Studies on HPV prevalence in the head and neck region of South Africans are sparse. Of the available reports in the literature, there were no studies on the association between HPV-DNA presence in the mouth and oropharynx in relation to high-risk behaviours such as oral sex practice or tobacco and alcohol use.  Following ethical clearance and informed consent, patients attending a regional HIV-management clinic and patients attending a dental hospital were recruited to this study. The participants completed an interview-based questionnaire obtaining demographic information, data on HIV serostatus, and behavioural data including sexual practices and tobacco and alcohol use, and a rinse-and-gargle specimen was taken. Specimens were analysed for HPV DNA on 3 separate PCR/qPCR platforms. Statistical analyses were performed for associations between the study group and categorical variables, HPV status, and data from the questionnaires. α-HPV DNA, and 1 (0.4%) had detectable β-HPV DNA in their rinse-and-gargle specimens. Two participants were from the HIV-management clinic and 6 from the general dental population .  Of 221 participants, 149 were from a general population and 72 from the HIV-management clinic. Smokers comprised 29.4% of the sample, and 45.2% of participants reported to have ever used alcohol. Open mouth kissing during teenage years was confirmed by 64.7% of participants, 40.3% have given oral sex with their mouth, and 44.8% confirmed to have received oral sex from their partner's mouth. Seven participants (3.2%) had detectable β-HPV types were detected. The low prevalence of 3.6% compares well to similar studies in different cohorts studied in South Africa and falls within the global oral/oropharyngeal prevalence spectrum. Only 4 participants, all from the HIV-management clinic, had palatine tonsils. No significant relationships were found between HPV presence and demographic data or sexual, oral sexual, tobacco use, or alcohol use, and no associations were seen with numbers of sexual and oral-sex partners. Five high-risk HPV, 2 low-risk HPV, and one  The human papillomavirus (HPV) is epitheliotropic and requires access to the basal cells of epithelium to initiate a complex sequence of events that additionally relies on specific host reactions and interactions to successfully infect the basal keratinocyte . MucosalTaking into consideration the high HIV prevalence in South Africa and the bidirectional risks in acquisition of either infection, HIV or HPV, in cases where the one precedes the other, the importance of providing prevalence data on oral and oropharyngeal HPV infection becomes urgent , 4. The The practice of oral sex (OS), open mouth kissing, having multiple sexual partners, and a compromised immune system increases the risk of acquiring oral/oropharyngeal HPV infection in general population groups –12. The Smoking has been shown to influence HPV clearance from the mouth, and in cases of HPV-positive SCC, prognosis is worsened with concomitant smoking , 14.This study describes the prevalence of HPV-DNA detected in the mouths and oropharynges of a dental clinic population and of an HIV-seropositive clinic population. High-risk sexual behaviours and habits that also include tobacco and alcohol use are reported.Patients who attended the University oral health centre and the HIV management clinic were recruited as a convenience sample. The dental patients attended the institution for treatment of oral health-related issues that were unrelated to HPV infection, and the patients attending the HIV clinic came for follow-up and treatment management visits, which were also unrelated to the study.An interview-based questionnaire was completed prior to oral/oropharyngeal specimen collection. The questionnaire was developed from a previous project  and exisParticipants rinsed for 15 seconds and then gargled for 15 seconds with 10 ml of saline and then spit the contents into a Thinprep® vial containing Preservcyt® solution. The specimens were stored at 4°C until they were transported to the laboratory. The material in the vial is fixed by Preservcyt® and can be preserved for 6 months at 15–30°C. For the purposes of this report, oral wash implies sampling of the oral and oropharyngeal mucosae. The vials were numbered with the corresponding questionnaire.⍺-papilloma viruses, and the AML β-human papillomavirus typing assay.All oral washes were tested for the presence of HPV with 3 different assays: Abbott HR-HPV assay and the RIATOL qPCR HPV genotyping assay, both focussing on the presence of m2000™ RealTime system was used for the qualitative HPV detection with the HR-HPV assay. This real-time multiplex PCR detection kit reports only limited HPV DNA typing results: presence of HPV 16, HPV 18, and other HR-HPV . The assay is optimized using probe specificity design and an internal human beta-globin control, which is a sample validity control for cell adequacy, sample extraction, and amplification efficiency. The assay is clinically optimized for cervical cancer screening. This clinically based assay cut-off is also used in this study for the detection of HR-HPV in oral washes.The Abbott μl) was determined in every sample. Samples with a DNA concentration below 0.12 ng/μl are considered as invalid and reported as inconclusive. Type-specific HPV positivity is defined as having a positive amplification signal for that specific HPV type, independently of the beta-globin signal.The Riatol qPCR HPV genotyping assay is an ISO certified, fully automated, clinically validated laboratory-developed test . This qPβ-papillomaviruses with the AML lab-developed assay. The assay involves a TaqMan real-time PCR containing type-specific primers and consensus probes capable of detecting multiple β-HPV types. In total, 5 multiplex consensus reactions are performed: three with double targets detecting HPV types 1/63, 3/10, and 7/41 and two with triple targets detecting HPV types 2/27/57 and 4/60/65.Specimens were also tested for a spectrum of Sample size estimation was based on the estimation of the proportion of HPV-positive patients in each study group, taking the general global and locally reported prevalence rates into consideration. Based on an overestimated HPV prevalence of 20%, 5% precision, and the 95% confidence level, a sample size of 246 would be required for each group .2 test was used to assess the relationship between the study group and categorical variables. Fisher's exact test was used for 2 × 2 tables or where the requirements for the Χ2 test could not be met. The strength of the associations was measured by Cramer's V and by the phi-coefficient, respectively.The Χt-test (or ANOVA for more than two categories). Data analysis was carried out using SAS (version 9.4 for Windows). The 5% significance level was used.The relationship between study groups as well as between age and lifetime sex partners was assessed by the This study received ethical clearance from the Sefako Makgatho Health Sciences University Research and Ethics Committee (SMUREC/P/86/2016). Individuals received an information sheet and provided informed consent prior to participation. Participants had the opportunity to ask questions and not one person was disadvantaged or prejudiced in any way in the event of opting not to participate in the study.A total of 221 participants were enrolled, 149 from the general population attending the dental clinic, and 72 attending the regional HIV-management clinic. The actual sample sizes of 72 (HIV clinic) and 149  used in this study correspond to a precision of 9.2% and 6.5%, respectively (rather than 5.0%), which could be a limitation of the study. For a sample size of 221, this study has the resolution to determine differences between these two datasets. The power value informed the degree of confidence to which our sample size is sufficient in order to see differences between these datasets. A power value of 90% is deemed to be an acceptable level of confidence. Based on these analyses, for a sample size of 221 with a comparison difference of 0.018, the power value is 97.8%. The sample size is, therefore, sufficient to determine any differences between this dataset and global data.Participants' ages ranged from 20 to 74 years with a mean age of 43.8 years. Majority of the participants were black and more than half were female .n = 36) of all participants (29.4%) were current smokers with no significant difference between the clinic groups in the number of cigarettes smoked per day. 54.8% of participants confirmed to have consumed alcohol. Of those who confirmed to have consumed alcohol (n = 121), 36.4% consumed alcohol in the week prior to the study and 33.1% reported consuming alcohol during the four months before the study (16.3% (he study . There wn = 144) confirmed they knew what OS was, and 40.3% (n = 89) reported to have given OS with their mouths and 44.8% (n = 99) reporting to have received OS. Only one of these men tested positive for oral/oropharyngeal HPV and was from the dental clinic group who indicated he did not give nor receive OS from his single OS partner during the past year. There was a significant association between age and number of lifetime OS partners within the dental clinic group  (Majority (64.7%) of the participants had open mouth kissed during their teenage years (15–19 years) with a significant difference between the two groups where more dental clinic participants practiced open mouth kissing at a very early age (10–14 years) . There wp=0.006) . Interesα-papillomavirus) prevalence for the dental clinic population (n = 149) analysed by the Riatol assay only was 1.4% (95% CI: 0.2–4.9%) (n = 2) compared to the 2.8% (95% CI: 0.3–9.8%) (n = 2) for the HIV clinic (n = 72) with no significant difference between these groups. All specimens except 2 had a positive beta-globin control. Only 3 participants from the dental clinic and none from the HIV clinic had HPV-DNA detectable by the Abbott platform. The combined proportion of oral/oropharyngeal α-HPV-positivity for this study (n = 221) was 3.2% (n = 7)  . This coα-HPV-positive participants (n = 7) had tonsils. Not a single participant presented with HPV-associated lesions during clinical examination.Palatine tonsils were absent in 100% of the dental clinic participants group and in 94.4% of the HIV-clinic participants group. None of the α-HPV DNA . No significant associations were found between HPV presence and any demographic factor or high-risk behaviour or with number of sex or of OS partners (supplementary file).