HPV16/18 detection may improve cervical cancer risk stratification and better guide which HPV-positive women warrant immediate colposcopy/biopsy. We estimated risks of cervical precancer and cancer by HPV genotype and cytology during the implementation phase of primary HPV testing in Norway.A total of 3111 women, aged 34–69 years, testing HPV-positive at baseline and undergoing cytology testing from February 2015 to April 2018 had data available for analysis. Risk estimates with 95% confidence intervals (95%CIs) of cervical intraepithelial neoplasia grade 3 or more severe (CIN3+) were estimated for cytology results and HPV genotypes .CIN3+ risks were higher for HPV16/18 than other high-risk HPV genotypes. Among women with any cytologic abnormality [atypical squamous cells of undetermined significance or worse], immediate risks were 57.8% (95%CI = 53.0–62.6%) for HPV16, 40.2% (95%CI = 32.3–49.2%) for HPV18, and 31.4% (95%CI = 28.7–34.3%) for other high-risk HPV. Among those with normal cytology, CIN3+ risks were 19.9% (95%CI = 15.0–26.1%) for HPV16 positives, 10.8% (95%CI = 5.6–20.5%) for HPV18 positives, and 5.5% (95%CI = 4.2–7.1%) for other high-risk HPV.The benefits and harms of managing women based on HPV positivity and cytology results can be better balanced by inclusion of HPV genotyping in screening and choosing more conservative management for other high-risk HPV compared to HPV16/18. HPV infection in women over 30 years old is more likely to be persistent, with a greater likelihood of the development of high-grade cervical intraepithelial neoplasia (CIN2/3) or cervical cancer.3Women with human papillomavirus (HPV) infections that persist for at least one5 HPV testing is also more sensitive than cervical cytology alone in detecting CIN2 or more severe diagnoses (CIN2+) and CIN3 or more severe diagnoses (CIN3+).8 However, primary HPV screening also detects transient HPV infections, which increases the number of positive screening results and potentially the number of colposcopies performed,9 the latter depending on the criteria for referral to colposcopy.Randomised controlled trials have demonstrated that primary HPV screening leads to a greater reduction in the overall incidence of cervical cancer compared to conventional cytology-based screening and it is now recommended to use HPV as a screening test for women over 30 years of age.10 This presents a major challenge to the limited colposcopy capacity in most healthcare systems.11 It is important to choose a colposcopy referral threshold based on the balance between benefits and harms.Particularly for HPV-positive women with cytology negative for intraepithelial lesion or malignancy (NILM) or low-grade cytology, over-referral to colposcopy can lead to a larger number of colposcopy/biopsy results negative for CIN2+.15 HPV16 and/or 18 infection are found in 70% of cervical cancers,16 and confer a higher risk than other high-risk HPV genotypes.18 Among women undergoing primary HPV screening, partial HPV genotyping in combination with cytology may better determine women who should be referred to biopsy compared to cytology alone. In a large US cohort study, women positive for HPV16 were at highest risk for CIN3+, followed by HPV18.19 Similar results were also found in a Japanese longitudinal study20 and a large clinical trial, ATHENA (Addressing the Need for Advanced HPV Diagnosis).21 However, observational studies and randomised controlled trials have specific enrolment criteria that may limit the applicability of results to whole populations. Such limitations include results reflecting subgroups of populations and possible under- or over-estimation of outcomes due to censoring and aggressive follow-up.22 Risk assessments using real-world or population-based data is needed to ensure “equal management for equal risk” for referral to colposcopy for populations with different patterns of care, compliance, testing, rescreening intervals, and risk factors.23 Consequently, national screening programs should evaluate and calibrate their screening algorithms based on accepted cervical cancer risk threshold for referring to colposcopy and based on the population’s real-world screening data and capacity.The optimal triage protocol for HPV-positive women in routine clinical setting is not yet determined but cytology is currently the general standard. The risk of CIN2+ and CIN3+ varies among oncogenic HPV genotypes.The Norwegian Cervical Cancer Screening Program (NCCSP) requires data generated within Norway using a real-world population base to modify national screening guidelines. This study aimed to estimate CIN3+ and CIN2+ risks by combinations of HPV16, HPV18, and other high-risk genotypes and cytological results among women in the Norwegian cervical cancer screening population.24 An exfoliated cervical specimen was collected during pelvic examination using a routinely available collection devices, usually a dry brush for collecting exfoliating cells, which was then rinsed immediately after collection into a ThinPrep  for HPV and liquid-based cytology (LBC) testing. Vials were then transported to one of three laboratories for pre-processing and evaluation. All participating laboratories followed a set of guidelines, approved by the Academic Panel for HPV screening implementation in Norway and described in the Quality Assurance Manual for the NCCSP, for allocation, conduction and follow-up of both screening strategies to unify implementation routines.Women aged 34–69 years and living in four Norwegian counties , were pseudo-randomly assigned by birthday to undergo 5-yearly primary HPV screening or continue to receive 3-yearly cytology screening , from February 2015 until April 2018.24 Briefly, the real-time polymerase chain reaction-based method detects 14 HPV genotypes. Results are reported separately for HPV16 and HPV18. The other 12 types  are reported concurrently as a pooled result referred to as other high-risk HPV.26HPV testing was conducted utilising the cobas 4800 HPV test  as previously described.0, the beginning of the observation period for each subject , low-grade squamous intraepithelial lesion (LSIL), atypical glandular cells of undetermined significance (AGC), atypical squamous cells-cannot exclude HSIL (ASC-H), high-grade squamous intraepithelial lesion (HSIL), adenocarcinoma in situ (AIS) and cervical carcinoma. If the cytology result was ASC-US or more severe interpretation, “immediate” colposcopy and biopsy were recommended.For HPV-positive women, a baseline cytological evaluation of the cervical sample was performed at T28If the baseline cytology test was NILM, then a 12-month follow-up period was recommended prior to repeating the HPV testing. If the repeat HPV test was positive, women were referred for colposcopy with biopsy. If the repeat HPV test was negative, then women return to routine screening and scheduled to be re-screened by HPV testing in 5 years.A unique identification number, allowing identification of every legal resident in Norway, was used to link the information on cytology, HPV tests and histology at an individual level. Relevant screening results were extracted from the databases at the Cancer Registry of Norway for 92,076 women aged 34–69 years who underwent primary HPV screening with no recorded abnormal HPV-tests, cytology or histology (CIN2+) the last two years before the baseline screening test Fig. . A total31 Histological diagnoses endpoints were CIN2+ and CIN3+ according to WHO guidelines.32The Cancer Registry of Norway receives reports of cancer and precancerous lesions with compulsory central registration of cervical cytology, histology, and all HPV test results (both positive and negative), as previously described.Follow-up started at the date of the baseline cytology test and continued for all women until the worst histologically confirmed lesion was identified. All other observations were censored after the end of recommended follow up (9 months for HPV-positive and ASC-US or worse results or 21 months for HPV-positive and NILM results), which was according to NCCSP guidelines  for those referred immediately to colposcopy and the subsequent CIN3+ risk for those undergoing follow-up HPV testing for a second time after 12 months, as per NCCSP. In the follow-up after 12 months, 222 (17%) out of 1278 HPV-positive and NILM women did not come to the second round of screening. We also compared risks for younger (35–43 years) and older (44–69 years) women stratified by the median age of the screened women, using the log-rank test of equality for failure functions to test for significant differences by age.42 All statistical analyses were done using STATA statistical software: release 15 .CIN3+ and CIN2+ risks with 95% confidence intervals (95%CI) for each screening HPV genotype/cytology result combination were estimated using the Kaplan–Meier failure function.Ptrend < 0.001). Older women were more likely to have a NILM cytology than younger women (P < 0.001).HPV genotype and cytology counts are given for all HPV-positive women, overall and stratified by median age Table . Categorn = 745) and CIN2+ risk was 30.0%  (data not shown).The overall cumulative CIN3+ risk after testing HPV-positive, regardless of cytologic interpretation or HPV genotype, was 23.9%  cytology was 38.2% (95%CI = 35.8–40.6%).CIN3+ and CIN2+ risks for pairwise combinations of HPV and ≥ASC-US categories are shown in Table n = 457) compared to older women (44–69 years)  (P < 0.001). Figure P < 0.001) and HPV16-positive ASC-US  and LSIL  (data not shown).Stratified on the median screening age of 44 years IQR: 38–52 years), overall CIN3+ risk was higher for younger women (34–43 years)  for HPV16, 10.8% (95%CI = 5.6–20.5%) for HPV18 and 5.5% (95%CI =  4.2–7.1%) for other high-risk HPV  re-tested HPV positive in a year. Among those who repeatedly tested HPV-positive, most  were positive for the same HPV genotype. Twelve-month CIN3+ risks were 29.5% (95%CI = 21.8–39.3%) among women who twice tested HPV16 positive, 15.6% (95%CI = 6.8–33.5%) among women who twice tested HPV18 positive, and 10.2% (95%CI = 7.7–13.6%) among women who twice tested positive for other high-risk HPV (Table 9.0% re-tP < 0.001) . When stratified for age, the results among HPV genotypes upon second HPV testing also were only significantly different among women who were overall HPV positive and women (P < 0.001) who were other high-risk HPV positive (P < 0.001). All other persistent HPV genotypes had P-values greater than 0.05 (data not shown).Among women