers of cervical cancer cases averted per 1000 vaccinated girls in most countries, with the greatest increases in eSwatini, Namibia, Lesotho, Burkina Faso, and Iceland. The greatest increases in the numbers of deaths averted per 1000 vaccinated girls were in Malta, eSwatini, Namibia, Lebanon, and Samoa, and in DALYs averted per 1000 vaccinated girls were in Iceland, Malta, Bahrain, Maldives, and eSwatini. These increases in cases, deaths, and DALYs averted are primarily due to the demography update of UNWPP 2019, which includes increasing life expectancy and population aging among women in these countries, and a relative increase in cervical cancer burden estimates from the GLOBOCAN 2018 database.The updated HPV vaccination impact estimates for cases, deaths, and DALYs averted per 1000 vaccinated girls are lower than the previous estimates for 49, 18, and eight countries, respectively. These decreases are primarily due to lower estimates of cervical cancer burden for these countries in the GLOBOCAN 2018 database compared with the GLOBOCAN 2012 database. The changes in vaccination impact between the different comparative scenarios (S2–5) compared with the base scenario (S1) for vaccination of 9-year-old and 12-year-old girls are provided in We updated PRIME with demography from the UNWPP 2019 revision, disability weights from the GBD 2017 study, and cervical cancer burden from the GLOBOCAN 2018 database. We assessed the effects of these updates on the health impact estimates generated by the model for bivalent or quadrivalent and nonavalent vaccination of 9-year-old and 12-year-old girls at 90% coverage during 2020–29 at the global, regional, and national levels for 177 countries. After these updates, we found that HPV vaccination provides larger health benefits than previously estimated, because the number of girls needed to be vaccinated to prevent a single case, death, or DALY are lower than previous forecasts.We found that the demography update from the WHO 2009 life tables to the UNWPP 2019 revision was the major driver for change in the health impact estimates of PRIME, in comparison with the updates to disability weights or cervical cancer burden estimates. Because UNWPP mortality estimates are declining over time and incorporate population aging (unlike static WHO life tables), using UNWPP 2019 estimates led to an increasing life expectancy among women and a subsequent increase in lifetime risk of cervical cancer incidence and mortality without vaccination. Therefore, HPV vaccination provides greater benefits in preventing new cases and deaths than previously estimated.HPV vaccination could have an even greater impact and be more cost-effective than predicted in this study, because some assumptions in PRIME are conservative and likely to underestimate the impact of vaccination. These conservative assumptions and study limitations are that herd effects are not considered; vaccination is considered to have no effect on women after sexual debut; cervical cancer incidence is assumed to remain constant in the absence of preventive interventions; and cross-protection against non-vaccine HPV genotypes is excluded. Other limitations are exclusion of transmission dynamics of HPV infection and natural history of precancer lesions; exclusion of changes in coverage and effectiveness of non-vaccine interventions ; and exclusion of changes in coverage and effectiveness of treatment and care of cervical cancer cases. The combined impact of vaccine and non-vaccine interventions are significant strategic considerations, and the working draft of the WHO 90–70–90 cervical cancer elimination strategy has set targets of achieving 90% coverage of HPV vaccination; 70% coverage for screening and 90% treatment of precancer lesions; and 90% treatment and care of cervical cancer cases.The structural uncertainty and variability between HPV vaccine impact models warrants a comparison of the models to identify key drivers of the conclusions on the effectiveness and cost-effectiveness of vaccination.Sexual debut data by age for 67 countries are included in this study and future directions include estimation for other countries. Although we have not explored the impact of changes in sexual behaviour, countries that are transitioning from traditional age-specific sexual behaviour to gender-similar age-specific sexual behaviour, with increases in the average number of partners and the number of partners women have before marriage, are likely to result in increased prevalence of HPV infection and incidence of cervical cancer.PRIME updates for demography, disability weights, and cervical cancer burden improve the fidelity of the projections of HPV vaccination impact at the global, regional, and national levels, and provide essential information for decision making in the era of achieving cervical cancer elimination. Given that cervical cancer elimination is likely to take many decades, HPV vaccine impact models must be continually updated to reflect new knowledge about parameter estimates and disease burden.


Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors , its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia , as well as rare cutaneous non-melanoma associated cancer. Human papillomaviruses (HPVs) are double-stranded circular DNA epitheliotropic tumor viruses that are causally associated with all cervical cancers, as well as a significant fraction of several other human malignancies arising from the mucosal squamous epithelia of the anogenital tract , oral, and oropharyngeal cavity  and, infrequently, from the cutaneous epithelium (skin) ,2,3. AccRecent findings indicate that about 60%–70% of the oropharyngeal cancers may be linked to HPV, although traditionally they were considered to be caused by tobacco and alcohol, or by a combination of these ,6. BasedAlthough HPV infection is usually solved by the immune system and the vast majority of the virus infections are transient and asymptomatic, persistent HPV infections have an increased chance to induce epithelial cell abnormalities that can ultimately cause cancer ,15. HR-HIn developing countries, cervical cancer is still the second leading cause of cancer death, while in the western world the number of deaths continues to decrease thanks above all to the introduction of an early diagnostic examination . One of The first models produced for investigating the HPV replication mechanisms were developed by transplanting infected foreskin explants into immune-compromised mice with the aim to propagate virion stocks from HR-HPVs (HPV16 and HPV18) ,46. HoweIt is now established that the stromal microenvironment contributes to tumorigenesis in HPV-related cervical cancer and that the TME sends signals that guide growth, tumor progression, and the formation of metastases, as well as resistance to anticancer drugs ,86. CAFsHead and Neck cancers (HNCs) represent a heterogeneous group of tumors that include cancer of squamous epithelial cells of the oral cavity (mouth and throat), nasal cavity (sinuses), larynx and pharynx, salivary glands and lymph nodes in the neck . ConnectTonsillar carcinomas are among the most frequent HNCs, arising in the reticulated epithelial cells of the crypts with immune cell infiltrations. HR-HPV16 persistent infections are related to oropharyngeal carcinogenesis . HoweverTo reproduce in vitro anogenital HPV-associated cancers in males and females, the main models developed are the 3D organotypic culture that allows to accurately mimic the life cycle of several cancer-associated HR-HPV types. In the previous sections, we focused on the culture system used to study HPV-related cervical cancer; here, we revised the in vitro model mimicking other organs of the human anogenital apparatus, both in male and female.Lower genital cancers are frequent malignancies in women prevalently associated with HR-HPVs infection. To date, some studies have reported that vulvar squamous cell carcinoma (VSCC) have different pathological pathways related, or not, to HPV infections. HPV-related VSCC with profound cellular atypia and basaloid/warty histology and VSCC non-dependent to HPV infection with basal atypia and keratinized histology ,137,138.Anal cancer is closely related to high-risk HPV persistent infection that occurs in the anal transformation zone, similar to what happens in the uterine cervix . HPV infFor several years, the foreskin cell line derived from circumcision has been used to study various HPV-related cancer due to the high availability of this skin fold. The penile urethra, that consists of pluri-stratified squamous cells without keratinization process alike the foreskin or the glans, is also routinely targeted by sexually transmitted viral pathogens such as HIV infection in the mouse model . LimitedAmong cutaneous malignancies, the non-melanoma skin cancer (NMSC) involves fair-skinned populations and are mainly correlated to solar ultraviolet irradiation . HoweverIn this review, we outlined the current knowledge on the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex 3D organotypic models focusing on HPV-associated cervical cancer disease platforms and we also reviewed the in vitro culture systems of human HPV-associated mucosal malignancies from the anogenital tract, oropharynx, and cutaneous epithelium. We highlighted the importance of using multicellular models, which involve the use of compartment-specific cell types at both the epithelial and stromal level, and a complex ECM capable of remodeling to fully reproduce the histomorphological features of the tissue in vivo. This review also addresses the issue of the cross-talk between the stroma and its microenvironment on HPV-infected epithelial, emphasizing the need for most promising in vitro models to study host-pathogen, as well as HPV-infected-TME interactions in cancer development in humans.


In 2013, recurrent reports of diverse symptoms occurring in girls after receiving HPV vaccination appeared in Japanese media. The Ministry of Health, Labor and Welfare quickly responded by announcing a temporary suspension of its recommendation for the vaccine. The HPV vaccination rate soon fell to almost zero. In the present study, we calculated the potential future numbers of cervical cancer incidence and death that will be increased by this policy decision. We have assumed that the number of yearly vaccinations is evenly distributed across a daily basis. Future incidence and death increased in females born in FY2000 are estimated to be 3651 and 904, respectively, 4566 and 1130 for those born in FY2001, 4645 and 1150 for those born in FY2002, and 4657 and 1153 for those born in FY2003. In FY2020, the large increase of risks to females born in FY2004 amounts to 12.0 females per day who will now be at a higher risk for acquiring of cervical cancer in their future, and 3.0 females per day newly at risk for future death from that disease in its progressive form. No one should be able to accept this situation. We sincerely ask the government to resume its recommendation for the vaccine as soon as possible. In January of 2019, a global strategy for doing just that was announced at the 144th Session of the WHO Executive Board2. They listed the known barriers to the worldwide elimination of cervical cancer, such as a Human Papilloma Virus (HPV) vaccine shortage, price , vaccine hesitancy, etc.In May of 2018, the Director-General of the World Health Organization (WHO) announced a global call-to-action towards the elimination of cervical cancer5. By April of 2013, HPV vaccination had become a ‘national routine immunization’, meaning that thereafter females aged 12–16 could receive the vaccine for free.In Japan, a stalled policy decision, initiated in 2013 by the Ministry of Health, Labor and Welfare (MHLW) to suspend its governmental recommendation for HPV vaccination, has become a major health issue. A brief history of