-HPV DNA . One parThere are limited data describing the spectrum of HPV genotypes infecting the oral and oropharyngeal mucosae of the general South African population, and there are similarly no reliable data describing the types of HPV present in oral/oropharyngeal HPV-associated benign and malignant lesions of South African populations. Previous studies from South Africa on oral/oropharyngeal HPV infection were further limited by smaller specific cohorts –22. The α-papillomaviruses because of the strong oncogenic association and transduction potential of E6/E7 oncoproteins. However, it is not strange to find HPV from other genera in the mouth and oropharynx and some even associated with benign lesions and more recently, potentially malignant lesions [α-papillomaviruses may have a part to play in the aetiology of head and neck SCC, being the first to report a positive association between infection with β- and/or γ-HPV subtypes. Only one participant from this study had demonstrable β-HPV DNA, and we can only speculate that this was a passenger-infection (background infection) as described in the literature [In general, HPV prevalence studies are centred around the  lesions –26. Howe lesions  suggesteterature , 21.Although this study did not interrogate multiple HPV-types in the individual specimens, it did look at HPV-16, HPV-18, and other HR-HPV types. The finding of multiple types infecting a single individual is not infrequently reported. It is entirely possible for one patient to have two HPV types infecting the same mucosal field and also possible for one patient to have one type affecting more than one mucosal field, e.g., orogenital as evident by numerous concordance studies for different anatomical locations lined by mucosa. No incidental finding of multiple HPV-type infections were made, and the 8 HPV-positive specimens in this study all had single HPV subtypes detected as determined by the three independent assays.Oral and oropharyngeal HPV prevalence studies report prevalence rates of up to 10.0% for patients considered otherwise healthy. Many of these do not present data on HPV genotypes. In the USA, prevalence of oral HPV infection in a group of 14–69 years old participants was 6.9% and higher among men. The study showed a HR-HPV prevalence of 3.7% and a low-risk HPV prevalence of 3.1% . AnotherA Swedish study of oral HPV infection in youths between the ages of 15 and 23 years demonstrated higher HPV prevalence rates for males and females at 9.3% and 9.5%, respectively. However, females with concomitant cervical HPV infection had significantly higher oral HPV prevalence rates (17.1%) when compared to those without cervical HPV infection . South AAn investigation of oral/oropharyngeal HPV presence in 128 South African male factory workers revealed an oral/oropharyngeal HPV prevalence of 5.6% . ChikandThe group led by Mbulawa  proposedHigh-risk sexual practices such as unprotected sexual intercourse, having multiple sexual partners, and the practice of oral sex/anal sex need to be investigated concomitantly with oral/oropharyngeal HPV prevalence because these are classic risk factors for HPV transmission. However, data on oral-sexual practice from South Africa are very limited , and no Global data suggests a much higher oral/oropharyngeal HPV prevalence for HIV-seropositive patients; and HPV infection rates of these mucosal sites are proportional to the level of immune suppression . This stIt is still unknown whether early or later HPV infection plays any significant role in the mouth/oropharynx nor has latency of HPV in humans been established conclusively . HoweverTobacco and alcohol use are considered the traditional risk factors in the development of oral/oropharyngeal squamous cell carcinoma. However, a slow decline in tobacco use globally has also been accompanied by a rise in oropharyngeal cancer incidence . A distiIn a report on trends regarding tobacco use in South Africa, the prevalence of smoking among black South Africans was 22.7% and 36.6% among Caucasians; and the trend reflected a decrease in smoking , 51. AnoHPV infection has a stronger association with tonsillar SCC than SCC of the mouth . The disAn interesting incidental finding from this study's population groups showed that 98.2% of participants had their palatine tonsils removed. None of the oral/oropharyngeal HPV prevalence studies we interrogated included data on the presence or absence of palatine tonsils. It is, therefore, conceivable that the absence of tonsils could play a preventive role in HPV-associated SCC of the head and neck. This needs further investigation.It could be that the oral cavity serves the passenger infection model as a reservoir to inoculate the tonsils and other more distant mucosal sites , 21. Theβ-papillomavirus DNA present in the rinse and gargle specimen.The overall prevalence of oral/oropharyngeal HPV DNA in dental clinic attendees and in attendees of an HIV-management clinic is low in this report. This data compares favourably to other South African papers in different cohort studies and also falls within the expected global prevalence range. One participant presented with Oral/oropharyngeal mucosal infection by HPV is not implied by the detection of any HPV-DNA in these participants. The possibility that these represent passenger infections that serve to inoculate other anatomical sites lined by mucosa must be considered. Single-site coinfection with multiple HPV types may be detected only as a singular HPV positivity using conventional PCR platforms and should be individually identified through sequencing . A signi


Human papillomavirus (HPV) infection rates in women vary regionally. This study analyzed HPV infection in women of different age groups in Hefei, China, performed follow-up on positive cases, and discussed infection prognoses.Samples  of exfoliated cervical cells were collected in Hefei and tested with an HPV assay kit against 27 HPV genotypes. Statistical software was used to analyze the data.P = 0.002), 21–30 and 41–50 (P = 0.0003), 21–30 and 51–60 (P = 0.00003), and 51–60 and >60 age groups (P = 0.046). High-risk infection (15.67%) and single infection (13.01%) were the main types of HPV infection. The dominant genotypes of high-risk infection were HPV 52 (2.42%), HPV 16 (2.01%), HPV 53 (1.43%), HPV 58 (1.32%) and HPV 66 (1.01%). We conducted follow-up on cases in 69 of 94 women who had a history of 1–4 years of positive infection, and in 18  patients, infection status turned negative (26.09%). Seventeen of the fifty-one women whose infections did not turn negative received treatment. Persistent infection was predominantly observed in high-risk genotypes (56 of 69).The total positive rate of infection was 17.13% , and the 51–60-year age group had the highest HPV infection rate (19.82%). There were statistically significant differences between rates in the 21–30 and 31–40 (The results recommend that women in Hefei improve health awareness and receive a 9-valent vaccine. Additionally, women with persistent infections should consult a gynecologist to prevent cervical lesions. Papillomaviruses are a family of viruses that infect animals and humans. Almost 200 human papillomavirus (HPV) types have been identified, 40 of which colonize the reproductive tract . They arHPV infections are mostly subclinical and transient. The median duration of infection is approximately 13 months for carcinogenic HPV types and 8 for non-carcinogenic types. Clearance time is also affected by HPV type: approximately 8 months for carcinogenic types and 5 months for non-carcinogenic types . AlthougThe incidence of cervical cancer among young women in China showed an upward trend from 2000 to 2014 . In 2012HPV DNA testing is currently a routine part of physical examinations for women. The US Preventive Services Task Force (USPSTF) recommended that women aged 30–65 years should undergo