with an HPV-positive/NILM result at baseline, those aged 34–43 years had a higher CIN3+ risk overall  than women aged 44–69 years   and low-grade cytology (ASC-US or LSIL) with surveillance.28 The switch to the more sensitive HPV test as the primary screening test can lead to over-referral to colposcopy, again leading to increased likelihood of morbidity.45 Although an HPV positive result alone does confer a high risk of cervical precancer and cancer compared to a negative result, not all HPV genotypes are equally likely to persist46 or develop into precancer or cancer.10In Norway, cervical cancer management has been based on results from cervical cytology primary screening.47 These differences might explain variation in reported proportion of those with ASC-US+ cytology (range 27–46%).50 Inter-laboratory differences for cytology results, are commonly observed in Norway, suggesting further that locally agreed diagnostic criteria and routines are applied.51 HPV testing in screening, however, demonstrated an excellent agreement between labs,24 suggesting wider application of molecular testing in the follow-up. Our study demonstrate that HPV genotyping provides further risk stratification and reduces an over-referral of all women who have an HPV positive and low-grade or NILM cytology result to colposcopy and biopsy.To increase the accuracy of the HPV screening, a cytological evaluation of HPV positives with divergent clinical management at threshold ASC-US+ has been recommended internationally. Although the Bethesda classification provide criteria for cytological definitions universally, systematic differences between pathology reviewers and countries has been reported in replicating cytological results.To avoid an over-referral of all women who have an HPV positive and low-grade or NILM cytology result to colposcopy and biopsy, HPV genotyping provides further risk stratification.53 With cytology testing included, the Portland-Kaiser study, a large cohort of ~20,000 women, found that HPV16 had a 2.7 times higher risk of precancer and cancer compared to other HPV genotypes across all cytology results.54The present analysis of HPV genotypes show consistent results with the POBASCAM and ATHENA trials, which showed higher precancer and cancer risk among HPV16/18 positive women (without cytology testing).55 It must be noted that, in this analysis, sample sizes among women testing for two different HPV genotypes upon repeat testing were small [i.e. HPV18 followed up by other high-risk HPV positivity (n = 4)]. Thus, these findings require replication in larger populations.Among women with baseline cytology of NILM and an HPV genotype of HPV16, 18, or other high-risk HPV type, women who continue to test positive for the same HPV genotype upon two screening rounds had an elevated risk compared to those with whole sub-cohort of women with HPV-positive and NILM cytology result at baseline. CIN3+ risk was highest for women with persistent HPV16 positive results (30%) and lowest for persistent other high-risk HPV-positive results (10%), indicating that the most of the 14% CIN3+ risk for women with NILM cytology and repeat HPV-positive test is due to persistent HPV16 infection. Similarly, an English pilot study found that HPV 16/18 triage of persistently high-risk HPV-positive and cytological negative women 12 months after primary screening added very little in terms of a clinical benefit such as additional detection of CIN2+ after baseline HPV genotyping.56 uses a screening (HPV genotype and cytology) result as a benchmark for clinical management decisions for HPV-positive women. Based on real-world screening results from the NCCSP, the risk threshold for a colposcopy referral among HPV-positive women has been equivalent to the risk for other high-risk HPV and LSIL result or ~37.3% for CIN2+ and 24.4% for CIN3+. Women above this threshold, such as those with high-grade cytologic abnormalities or low-grade cytologic abnormalities and test HPV16 or HPV18 positive, should immediately be referred to colposcopy and biopsy. Women with one of these risk markers, other high-risk HPV positive with low-grade cytologic abnormalities or test HPV16 or HPV18 positive with NILM have a risk for CIN3+ below 24.4% and can be re-tested in 12 months. Women with neither marker, i.e., NILM cytology and positive for other high-risk HPV, can be retested in 24 months as they have less than half the precancer and cancer risk of women with one of these risk markers.The conclusions drawn from the genotype results for the Norwegian HPV screening implementation provide the basis for “equal management for equal benchmark risk” approach as performed in the US and certain European countries. This risk-based approach56 It must also be noted that the threshold at which the risk of cancer in an individual patient outweighs any disadvantage of management and treatment varies according to the patient’s characteristics and local service considerations. In a country like Norway, with an established screening program and with high follow-up of screen positives, the threshold for colposcopy referral and treatment may be set higher safely, due to the relatively low risk among women with negative test results. Taking these factors under consideration, these results should be interpreted as a basis for the incorporation of HPV genotyping into primary screening as well as a rationale for risk reductions following over-referral of women to colposcopy and biopsy after primary HPV screening using real-world screening data.This analysis focuses on immediate risks of CIN3+ by HPV genotype, including results for women with HPV+ and NILM cytology on their baseline test that are retested after one year. The negative predictive value of this follow-up regimen can be confirmed in long-term follow-up only. The relative rank order between the risk groups observed in this study is consistent with those HPV genotype/cytology screening results reported elsewhere.57 It has also been shown that precancer and cancer risk for women with an NILM cytology and HPV-positive result is 5.9%, making the likelihood of undetected precancer and cancer in a subset of these women quite low.49There were several limitations. First, there was only a short follow-up time for this cohort. Second, estimates of cervical disease (CIN2+ and CIN3+) were calculated based on women who underwent concurrent colposcopy and biopsy. Women with an NILM cytology and HPV-negative result are not typically referred to colposcopy and biopsy and therefore, their cervical histology status is not known. Women who do undergo immediate colposcopy and biopsy with an NILM cytology result, which is against NCCSP and most other cervical cancer screening guidelines, likely represent a biased sample.58 Further, data were collected uniformly, and laboratory tests were performed within an organised cervical cancer screening program with relatively high-population coverage. Results of this analysis can, therefore, be generalisable to a larger population-based cervical screening program, which is a prerequisite for advocating population-wide cancer screening management by HPV genotype. While not generalisable to certain other populations with different patterns of care, compliance, testing, rescreening intervals, or risk factors,12 the results of this study have altered the management protocol for HPV-positive women living in Norway,59 providing an example of using population-based screening data as a basis for which to implement screening (Supplementary Fig. Despite these limitations, this study has notable strengths. This is the first study to investigate the role of HPV partial genotyping and cytology in an HPV-based screening programme in Norway. Norway has a population-based registration system of cytology results and biopsies and all results are included even if women chose to attend private healthcare. This system reduces loss to follow-up risk and selection biases and has shown to be highly reproducible.59In conclusion, we found that HPV16 and HPV18 have a higher risk for CIN3+ and CIN2+ and partial HPV genotyping, in combination with cytology results, can better differentiate HPV-positive women at higher and lower risk for cervical precancer or cancer than cytology alone. This is crucial for women who are at too high risk for surveillance alone, such as those with HPV16/18 and low-grade cytology, while limiting unnecessary colposcopy and biopsy for women with lower precancer or cancer risk, such as those positive for other high-risk HPV genotypes and low-grade cytology. These findings provided the evidence for and adoption of the use of partial HPV genotyping for the management of HPV-positive women aged 34–69 years in Norway.Supplementary material


Pseudomonas aeroginosa bacteremia secondary to acute folliculitis and epididymo-orchitis. He was given methylene blue for reversal of septic shock but he did not respond and expired. Autopsy findings were significant for bluish-green discoloration of organs, especially the heart, lungs, and brain during prosection secondary to methylene blue treatment. It is important to recognize artifacts of treatment and to discern them from changes due to putrefaction or the classic green pigmentation associated with Pseudomonas aeruginosa infection, such as chloronychia. The case report illustrates that circulating methylene blue and its metabolites can accumulate in the organs in a dose-related fashion, imparting an interesting turquoise to dark blue-green pigment during the autopsy. Additional studies are warranted to enable pathologists to differentiate among the pigmentation associated with Pseudomonas aeruginosa bacteremia, putrefaction, and methylene blue treatment.Septic shock can result from the dissemination of infections and can lead to hypoperfusion secondary to vasodilation. Methylene blue can help stabilize blood pressure refractory to other measures in shock. We report a case of a 58-year-old male who died of septic shock due to  Septic shock has a mortality rate of around 20%. It is the most common cause of death in intensive care units (ICU). The outcomes of septic shock vary depending on the extent of the infection and the presence of comorbidities. It is the result of a systemic immune response to infections. In response to the infectious insult, inflammatory mediators cause systemic vasodilatation, manifesting as low blood pressure, leading to tissue hypoperfusion and the vascular congestion seen histologically . AnotherMethylene blue has been used