The interest in edible insects as food is growing, both in traditional and non-traditional insect-eating countries given their advantages in terms of sustainability and nutritional content. However, only a few studies have conducted cross-country investigations on the acceptance of including processed or whole insects in the diet. Thus, this study aimed to examine to which extent consumers were accepting (i) whole and visible mealworms, (ii) processed mealworms in their diet and (iii) to explore the factors affecting the acceptance level of consuming mealworms in countries with and without entomophagy tradition. An online survey was applied to collect responses  from five countries–i.e., Belgium, China, Italy, Mexico, and the US–using a quota sampling method. Moreover, an information treatment was included with about half of the participants receiving information about the advantages of edible insects as food (ingredient) and the presence of food safety regulations. Across countries, gender was the main factor affecting acceptance level as men accepted mealworms more than women. Entomophagy tradition mainly explained the differences among countries. Countries with entomophagy traditions (Mexico and China) showed higher acceptance of including whole or processed mealworms in the diet compared to countries with no entomophagy traditions . While information and age did affect differently the acceptance of including processed mealworms in countries with entomophagy traditions showing that consumer acceptance was affected by information in Mexico and by age in China. Whereas it was found that younger people (below 42 years old) in countries without entomophagy tradition were more open to accepting processed mealworms in their diet. Moreover, across countries, the acceptance of including processed mealworms was higher compared to whole mealworms. These findings provide insights into which consumer segments to target and the potential impact of information when introducing new insect-based foods in countries with and without entomophagy traditions. Tenebrio molitor, FCR of 3.8–5.8) is higher than for black soldier fly larvae  and can be similar to that of pigs (FCR of 3.1); hence, mealworms are not as efficient in converting feed into body weight compared to black soldier fly larvae. In addition, the energy expenditure for mealworm production could be higher than that of milk or chicken if produced in cold regions [The growing world population puts additional pressure on food security and the environment. This situation has urged the search for novel protein sources that can be produced under sustainable food production systems. As such, there is a growing interest in the use of edible insects, either as whole insects or as food ingredients, as they are considered more sustainable alternatives to traditional animal-based food sources , 2. Alth regions . Several regions .et al. [et al. [et al. [et al. [Mealworms, like other edible insects, are not part of the traditional diet in many countries, especially in the Global North . Meanwhiet al.  showed t [et al.  found th [et al.  and Tan  [et al.  found th [et al. , 35.et al. [Processing insects to make them invisible in food is seen as the most important single strategy to promote positive appearance evaluations , 25, thuet al. , found tet al. . Finallyet al. , there iet al. .Therefore, this study aimed to examine to which extent consumers are willing to accept mealworms as food, either whole and visible or processed. The study included five countries  with high and low entomophagy traditions. The acceptance to include whole and processed mealworms in the diet was evaluated based on their country of origin, their gender , their age (18–41 years old vs 42 years old and older), and the macro-region within a country . We also measured whether providing information about (i) the advantages in terms of sustainability and nutrition of using edible insects as food and (ii) presence of food safety regulations would have an impact on their acceptance. The results of this study could be of interest for scholars exploring consumer acceptance and attitudes towards insects as food as well as for practitioners in the food industry to develop appropriate marketing campaigns.An online survey was conducted in Belgium, China, Italy, Mexico, and the US between February and March 2022. The selection of the countries was done for several reasons. First, these countries represent the region where the authors currently work and therefore they are familiar in terms of population, culture, insect market. Second, the five countries represent a diversified target of consumers and the tradition of entomophagy: Third, countries were selected in order to include both regions with a strong entomophagy tradition  and without an entomophagy tradition . Moreover, each country has unique food habits and attitudes towards foods, including countries–like Italy–with a strong and long-standing gastronomic tradition  and otheRespondents electronically approved an informed consent form before their participation. The study was granted ethical approval by the University of Pisa Ethics committee (Committee on Bioethics of the University of Pisa—Review No. 26/2021).The survey consisted of several question groups, including general eating habits, and demographics. Following a random assignment, a between-subjects design was implemented. Approximately half of the participants received the information treatment before the question was displayed, whereas the rest of the sample represents the control group with no information. The information was adapted from the Report “Looking at edible insects from a food safety perspective. Challenges and opportunities for the sector” published by the FAO in 2021. The information provided included several elements regarding the potential benefits of using edible insects as food , as well as the presence of food safety regulations see .T. molitor). Respondents were asked about their level of acceptance of including whole and processed mealworms in their diet. The two questions presented were as follows: i) “Would you accept including whole mealworms in your diet?”; ii) “Would you accept including processed food  that contains mealworm powder in your diet?”