either cytology testing alone every 3 years or high-risk HPV types testing alone every 5 years to reduce cervical cancer incidence and mortality . This stThe samples  of exfoliated cervical cells for the HPV genotype assay were obtained from the Health Management Center of the First Affiliated Hospital of Anhui Medical University from January 1, 2016 to October 31, 2019. After analysis, it was found that some women had multiple HPV tests during this period. The samples were taken from 6,234 women with mean age of 43 ± 10 years who underwent physical examinations. We requested an exemption from informed consent from the Ethics Review Committee for research using medical records or biological specimens obtained in previous clinical diagnosis and treatment, and applied for exemption from informed consent. This study was approved by the Ethics Review Committee of the First Affiliated Hospital of Anhui Medical University, Anhui, China (reference number: Quick-PJ 2020-03032).Shanghai Tellgen HPV assay kit against 27 genotypes along with sterile collector, cell preservation solution, DNA extraction reagent, amplification reagents, and hybridization reagents ; LifeECO PCR platform ; Luminex 200 analyzer ; ThermoMixer C metal bath and 5424R centrifuge .A disposable, sterile collector was used to collect the extirpated cervical cells and put them into the cell preservation solution. Specimens were sent for inspection at room temperature within 2 h or stored at −20 °C (retention period 60 days).The samples were shaken for 20 s, and 200 µL of the mixture was placed into a 1.5 mL tube. The tube was centrifuged at 15,000 rpm for 5 min, and the supernatant was removed. DNA extraction reagent (200 µL) was added to the tube and mixed well. The tube was incubated at 100 °C for 15 min in the a metal bath. The tube was then centrifuged at 15,000 rpm for 5 min, and the supernatant was transferred to a new tube for PCR amplification or stored at −20 °C.The DNA amplification reagents and extracted DNA retained in the supernatant after DNA extraction were brought to room temperature. The PCR reaction mixture was prepared with 10 µL of master mix, 5 µL of primer, and 0.8 µL of Taq enzyme per sample. Using PCR tandem tubes, 15 µL PCR reaction mixture and 5 µL extracted DNA were added to each tube. The following amplification program was run: 95 °C for 5 min, one cycle; 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, 5 cycles; 95 °C for 30 s, 55 °C foe 30 s, 72 °C for 30 s, 35 cycles; 72 °C for 3 min, one cycle; 4 °C for storage until analysis.Microsphere hybridization solution (22 µL) and PCR amplification product (3 µL) were added to each well of a 96-well plate. Contents in each well were mixed and sealed. The hybridization program was conducted as follows: the first step was at 95 °C for 5 min, the second step was at 48 °C for 30 min, the third step was at 48 °C for 15 min, and the fourth step was at 48 °C until testing. At the end of the second step, the wells were opened, and 75 µL of streptavidin-phycoerythrin was quickly added. After the third step, the 96-well plate was removed and placed on a preheated metal plate at 48 °C before testing with the Luminex 200.The data were analyzed using the Tellgen Technology 3.2 software provided with the HPV assay kit.According to personal information collected during physical examinations, women with positive HPV infection history  in the Health Management Center of our hospital were selected. The following two groups were selected for telephonic follow-up: (1) women with positive infection history that were positive after their latest HPV genotype assay, and (2) women with positive infection history that had turned negative after their latest HPV genotype assay. Telephonic follow-up included discussion of spontaneous symptoms during HPV infection, treatment measures and prognosis, and ThinPrep cytology test (TCT) results or colposcopy reexamination.χ2) was used to analyze qualitative data. The test level is α = 0.05, and P < 0.05 was considered statistically significant.Statistical analysis was performed using Excel 2016  and SPSS 16.0 . The chi-squared test (P = 0.002), 21–30 and 41–50 (P = 0.0003), 21–30 and 51–60 (P = 0.00003), and 51–60 and >60 age groups (P = 0.046), as determined by a paired χ2 test of the five groups; there were no other statistically significant differences between groups. As age increased, the infection rate increased until 60 years of age. The highest infection rate was in the 51–60-year age group . The number of high-risk HPV infections was 977 . The rate of high-risk HPV infection was 15.67% (977/6234), and the rate of low-risk infection 7.20% (449/6234). There were no significant differences between rates of high-risk and low-risk infections in different age groups (P = 0.520). As shown in P = 0.075). HPV infection was dominated by single infection, with a rate of 13.01% (811/6234). Quintuple infection was the rarest, with a rate of 0.08% (5/6234).This article analyzed 7,222 exfoliated cervical cell samples from 6,234 women divided into five groups by age . The age distribution of HPV infection is shown in The 27-genotype HPV assay was routinely employed in examinations of the 6234 women. The distribution of results for each genotype is shown in Follow-up was conducted for 94 women who received multiple HPV tests and had a history of positive infections, 25 of which were lost to or refused follow-up, and 69 of which were assessed successfully. The history of positive HPV infection among the follow-up cases in women is shown in Hefei is located in eastern China, on the western end of the Yangtze River delta. It is the capital city of Anhui Province, with a large population . Screening and prevention of HPV-related diseases are vital to women’s health and economic development in Hefei.Globally, the rate of HPV infection among women without cervical lesions is 11.6%–11.7%, with the highest rates in sub-Saharan Africa (24%), Eastern Europe (21.4%), and Latin America (16.1%) . This stThe prevalence of HPV infection in 37 cities in China shows a “two-peak” pattern, with one peak in the 15–19-year age group, followed by a second peak in the 50–60-year age group . The risThe five most common high-risk HPV genotypes worldwide are HPV 16 (3.2%), HPV 18 (1.4%), HPV 52 (0.9%), HPV 31 (0.8%), and HPV 58 (0.7%) . In mainThe development of the HPV vaccine was a leap forward in the prevention of cervical cancer. The HPV vaccine, which has been available in the United States since 2006, has been shown to prevent genital warts as well as cervical, vulvar, vaginal, and anal cancer. The latest version of the 9-valent vaccine  can prevent 90% of cervical cancer. It was initially recommended for women aged 9–26 years, but this recommendation was recently extended to women up to 45 years old . HPV 52 The present study represents the first collection of a large number of physical examination samples for the analysis of HPV infection in women in Hefei and follow-up of case in women with 1–4 years of continuous infection. However, it has the following limitations. First, the HPV genotype assay is usually not performed in women under 20 years of age due to invasive injuries caused by the collection of exfoliated cervical cells. Thus, this study lacks data for women under 20 years of age. Second, the specimens selected in this article are all from the Health Management Center, which examines relatively healthy people. Therefore, the HPV infection rate may be lower than the true population rate and may not present a full picture of the HPV infection of women in the region or country. Third, participants with HPV infections may change jobs or choose other hospitals for physical examinations, making follow-up difficult. Thus, a comprehensive analysis of every patient at our hospital was impossible in this study. In the future, we hope to expand the sample to the entire Hefei area to analyze HPV infection and prevention comprehensively.In summary, the results of this research suggested that the positive rate of HPV infection in women of Hefei was 17.13%, and the negative conversion rate in women with continuous positive infection was low. The most common high-risk HPV genotypes are HPV 52, 16, 53, 58 and 66, and HPV genotype assays combined with TCT and colposcopy can be used to confirm diagnoses if necessary. Early vaccination, early diagnosis, and early treatment measures are necessary to prevent the occurrence of cervical cancer and other HPV-related lesions.10.7717/peerj.10179/supp-1Supplemental Information 11068 women had HPV infections, including single infection and multiple infections.Click here for additional data file.10.7717/peerj.10179/supp-2Supplemental Information 2P = 0.002), 21–30 and 41–50 (P = 0.0003), 21–30 and 51–60 (P = 0.00003), and 51–60 and < 60 years groups (P = 0.046); there were no other statistically significant differences between groups.There were statistically significant differences between HPV infection rates in different age groups, including the 21–30 and 31–40 , 6 drug,11 untreated] had infections turn negative. Seventeen of the 51 women whose infections did not turn negative received treatment.Click here for additional data file.