therapeutically dating back to the late 18th century for the treatment of conditions ranging from malaria to carbon monoxide poisoning, and even as a urinary analgesic. Modern use revolves around its mechanism as a reducing agent in the treatment of methemoglobinemia; however, there is also experimental and clinical evidence supporting methylene blue as a treatment for shock refractory to fluid administration, vasoconstrictors, and inotropic agents because it has shown to induce systemic and pulmonary vasoconstriction through its action as a selective inhibitor of guanylate cyclase, a second messenger involved in nitric oxide-mediated vasodilation in patients with septic shock, without a significant decrease in cardiac index -4.Pseudomonas aeroginosa. He was intubated and required multiple vasopressors and broad-spectrum antibiotics. Methylene blue was also used to elevate his blood pressure approximately 27 hours prior to death. Due to the poor prognosis, the family chose to pursue comfort care measures only, and the patient expired shortly thereafter due to septic shock.A 58-year-old male with a history of congestive heart failure, hypertension, type II diabetes mellitus, recurrent deep vein thromboses (status post inferior vena cava [IVC] filter placement), pulmonary embolism, and hypothyroidism was admitted with neurologic symptoms in May 2020 related to a T4/T5 mass. Clinical features included bone pain, recurrent infection, spinal cord compression, and peripheral neuropathy. He underwent laminectomy and tumor debulking in June 2020, during which a biopsy of his lesion was taken that revealed a plasmacytoma. Bone marrow biopsy showed no definitive evidence of a monoclonal plasma cell population, but the patient met the criteria for multiple myeloma (plasmacytoma and lytic lesions) and was started on multiple chemotherapeutic agents. After developing pancytopenia and fever post-chemotherapy, he developed septic shock that manifested as lactic acidosis, disseminated intravascular coagulation (DIC), renal failure, and elevated transaminases. He was transferred to the ICU. Blood and scrotal wound cultures were sent that grew An unrestricted autopsy was requested by the family and treating clinicians. The post-mortem examination revealed a full-thickness scrotal ulcer, and an edematous, hemorrhagic left testicle. Gross findings also included superficial sacral decubitus ulcers, severe calcific coronary, and systemic atherosclerotic cardiovascular disease, cardiomegaly, pulmonary edema, bilateral pleural effusions, and thromboemboli entangled in the IVC filter Figure , 1B. HisPseudomonas aeroginosa bacteremia secondary to acute folliculitis and epididymo-orchitis (perforated/ulcerated scrotum with areas of hemorrhagic necrosis)  Figure . This diShock is the result of several potentially lethal clinical entities, including infections, trauma, and cardiovascular collapse. Clinically, shock manifests as systemic hypoperfusion due to either decreased cardiac output, reduced circulating volume, or vasodilation, which results in eventually irreversible tissue injuries in multiple organ systems . Our patPseudomonas aeruginosa diagnosis depends on laboratory identification and isolation on media. It grows well on most laboratory media and is commonly isolated on blood agar plates or eosin-methylthionine blue agar. It is identified as a gram-negative lactose non-fermenter with a positive oxidase reaction, a fruity odor, and its ability to grow at 42°C [Pseudomonas aeruginosa colonization. It can be confusing to differentiate the discoloration caused by two entirely separate etiologies, i.e. methylene blue and Pseudomonas aeruginosa. at 42°C ,9. It isOur patient was immunocompromised and acquired the infection post-chemotherapy. In short, his multiple myeloma treatment contributed to the septic shock by making him more susceptible to otherwise manageable infections. A plasma cell myeloma is a clonal proliferation of plasma cells or their precursors causing tumor formation, excess immunoglobulin, and osteolytic bone disease. The median survival is three years using standard therapy, and five years with dose-intensive therapy and stem cell transplantation .Pseudomonas aeruginosa infection associated green pigmentation.Methylene blue accumulation in organs in a dose-related fashion can give interesting turquoise to dark blue-green shades during the autopsy. Therefore, it is essential to identify these features of methylene blue treatment and to distinguish them from changes due to putrefaction or


Background: Cervical cancer is one of the most common cancers in women and about 90% of cervical cancer can be reduced by regular screening. The Pap smear has been well in place as a primary cervical screening method since 1950s; however, coverage is still not optimal. This study explored the feasibility of HPV self-sampling in two under-screened population groups in Hong Kong (HK): never screened and not regularly screened females, to estimate the uptake rate and preference rate in the future. Materials and Methods: This was a cross-sectional study to explore the acceptability and feasibility of HPV self-sampling in two age groups: aged 25–35 and aged ≥45, which were reported as the highest proportion of the under-screened population in HK between 2017 and 2018. The study invited eligible women from an HPV study cohort to perform HPV self-sampling at home by themselves. The number of specimens returned from participants was recorded and used to determine the feasibility of HPV self-sampling in the community. The participants were asked to fill in the questionnaires before and after HPV self-sampling to indicate their attitudes, acceptability, and future preference for HPV self-sampling as an acceptable alternative primary cervical cancer screening method. Results: A total of 177 subjects participated in the present study and have achieved a good overall uptake rate of 73% (129/177) who returned the self-collected cervicovaginal sample for HPV testing. Among the under-screened population, there was a higher response rate in aged ≥45 than those aged 25–35. The findings also revealed that women who were under-screened, including those who have never been screened, were more likely to prefer HPV self-sampling than those who had regular screening. This study found that the acceptability of HPV self-sampling was fairly positive among the respondents. The findings also indicated that HPV self-sampling was not only beneficial to enhance their health awareness but also to promote the cervical cancer screening uptake rate, especially among the under-screened or never screened populations. Conclusions: HPV self-sampling would be a solution to overcome the perceived barriers in clinician-based screening. The findings also indicated that it could be feasible to use as an alternative primary cervical cancer screening. According to the World Health Organization, cervical cancer is one of the most common cancers in women and about 90% of cervical cancer can be reduced by regular screening. The association between Human Papillomavirus (HPV) and cervical cancer is well established. Due to the slow growing nature of HPV, cervical cancer is regarded as highly preventable and treatable if it can be detected earlier . The PapWith the emerging of supportive findings in the causal relationship between persistent infection of high risk HPV and the development of cervical cancer, additive testing for HPV was implemented as co- or standalone- testing so as to increase the sensitivity and specificity of population-based cervical screening ,11. To oIn HK, the Guidelines for Cervical Cancer Prevention of The Hong Kong College of Obstetricians and Gynaecologists were revised in 2016  by addinThis was a cross-sectional study to explore the acceptability and feasibility of HPV self-sampling in two age groups: aged 25–35 and aged ≥45, which were reported as the highest proportion of under-screened population in HK between 2017 and 2018. It was an ongoing community-based project to promote using HPV self-sampling as a cervical screening tool in the community and invite women to do HPV self-sampling by themselves at home. If the participants agreed, their uptake rate of HPV self-sampling and cervical cancer screening records would be tracked in subsequent years. This paper aimed to report the return rate and feedback of the participants after the first attempt of conducting HPV self-sampling in the study population.Participants were recruited from two different sources for corresponding types of the under-screened population. First source: women aged 25–35 were recruited from graduates of one of the selected local universities through the HPV Vaccination Campaign Registry or their peer network. A total of 1328 HPV-vaccinated female registered with the campaign were approached by a telephone survey using a structural questionnaire with a response rate of 49% (651/1328), in which 117 participants indicated their interest to perform the HPV self-sampling. Upon agreeing to participate in the study, participants were given guidance on the use of HPV self-sampling kits by phone first and an HPV self-sampling package was sent to their mailing address for self-completion. A complete written informed consent and a post-questionnaire survey were returned, together with the self-collected samples once they had performed the HPV self-sampling. Second source: women aged ≥45 were recruited through community centres in Hong Kong. Most subjects recruited from this group would have not received the HPV vaccination as the vaccine was not available until 2014 in Hong Kong. With the facilitation of local district officers, interested participants from the community were invited to join the study with a face-to-face interview and a take home HPV self-sampling package was provided at the end. Both study groups were also presented with a link to video instructions to perform the self-sampling at home.Due to the applicability to perform the HPV self-sampling, women who were pregnant, or possibly pregnant; with hysterectomy; with cancers of the reproductive system; with exposure to hormonal therapy (long-term steroids user); and with immune function disease or on immune therapy were excluded from the studies. Immunocompromised is considered to be the prerequisite for disease progression in such a way that it essentially facilitates persistent hr-HPV infection and cervical lesion progression. In order to make a generalization of the populations, those who are almost certain to acquire HPV infection were excluded from the study.