the current situation for HPV vaccination in Japan shows that public subsidies for the Human Papilloma Virus (HPV) vaccine started in Fiscal Year (FY) 2010, when females aged 13–16 could be immunized with only a small out-of-pocket fee. The HPV vaccination rate soared to nearly 70% in targeted females5. This has led to immunization rates in Japanese females that stretch from near 70% to nearly 0%, depending completely on a girl’s FY of birth.With the success of such massive numbers of immunizations, problems were sure to evolve. There began to appear repeated media reports of a diverse group of symptoms occurring in young girls after their HPV vaccination. In an understandable precautionary response, the MHLW announced a temporary suspension of its recommendation for the vaccine in June of the same year. The HPV vaccination rate in Japan soon fell to almost zero, where it has since languishedFemales born in Japan in 1993 or earlier were not vaccinated for HPV, as the vaccine had not yet been introduced, and public subsidies did not begin until FY2010, by which time even the youngest of them were already 17. Females born from 1994 to 1999 were immunized under public subsidies for the vaccine, and achieved a 70% vaccination coverage rate for this cohort. Following the MHLW’s negative policy change in 2013, the vaccination rate reverted to nearly zero for girls born in 2000 and thereafter, as they refused to be vaccinated under the cloud of suspicion surrounding its adverse events.7. For example, because the recommendation of HPV vaccine was not restarted at the end of FY2019, those females born in 2000, 2001, 2002 and 2003 are now over the targeted ages of 12–16. It is not realistic to think that these unvaccinated girls will never experience sexual intercourse. We can say that the risk for these females is ‘fixed in place’. If the resumption of the recommendation is delayed for one more year, the relative risk of future HPV-16/18 infection in females born in FY2004 in Japan will be at least like the risks that existed before the HPV vaccine was first introduced.We have previously calculated the relative risk of infection with HPV-16/18 at age 20, and the resulting lifetime incidence of HPV-caused cervical cancer, and we predicted these risks would differ greatly in Japan, depending on a girl’s FY of birth—because of the lapse of protective vaccinations that has occurred since June of 2013Here, we have estimated the future daily numbers of cervical cancer incidence and death increased by the stalled policy decision of the suspension of the governmental recommendation for the HPV vaccine.8 to reflect the real cumulative vaccination rate for each birth FY9. In summary, the cumulative initial vaccination rate for each birth year: Born in FY1994: 55.5%, Born in FY1995: 73.5%, Born in FY1996: 78.2%, Born in FY1997: 78.8%, Born in FY1998: 78.7%, Born in FY1999: 68.9%, born in FY2000: 14.3%, born in FY2001: 1.6%, born in FY2002: 0.4%, born in FY2003: 0.2%, born in FY2004: 0.1%, born in FY2005: 0.0%. Notably, the vaccination rate for females born in FY2000 has been corrected from 42.9% to 14.3%.The data used in the present study was modified from those shown in the MHLW council meeting7, modified as follows: (1) One vaccination shot is considered to be sufficiently effective for preventing HPV infection (this decision was needed because in most cases only data for the first inoculation could be obtained). (2) Lifetime incidence risk is proportional to lifetime HPV infection risk. (3) Cervical cancer is caused predominantly by HPV infection, and of them, the proportion caused by high-risk HPV-16/18 sub-type infections (targeted by the most common vaccine) is 60%. HPV-16/18 sub-types and other types of mixed infection are prevented by the vaccine. (4) Even when vaccination is given, the incidence of cervical cancer caused by HPV other than HPV-16/18 will not change. (5) The population per birth year does not change significantly in Japan. (6) Unless the vaccine is present, the incidence of cervical cancer does not change. (7) The presence or absence of vaccination and sexual activity are independent of each other, and sexual activity does not increase significantly following vaccination (unpublished findings). (8) The number of yearly vaccinations is evenly distributed across a daily basis. (9) A female who receives vaccination and experiences sexual intercourse in the same year is vaccinated before having that sexual intercourse. (10) HPV infection risk is calculated based on the rate of having sexual intercourse without HPV vaccination at every age, and over a lifetime. (11) Sexual activity and cervical cancer prevention behaviors of the females who are over the target age without vaccination don't change. (12) In the case of when a governmental recommendation is resumed, within the resumed year the vaccination rate for each birth year will recover to 68.93%, the same as the vaccination rate for those born in FY1999. (13) The rate of experiencing sexual intercourse at the age of 12, 13, 14, 15, 16 and 17 is assumed to be 0%, 1%, 2%, 5%, 15%, and 25%, respectively. The rate of experiencing sexual intercourse throughout a lifetime is assumed to be 85%10.The relative risk of cervical cancer incidence was calculated using conditions from our previous predictions11.We have also assumed that the number of people suffering from cervical cancer and the number of deaths will remain unchanged by any conditions other than the vaccination rate. The increased numbers of future incidences of cervical cancer and death, in each birth FY, compared to those of females born in FY1999, most of whom received HPV vaccination with public subsidies before the suspension of the recommendation, were calculated by multiplying the increased relative risk, compared to the risk for females born in 1999, by the number of affected females, 11,293, in the latest statistics for FY2014, and the number of deaths, 2795, in the latest statistics for FY2017All methods were analysis of statistical data not obtained from human participants (including the use of tissue samples), and informed consent was not required. The study was performed in accordance with the relevant guidelines and regulations. All the data used in the present analysis were publicly available or derived from previous publications shown above.This study was approved by the Institutional Review Board and Ethics Committee of the Osaka University Medical Hospital.Theoretically, if the suspension of the governmental recommendation hadn't been implemented, and if the vaccination rate of females born in FY1999 had continued, at the same rate as then, with all females born thereafter, then the relative risk of cervical cancer incidence and death for each birth FY would have remained flat, at 0.59  cases and around 600–700 (5000–5700 /8) deaths in each birth year. These numbers were similar to, but a little lower than the predicted in our study. One of the reasons is the difference in models used. They conducted a precise prediction by taking various factors into account using ‘Policy1-Cervix’, an extensively validated dynamic model of HPV transmission, vaccination, type-specific natural history, cancer survival, screening, diagnosis, and treatment. However, our estimation was a simple one. We just predicted the cumulative risk of cervical cancer using certain conditions that lifetime incidence risk is proportional to lifetime HPV infection risk and HPV infection risk is calculated based on the rate of having sexual intercourse without HPV vaccination.Recently, Simms K et al. reported the impact of HPV vaccine hesitancy on cervical cancer in JapanAnother possible reason is that we used the corrected vaccination rate in the females born in FY2000. The rate shown in the MHLW council was 42.9%, however, the rates was incorrectly calculated. We used the corrected rate, 14.2%, in our present study, and the risk of future cervical cancer incidence and death in those born in FY2000 was estimated to be higher than their estimation.These unvaccinated females have now reached the age where sexual intercourse, and the risk of HPV exposure, naturally begins. Their future of acquiring and dying from HPV-caused cervical cancer is now a predetermined risk for them—unless they refrain from all sexual intercourse to avoid infection and cervical cancer – which, we can all agree on, is unrealistic. From this viewpoint, their future for incidence and death from HPV-related diseases is being predestined on a daily basis.Who will take responsibility then for this tangible harm? We sincerely ask the government not only to resume its recommendation for the vaccine as soon as possible, but also to provide an easily accessible and highly affordable opportunity for immunization to those females who already missed getting immunized during the vaccine recommendation hiatus. Cervical cancer screenings should be also be more strongly recommended for these females, as they are at much higher risk. The nine-valent vaccine, which has not yet been introduced in Japan, and immunization for young boys, should both be strongly considered. With discontinuation of recommendations, it is important to keep in mind that the future risk of cervical cancer is rising every day in individual females. No one should be able to accept this situation.Supplementary file1


Particularly, in men with lesions and risky sexual contact was significant only for low impact . In partners with positive females was significant for low impact (26.3% > 15.1%) and high impact (33.7% > 23.2%). Nonavalent vaccine offers broader protection in men with HPV positive partners, who would have a potential role in the transmission of the infection.Human papilloma virus infection (HPV) is the most common sexually transmitted disease. Little is known about male infection. Nonavalent vaccine against types 6/11/16/18/31/33/45/52/58 was approved and neutral gender immunization programs have been proposed. This study evaluates the potential impact of nonavalent vaccine compared to quadrivalent in male living in Sicily . 58.7% of samples were HPV positive and forty-four types of HPV were identified. A significant higher estimated coverage of nonavalent vaccine than quadrivalent was observed (64.3% vs. 45.8%), with absolute and relative additional impact of 20.1% and 47.2%, respectively. Low impact of the vaccine were calculated as the empirical probability of HPV genotypes 6/11/16/18/31/33/45/52/58 alone or in combination; the high impact as empirical probability of HPV6/11/16/18/31/33/45/52/58 genotypes alone or in association with other genotypes. The potential impact of the nonavalent vaccine  There are more than 200 different types of human papillomaviruses, and around 40 that affect the anogenital area2. Most of the time HPV infections are asymptomatic, transient and disappear without treatment in a short time. Some infections, however, can persist for many years, causing cellular changes that, if left untreated, have the potential to develop into cancer of the anogenital area 8 or in benign proliferative lesions, warts and anogenital papillomas, oral papillomas, and forms of recurrent respiratory papillomatosis in men and women12. In contrast to the wealth of information available on the rate of and risk factors for acquisition of HPV infection in women, much less is known about such factors in men. There is great variation in the prevalence depending on anatomical sites sampled, sampling methods, and HPV DNA detection assays15. HPV infection prevalence varies greatly in the men population and reaches 65.4% between 18 and 40 years16. Researchers have documented that 50–77% of steady male partners of women with HPV infection, cervical neoplasia, or both have subclinical HPV infection18, in addition to being an indirect causal factor for cervical cancer via men serving as reservoirs for HPV transmission. Therefore, monitoring for the presence of various HPV genotypes might be important, especially in those patients with known high risk HPV (hrHPV) type19.Human PapillomaVirus (HPV) infection is the most common sexually transmitted disease in the world20 and a universal immunization program has been proposed22. The quadrivalent vaccine  protects against 4 HPV types: 6, 11, 16, and 18. Gardasil prevents persistent infections and genital diseases caused by these HPV types in females. Moreover, it has been demonstrated a protective effect of quadrivalent among heterosexual males (92% efficacy) and its use was licensed in young males to prevent anogenital condyloma. In 2014 the U.S. Food and Drug Administration has approved the nonavalent vaccine . It is a vaccine given to individuals 9 through 45 years of age to help protect against diseases caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52, 58. Since 2018 all EU/EEA countries has introduced HPV vaccination in their national immunization programs23.To prevent HPV infection, a widespread immunization campaign has been approved in adolescent females around the world. More recently, the implementation of vaccination has been recommended in boys and young men as well24. Capra et al. showed, by high estimate calculations, that the absolute additional impact of the nonavalent HPV vaccine is substantial for both low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL), with a 23.8–32.8% increase in the proportion of cases effectively prevented by the second generation HPV vaccine 25.The switch from a first generation HPV vaccines to a nonavalent vaccine increases the prevention of high grade cervical lesions up to 90% of casesThe aim of this study was to assess HPV genotype distribution among men with and