The human virome, which is a collection of all the viruses that are present in the human body, is increasingly being recognized as an essential part of the human microbiota. The human gastrointestinal tract and related organs —composing the gastrointestinal (or digestive) system—contain a huge number of viral particles which contribute to maintaining tissue homeostasis and keeping our body healthy. However, perturbations of the virome steady-state may, both directly and indirectly, ignite/sustain oncogenic mechanisms contributing to the initiation of a dysplastic process and/or cancer progression. In this review, we summarize and discuss the available evidence on the association and role of viruses in the development of cancers of the digestive system. The human microbiota represents one of the most complex ecosystems on Earth, spanning bacteria, archaea, fungi, and viruses. Such microbes have coevolved with humans for millions of years, influencing our development and shaping, in particular, our immune defenses . DespiteThe human virome, which is a collection of all the viruses that are present in the human body , is incr15) [The human gastrointestinal tract and related organs —composing the gastrointestinal (or digestive) system—contain a huge number of viral particles (estimated to be more than 1015) ,7, which15) . HoweverIn this review, we summarize and discuss the available evidence on the association and role of viruses in the initiation and development of cancers of the gastrointestinal system.A virome includes all of the nucleic acids  belonging to the viruses associated with a particular ecosystem . Such viFusobacterium nucleatum [Clostridium difficile faster when placed in a culture of human colon cancer cells [As bacterial predators, phages extensively contribute to the abundance and composition of the gut microbiota and have been mainly studied for their impact on intestinal inflammation, as in the case of Crohn’s disease and ulcerative colitis ,15. To ducleatum . Alternaucleatum . Phages ucleatum . In the ucleatum . Such geucleatum ,22,23. Sucleatum ,25 and pucleatum . In thisucleatum ,28. Inteer cells . Furtherer cells . OverallViruses can infect all the cells of our body and other eukaryotic organisms colonizing our digestive tract. The diversity of such viruses is commonly evaluated by determining the viral families or species in a given sample. This is usually expressed as the number of reads as a surrogate of their relative abundance. Among the DNA viruses, some can establish persistent infections, such as those belonging to the Herpesviridae family, whereas others, such as Anelloviruses, have not been associated with a specific pathology . In contIn the last two decades, researchers have attempted to unravel the potential link between virus infection and cancer development. While most of the studies in this regard only aimed at detecting the presence of viral particles in cancer patients, some aimed to shed light on the interaction/causality between putative tumor viruses and transformed cells. The associations of specific viruses with cancers of the gastrointestinal tract and related organs, as well as the virus-driven molecular mechanisms , are summarized in Compared to other cancers of the digestive system, oral cancer (OC) has a lower worldwide incidence . HoweverInterestingly, the percentage of HPV-positive OC patients without any history of oral tobacco use was more than 31%, whereas only 10% of OC patients with previous oral tobacco use tested positive for HPV infection .Mechanistically, recent research has been focusing on elucidating potential viral interactions with oncogenic signaling pathways. For instance, the overexpression of p16—a tumor suppressor protein—has been detected in OC samples and suggested to act as a possible surrogate marker for infections with high-risk HPV strains . SeveralEsophageal cancer (EC) usually manifests as either esophageal adenocarcinoma (EAC) or esophageal squamous cell carcinoma (ESCC) . The notHPV E6 and HPV E7 genes contribute to ESCC pathogenesis via DNA demethylation and upregulation of the human leukocyte antigen (HLA)-DQB1 [Although the mechanisms of conversion of normal squamous cells to carcinoma cells have not been completely elucidated, some studies have highlighted the role of several viruses in such an event. For instance, it has been found that the LA)-DQB1 . MoreoveLA)-DQB1 ,100. BesLA)-DQB1 .Some ESCC cases may arise as a combination of genetic susceptibility and HPV infection. Yang et al. managed to identify two risk loci, which were found to not only increase the risk for ESCC, but also interact with the HPV serological status . FurtherDespite all of the data presented, two issues must be stressed. Firstly, most of the studies were observational (either prognostic or retrospective) and thus lack causality. Secondly, all of the studies were designed to detect the presence of either HPV or EBV in EC samples and, to the best of our knowledge, there is no study analyzing a complete virome panel in EC. Therefore, the answer to the question of whether viruses lead to a higher risk of EC may be even more complex than previously thought.Data from epidemiological studies performed across the globe do not unambiguously unravel the potential role of HPV in the pathogenesis of gastric cancer (GC) ,108. DatRecently, a number of studies have tried to shed light on the molecular mechanisms underlying the effects of EBV on GC pathogenesis. Zhang et al.  focused In 2012, 770,000 cases of liver cancer occurred worldwide, of which 430,000 (56%) and 150,000 (20%) were attributed to hepatitis B virus (HBV) and hepatitis C virus (HCV), respectively . Most ofAlthough the above-mentioned viruses and factors account for the majority of HCC cases, other viruses, such as HPV and EBV, may play a role in liver carcinogenesis. So far, a very limited number of studies have focused on elucidating the potential role of HPV in HCC. A study dated to 1992 found HCC samples infected with either HPV-16 or HPV-18 and suggested that HPV could act synergistically with HBV to promote HCC development . HoweverThe TT virus (TTV), first isolated in 1997 from a Japanese patient and named after his initials , is anotIntrahepatic (IHCC) and extrahepatic cholangiocarcinoma (EHCC) represent the two most common forms of bile duct cancer . There aBased on the available literature, a limited number of viruses are supposedly involved in the development of pancreatic cancer (PC), with HBV and HCV as the most likely contributors. The available studies/meta-analyses do not clearly prove whether HBV and HCV infections are associated with a higher risk of developing PC. A meta-analysis by Wang et al. suggested that HBV infection may increase the risk of PC . In contTaken together, despite certain advances that shed some light on the role of hepatitis viruses in the development of PC, more research is needed to uncover the putative molecular mechanisms governing the pancreatic carcinogenesis associated with viral infections.Colorectal cancer (CRC), defined as a cancer arising in the human colon and/or rectum, is one of the most common and studied types of malignancy. In 2018, CRC was the third most frequently diagnosed cancer (with more than 1.8 million cases) and the second in terms of mortality, accounting for almost 900,000 deaths worldwide . The humIn contrast to their role in promoting liver carcinogenesis, hepatitis viruses  seem to decrease the risk of colorectal liver metastasis in CRC patients ,166,167.The first association between HPV infection and CRC is dated to 1990, when, by immunohistochemistry and in situ hybridization, Kirgan and co-workers found that the HPV antigen was present in 60% of benign tumors and 97% of carcinomas, compared with 23% of normal colon specimens . The autSeveral studies have attempted to uncover the mechanisms underlying the possible link between HPV infection and CRC. In this regard, Qiu et al. performed a gene expression analysis and found four upregulated and differentially expressed genes in HPV-positive CRC samples compared to HPV-negative tissues . These gIn a similar fashion, c-Myc acts as a protooncogene which has been shown to be constitutively expressed, besides CRC , in otheK-Ras [Another study reported a correlation between HPV infection and worse clinical stages of CRC . NotablyK-Ras . AlthougK-Ras ,190.Taken together, despite a considerable amount of data pointing toward the notion of a carcinogenic effect of HPV infection, more studies are needed to elucidate whether such infection arises before, during, or after CRC development and whether the aforementioned changes in gene expression are strictly dependent on HPV, rather than driven by other factors.Studies concerning the association of EBV with CRC are limited. One of the first reports on the potential carcinogenic effect of EBV comes from Song and colleagues, who detected the presence of EBV in almost 80% of CRC specimens . Next, FAnother potentially important player in CRC carcinogenesis may be human CMV, which belongs to the Herpesviridae family of viruses. CMV has been shown to preferentially infect CRC lesions compared to normal healthy tissue ,195,196.Multiple mechanisms have been proposed to contribute to the potential carcinogenic effect of CMV in CRC. For instance, it seems that genetic polymorphisms in the CMV genome may affect the outcome of CRC. Specifically, Chen et al. detected the presence of CMV in 47.8% of the CRC specimens analyzed. Among these, three different polymorphisms of the viral immunomodulatory gene UL144  were identified. The presence of genotype B, occurring in 30.2% of the CMV-positive samples and characterized by the highest UL144 expression, was associated with shorter disease-free survival and independently predicted tumor recurrence . Other dHuman polyomavirus 2, more commonly known as John Cunningham virus (JCV), which