® Brush  was the self-sampling brush device used in the study to collect a cervicovaginal sample for subsequent high-risk HPV testing. The instructions described how to insert the brush device into the vagina to a depth of at least 2 inches and make 3–5 full rotations, then, remove the device from the vagina and place the cap back on before putting inside the packaging. The instructions also suggested the method of returning the specimens using the pre-stamped self-addressed envelope to our office. For those participants who did not want to mail back their specimens, there was an option for them to return them to our research team in person at the community clinic.For HPV self-sampling, the consented participants were provided with the self-sampling package either on the recruitment site or by mailing upon the request of participants. The package included a HPV self-sampling kit, written instructions for use in Chinese with diagrams, and a verbal explanation so that the materials and procedure of self-sampling were fully understood. The EvalynThe self-collected cervicovaginal samples were transported to the laboratory for HPV testing with DNA extraction using the QIAamp DNA Mini Kit . Two recThe number of specimens returned from participants were recorded and used to determine the feasibility of HPV self-sampling in the community. The participants were asked to fill in the questionnaires before and after HPV self-sampling to indicate their attitudes, acceptability, and preference for HPV self-sampling as an acceptable alternative primary cervical cancer screening method in the future.All participants completed the baseline standardised questionnaire, which included information on age, sex, lifestyle risk factors , frequent sexual activity, and no use of contraceptive method), and cervical cancer screening behaviour. Laboratory results of genotypes of HPV self-sampling including high risk (hr) and low risk (lr) were also reported.p-value < 0.05 was considered to be statistically significant.Descriptive statistics (numbers and proportions) were used to report the socioeconomic characteristics of the women who received the HPV self-sampling in the studies. The response rate (uptake rate) of HPV self-sampling (specimens successfully returned for laboratory testing) in each age group and the overall and subgroup proportions according to their cervical cancer screening behaviour were reported. The prevalence of HPV infection with different genotypes (HR or LR) of HPV was reported. Furthermore, to determine if the uptake rate of HPV self-sampling was modified by the socioeconomic status and lifestyle risk factors, both univariate and multiple regression models were applied. Chi-square test or Fisher’s exact test and the t-test or ANOVA were used when comparing categorical and continuous variables, respectively. Univariate logistic regressions were further applied to study the adjusted association of socioeconomic status and lifestyle risk factors  if necessary. All analyses were performed using IBM SPSS Statistics Version 24   and p-vaThe study was conducted in accordance with the Declaration of Helsinki and ethical approvals were obtained from The Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee (The Joint CUHK-NTEC CREC). For the respondents who agreed to participate in the study using the self-collected HPV testing, written consent was obtained. All the respondents were informed of the study purpose, study details, research procedures, and its potential risks and benefit, and the right of refusal or withdrawal from the study. All information collected from respondents were treated with strict confidentiality.A total of 177 participants were recruited between September 2016 and September 2017 for those aged ≥45 (60 cases) and between March 2018 and November 2018 for those aged 25–35 (117 cases). The demographic characteristics of the two groups are shown in Among the 177 participants, those who showed initial interest in HPV self-sampling were invited to perform HPV testing at home by taking the self-sampling package away from the recruitment site/mailing to their address. At least three reminders were sent via telephone if the participant had not returned their HPV specimen. The most common reasons for not returning included having no time, not confident in the sampling procedure, and some might have simply failed in their attempt to collect the sample. The overall response rate of HPV self-sampling among the participants was 73% (129/177). The response rates among the two age groups were similar—74% for aged 25–35, while 72% for aged ≥45. The uptake rate was considerably higher among those who had never been screened or were under-screened  than those with regular cervical cancer screening . The details are shown in HPV infection was detected in 3 of the 129 respondents (2.3%). The overall prevalence of HPV infection among women aged ≥45 was 4.7% (2/43), which was higher than those aged 25–35 . Among the three HPV-positive cases, they were all either under-screened or never screened. Furthermore, the prevalence of hr-HPV genotype infection among respondents ≥45 was 100% but none was detected among women aged 25–35 (only the lr-HPV genotype was found). However, the low hr-HPV prevalence among the younger age group may be due to the protection of the HPV vaccination.The results showed that only having religious belief was significantly associated with the HPV self-sampling uptake among those aged 25–35 years. Overall, women who were under-screened or never screened with the Pap smear  were generally more likely to return the HPV self-sampling kit than those had regular Pap screening . In particular, under-screened/never screened women aged ≥45 were more likely to uptake HPV self-sampling, whereas women aged 25–35 had a higher uptake rate among those who were regularly screened than under-screened. No statistical significance was detected in either under-screened/never screened women or those with regular screening. The details are shown in The acceptability of HPV self-sampling was measured in five domains using the post-questionnaire: (1) ease of use; (2) trusting the test results; (3) handling procedure; (4) beneficial to health; and (5) convenience. Overall, more than half of the respondents gave a positive response  in all five domains. The majority of respondents reported that it was convenient (86% and 95%) and beneficial to health (94% and 95%) among both aged 25–35 and aged ≥45, respectively. However, there was no statistical difference between the two groups. The details are shown in p > 0.05) between the proportions of HPV self-sampling preference among the two study groups. The overall preference for HPV self-sampling with different screening patterns among those who had completed the follow-up study is shown in In the overall study population, the proportion of preference towards HPV self-sampling as an alternative primary screening for cervical cancer was 69% (86/124). For analysis, options with not able to decide between the Pap smear and HPV self-sampling or preferred both were also included in the preference for HPV self-sampling in the present study. Preference among age groups was presented in This is the first study to explore uptake rate of HPV self-sampling and its acceptability and feasibility among the under-screened population in HK. This study’s findings report an encouraging uptake rate of HPV self-sampling with 73%, considerably higher in the under-screened/never screened population, in particular, among aged ≥ 45. This study found that the acceptability of HPV self-sampling was very positive among both women aged 25–35 and aged ≥45, indicating that it could be a solution to overcome the barrier of the Pap smear and feasible to use as an alternative cervical cancer screening to increase the coverage of cervical cancer screening. High response rate was observed in under-screened and never screened populations, indicating that the acceptability and feasibility of HPV self-sampling as an alternative primary screening are promising in Hong Kong. Our findings align with other jurisdictions that focused on the feasibility and evaluation on HPV self-sampling in cervical cancer screening due to its high sensitivity, high acceptability, and cost-effectiveness in health settings ,25,26. IIn our study, religious belief was significantly associated with the high uptake rate of HPV self-sampling among younger women. In other words, religion significantly determined knowledge and prevention behaviour that influenced their decision in the uptake of cervical cancer screening. As not many studies have explored factors associated with the uptake of HPV self-sampling but Pap smear testing , it wasPerceptions and experience of HPV self-sampling were evaluated in five domains, including ease of use, handling procedure, trusting the test results, beneficial to health, and convenience, which all have been considered as key attributes of acceptability . OverallAmong the under-screened women with preference for HPV self-sampling, a considerably larger proportion was found among those aged 25–35 than those aged ≥ 45, indicating that there was an age-related acceptability of HPV self-sampling in cervical cancer screening . Our resThe most common reasons for women not attending the clinician-based cervical cancer screening included fear of pain, lack of control, embarrassment, privacy concerns, and unpleasant experience of screening in the past . The preAccording to the latest WHO guidelines for the prevention and control of cervical cancer , HPV tesOverall, around 70% of the respondents reported preference for HPV self-sampling in our study, which is consistent or even more feasible than other studies, where the pooled estimate of women who preferred self-sampling was 59% . FurtherThis is among the first to conduct HPV self-sampling in the under-screened population in Hong Kong and is an on-going project to keep track of the uptake rate of HPV self-sampling. Such data provide support for evidence-based decisions in health policy making to incorporate self-sampling strategies into the organised cervical screening programme.Our study forms a basis for promoting cervical cancer screening and increasing the knowledge of HPV and cervical cancer. Lack of self-competency and testing anxiety arose with uncertainties while women tested in their homes, as HPV self-sampling was still fairly new to the majority of Hong Kong women, thus public education is required.A larger study is needed to improve the validity of the research and to provide results generalisable to the Hong Kong population.HPV self-sampling as an alternative primary cervical cancer screening is feasible and acceptable to under-screened women in Hong Kong. It could effectively increase screening coverage among those under-screened populations. Considering the local organised cervical cancer prevention programme in Hong Kong, the HKCOG Guidelines for Cervical Cancer Prevention and Screening  suggeste