without clinical manifestations, and to evaluate the potential impact of the nonavalent HPV vaccine on HPV infection compared with the previously utilized quadrivalent HPV vaccine, in a male population living in Sicily, Southern Italy.The subject of this study is the male population living in Sicily, Southern Italy. The analysis involved 975 consecutive men who had come to the Virology laboratory at the of the Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties-ProMISE  between January 2015 and December 2019, with a request for HPV testing.The common reasons for requesting an HPV test included men with clinical lesions ; men having a risky sexual contact in the prior 2 months  evaluation);men having an HPV positive partner.Group I: 200 genital samples of men with presence of clinical symptoms;Group II: 309 genital samples of men with a risky sexual contact;Group III: 466 genital samples of men with HPV positive partner.A visual inspection (hand-held lens) was carried out to assess the presence of clinical symptoms such as atypical lesions or warts on the external genitals. After HPV testing and evaluation of clinical symptoms three series of genital samples were selected and examined as follows:All patients who met the following inclusion criteria were enrolled: men with age between 18 and 77 years; men with clinical symptoms; men with risky sexual contact; men positive partner for hrHPV. The exclusion criteria were: varicocele; cryptorchidism; other genital infections; patients treated with different medical therapies (chemo/radiotherapy), patients with clinical characteristics that did not allow exclusive inclusion in a single study group. These patients were excluded from the study.All study subjects provided written informed consent. Study participants authorized the research responsible to use biological specimens collected for scientific purposes. They also expressed their agreement to participate to medical and sexual interviews. The research was conducted in accordance with the Helsinki Declaration; the informed consent and the study protocol were approved by the Institutional Review Board at the Policlinico, University of Palermo . The study did not provide financial compensation for the participants. The authors are grateful to the host institution:University Hospital Polyclinic, who made the fund available PSN2018-4.23, entitled "Vaginal microbiota in physiological and pathological conditions with particular reference to HPV infections". 13, with some modifications. In brief, for penile brushing, cells from the dorsal/ventral surface of the shaft were collected by a standard-sized, dry DACRON swab first, and then by a saline-wetted Dacron swab. Cells from the inner foreskin, coronal sulcus, frenulum and glans were also collected using a saline-wetted cytobrush. Five to six back-and-forth movements of the swab/cytobrush were performed at each penile site. For urethral sampling, a very thin, saline-wetted cytobrush was inserted 1.5 cm into the urethra, rotated 360 degrees, and removed. The urethral cells were placed in a separate vial with 3 ml of phosphate-buffered saline. The samples were processed for DNA extraction immediately following collection.Samples were collected from penis and urethra. For HPV sampling, patients were instructed to avoid washing their genitalia the day before the examination and to observe 2 days of sexual abstinence before the examination. Genital sampling was performed as described previously26.Cells obtained from penile and urethral brushing were spun down at 13,000 rpm for 5 min and total DNA was extracted with the use of the High Pure PCR Template Preparation kit , following manufacturer’s instructions. DNA quality and the absence of inhibitors were confirmed by testing for the human β-globin gene, as described elsewhere. Amplifications were carried out in a DNA thermal cycler  and the PCR products were analyzed in 8% polyacrylamide gel27 and the LiPA type-specific assay identifies thirty-two types. 20 hrHPV , 12 low-risk HPV (lrHPV): HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV62 HPV81, HPV83 and HPV89). Due to the higher number of HPV types detected by the SPF10 primers compared to the LiPA assay, some samples yielded SPF10-positive/LiPA negative results. These HPV types were subsequently amplified by a highly sensitive nested PCR assay, consisting of a first step of amplification with the PGMY09/11 primer pair, followed by the second step with the GP05 + /GP06 + primers, as previously described28. The HPV genotyping procedure was based on the direct sequencing of GP-PCR fragments, utilizing consensus nested primers as sequencing primers, as described elsewhere29.The presence of HPV DNA was detected using two HPV assays. The INNO-LiPA HPV Genotyping Extra II kit , based on the combined use of SPF10 PCR and LiPA hybridization, was employed. The SPF general primers detect at least 43 different HPV genotypes30.To individualize a sample sizes statistically significant for this study, we considered a Bernoulli sampling31.The minimum sample size was estimated equal to 350 patients HPV-positive. It was obtained considering a statistical z-score at 95%, an error ε = 5% and hypothesizing a prevalence π of 35.4%, obtained from penile and urethral samples, including patients with presence of clinical symptoms,with a risky sexual contact, and with HPV positive partner, according to Barzon, in this way we estimated a prevalence range equal to 30.4–40.4%Consequently, the minimum sample size for each group considered in this study was estimated equal to 117 patients. Finally, the sample size for each group was enlarged considering the possibility of unexpected events and consequently the possibility of patients data loss, and to minimize possible statistical biases. Therefore, we consider Group I, Group II and Group III composed by 138, 137 and 297 patients HPV-positive.Data are presented as number and percentage for categorical variables, and continuous data expressed as the mean ± standard deviation (SD) unless otherwise specified. In order to compare the impact of nonavalent HPV vaccine with the quadrivalent vaccine we used the following statistical tests.2 test was performed to test the differences between two unpaired proportions or percentages. The McNemar’s exact test was used to test the difference between two paired proportions or percentages.The χMultiple chi-square test was used to define significant differences among independent groups. In this case, if the chi-square test was significant (p < 0.05), a post hoc Z-test was performed to identify the highest or lowest significant frequency. In addition tests on continuous data were performed with one-way ANOVA test to evaluate significant differences among independent groups. If the ANOVA test was positive, post hoc test with Scheffé's method was