is a member of Polyomaviruses named after a patient with progressive multifocal leukoencephalopathy from whom it was isolated , has alsJCV has been proposed to promote colon carcinogenesis in a number of ways. Firstly, a possible link between the expression of T-antigen (T-ag)—an early JCV protein which is believed to mediate the oncogenic potential of the virus—and chromosomal instability has been found ,217. In While the body of evidence strongly supports a role for JCV in contributing to CRC carcinogenesis, future studies should be aimed at investigating the conditions under which such cell signaling regulation can happen.INK4a, which is a surrogate marker for HPV infection that appears to have prognostic value in AC [INK4a was expressed in HPV-positive AC patients, better overall survival was observed [INK4a and p53 in AC patients. The authors described an inverse trend in the association between the viral load and p16INK4a expression status [While colon and rectal cancers share similar features and are often referred to collectively as CRC, anal cancer (AC) differs in significant ways, including the tissue where neoplastic transformation begins (similar to that lining the woman’s cervix), pathogenesis, and treatment . Althougue in AC . Despiteue in AC . In contobserved ,233 and observed . Of partn status . In addin status ,237. In n status . These Hn status ,239,240.n status .Apart from acting alone, HPV may cooperate with human immunodeficiency virus (HIV) to increase the probability of AC development. This is not surprising, as HIV patients bear a weakened immune system and thus have a higher chance of developing malignancies compared to the general population . StudiesThe study of microbial entities that may be involved in the development of malignancies of the alimentary tract has mainly concentrated on the bacterial component of the microbiome . AnotherMost of the studies described herein have focused on the prevalence of infection with a certain virus in a type of cancer in a given geographical location. While data gathered in such a manner can be undoubtedly seen as a necessary first step, it is important to point out that such studies do not carry the collective knowledge beyond the level of association and may be hampered by methodological flaws. Indeed, research on patients usually contains many potentially confounding factors  that may reasonably impact the structure and function of the virome in the short or long term. The use of related individuals living in the same household/environment, rather than unrelated randomly selected individuals, may be useful for reducing such a bias. Another concern relates to cross-study comparisons of datasets, which are almost impossible due to differences in sample collection, handling, storage, and processing . The development of standardized protocols, as well as the establishment of Gold Standard procedures, would help address this issue. Finally, as the virome composition may differ from early to late stages of disease, research in this area should take into account a rigorous analysis of tumor stadiation. Discovering molecular mechanisms behind virus–cell communication may be of particular importance in the future. To elucidate the potential carcinogenic role of a given virus, functional studies involving cell cultures and animal models should be employed to a greater extent. However, determining the functional impact of a virus as a cancer agent requires effective vaccines or antivirals. If eliminating the infection also eliminates the cancer, then the link is clear. Unfortunately, such an approach requires specific drug and vaccine development, which only occurs once a virus is already established to cause a disease or cancer, and there is sufficient economic interest on the part of biopharmaceutical companies. In 2018, about 2.2 million cancer cases, or 13% of the global cancer incidence, were attributable to infection  . In accoA sector of the virome that remains inadequately investigated is the phageome. This is particularly true for the colon, which harbors conditions such as a mild pH and nutrient-rich environment, allowing for the abundant occurrence of bacteria and thus phages. Although research in the field has made great strides, whether phages play a role in the development of gastrointestinal cancer remains an unanswered question. To date, publications considering the possibility that phages may be a driving force of carcinogenesis in the gut are lacking . CertainWhile some of the potential virome-mediated carcinogenic effects occurring in the gut can be induced by a direct interaction of viruses with the host cells, others may require the interplay of viruses with other components of the microbiome . The above-mentioned interactions may take place regardless of whether the enteric viruses replicate locally in the intestine or whether they use the gut as a point of entry to the host, showing their pathologic effects in organs other than the gastrointestinal tract . To helpNotwithstanding, it is important to point out that viral infections are generally not sufficient for cancer development, as cancers are evolutionarily dead-end events that threaten the viral agent as much as the host. Indeed, viral-mediated tumors only occur in a minor fraction of infected individuals when additional events  and/or concomitant host factors  exist.Despite the recent increase of the awareness about the relationship between specific viruses  and the pathogenesis of oral, hepatic, colorectal, and anal cancers, the human virome remains largely uncharacterized and we still have a poor knowledge on how it may influence the risk of developing cancers of the digestive system. Further mechanistic studies using experimental models and multicenter observational cohort studies—designed to pay attention to the potential biases—would hopefully help with further unravelling such a complex topic.


The role of two intracellular DNA sensors, which belong to the PYHIN family (interferon-inducible protein 16 (IFI16) and absent in melanoma 2 protein (AIM2)), has been analyzed in relation to HPV infection and head and neck carcinogenesis. In particular, IFI16 and AIM2 expression depends on HPV infection in HNSCC. They represent viral restriction factors and are key components of the intrinsic immunity activated against different viruses, including HPV. This review analyzed and summarized the recent findings about the role of PYHIN proteins in HPV Head and neck squamous cell carcinoma (HNSCC) represents the sixth most prevalent type of cancer, including about 6% of all cases of tumors worldwide. In the male and female population, its incidence is of 15.2 and 4.6/100,000 people, respectively, accounting for approximately 650,000 new cases and 350,000 deaths per year [Although the two major risk factors for HNSCC are alcohol and tobacco abuse, which act synergistically, the human papillomavirus (HPV) has recently emerged as one important independent risk factor for the development of these tumors, especially at the level of the oropharynx, with the tonsils and the base of the tongue being the most vulnerable sites. In particular, HPV infection has been directly associated with increased incidence of oropharyngeal tumors in men younger than 50 years without a prior history of tobacco and alcohol consumption . HPV infThe PYRIN and HIN domain(PYHIN) family consists of five interferon-inducible proteins that have recently emerged as intracellular DNA sensors. These include the interferon-inducible protein 16 (IFI16), absent in melanoma 2 protein (AIM2), myeloid cell nuclear differentiation antigen (MNDA), pyrin and HIN domain family 1 (PYHIN1 or IFIX), and PYRIN domain (PYD)-only protein 3 (POP3) ,6. The gIn this review, we will describe our current understanding of the emerging role of PYHIN proteins in HNSCC pathogenesis, focusing in particular on IFI16 and AIM2, whose expression depends on HPV infection ,12,13,14HNSCC arises from mucosal epithelial cells of the upper airways . Its complex anatomical origin, as well as its different etiologies and molecular alterations, makes this tumor intrinsically heterogeneous ,3.+) and HPV-negative (HPV−) tumors, each with distinct clinical presentation, molecular profiles, and prognosis [+ HNSCCs show a better response to chemoradiotherapy and survival [Although alcohol and smoking abuse represent classical risk factors for HNSCC, infection by high-risk HPV serotypes has increased the prevalence of this tumor, mainly in the oropharyngeal area . Indeed,rognosis ,18,19,20survival .+ HNSCC more often affects young non-smoking and non-drinking individuals compared to HPV− HNSCC. Moreover, HPV+ HNSCC generally presents itself as a small primary tumor with large nodal metastases [+ vs. HPV− oropharyngeal squamous cell carcinoma, the tumor-node-metastasis (TNM) staging for oropharyngeal tumors has been recently changed by the American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC) to better represent this emerging feature [HPVtastases . Due to  feature .+ tumors comprise approximately 22%–47% of all oropharyngeal HNSCCs [+ HNSCC are positively associated with oral sex [HPVl HNSCCs , with thl HNSCCs . The fraoral sex .+ HNSCCs were negative for E6 and E7 gene expression [E6/E7 mRNA in 3.9% and 3.1% of oral and laryngeal tumors, respectively [HPV infection in non-oropharyngeal HNSCC is less frequent. A systematic review reported an HPV DNA prevalence for oral and laryngeal tumors of 23.5% and 24.0%, respectively . Howeverectively .NOTCH1, p16, and TP53) or genes regulating the cell cycle  have been identified in HNSCC, with NOTCH1 and TP53 being the most frequently found mutated genes [− and HPV+ HNSCC [1/S transition. Consequently, p16 is generally regarded as a bona fide molecular marker of HPV infection, which has recently led to modification of the TNM staging to include p16 positivity as a surrogate for HPV status [In the last few decades, a number of molecular changes mainly involving oncogenes and tumor suppressor genes  [NOTCH1 in HNSCC has led to the hypothesis that this protein acts as a tumor suppressor rather than as an oncogene in this type of tumor [− HNSCC include the epidermal growth factor receptor (EGFR), often amplified in HNSCCs and related to cell cycle control [Met, a receptor tyrosine kinase often associated with enhanced migration, invasion, and angiogenesis when overexpressed in cancer [f cases) . The preof tumor . Other g control , and Metn cancer .