People living with HIV/AIDS (PLWHA) have an increased incidence of anal squamous cell carcinoma. Since high-risk human papillomavirus (hrHPV) is the primary cause, hrHPV DNA testing may play an important role in anal cancer screening. This study aims to determine the negative predictive value (NPV) of hrHPV testing in PLWHA as well as factors that may lead to false-negative results.  Anal swabs were collected for cytology and Cobas® 4800 HPV test for 14 hrHPV types. Patients underwent concomitant high-resolution anoscopy (HRA) examination and biopsy. High-grade squamous intraepithelial lesions  detected in Cobas-negative patients were genotyped for 22 HPV types using BioPerfectus Multiplex Real-time PCR. p ≥ 0.02). Genotyping HSIL tissue derived from Cobas-negative patients revealed hrHPV (n=7), possibly carcinogenic HPV53, 67, 73, 82 (n=12), or absence of hrHPV (n=4).  156 PLWHA tested negative for hrHPV on anal swab samples . HRA-guided biopsy detected HSIL/AIN3 in 13 patients , HSIL/AIN2 in 5 patients (3%), low-grade squamous intraepithelial lesions in 82 , or benign findings in 56 (36%). No cancer was found. The HSIL group was similar to the LSIL/benign group regarding age, gender, race/ethnicity, clinical HIV parameters, cytological diagnoses, history of receptive anal sex, and smoking ( In this series, anal hrHPV DNA testing offered 92% NPV for PLWHA; in other words, a 8% risk of occult precancer remains for those who test hrHPV negative on anal swab samples. Human papillomavirus- (HPV) associated anal cancer has been on the rise with a projected incidence of 8,300 new cases and 1,280 deaths in the United States in 2019 , 5. AccoIn the case of cervical cancer screening, both exfoliative cytology and high-risk HPV (hrHPV) testing have proven largely successful . Given tBy contrast, recent research has demonstrated the important prognostic value of oncogenic HPV in anal carcinogenesis, particularly HPV16, reinforcing the role of hrHPV testing in anal cancer screening . StudiesNegative predictive value (NPV) is an important performance measure for screening tests, especially those used in populations with high disease prevalence . Tests wThe Mount Sinai anal dysplasia program, part of a large urban HIV clinic system, serves as a referral center for the diagnosis and treatment of PLWHA with HPV-associated anal precancer and cancer. According to current practice guidelines endorsed by IDSA, PLWHA with risk factors for anal cancer is screened by primary care or infectious disease physicians using anorectal cytology. If anorectal cytology is abnormal, defined as Atypical Squamous Cells of Undetermined Significance (ASCUS) or worse, patients are referred to the anal dysplasia program for high-resolution anoscopy (HRA) examination and biopsy. At the time of HRA, we routinely repeat anorectal cytology and hrHPV testing.After obtaining Icahn School of Medicine Institutional Review Board approval, we searched the HRA database from January 2015 to December 2018 for PLWHA who tested negative for hrHPV at the time of HRA and biopsy. The following demographics were collected from the electronic medical record: age, gender, race/ethnicity, sexual behavior, history of receptive anal sex, HIV status, CD4+ T-cell count, HIV-1 viral load, and smoking history.Anal swabs were performed using a moistened cytobrush to survey the anal canal mucosa from the anal verge to right above the squamocolumnar junction. Samples were preserved in liquid-based cytology medium for ThinPrep® Pap Test  and stained with the Papanicolaou stain. Cytopathologists from the Mount Sinai Hospital reported all cases using the 2001 Bethesda System: unsatisfactory (≤two nucleated squamous cells/high-power field); benign; ASCUS; low-grade squamous intraepithelial lesion (LSIL); atypical squamous cells, cannot exclude HSIL (ASC-H); and HSIL . RegardlAnal HSIL biopsy samples derived from Cobas-negative patients were genotyped using the BioPerfectus Multiplex Real Time (BMRT) PCR assay. DNA was extracted from biopsy samples using QIAamp DNA mini kit  according to the manufacturer's instructions. PCR was performed using the fluorescence-based multiplex HPV DNA genotyping kit  that is designed to identify 19 high-risk or possibly carcinogenic HPV types  and 3 low-risk HPV types  [First, anal swabbing was performed to collect samples for cytology and Cobas HPV test. Subsequently, HRA and biopsies were performed following standard protocols . After tThe authors YL and WZ independently reviewed H&E slides for all cases and confirmed the diagnoses. Both authors are specialized gynecological pathologists with more than ten years experience in diagnosing HPV-associated anogenital disease. Only those cases with consensus diagnosis were included in the study. p16 immunohistochemistry (IHC) was performed on a subset of lesions. For small lesions, p16 IHC was not performed in order to preserve sufficient tissue for DNA isolation and HPV genotyping.Differences in patient demographics between subjects with benign findings vs. LSIL vs. HSIL were compared using the chi-square test or Fisher's exact test for categorical or binary variables as well as the Wilcoxon test for continuous variables , as appropriate. All analyses were performed using STATA 15 .The study flow is shown in All Cobas-negative individuals underwent HRA examination and biopsy . Histological examination of biopsies revealed HSIL/AIN3 in 13 individuals , HSIL/AIN2 in 5 patients (3%), LSIL in 82 (53%), and benign findings in 56 (36%). In the HSIL group, 14 individuals had a solitary high-grade lesion, 3 had two high-grade lesions, and one individual had three high-grade lesions. In total, 23 biopsy-proven HSILs were identified including 13 AIN3 and 10 AIN2. Two experienced pathologists confirmed HSIL diagnosis for all cases through independent review. p16 IHC was performed on 8 lesions and revealed block-positive staining, supporting the HSIL diagnosis.p ≥ 0.02).Demographics were compared between the benign, LSIL, and HSIL groups . There wn=5) and HPV82 (n=5). Seven lesions were positive for hrHPV types included in the Cobas® panel . Twelve lesions were positive for possibly carcinogenic HPV types 53, 67, 73, and 82 classified as the International Agency for Research on Cancer (IARC) category 2B and not included in the Cobas® panel [The 23 HSILs derived from Cobas-negative patients were genotyped for HPV using BMRT assay. As shown in s® panel . Four leIn this retrospective study, using biopsy-proven anal HSIL/AIN3 as an endpoint, we found that the hrHPV DNA test had a NPV of 92% for PLWHA undergoing anal cancer screening at our clinic. Thus, there is a low, but nonnegligible, risk (8%) of occult HSIL/AIN3 for subjects who test negative for hrHPV on anal swab.Our cohort represents a high-risk population with known risk factors for anal HPV infection, including HIV infection, MSM, women with HPV disease at other genital sites, and a history of receptive anal sex. During the same study period, a total of 1,140 PLWHA had anorectal cytology and HPV contesting at our clinic. As anticipated, the majority tested positive for anal hrHPV . In this substudy, we focused on the smaller subset of patients (14%) who tested negative for anal hrHPV.In the context of cervical cancer screening, the Cobas® 4800 HPV test was reported to have a false negative rate of 0.7% in the multicenter, prospective ATHENA study . The HybAnal squamous cell carcinoma is the major histologic type, and over 90% of cases are associated with HPV infection . In analNotwithstanding such optimal outcomes, histological diagnosis is still the gold standard and provides definitive evidence of disease. While HSIL generally entails persistent oncogenic HPV infection, HPV clearance can occur spontaneously and thereby account for a subset of negative HPV results. In the SUN study, anal HPV16 clearance and HPV18 clearance were 31% and 60% at 48 months in MSM, respectively . Apart fThe first cause relates to anal HSIL induced by HPV types that are not routinely screened for. Twelve of our high-grade lesions tested positive for HPV53, 67, 73, or 82 . Unlike the WHO-defined hrHPV types , these HPV types are currently designated as “possibly carcinogenic,” falling within the IARC Group 2B due to their low prevalence in cervical cancer (≤1% each) and the lack of biological data . AlthougSecondly, patients with solitary and localized HSILs are likely to have negative HPV results via anal swab. Since anal swabbing is performed blindly, the large, corrugated anal canal surface is difficult to survey thoroughly; it is therefore easy to miss small, localized lesions. These factors could explain why seven of our false negative cases (patients 1–7) tested hrHPV negative on anal swabs but positive for hrHPV on biopsy specimens. All seven patients had only one localized high-grade lesion presumably missed by anal swabbing.The third cause pertains to the methodology of HPV testing. Both the Cobas®4800 system and BMRT PCR assays are designed to target the HPV L1 gene, a region that can be lost during integration of viral DNA into host genomic DNA . ConsequThe strength of our study is that concomitant HPV testing and HRA-guided biopsy allowed for analysis of false negative HPV results. The study is limited in that random biopsies were not performed; as a result, disease burden may have been underestimated. Furthermore, as our cohort represents a population receiving effective antiretroviral therapy at a specialized HIV clinic, it may not be generalizable to patients in less rigorous clinical settings.In conclusion, we found that anal HPV testing offered 92% NPV for PLWHA, in line with other comparable studies. There remains a low, but not negligible, risk (8%) of occult precancer. Our results should aid in the implementation of HPV testing in anal cancer screening programs. Future studies are needed to explore the optimal use of HPV testing whether alone, as cotesting with cytology or as a reflex test.