performed for pairwise comparison of subgroups.24, particularly, we considered the empirical probability evaluated on positive cases only. In this way, we define low impact, as the empirical probability of HPV genotypes , alone or in association, by excluding the presence of any other HPV type; while with high impact, the empirical probability of HPV genotypes  alone or in association, also in the presence of any other HPV type. These parameters have been defined in order to be able to optimally compare the effectiveness of the two vaccines.. Furthermore, sample size estimation was performed to ensure an adequate group size to obtain a statistically significant and robust study.About vaccine impact, we used similar parameters adopted by Riethmuller et al.nnonavalent-nquadrivalent)/N] × 100, with n being the number of lesions/infections potentially prevented and N the total number of lesions/infections. Instead, the relative additional potential impact of the nonavalent vaccine compared to the quadrivalent vaccine was calculated as [/nquadrivalent] × 100 with n representing the number of potentially prevented lesions/infections.24.In addition, we computed both absolute and relative additional potential impact of the nonavalent vaccine compared to the quadrivalent vaccine. Particularly the absolute additional potential impact of the nonavalent vaccine, i.e., the proportion of additional cases potentially prevented by the nonavalent vaccine compared to the quadrivalent vaccine, was calculated as [ < 0.05 were considered significant. The statistical analysis was performed using the Matrix Laboratory (MATLAB) analytical toolbox version 2008 . for Windows at 32 bit.Of 975 genital samples β-globin-positive 572 (58.7%) samples tested were HPV positive, characterized by mean ± SD age equal to 36.0 ± 10.11 (range 19–77 years).In Table Forty-four different HPV types were identified, of which 20 were hrHPV types and 24 lrHPV. The genotype most frequently identified was HPV6 (31.9% of HPV positive patients), HPV16 (20.3%) and HPV66 (14.5%) in Group I; HPV16 (22.6%) HPV51 (17.5%) and HPV53 (15.3%) in Group II, and HPV16 (21.2%), HPV51 (18.5%) and HPV31 (16.8%) in Group III. The percentage of identified genotypes in the three groups were reported in Figs. As expected, about hrHPV, we found a significant relationship with groups (p = 0.0015). Particularly, in Group I was significant less frequent  and lrHPV was significant more frequent .Multiple HPV type infections were shown in 51.2% (293/572) of the samples: 45.6% (63/138) of the Group I, 53.3% (73/137) of the Group II, and 52.9% (157/297) of the Group III, respectively. Oncogenic types were found in 79.4% (454/572); in particular, in 69.6% (96/138) of the Group I, in 78.1% (107/137) of the Group II and in 84.5% (251/297) in the Group III. Prevalence of oncogenic types, and the distribution of single and multiple infections in the different categories of men are shown in Tables From Table .In addition, for low impact, Absolute Additional Impact (AAI), and Relative Additional Impact (RAI) a significant relationship with groups was found . Particularly, the Group I was significant more frequent for low impact , while was less frequent for AAI and RAI . Finally, the Group III was more frequent only for RAI , as reported in Table 2vs. 38.1%, p < 0.0001).In Table Finally, Table On the whole sample of 572 positive males, the potential impact of the quadrivalent and nonavalent vaccine varied significantly for the low impact between 19.8% and 29.7% (p < 0.0001) and for high impact between 26.6% and34.6% (p < 0.0001).vs the quadrivalent vaccine was only effective for the low impact 35.5% > 29.7 (p = 0.0078) for Group I; and 31.4% > 19.7% (p < 0.0001) for Group II. In the first Group the absolute and relative additional impact of the nonavalent vs quadrivalent vaccine was 10.1% and 17.5%; in the second Group the absolute and relative additional impact of the nonavalent vs quadrivalent vaccine was 18.2% and 35.7%, respectively, Tables With regards to the sample of men with lesions and with STD evaluation (Group I and II), the potential impact of the nonavalent vaccine With concern to 297 partners of positive females (Group III), the potential impact of the nonavalent vaccine against quadrivalent vaccine was effective for both the low impact 26.3% > 15.1% (p < 0.0001) and the high impact 33.7% > 23.2% (p < 0.0001). The benefit of the nonavalent vaccine compared to quadrivalent vaccine, as shown by the absolute and relative additional potential impact, was 21.5% and 56.1%, respectively.32.The current study provides an important overview on HPV-DNA prevalence in men with and without clinical manifestations and on the evaluation of the potential impact that nonavalent HPV vaccine has on HPV infection compared to the previously used quadrivalent HPV vaccine. The 58.7% of the samples tested were positive for HPV, with significant differences depending by considered group. In fact, 69% of men with clinical symptoms, 44.3% of those with a risky sexual contact (STD), and 63.7% of men with positive partners, were positive results to the HPV infection. HPV infection is very common in men, with variations reflecting the selection criteria of the studied populations and sampling methods used33.The prevailing goal is to try to understand the relationship between HPV infection and disease in men, including the development of genital warts, penile intraepithelial neoplasia and invasive penile carcinomas. Genital warts are caused by HPV6,11 and are the most common clinical manifestation of HPV in men. Although they are benign and not associated with mortality, they are a source of psychosocial and physical discomfortFrom the statistical analysis of the data it is highlighted that in group II, of subjects with sexual contacts at risk, a higher age was observed, suggesting once again to extend the vaccination also to adult men. This vaccination policy, in Sicily, is also applied to women. The vaccine is free for women up to 45 years of age.Variations emerged also regarding the most frequent genotypes in the three groups, confirming how the selection of the target population is important in epidemiological assessments.34, while the another one found strong associations between high grade squamous intraepithelial lesions and the number of HPV-types35. Furthermore, in a case–control study, women with cervical intraepithelial neoplasia 1 or worse were more likely to have more HPV types than women with normal cytology36. The role of multiple HPV infection in male health is not yet clear37.In our study, multiple HPV-type infections were shown in 51.2% without significant differences between the groups. Little is known about the prevalence of multiple infection HPV and associated factors in men. In women, two cohort studies showed contradictory