− HNSCCs are often unaltered in their HPV+ counterparts [TP53 as HPV+ HNSCCs express this gene in its wild-type conformation, whereas HPV− HNSCCs generally harbor a mutated form [+ HNSCCs have a lower average number of mutations per tumor, and rarely display p16INK4A loss-of-function compared with HPV− HNSCCs [On the other hand, a number of epigenetic and genomic studies have shown how the majority of commonly altered genes in HPVterparts . This isted form . Moreove− HNSCCs ,34,37,38PIK3CA mutation hotspots. PIK3CA encodes p110α, a catalytic subunit of phosphoinositide 3-kinase (PI3K), which activates the v-akt murine thymoma viral oncogene(AKT) signaling pathway. HPV+ HNSCCs carry mutations in the helical domain of this protein, whereas HPV− tumors harbor mutations throughout the entire gene, albeit those in the helical and kinase domains are more frequently observed. In particular, PIK3CA amplification and/or mutation can be found in 34% of HPV− and 56% of HPV+ HNSCCs [Another interesting difference concerns + HNSCCs .+ HNSCC [+ tumors [− HNSCC presents a smoking-associated mutational signature, while reduced exposure to exogenous carcinogens in HPV+ HNSCC favors the emergence of tumors carrying APOBEC-mediated driver mutations. Finally, Henderson et al. reported that APOBEC activity is responsible for creating driver mutations in the helical domain of PIK3CA gene across multiple cancers. They implicate APOBEC activity as a key driver of PIK3CA mutagenesis and HPV-induced transformation [+ HNSCC could be identified basing on gene-expression data: immune related (cluster 1), epithelial–mesenchymal transition related (cluster 2), and proliferation related (cluster 3). This stratification has a prognostic relevance, with cluster 1 having the best outcome, cluster 2 the worst, and cluster 3 an intermediate survival rate [More recently, our group has shown that the apolipoprotein B mRNA-editing enzyme catalytic subunit (APOBEC) family of cytidine deaminases plays a role in HPV+ HNSCC , in line+ tumors . APOBEC + tumors . Interesormation . Moreoveval rate .The five interferon-inducible proteins belonging to the PYHIN family have recently emerged as an important group of intracellular DNA sensors ,6. The NPYHIN proteins participate in the innate immune response as pattern-recognition receptors (PRRs), acting as whistleblowers of pathogen-associated molecular patterns (PAMPs) ,43. In pMNDA, the first human PYHIN family member to be identified, is a 55 kDa protein predominantly expressed in the nucleus of myeloid cells . MNDA haTP53 and c-myc [The second identified human PYHIN family member, IFI16, can be found in three alternative spliced isoforms  clustered at 85–95 kDa ,53,54. Tnd c-myc . In addind c-myc . In contnd c-myc . Finallynd c-myc ,64,65,66The antiviral role of IFI16 has been reported for both DNA and RNA viruses. The HIN domains allow this protein to bind both double-strand (ds) and single-strand (ss) DNA in a sequence-independent manner . In partSTING)/ TANK-binding kinase 1 (TBK-1)/ interferon regulatory factor 3 (IRF3) pathway to induce cell death [More recently, the cellular DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to be another important interactor of IFI16, albeit these two proteins display dissimilar functions. Specifically, upon herpes simplex virus type I (HSV-1) and HCMV infections, IFI16 interacts with cGAS through the PYRIN domain , but whell death ,71. An all death . Finallyll death . In respll death . Similarll death .Similar to IFI16, the PYHIN protein AIM2 plays an important role as a gatekeeper of host innate immunity, even though before being classified as a DNA sensor, it was initially identified among a set of genes associated with the reversal of the malignant phenotype of melanoma cells (absent in melanoma)  Table 2Table 2 ,76. AIM2Another important function of AIM2 relies on its ability to suppress neoplastic progression . Indeed,PYHIN1, also known as IFIX, is among the most recently identified PYHIN proteins. As a result of alternative mRNA splicing, it can be found in six isoforms . Being the most abundant, the isoform α1 is generally regarded as the prototype of this group. Due to the presence of an NLS, IFIX is mainly found in the nucleus , and likThe most recently identified member of the PYHIN family is POP3, whose genomic sequence is comprised between PYHIN1and IFI16 on the PYHIN locus. Although POP3 does not harbor a HIN domain responsible for DNA binding, it has a PYD domain, which allows it to interact with both AIM2 and IFI16, thus modulating their functions in innate immunity .In recent years, restriction factors (RFs) have emerged as key components of intrinsic immunity in response to different viruses, especially HPVs . All RFsIFI16 is a sensor of exogenous DNA and acts as a viral restriction factor against HCMV, HSV-1, KHSV, EBV . In a re+ oropharyngeal tumors are caused by HPV16 or -18, vaccination against HPV is likely to play a fundamental role in preventing HPV-related oropharyngeal cancer in the years to come [The role of IFI16 as a restriction factor for HPV is of particular interest for HNSCC, since HPV has been detected in 22%–47% of oropharyngeal squamous cell carcinomas ,12,13,14 to come .Even though the role of AIM2 in HPV infection has been only marginally addressed, it is well established that the AIM2 inflammasome is crucial for the immune response to viral and bacterial infections. In particular, AIM2 is involved in DNA and RNA virus infection, such as CMV, hepatitis B virus (HBV), HPV, and influenza virus . With re+-dependent protein deacetylase Sirtuin 1 (SIRT1) has recently emerged in HPV-induced cervical cancer. In these tumor cells, aberrant expression of SIRT1 inhibits NF-κB-mediated transcription of AIM2 through destabilization of v-rel reticuloendotheliosis viral oncogene homolog B (RELB) mRNA. Moreover, knockdown of SIRT1 not only result in pyroptosis of HPV-infected cervical cancer cells, such as SiHa cells, but it also induces pyroptotic cell death of naïve cervical cancer cells by releasing extracellular vesicles carrying AIM2 inflammasome proteins , harboring a mutated p53, failed to exert antiproliferative activity, suggesting that IFI16-mediated inhibition of cell growth probably depends on the presence of a functional p53 protein. In the same study, the authors observed that IFI16 restoration in HNO136 increased doxorubicin-induced cell death through G2/M phase arrest. Specifically, fluorescence-activated cell sorter (FACS) analysis revealed that ectopic expression of IFI16 in HNO136 cells caused a more pronounced G2 arrest in comparison with their non-transduced counterparts. The lack of caspase-3 activation and p53 transcriptional activity in these cells ruled out the possibility that doxorubicin-treated cells were dying from apoptosis.By the means of in vitro studies, it was observed that ectopic expression of IFI16 in an IFI16-null HNSCC-derived cell line (HNO136) suppressed both proliferation and transforming activity of these cells in vitro, indicating that IFI16 exerts a potent antiproliferative effect in HNSCC . Congruo+ cells at the stromal–epithelial junction, the authors performed chemotaxis experiments in vitro to determine whether IFI16 was directly involved in blood cell recruitment. The supernatants from IFI16 transduced HNO136 cells showed chemoattractive activity on mouse macrophages. This result was confirmed by the detection of CD68+ or CD14+ macrophage infiltration in the xenografts by immunohistochemistry. Overall, these data indicate that IFI16 exerts an anti-tumoral activity in vivo by stimulating apoptosis of tumor cells, impairing neo-vascularization, and releasing chemotactic factors, which in turn recruit macrophages . In HPV+ tumors, IFI16 expression positively correlated with APOBEC3A expression. Furthermore, the upregulation of IFI16 positively associated with a lower incidence of nodal metastases in HPV− HNSCCs. In contrast, the HPV+ group did not show any statistically significant correlation with clinical characteristics. This study was the first demonstration of a positive role of IFI16 in modulating a clinical feature in HPV− tumors. Lastly, IFI16 or AIM2 low expressors displayed a worse prognosis, especially with regard to overall survival. However, the correlation between nodal metastases and IFI16 may be a confounding factor due to the better prognosis of patients without nodal metastases. Based on the aforementioned data, Riva et al. have more recently shown a protective role of IFI16 in HNSCC patients . Specifi− vs. HPV+ HNSCCs may be partly ascribed to the emerging etiopathological role played by PYHIN proteins in these tumors. In this regard, particular attention has been paid to the functions of the two PYHIN family members IFI16 and AIM2. On the one hand, IFI16 appears to be mainly overexpressed in HPV+ tumors compared to their HPV− counterparts, which is consistent with its well-established role of viral restriction factor. On the other hand, AIM2 seems to be predominantly overexpressed in HPV− HNSCC. It is however important to point out that in 50% of HPV+ tumors AIM2 expression levels are unchanged, supporting the hypothesis that this intracellular sensor of viral DNA might be differentially regulated in HPV+ vs. HPV− HNSCC, an exciting possibility that should be further explored.In conclusion, from the literature reviewed above, it appears that the different molecular profiles and prognoses of HPV− HNSCCs, suggesting a protective role against the lymphatic diffusion of tumor cells also in the absence of HPV infection. This is probably due to the ability of IFI16 to orchestrate multiple transcription factors, as previously demonstrated during B-cell differentiation [Interestingly, IFI16 is inversely associated with nodal metastases in HPVntiation . Further+ patients.Overall, patients with low levels of IFI16 and AIM2 expression seem to have a worse prognosis. However, the correlation between nodal metastases and IFI16 may represent a confounding factor in such survival analyses. Thus, the prognostic value of PYHIN proteins in human HNSCC awaits additional studies on larger patient cohorts. Such studies should allow the identification of new druggable targets and provide the rationale for de-intensification trials on HPV