INK4a immunohistochemistry. Data were collected from medical records. Factors associated with HPV positivity and time trends were evaluated with multivariable Bayesian models. The adjusted prevalence of HPV-related cases in 864 patients with a valid HPV-DNA result was 9.7%, with HPV-DNA/p16INK4a double positivity being considered. HPV-related oropharyngeal cancer was likely to occur in non-smokers and non-drinkers, to be located in the tonsil or diagnosed at advanced stages. Time-trend analysis showed an increasing risk of HPV-related oropharyngeal cancer in the most recent periods (5-year period increase of 30%). This increase was highest and with a clear increasing trend only in the most recent years (2012–2016). The prevalence of HPV-related oropharyngeal cancer started to sharply increase in the most recent years in our setting, as occurred two decades ago in areas where most oropharyngeal cancer cases are currently HPV-related. Our results provide a comprehensive assessment of the epidemiological landscape of HPV-related oropharyngeal cancer in a region of southern Europe.The incidence of human papillomavirus (HPV)-related oropharyngeal cancer is increasing in some regions. Nevertheless, the epidemiology of this disease has not been extensively investigated in southern Europe. We conducted a retrospective cohort study of patients diagnosed with primary oropharyngeal cancer from 1991 to 2016. Cancer tissues underwent histopathological evaluation, DNA quality control, HPV-DNA detection and p16 Currently, 29,000 new HPV-related OPC cases  are estimated to occur every year worldwide2, with marked differences between geographical regions. HPV-related OPC is increasing in some regions of the world such as North America3 and northern Europe7, where most OPC cases are currently HPV-related. The increasing trends are thought to be related to a decrease in smoking and increased numbers of sexual partners and oral sex practices9. The role of HPV in OPC has important clinical implications given the distinct nature and marked prognostic differences between HPV-related and non-related patients9. Nevertheless, the epidemiology of the disease in southern Europe, where current estimates of HPV prevalence are low (9–24%)10, has not been extensively investigated. In Spain, only two studies on HPV involvement in selected OPC cases were available12 until our group recently reported the results of a multicenter retrospective cohort of 788 patients with primary OPC consecutively diagnosed between 1991 and 201313. The study identified double positivity for HPV-DNA/p16INK4a as the biomarker with strongest diagnostic accuracy and prognostic value for HPV-related OPC patients and found an HPV prevalence of 7.4% when considering HPV-DNA/p16INK4a double positivity13. A recent Italian study also evaluated the role of HPV in patients with newly diagnosed OPC during the period 2000–2018, reporting a prevalence of HPV-driven OPC of 32.3% and a higher prevalence in the most recent years14.The epidemiology of oropharyngeal cancer (OPC) has dramatically changed in the last two decades after the incursion of human papillomavirus (HPV) in the etiological arena of the diseaseIn this study, we aimed to provide an updated assessment of the time trends of HPV prevalence estimates in OPC, as well as to evaluate further its epidemiological features compared with other high-burden regions.We designed a retrospective cohort study of all new patients diagnosed with primary OPC in four hospitals in Catalonia (Spain) from 1991 to 2013 . We also included all patients diagnosed with primary OPC in the latter setting through 2016.10. Briefly, cases were identified from medical records/pathology reports of the centers of origin. We included cases fulfilling the following criteria: diagnosis of primary invasive cancer of the oropharynx, any histology, codes from the International Classification of Diseases for Oncology version 3: Tonsil-C09, C02.4, Base of tongue-C01 and Others ; and available access to medical records on demographic, toxic habits and clinical and follow-up information. Formalin-fixed paraffin embedded (FFPE) primary tumour samples from the diagnosis prior to treatment were retrieved when available.The procedures followed have been previously describedINK4a expression, which was performed at Hospital General de l’Hospitalet (Spain) under the manufacturer’s standards . A strong and diffuse nuclear and cytoplasmic staining of > 70% of the cancer tissue was considered p16INK4a-positive15. Since our previous study showed high diagnostic accuracy and prognostic value for double positivity for HPV-DNA/p16INK4a13, which is an easier diagnostic algorithm to implement in clinical practice than HPV-mRNA testing, we did not further use HPV-mRNA positivity for the analyses herein presented. A block was classified as adequate for HPV testing if invasive cancer was observed in the two H&E stained sections of the specimen.All specimens processing was centralized at the Catalan Institute of Oncology (ICO). FFPE blocks were re-embedded whenever necessary. The first and last sections were used for histopathological evaluation after hematoxylin and eosin (H&E) staining. Two in-between sections were used for HPV-DNA testing and genotyping; one additional slide was obtained to assess p16INK4a results, following a pre-established algorithm for diagnostic consensus involving three pathologists, as reported elsewhere10.Pathology review was based on the WHO pathological criteria for head and neck cancer and was performed blind with respect to the original local diagnosis and HPV-DNA/p1613. HPV genotyping was performed using a reverse hybridization line probe assay (LiPA25_v1) on all samples testing positive for viral DNA, targeting 25 HPV types with different oncogenic risk . DNA quality was evaluated in all HPV-DNA negative samples by testing for the tubulin-β gene. All DEIA and LiPA25_v1 assays were performed at the ICO.We used a PCR with the consensus primers SPF10 PCR and a DNA enzyme immunoassay (DEIA) to test for the presence of HPV-DNAINK4a and p16INK4a-positivity only. Crude and adjusted odds ratios and their 95% credibility intervals , were estimated. Moreover, crude and adjusted prevalences were estimated, the latter to account for other covariates that may have an effect on the prevalence estimates. Adjusted prevalences were calculated with R “prediction” function of “prediction” package16 based on the adjusted models. Histological variables were not included in the multivariable analyses as they were considered to be intermediate variables in the carcinogenic process, as previously described17. Bayesian methodology was chosen in all analyses with non-informative prior distribution due to lack of data on the distribution of the parameters19 and, moreover, because Bayesian methodology is useful to (1) avoid model fitting problems in parameter estimation due to small counts, and (2) produce robust estimators20.OPC samples testing negative for both viral and human DNA were excluded from the analyses. Descriptive, bivariate and unconditional logistic regression analyses were performed to identify independent factors associated with HPV etiological involvement in OPC according to double positivity for HPV-DNA/p16INK4a and prevalence risk ratios (RR) were obtained, with their 95% CI. Patients from 1991 were included in the first 5-year period (1992–1996) in order not to lose sample size. Multivariable models were adjusted for confounders: age, 5-year periods, gender, tobacco/alcohol consumption, subsite and tumour stage.To more specifically assess time trends in HPV-related OPCs in Spain, a log-binomial regression model was defined for double positivity for HPV-DNA/p1619 in R. All possible interactions between variables were assessed and stratified by anatomical subsite.All analyses were performed using the “bayesglm” function of the “arm” packageThis study was performed in accordance with the Declaration of Helsinki. The study had formal approval by the ethics committees of the four participating hospitals . Adequate measures to ensure data protection, confidentiality, patients’ privacy and anonymization were taken into account in compliance with European and Spanish current laws and regulations. Informed consent was not available due to the retrospective design of the study and the large proportion of deceased and untraceable patients. Thus, and in accordance to current regulations, informed consent was waved off by the ethics committees for patients diagnosed up to 2013. Informed consent was obtained for patients diagnosed in 2014–2017.13.Supplementary Fig. INK4a. Patients had a mean age at diagnosis of 60 years; they were mostly male (88.9%), heavy smokers (74.4%) and heavy drinkers (49.7%). The most common anatomical subsite was the tonsil (40.5%). Most tumours (63.3%) were squamous cell carcinoma (SCC) with conventional keratinizing morphological features and the most common stage was IVa according to the 7th TNM edition (45.6%). The adjusted prevalence of HPV-related patients was 9.7%.Table INK4a positivity alone are presented in supplementary Table After adjustment for confounders, HPV-related patients were more likely to be younger than 60 years, non-smokers and non-drinkers, to have tonsillar carcinoma, and to be diagnosed in most recent periods or with stages III–IVa Table . The equINK4a double-positivity and p16INK4a-positivity alone in supplementary Tables The associations of HPV-positivity with the demographic and clinical characteristics of OPC patients were also examined, stratified by three major anatomical subsites , followed by HPV33 (5.9%) and HPV35 (4.0%),  compared with early stages (I and II).Observed HPV prevalences were about 10%, as defined by the percentage of cases that were double positive for HPV-DNA/p1621. These results, together with our own, support the hypothesis that these increasing trends for OPC are caused by HPV infection. Of note, age-standardized incidence rates for OPC were estimated in 2018 at 1.4 in Spain and 1.3 in southern Europe, respectively22.Our results indicate that HPV prevalences in OPC have started to increase sharply in the most recent years in our setting. However, our results reporting HPV prevalence estimates are based on primary OPC tumours from four hospital-based registries and do not necessarily imply an increasing incidence of HPV-associated OPC in our region. Nevertheless, a previous study estimating the incidence trends of OPC in Spain by using data from population-based registries predicted statistically significant increasing trends for OPC over the period 2003–20173. This two-decade gap is in agreement with the calendar period differences between smoking drops in the US and Spain  than male  patients. A systematic review on geographical differences in the proportion of HPV prevalence in OPC between men and women revealed that those differences were mainly a consequence of the vast international variation in male smoking habits27. Notwithstanding, a population-based study describing time trends of cancer incidence and mortality in Catalonia during the period 1993–2007 detected a rising trend in oral cavity and pharyngeal cancers among women but not among men, presumably explained by a