results; one did not find any association between the number of HPV types and the cytological changes shown38 and have been associated with a longer duration of infection39. In general, HPV infection is complicated by the existence of multiple infections; therefore, the potential benefit of the HPV vaccine is not easily assessable.Infections with multiple genotypes seem to favour the persistence of HPVOncogenic genotypes have been found in almost 80% of the men included in our study. The prevalence of hrHPV in the three different groups was statistically significant. In 84.5% of partner men of HPV-positive women, hrHPV was shown, suggesting a possible role of man in the transmission of the infection. Furthermore, high and significant prevalence was observed also in men with risky intercourse, again suggesting the need to make vaccination offerings as gender-neutral as possible.40. This occurs in these countries due to the immunocompromised caused by HIV infection41. In contrast, in women a decrease in dysplasia has been observed thanks to screening programs but overall thanks to vaccination40.We should also note that no screening tests are currently available for men, and that persistent penile HPV infections have been shown to be associated with penile intraepithelial neoplasia (PIN) development in younger men. These generally resolve within 2 years; however, a small minority of cases can progress and cause invasive cancer. Penile cancer in the United States has an incidence rate of around 1/100,000, while the incidence in some developing countries, such as Uganda, can be much higher 24, but we consider positive HPV patients only, to evaluate the effectiveness of the two vaccine.Impact of the vaccine was measured according to low and high impact parameters and defined in statistical analysis section. These parameters were similar to parameters adopted by Riethmuller et al.25.On total HPV positive sample we observed that nonavalent vaccine had a greater impact than the quadrivalent vaccine. In fact, the absolute additional impact of the nonavalent vaccine compared to the quadrivalent was of the 20.1%, while the relative additional potential impact was 47.2% , while the nonavalent is suitable for all three groups, confirming that the transition from quadrivalent to nonavalent is an excellent prevention strategy . This is probably due to the fact that the nonavalent vaccine contains, in addition to HPV16 and HPV18, five other high-risk genotypes, which together are responsible for about 90% of cancers worldwideAs for the partners of positive women, the nonavalent vaccine had a significant impact for both the low and high impact . With regard to precancerous lesions, it is estimated that over 1000 cases of AIN2/3 are diagnosed in men each year44.Head and neck cancers also constitute a heavy burden, particularly in men, with an estimated 11,000 cases diagnosed annually. Further, increasing trends in the incidence of HPV-positive head and neck cancers have been consistently observed in the last decade in concomitance with the decline in tobacco use45.There is evidence to suggest that where there is a high level of vaccination coverage in women, males are indirectly protected from infection. In Australian heterosexual men under the age of 25, the prevalence of HPV16,18, 6,11 decreased by 78% from the pre-vaccination period46. For reasons of fairness, some believe it is preferable for both males and females to have access to the same vaccination opportunities47. In conclusion, the present study on a male population in Sicily  confirms that the transition from quadrivalent to nonavalent HPV vaccine offers much wider protection even in men, although with some differences between the analysed groups. The nonavalent vaccine respect to quadrivalent is to be preferred especially in men with positive HPV partners, who would have a potential role in the transmission of the infection, and in the eventual relapse after treatment. However, it should also be emphasized, that a significant reduction in HPV related infections, both in men and women, can be achieved only if the vaccination coverage reaches and exceeds 80%48.In the context of female-only vaccination, the indirect advantage of herd protection among men who have sex with men are limited49.Adequate vaccination schedules could therefore become an economically viable approach to prevent public healthThe implementation of vaccination programs and gender-neutral vaccination represent the main way to go in the prevention of HPV related diseases in both men and women.


Further studies are needed to confirm the hypothesis that beta HPVs are transmitted to breastfeeding infants through shedding of viruses in the breast milk or on the external breast epithelium.Vertical transmission of human papillomaviruses (HPVs) from mother to infant is known to occur during labor, delivery or breastfeeding. Infection with mucosal HPV 6 and 11 may cause recurrent respiratory papillomatosis in children, which is a rare and severe respiratory disease. The cutaneous HPV genotypes have also been described to be transmitted from mother to newborn through skin-to-skin contacts and during breastfeeding. To investigate the perinatal transmission of alpha and beta HPVs we collected nasopharyngeal specimens from 0–12-months-old infants born by vaginal delivery and breastfed at the time of sample collection. The mucosal and cutaneous HPVs were searched by nested PCR using the MY09/11-MGPs and CP65/70-CP66/69 primer sets, respectively, and genotypes identified by direct sequencing analysis. Fourteen out of 113 (12.4%) samples tested positive for HPV and sequence analysis allowed us to identify eight beta genotypes . Moreover, we performed a comprehensive review of published studies on the prevalence of mucosal and cutaneous HPVs among 5126 newborns and observed that 10% and 53% were positive for alpha and beta HPVs, respectively. In all studies there was an inverse correlation between the rate of alpha HPV positivity and age, while a significant positive trend was observed in beta HPV detection and age with the highest rate among children older than 12 months (Χ More than 200 human papillomaviruses (HPVs) have been fully characterized and new genotypes are often identified in metagenomic libraries obtained by the application of highly sensitive techniques ,2. DepenHPVs have a circular double-strand DNA genome of approximately 8000 base pairs that is divided into three major regions: (1) the “upstream regulatory region” (URR), also named “long control region” (LCR), which contains the enhancer and other regulatory elements; (2) the early gene region containing the E1–E8 Open Reading Frames (ORFs) that encode proteins involved in viral genome maintenance and replication; and (3) the late gene region that contains the L1 and L2 ORFs expressing