Background: The epidemiological feature of human papillomavirus (HPV) infection is distinctive in China. We aimed to investigate the multi-infection patterns and co-infection preference of 27 HPV types among gynecological outpatients across China.Methods: Overall 137,943 gynecological outpatients were recruited from eight tertiary hospitals located in seven regions of China, between July 1st, 2014 and December 31st, 2016. The overall, region-specific, age-specific and type-specific prevalence of HPV infection were calculated, respectively. The pattern of HPV infection was also evaluated. Furthermore, rate ratio was calculated to evaluate the co-infection preference of any two HPV genotypes.Results: The overall prevalence of 27 HPVs' [17 high-risk (hr)/10 low-risk (lr)] infection was 23.5%. The age-specific HPV prevalence showed a “U-shaped” pattern. The most prevalent hrHPV genotypes were 16, 52, and 58. Multiple infections were detected in 25.8% of the HPV-positive women, in which dual infection was more prevalent. HPV 16/18 were likely to co-infected with HPV 31 but unlikely with HPV 52/58, i.e., the co-infection of HPV 16 with HPV 31 was high (3.5-fold), but low for HPV 58 (1.8-fold), and 52 (1.2-fold), while the co-infection of HPV 18 with HPV 31 was high (4.3-fold), but low for HPV 52 (1.9-fold), and 58 (1.7-fold).Conclusions: We found age-specific prevalence of HPV infection showed a “U-shaped” pattern for high and low risk HPV, suggesting the importance of screening among younger women and the necessary of detection among older women. We found a novel co-infection preference of HPV 16/18 with 31, 52, and 58, suggesting a need of developing and marketing prophylactic HPV vaccines that protect against more genotypes in China. Cervical cancer, being the 4th most frequent cancer among women, is major public health issue worldwide . It's esIt is well-known that persistent infection with oncogenic types of human papillomavirus (HPV) causes cervical cancer. More than 200 genotypes of HPV have been identified and among them, 13 types were classified as oncogenic . HPV genProphylactic HPV vaccine is the best approach for prevention and control of cervical cancer burden, which is widely used in more than 160 countries . CurrentTo the best of our knowledge, investigations on the co-infection preference of specific genotypes of HPV in Chinese population are scarce. Updated information on the prevalence and distribution of type-specific HPV infection, and multi-infection patterns among Chinese population is highly warranted for the choice of appropriate HPV vaccines, and timely assessment of vaccine efficacy and cost-effectiveness. Therefore, we aimed to investigate the prevalence and distribution of type-specific HPV infection among gynecological outpatients across China and to further evaluate the multi-infection patterns and co-infection preference of 27 HPV genotypes.We only recruited gynecological outpatients who attend tertiary hospitals across China. Eight tertiary hospitals from seven regions of China, including four regions  located in Eastern China, one region (Hunan) in Central China and two regions  in Western China , particiCervical exfoliated cell samples were collected from all participants by gynecologists according to the standard operation procedure. Cell samples were collected in 2.5 ml of cell preserve solution  for HPV DNA testing.The Tellgenplex™ HPV DNA Test was performed using a Luminex-based suspension beads array to identify HPV types. The experimental protocol includes DNA extraction, PCR amplification, bead-coated hybridization, and digital signal processing. The Tellgenplex™ HPV DNA Test can identify 27 HPV types, including 14 hrHPVs , three potential high-risk HPVs  and 10 lrHPVs .We calculated overall, region-specific and age-specific prevalence of HPV infection, respectively. Difference in the prevalence of HPV infection between each age group  was analyzed using Chi-square test or Fisher's exact test. Subsequently, the prevalence and distribution of type-specific HPV infection were calculated and, further analyses were stratified by region and infection pattern . Furthermore, the infection pattern of 27 HPV types was evaluated, i.e., the prevalence of single, dual and multiple infection was calculated, respectively. The age-specific infection pattern of 27 HPV types was also evaluated. Moreover, the number of co-infections of 14 hrHPVs  and 2 lrHPVs (HPV 6 and 11) was calculated, respectively. Finally, in order to further investigate the co-infection preference of any two HPV types (such as HPV A and HPV B), we selected the aforementioned HPV types  to calculate the rate ratio, using the real infection rate of HPV A/B as the numerator and their theoretical infection rates (multiplication of the single infection rate of HPV A and HPV B) as the denominator.P < 0.05 was considered statistically significant.All statistical analyses were performed using the SAS statistical software, version 9.4 . Graphs were generated using R version 3.4.2 . Two-sided Overall 137,943 gynecological outpatients were included in the analyses  . The oveOverall age-specific prevalence of HPV infection showed a “U-shaped” pattern . Two peaType-specific HPV infection rate is presented in Variations by regions were found . For eacThe prevalence of multiple infections is shown in In the single infection group, HPV 16 was the most prevalent genotype (with infection rate 3.0%), followed by HPV 52 (2.2%) and 58 (1.6%) . In the Age-specific multiple infections of 27 HPV genotypes are shown in The co-infection of any two genotypes is presented in We investigated, for the first time systematically in China, the prevalence and distribution of type-specific HPV infection among gynecological outpatients, and also the multi-infection patterns and co-infection preference of 27 HPV types, using data from 137,943 Chinese gynecological outpatients across seven regions of China. We found the overall prevalence of HPV infection (27 HPVs) reached 23.5%, with 19.4% for hrHPV group and 7.0% for lrHPV group. The age-specific prevalence of HPV infection showed a “U-shaped” pattern for hrHPV and lrHPV, with two peaks for age ≤19 years and for age ≥60 years . The overall prevalence of hrHPV infection was 19.4% and the most prevalent hrHPV genotypes were HPV 16 (4.4%), followed by HPV 52 (3.5%) and 58 (2.6%). Multiple infections were found in 25.8% of HPV-positive women, in which dual infection was more prevalent. HPV 16/18 were likely to co-infected with HPV 31 (the co-infection of HPV 16 with HPV 31 was 3.5-fold elevated and the co-infection of HPV 18 with HPV 31 was 4.3-fold elevated) but unlikely with HPV 52/58.Our finding of overall prevalence rate of hrHPV infection (19.4%) was slightly lower compared to a pooled analysis 23.2%) , but muc3.2% , buOur finding of a “U-shaped” pattern of age-specific prevalence for hrHPV and lrHPV group (with two peaks for young women aged ≤19 years and for older women aged ≥60 years) is generally consistent with previous similar investigations in China , 25–28, We found that HPV 16, 52, and 58 were the predominant genotypes, in line with previous investigations in China , 32–35. We found lrHPVs were also detectable in the cervical exfoliated cell samples, indicating the necessary of detecting hrHPVs and lrHPVs at the same time, particularly 14 hrHPVs with 2 lrHPVs (HPV 6 and 11). The prevalent genotypes found in our study suggested that prophylactic HPV vaccines protecting against additional HPV genotypes  may offer a better protection efficacy for Chinese and other Asian populations.We found that single infection was more common among the HPV-positive women and multiple infections accounted for a small proportion (25.8%), consistent with previous investigations in Chinese population , 37–39 aThere is no consensus on the association of multiple infections with occurrence or progression of cervical cancer. For instance, Fife et al.  showed tWe found that HPV 31 was most likely to co-infected with HPV 16/18, consistent with the cross-protective efficacy of bivalent vaccine against HPV 31 and with findings in other populations , 45. We Our study has a number of strengthens and limitations. Firstly, we systematically investigated multi-infection patterns and co-infection preference of 27 HPV types (17 hrHPVs/10 lrHPVs), using data from 137,943 Chinese gynecological outpatients across seven regions of China. Secondly, our study focused on gynecological outpatients who were identified as a high-risk population of HPV infection, which could help the clinicians to better understand the prevalence and distribution of type-specific HPV infection. Thirdly, the association between the co-infection preference and cross-protective efficacy of HPV vaccine was revealed for the first time, which might help to better understand the mechanisms of cross-protection of vaccines and to provide guidance on HPV vaccine development and application in the future. Major limitation concerns sample source. Subjects in the youngest (0.3%) and oldest (4.8%) age group accounted for a small proportion, which might over-estimate age-specific prevalence of HPV infection.In summary, using data from 137,943 Chinese gynecological outpatients across China, we found the overall prevalence of HPV infection reached 23.5% and genotype distribution varied by regions, suggesting the prevention of HPV infection in a region-specific pattern. We found age-specific prevalence of HPV infection showed a “U-shaped” pattern for hrHPV and lrHPV (with two peaks observed in younger and older women), suggesting the importance of screening among younger women and the necessary of HPV detection among older women. We found that a novel co-infection preference of HPV 16/18 with 31, 52, and 58, suggesting vaccines could also protect against HPV 52/58 are highly warranted in China. Our findings may help to reveal the mechanisms of cross-protection of HPV vaccines and to indicate the necessary of developing and marketing prophylactic HPV vaccines that protect against more genotypes (particularly HPV 52 and 58), which may offer better protection efficacy for Chinese population.