higher and earlier rate of smoking cessation among men than among women28. In addition to smoking differences between men and women, HPV transmission appears higher from women to men among heterosexual partners29 and differences in sexual behaviours between geographical regions have not been analysed at this level of detail. Intriguingly, although this study was not powered to precisely evaluate this issue due to the low number of female patients, increasing trends in HPV prevalence were suggested to be higher in women than in men  versus the oldest women (56–65 years)31. The CLEOPATRE results could thus also partially account for the recent increasing trends in HPV prevalence observed in the present study if it is assumed that changes in oral sex practices have also taken place.The gender and calendar differences in OPC HPV prevalence between high-burden regions and our setting could also be partially explained by differences in sexual behaviour, which is a clear risk factor for oral HPV acquisition and HPV-related OPC. Indeed, sexual behaviour greatly varies across regions with proportions of ever having oral sex in the US being higher than 65% compared with less than 20% in countries in southern Europe such as Spain32. However, other studies did not find that the observed significant increases in OPC were related to declines in tonsillectomies34 reinforcing increased oral HPV exposure as the likely cause.Different trends in tonsillectomy rates could also have contributed to the observed geographical heterogeneity. A previous study from Denmark reported a decrease in tonsillectomy rates concomitant to a simultaneous increase in the risk of OPC35 concluding that HPV prevalence differs markedly between OPC subsites and that its role in sites “other” than tonsil or base of tongue is uncertain and needs further evaluation.The differences in the role of HPV in OPC between anatomical subsites observed in our study are consistent with the results of a recent systematic reviewINK4a positive cases were HPV-DNA negative, representing 3.5% of the total sample, despite definition of p16INK4a positivity as staining above > 70%. This percentage is higher than the 10%-20% observed in most studies irrespective of the method of HPV-specific testing applied36, although it is lower than the 47% reported in an Italian study that considered a case to be p16INK4a positive if strong nuclear and cytoplasmic staining was present in > 50% of the tumour cells37. Classification and potential management of p16INK4a-positive—HPV-negative cases is still controversial39 although we13 and others39 have shown that these cases display similar survival to p16INK4a-negative cases. Moreover, molecular characterization of p16INK4a-positive—HPV-negative OPC cases has shown a similar genetic profile to HPV-negative tumours40. In this regard, we have observed epidemiological differences between OPC cases that were double positive for HPV-DNA/p16INK4a and cases that were p16INK4a-positive only, such as the loss of association with age and calendar period. Age discrepancies could be explained by a higher accumulation of mutations with age in the case of p16INK4a—positive—HPV-negative patients. A powered study aiming to perform an in-depth comparison between p16INK4a-positive—HPV-positive cases and p16INK4a-positive—HPV-negative cases is warranted to elucidate these questions.A total of 30 out of 108 (27.7%) p1641, about one third of OPC cases diagnosed each year in Catalonia are seen at the four hospitals of the study, and our sample represents approximately 16% of all incident OPC cases in the region. The retrospective nature of our cohort may have hampered thorough characterization of patients according to risk factors such as tobacco or alcohol use, since this kind of information could only be partially obtained from medical records. The more recent patients were only available for inclusion in the study for Sant Pau Hospital. However, when we addressed this limitation by performing a sensitivity analysis only considering Sant Pau patients, we obtained equivalent results. In addition, paraffin blocks were not available at diagnosis for a substantial number of patients, notably those with base of tongue carcinoma, a location particularly difficult to biopsy, as well as for patients from older periods. Of note, a fraction of these patients  had fine needle aspiration samples. However, these were not included according to our exclusion criteria. Our classification of subsites other than tonsil or base of tongue comprised many different locations, including oropharynx unspecified or overlapping lesions that could also include tonsil and base of tongue.Our study has several limitations. Selection of patients was not population-based and thus our results cannot be extrapolated to the general population. Moreover, the modest sample size does not allow firm conclusions to be drawn regarding associations with HPV positivity by subsite or gender, for instance. However, according to preliminary estimates of the OPC incidence in Catalonia using data from population-based registriesOur findings indicate that HPV prevalence in OPC has started to sharply increase in the most recent years in our setting. These results, together with previous estimates of increasing trends of OPC in Spain, suggest that HPV-related OPC has started to increase as occurred two decades ago in areas where most OPC cases are currently HPV-related. However, there are some differences between the epidemiology of the disease in our setting and other high-burden regions and we do not yet know whether they may change in the near future to approach the epidemiology of HPV-related OPC in high-burden regions.Importantly, our results strongly suggest that current estimates of HPV prevalence in southern Europe (probably other regions beyond high-burden areas) are outdated and warrant updated studies in selected populations. Continuing surveillance of sexual behaviours, alongside HPV vaccination status, is warranted to predict future HPV-related OPC burden.Supplementary information


This study aims to discuss audiences’ interaction and engagement with live streaming from a sociological perspective to investigate the different effects of information factors on audiences’ real-time interactions. Based on the interaction ritual chains theory, a data crawler was written using Python to collect data on the Huajiao platform for 1090 groups from 480 video game live rooms and 610 talent show live rooms. The results show that audiences’ commenting was mostly affected by the number of viewers, the gender of streamers, the number of likes, the number of gifts, and the duration of the live stream. Group comparison found that the effect of the number of viewers was significantly stronger in video game streaming, while the number of likes had a negative effect in video game streaming and a strong positive effect in live talent shows. In recent years, social media have overwhelmed the Internet experience of users and academic researches. “Social media” refers to various online applications that enable the content created by an individual to be shared with others . Social Live video streaming has prospered since a growing number of people are immersed in this new type of social media. Diverse live streaming platforms also create a free and open environment for users to enjoy this type of interaction  .Live video streaming has achieved remarkable progress in practice. However, it has received less attention in academic fields. Most studies of live video streaming have focused on the computer science and Information Systems realms , or examined scale and reliability problems –10. RareHowever, no study has examined which specific information provided in a live streaming room generates the most real-time audience interactions. Some studies have analyzed the characteristics of the content and posts in knowledge sharing platforms or some professional groups based on linguistic theories –15. In cCollins proposed interaction ritual chains (IRC) theory as a combination of Durkheim’s concept of collective enthusiasm and collective signs and Goffman’s theory of situational small-scale study of human behavior , 16.IRC consists of four basic elements . Collins illustrated that the critical point represents a two-way recreation, and consciousness will generate a collective reaction of cognition. On that basis, there are four results of IRC  . Thus, IThis theory perfectly interprets live streaming through certain theoretical mechanisms. First, audiences gathered in a common live room form a virtual co-presence with a barrier against outsiders. The streamer is the common focus of the audience and audiences can share the same emotion via real-time interactions . As a result of the ritual, the streamer and audience build an impromptu community with high group solidarity and a sense of morality. Members in this group gain emotional energy as an independent entity, feeling confident, empowered, and passionate, and they initiate interactions. Also, symbols are generated based on those rituals .A substantial amount of spiritual energy and two-way consciousness on a shared subject could improve participation, which is particularly true when people can play a crucial role in the interaction ritual instead of being auxiliary. Participation in rituals that allow individuals to be “performers” will lead to higher levels of emotional energy and greater involvement .Cognitive cost theory is vital to behavioral decision theory and creates a way to better understand consumers’ decision-making . The cogThis study aims to explore consumer interactions in live streaming rooms in the form of real-time comment (bullet curtain) based on streamers’ information , live timing information , and audience information . This study also extends the types of live content category to video games and talent shows.As mentioned above, live streaming fosters interaction ritual chains among the streamer and audience, and the audience generates a collective emotional effervescence based on a common focus or emotion. However, not all information in live streaming rooms has the same or equal effects on audience attention. Those live streaming platforms that provide particular communal circumstances exhibit an excessive capacity to influence respondents as they perform this interaction ritual chain based on anonymity, continuity, intimacy, and immediate accessibility . DerivedViewer information is critical in the conditions under which audiences initiate interactions. In accordance with Grice’s influential theory of conversation, the relevance of a conversation to group members may depend on who is the conversation starter . Users aThe choice over time is significantly affected by the opportunity costs of watching and making comments on live streams. Such costs may impact audiences’ interactions differently. Some studies found users have round-number goal, and they prefer to purchase near a half-hour interval online  , 33. LikIRC posits that feelings of effervescence will generate higher emotional energy via interaction ritual chains. Thus, the reactions of the whole group will impact the next effervescence more significantly . In addiSome researchers have proposed