the major and minor capsid proteins, respectively. The transforming ability of high risk alpha HPVs is mainly due to the constitutive expression of E5, E6, and E7 oncoproteins that interact with multiple cell factors, such as p53 and pRb oncosuppressors, thus causing uncontrolled cell replication . The rolTransmission of HPVs from mother to child has been reported by several studies. The virus may be transferred during pregnancy, through transplacental or perinatal transmission, or by nursing after delivery ,12,13. IIt has been suggested that HPV can be transferred vertically by breastfeeding . De VillThe studies on HPV vertical transmission are still controversial and scant. Our study aimed to analyze the mucosal and cutaneous HPV prevalence in nasopharyngeal samples obtained from children up to 1 year old who were born by vaginal delivery and breastfed. We also performed a meta-analysis of published studies to investigate the overall prevalence of alpha and beta HPV infection in nasopharynx of the children. Nasopharyngeal aspirates were collected from 113 breastfeeding infants, aged between 12 days and 12 months of life, seeking care for respiratory problems at the Santobono hospital in Naples from September 2018 to January 2019. All children included in the study were born by vaginal delivery. The study was designed as a retrospective analysis, it is in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Scientific Board of the Istituto Nazionale Tumori IRCCS “Fondazione G. Pascale”.Genomic DNA was extracted from nasopharyngeal specimens by digestion with proteinase K (150 μg per mL at 37 °C for 30 min) in 100 μL of lysis buffer , followed by extraction with phenol–chloroform–isoamyl alcohol (25:24:1) and ethanol precipitation in 0.3 M sodium acetate (pH 4.6). The quality and quantity of isolated nucleic acid was spectrophotometrically assessed with Nanodrop 2000C . All samples with a ratio of absorbance at 260 and 280 nm equal or above 1.8 were included for further analyses.Epidermodysplasia Verruciformis (EV)-related HPV types . HP. HP27]. PV types . A negatPublished articles whose title, abstract or keywords referred to the detection of HPV DNA in newborns were searched in Medline using the terms (“Human Papillomavirus” OR “HPV”) AND (“infants” OR “newborns”) AND (“transmission”) . The seaThe criteria for inclusion of the articles in the present meta-analysis were as follows: (1) explicitly provided information on HPV DNA detection method, such as PCR-based methods  or by non-PCR methods ; (2) necessary data could be directly extracted or calculated from the original article. Articles containing duplicated data were integrated and review articles, studies in languages other than English as well as articles with low sensitive detection methods (before the year 2000) were excluded.For each article included in the meta-analysis, sample types, percentages of HPV-positive samples, detected HPV types, delivery type and HPV detection methods were reported. All the data regarding newborns, their mothers and fathers (if available) were tabulated separately in a Microsoft Excel sheet .2-test for trend was used for comparison of categorical data. Differences were considered statistically significant when p values were less than 0.05.The statistical analyses were performed using GraphPad Prism (version 6) software. The ΧOverall, HPV DNA sequences were detected in 14 out of 113 (12.4%) nasopharyngeal samples . Beta HPp < 0.001).A comprehensive analysis of published studies allowed us to evaluate HPV prevalence among 5126 newborns at different times from delivery. Specifically, alpha HPVs have been detected in 8.5% (282/3298) of samples obtained from infants at birth (T0), in 15.7% (108/689) of the 1 day–1 month old group (T1), in 12.7% (90/708) of >1 month–12 months old group (T2) and in 9.1% (7/77) in the group of >12 months old infants (T3). Beta HPVs have been detected in 11.1% (2/18) of samples from infants at birth (T0), in 66.7% (24/36) of the 1 day–1 month old group (T1), in 51.4% (18/35) of >1 month–12 months old group (T2) and in 63.6% (21/33) of infants >12 months old (T3), , Figure. StatistHPV infection is known to have a sexual transmission, but other modalities of transmission have been described ,32. SeveIn our study we selected breastfed infants born by vaginal delivery that sought medical care for respiratory illness and underwent a nasopharyngeal aspirate. Ten out of 113 infants were diagnosed with respiratory syncytial virus (RSV) by RT-PCR. The nasopharyngeal samples were also analyzed by using a broad-spectrum PCR designed for the detection of mucosal and cutaneous HPVs. None of the nasopharyngeal samples contained alpha HPV DNA sequences. Conversely, the rate of cutaneous HPV detection showed an age-dependent positive trend ranging from 5.3% to 16.9% in infants less than or more than 2 months old, respectively, and no association with RSV infection. We observed a decreasing rate of HPV positivity in the group of children older than 6 months (10%). The reason for such a decline may be due to multiple factors including the ability to contain infections following the maturation of children’s immune system or changes in the gut microbiota at weaning preventing pathogens growth.Cutaneous HPVs all belonging to the beta genus included HPV 5b, HPV 20, HPV 25, HPV 100, HPV 107, HPV 124, RTRX7 and HPV 152. Particularly, the HPV 5b, HPV 20 and HPV 25 of the species beta 1 HPVs are possibly carcinogenic and frequently detected in cutaneous lesions ,4,26,34.It was reported that beta HPVs colonize the individual immediately after birth  and therThe meta-analysis showed that detection of beta HPVs is less frequent at the birth and that it increases over the time >12 months), as shown in  months, Various factors may contribute to the risk of HPV transmission, such as the presence of cervical warts in the mother  , maternaA limitation of the present study is represented by the relatively small sample size. However, the use of broad-spectrum PCR and direct sequence analyses allowed us to identify uncommon HPV genotypes partially different from those described in the literature until now. Since the samples were collected quite far from birth, we may suppose that infection has occurred postnatally. Hence, the prevalence of HPV 6 and 11 is shown to be strongly predictive of a JoRRP and the presence of condylomata acuminata in women, caused by HPV 6 and 11, is one of the factors involved in the onset of RRP in the infants; we wonder about the role of the uncommon viral genotypes  isolated in our study. So, we ask if they could cause some type of disease either in younger or in adult individuals. Recently, it was shown that the presence of some beta HPV types in the oral cavity is associated with an increased risk to develop head and neck cancer . It is o