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of West China Second University Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MX and TC were responsible for the study concept and design. TC, GL, and XJ obtained funding. ZW, HZ, YM, WX, HX, and WC acquired data. GL, XJ, BS, HT and JJ analyzed and interpreted data. GL, XJ, BS, and TC drafted the manuscript, and all authors revised it for important intellectual content. TC and MX are the guarantors of this work.BS was employed by the company Tellgen Corporation, Shanghai, China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Information on HPV-associated sinonasal squamous cell carcinoma (SNSCC) is very limited in China. The aim of this study was to determine the prevalence of HPV in a large cohort of SNSCC patients in China.Clinical records and formalin-fixed and paraffin-embedded tumor specimens from 30 SNSCC patients with associated inverted papilloma (IP-SNSCC) and 84 de novo SNSCC (DN-SNSCC) patients were retrieved between 2010 and 2017. HPV status was determined for each specimen using a combination of p16 immunohistochemistry and GP5+/6+ PCR.p = 0.150). Among 18 p16-positive SNSCC patients, only three were HPV DNA-positive. Furthermore, only three of nine HPV DNA-positive tumors exhibited high p16 expression. In IP-SNSCC patients, only one of six HPV DNA-positive tumors exhibited high p16 expression. In DN-SNSCC patients, two of three HPV DNA-positive tumors exhibited high p16 expression. The positive rates for both HPV DNA and p16 in IP-SNSCC patients and DN-SNSCC patients were 3.3 and 2.4%, respectively.Immunohistochemistry for p16 was positive in two IP-SNSCC patients  and in 16 DN-SNSCC patients . HPV DNA was detected in six IP-SNSCC patients  and in three DN-SNSCC patients . Expression of p16 was not correlated with the presence of HPV DNA (Immunostaining for p16 is not a reliable surrogate marker of HPV status in SNSCC. The presence of HPV is rarely detected in DN-SNSCC patients in Eastern China. IP-SNSCC patients frequently lack of p16 overexpression despite the presence of high-risk HPV DNA. Infection with human papillomavirus (HPV), particularly high-risk types of HPV, has been acknowledged as an etiological factor in a subset of head and neck cancers . Head anThe presence of HPV has also been reported in SCCs of the sinonasal tract, but HPV detection rates in sinonasal SCC (SNSCC) patients are widely disparate and range from 0 to 100% . There aInverted papilloma (IP) is the most common benign tumor of the sinonasal tract, with a 5–13% incidence malignant transformation . The mosTo date, the clinical significance of HPV status in SNSCC is not well established and information on HPV-associated SNSCC in China is very limited. Here, we analyzed the presence of HPV infection in DN-SNSCC and IP-SNSCC patients in a large Eastern China population.Formalin-fixed and paraffin-embedded (FFPE) tissues were retrospectively collected from SNSCC patients who underwent surgical treatment at the Eye and ENT Hospital of Fudan University between 2010 and 2017. Hematoxylin and eosin-stained sections were reviewed and histological diagnosis was confirmed by an experienced head and neck pathologist. FFPE tissues without tumor cells were excluded. In this study, specimens from 30 IP-SNSCC patients and 84 DN-SNSCC patients were obtained for HPV detection. Clinical information was extracted from medical records. Tumor node metastasis (TNM) stage was assessed according to the 7th edition of the American Joint Committee on Cancer staging system.The study was approved by the Institutional Review Committee of the Eye and ENT Hospital of Fudan University. Informed consent was obtained from all patients. All procedures were conducted in accordance with the Declaration of Helsinki.Immunohistochemical analysis for p16 was performed on 4-μm thick tissue sections cut from the FFPE tissue. A monoclonal antibody to p16  was used with a BenchMark Autostainer  following the manufacturer’s recommendations. Expression of p16 was considered positive if strong and diffuse nuclear and cytoplasmic staining was detected in ≥70% of the tumor  Two headFFPE tissues were cut into five 10-μm thick sections on glass slides, and DNA was extracted using a commercial kit  according to the manufacturer’s instructions. The quantity and quality of extracted DNA was determined via NanoDrop ND-1000 spectrophotometry . Isolated DNA was stored at − 20 °C until further use.HPV DNA detection was carried out utilizing general primers GP5+/6+. Validation of the DNA quality and efficacy of the PCR was assessed using the housekeeping gene β-globin. DNA from HPV-positive uterine cervix tissue was used as a positive control and reactions without a template were run in parallel as controls. The amplified products were subjected to electrophoresis in 2% agarose gels and observed by nucleic acid dye. Tissues positive for HPV DNA were then further analyzed using a commercial HPV genotyping kit (Chaozhou Hybribio Limited Corporation) . This kip-values were two-sided, and p-values < 0.05 were considered statistically significant.All statistical analyses in this study were performed using SPSS 22.0 software . The chi-squared test or Fisher’s exact test were used to analyze categorical variables. The student’s t-test or Mann-Whitney U tests were used to analyze continuous variables according to application conditions. All In our study, 94.7% 108/114) of patients were from Eastern China and were mainly distributed in Shanghai, Zhejiang province, Jiangxi province, Jiangsu province, and Anhui province. The remaining patients were from other regions of China. The clinicopathologic data of patients are listed in Table 08/114 ofp = 0.014). In IP-SNSCC patients, only one of six HPV-positive tumors exhibited high p16 expression. The positive rates of  both HPV DNA and p16 in IP-SNSCC patients and DN-SNSCC patients were 3.3 and 2.4%, respectively. The statistical results are listed in Table Immunohistochemistry analysis showed p16-positive tissues in two IP-SNSCC patients 2/30, 6.7%) and 16 DN-SNSCC patients . Representative immunohistochemical staining of p16 is shown in Fig. /30, 6.7%The genotyping result of HPV-positive patients is shown in Table The ‘gold standard’ for transcriptionally active HPV detection in cancer patients is the analysis of HPV E6/E7 mRNA in a tumor specimen, but this becomes technically challenging when using low-quality mRNA from archival FFPE samples . For FFPTo the best of our knowledge, the present study is the largest study completed in China to detect the rate of HPV infection in SNSCC. The prevalence of HPV infection in SNSCC patients that was identified in our study is significantly lower than previous studies in the literature. Previous studies using strict testing methods and large samples found overall HPV infection rates of 20 to 26% in SNSCC patients , 17. A rHPV types are classified as high-risk or low-risk with respect to their tendency to cause malignant neoplasms. The most common high-risk type is HPV-16, which accounts for more than 80% of HPV-positive head and neck tumors . SimilarAs p16 immunohistochemistry is cost-effective and practical, it has been widely used to define HPV status. However, the association between HPV infection and p16 overexpression seems to be site-specific . Althougp = 0.014). However, only one of six HPV-positive IP-SNSCC patients exhibited high p16 expression. Lack of p16 overexpression, despite the presence of high-risk HPV DNA, indicates that viral DNA may be a bystander rather than a driver in IP-SNSCC progression. In agreement with our study, no transcriptionally active HPV has been previously reported in patients with IP-SNSCC [HPV has been implicated in the pathogenesis of IP and high-risk HPV types appear to increase the risk of developing SCC . HoweverIP-SNSCC . We specThe improved prognosis for HPV-positive oropharyngeal tumors has been well established. However, there is not sufficient evidence to demonstrate better survival rates with HPV-positive SNSCC. In our study, we were not able to analyze the association between disease prognosis and HPV status due to a limited number of HPV-positive patients. Although some previous studies have reported a better outcome for HPV-related SNSCC, others did not find a difference in survival , 34, 35.In conclusion, immunostaining for p16 is not a reliable surrogate marker of HPV status in SNSCC. The presence of HPV is rarely detected in DN-SNSCC patients in Eastern China. We found that the presence of HPV DNA was significantly higher in IP-SNSCC patients than in DN-SNSCC patients. However, IP-SNSCC patients frequently lack of p16 overexpression despite the presence of high-risk HPV DNA.