that what is contained in a video can affect how consumers behave when they view, endorse, and comment on it , 38. VieIn video game streaming, streamers share live video content of their ongoing games composited with a video feed of themselves. Streamers can chat with the audience and show their playing skills or experience simultaneously. Audiences in video games have more specific and centralized interests and knowledge of certain games than those in talent shows. Due to this similar interest and background, audiences in video game rooms are more likely to communicate actively and frequently.Live streaming of talent shows is another major type of live streaming. Streamers in those channels primarily focus on various types of performance . This type of live usually exhibits higher-level interactions among the streamer and audience members. The admiration of the viewers for the streamers will stimulate communication and consumption. Streamers will receive more gifts due their excellent performance or skills than in video game streaming.Our methodology should fit the characteristics of our data. First, the quantity of real-time comments triggered in a live streaming room is a positive integer. Second, the quantity of real-time comments shows overdispersion  in different live streaming rooms. Third, in practice, the variance of real-time comments is several times the mean value of real-time comments. Thus, we adopt a count data model assuming the distribution of the underlying data to be Negative-Binomial .live stream i, Yi,We assume that the number of real-time comments to Yi is the number of real-time comments to live stream i , with N the number of live streams, andΓ is the gamma distribution.λi and θ. The expected number of real-time comments to live stream i, E(Yi), is equal to λi. The corresponding variance, Var(Yi), is equal to λi(1 + θ). The parameter θ is often referred to as the overdispersion parameter. Larger values of θ represent greater overdispersion of the underlying data.The Negative Binomial distribution is a two-parameter distribution. The two parameters are respectively i is:Next, we relate the lambda parameters to the explanatory model, and the model for the number of real-time comments (RECOM) to live stream i refers to the level of streamers in live stream i;LEVi refers to the number of fans in live stream i;lnFANi refers to the gender of streamers in live stream i;GENSTREi 1 if the live stream i is start near round-number time, 0 otherwise;STARTi refers to the duration of live stream i;DURAi refers to the number of audiences in live stream i;lnAUDi refers to the number of likes in live stream i;lnLIKi refers to the number of gifts in live stream i;lnGIFThis study draws on data from two genres of live streaming . The data for these two types of live streams come from Huajiao, one of the most famous live platforms in China. According to an Aimei Report at the end of 2018, Huajiao live platforms had an average 23.9 million active users a month, higher than other platforms.All the variables can be measured straightforwardly in the live room interface. Thus, a data crawler was written using Python according to the robots.txt to collect data on the Huajiao platform, and the data for 1090 groups were collected from 480 video game live rooms and 610 talent show live rooms.For starting time, if the live stream starts near the round-number hours, we take it as “1” . Otherwise, we take it as “2” . For the duration of the live stream, we describe it in minutes and take every 30 min as 1 unit . For streamers’ gender, we took male as “1” and female as “2.”Data analysis was conducted using STATA 14.1. To judge whether there is an issue of multicollinearity, Variance Inflation Factors (VIFs) were calculated. VIF ranges from 1.00 to 6.62, and all indexes are below 10. Thus, as multicollinearity was unlikely to arise as a severe problem in this study, we proceeded to test the hypotheses.χ2(8) = 3196.50, and p = 0.000. The Pseudo R2 of the number of real-time comments is 20.24%, indicating good fit of the model. Also, 29.08% and 9.06% of the variance was explained in video game and talent show live streaming, respectively. The Negative Binomial model of full sample is significant with According to the factors in With respect to other variables, female streamers received more real-time comments than male streamers, supporting H3. However, contrary to our hypothesis, the duration of a live stream shows little negative effect on real-time comments (H4). Moreover, the numbers of viewers, likes, and gifts all show a positive effect on real-time comments, supporting H6, H7, and H8.χ2 and p-values of different variables are listed in To verify the effect of live streaming genres, we conducted the subgroup comparison method according to Keil et al. . Tables Consistent with the results for the full sample, the duration time has negative effect and the number of gifts has positive effect in both genres. And the level of streamer and the number of fans show negative effect on real-time comments in game video streaming while the male streamers shows positive effect on real-time comments in talent show streaming.According to the two sub-groups comparison, the positive effect of the number of audiences on real-time comments is greater for game video streaming than talent show streaming. In addition, the number of like in talent show seems to be a strong stimulation on real-time comment rather than negative influence in game videos.This study sought to investigate consumer engagement in live streaming viewed from sociological and participation perspectives. For this study’s theoretical contribution, we focus on the interactive framework. This study provides insight into how interaction ritual information affects users’ interaction.First, live streaming has not received sufficient attention in the academic realm; only a few studies have explored the operation of live streaming systems. As far as we are aware, this is a pioneering study of the audiences’ real-time interactive conduct in live streaming in terms of social psychology and communication perspectives. More specifically, this paper explains the mechanism of the interaction of the streamer and audience in a live streaming room in accordance with interaction ritual chains theory (IRC). Our explanation of real-time users’ interaction in such an impromptu community incorporates communication theory into this new type of real-time social media platform.Second, our study illuminates the users’ interaction mechanism in real-time interaction on social media. Some research has concentrated on exploring the impact factors of regular comments or reviews in traditional social media, such as Twitter and Facebook. Several papers have focused on how features and characteristics of a post drive the number of comments it receives on some UGC forums from a semantic perspective. Recently, some studies have verified the users’ continuous intention of live streaming from the perspective of social identification. Based on this research, we sought to further examine users’ interaction triggers on live streaming. Thus, we performed an experiment to analyze the characteristics that impact the number of real-time comments that a streamer receives in one live streaming room. Data from live streaming rooms were collected from one of the most famous live streaming platforms in China, Huajiao. The features of streamers’ level, streamers’ gender, starting time, duration of live streaming, and numbers of fans, audiences, likes, and gifts were verified in this study. The test findings suggest that (1) female streamers have greater advantages than male streamers, (2) overlong show times will negatively affect the number of real-time comments, and (3) the numbers of viewers, likes, and gifts could contribute to the number of real-time comments. Thus, this study constitutes a profound analysis of data on how users of the live streaming behave.Third, two major genres of live were compared and analyzed. First, the effect of those characteristics is examined in video game and talent show live streams. Most hypotheses received similar results to those of the full sample. Second, the two groups were compared based on sub-group coefficient test to show that male streamers have an advantage in game video streaming, females in talent show streaming. For the number of viewers, audiences of video game streaming made more contributions than those of talent shows. This finding implies that different genres of live streaming have different patterns of user behavior that may impact audience interaction styles, suggesting that platforms can implement different and customized strategies in certain streaming types.This study suggests implications of the new types of social media platforms from the perspective of marketing communication, especially regarding how online social media communities can gain more user-to-user interaction.First, this study extends our understanding of the emerging media through research on connections and high user-interaction platforms . Live stSecond, live streaming platform operators should consider the features that impact users’ participating behavior effectively. UGCs are usually considered indicators of consumer activities that could be used to predict users’ interaction. However, treating these interactions as homogeneous does not take the observed behavioral differences into account. Also, different information indicators in live streaming are analyzed, and only several specific influencing factors were found. On the one hand, managers in live streaming should control for an appropriate gender ratio since female streamers are more attractive than male streamers. On the other hand, users’ individual behavior , i.e., user-streamer interaction, can stimulate user-user interaction.In addition, the comparative analysis of two major types of live streaming revealed that platform managers should differentiate the operation and marketing of different genres of live streaming. For instance, platforms can promote more female streamers in live talent shows or introduce targeted marketing on different channels. Customized strategies can be implemented in different genres of stream lives. For talent show live streams, platforms could provide some competitions . Also, gathering “likes” can be set up as an activity to help streamers enhance their competitiveness.First, our results are limited by the fact that the data were collected from one Chinese live streaming platform. Future studies should extend the scope of data to other platforms.Second, all the data we collected are direct and visual. However, there is other critical information in live streaming rooms  that contains indications of users’ attention and other preferences. Thus, in future studies, appropriate methods should be employed to obtain and process these types of data.Third, only two major genres of live streaming  were compared. In fact, many other types are available to audiences . Also, how consumers behave might differ. Thus, future research ought to integrate diverse playing types and perform specific comparative studies to better comprehend the effect of what is streamed.S1 Data(XLSX)Click here for additional data file.