. The two questions were divided into two separate sections of the survey to facilitate the reply.Based on the concept that consumer acceptance of insects is not only trying once the product but being willing to incorporate it into the diet, we developed two specific questions related to one of the currently authorized insect species on the market , a post hoc test (z-test) was performed to determine which of the independent variable differ in terms of the four categories of the dependent variable . In the post hoc test, Bonferroni’s correction was used to correct for multiple comparisons (P < 0.0125) decreasing the chance of committing Type I errors. Every following comparison was performed in a similar way.First, the effect of gender , treatment (informed vs uninformed), age (18–41 years old vs 42 years old and more), country , product , and macro-region within each country was tested using ChiThen, the countries were divided into two groups, according to their acceptance to include mealworms in the diet, namely high (Mexico and China) vs low acceptance . This categorization followed statistical tests between the two formed groups and within each group to check if the categorization was correct and if differences within countries in a category existed. Statistical analyses were performed using SPSS software .Across countries, gender was the leading factor affecting the acceptance level of mealworms in the diets . In counNo, because…”) towards including whole mealworms in their diet (P < 0.001) and Belgium . Moreover, in Mexico and the US, there was a significant increase in the acceptance  of whole mealworms by male participants. In Belgium, males answered “Maybe, if…” more frequently than women (+8%). Meanwhile, in Italy and China, men rejected  whole mealworms less often  compared to women.Males showed the lowest rejection rate (“eir diet . The difP < 0.125)   . WhereasMaybe, if…”). This level of acceptance increased when information about the low ecological footprint associated with insect production, their nutritional value, and information on the authorization of mealworms as food by the European Food Safety Authority (EFSA) was given to the participants.In the countries under study, there was no significant effect of information on the acceptance of including whole mealworms in the diet . RegardiNo, because…”) towards processed mealworms than older participants (≥ 42 years old) . In Belgium, older participants (≥ 42 years old) doubted more  about accepting processed mealworms in their diet compared to younger participants. Only in the US, age affected the acceptance to incorporate whole mealworms into their diet. In this country, younger participants (18–41 years old) showed an increase in the acceptance level of whole mealworms (+11%) and a decrease in rejection (-11%).In countries with low acceptance , age significantly affected the acceptance of the inclusion of mealworms in the diet . This ef“No, because…” remained. The region in Mexico showing the least rejection towards processed mealworms was the South/Southeast (14%) compared to the North (25%) and the Center/Center North (17%).In the countries under study, there was no significant effect of macro-regions on the acceptance of including whole or processed mealworms in the diet, except for Mexico where a significant difference was found for both mealworm products . HoweverThe five countries involved in the study had two distinctive acceptance levels of including mealworms and processed mealworms in the diet , i.e., oP < 0.001) between these two types of countries was found for almost every level of acceptance of including whole or processed mealworms  include whole mealworms in their diets.Comparing the countries with a high acceptance level of including mealworms in the diet, Mexican consumers showed the lowest rejection attitude of whole or processed mealworms . CompareP < 0.05), which showed the strongest rejection for both types of mealworm products  than in Italy (5%)  . MoreoveYes, because…”) increased from 6 to 17% compared to whole mealworms  drastically decreased by an average of 20.5% compared to whole mealworms  increased significantly (average +6.8%) compared to whole insects  and safety of this novel food source does not change the acceptance of consuming whole or processed mealworms , which iet al. ) to a shet al. . The conet al. . While set al. , this stAs expected, countries with entomophagy traditions (Mexico and China) showed a higher acceptance, regardless of their processing condition . In thesInterestingly, no difference in acceptance levels exists among macro-regions within countries. We initially hypothesized that the acceptance level would have differed within macro-regions, especially in sizable countries like China, Mexico, or the US., as differences in entomophagy tradition exist within a country. For instance, in Mexico, the South and Southeast regions are particularly rich in entomophagy traditions, followed by the central part of the country , 57–62. The results of the study showed there is a significant difference between Chinese and Mexican respondents, as the Mexican population had a higher acceptance of both whole and processed mealworms. These results are in accordance with a previous study where the willingness to eat an insect-based food product was tested in several countries including China and Mexico . RemarkaThe high acceptance level in Mexico can be explained by the fact that in this country insects are associated with a positive image since they are a delicacy sold either in local markets or served in high-end restaurants , 66. In Our results from China are aligned with the solid entomophagy tradition of this country, which includes raising and breeding insects for human consumption, but also for medical purposes , 24. ManIn Belgium, the US, and Italy, the acceptance to include mealworms in the diet was significantly lower than in China and Mexico . Our resWillingness to consume mealworms is one of the first steps toward increasing the consumption of edible insects. The first tasting should be accompanied by positive sensorial and emotional experiences, otherwise, repeated consumption will not be achieved , 34. FurThere are two primary limitations to our findings. First, this is a hypothetical survey and therefore we cannot ascertain whether the reported behavior would translate into a real behavior of willingness to consume whole or processed mealworms. Second, even if we used a quota sample based on national statistics, the representativeness is limited to the ones who have computer access and to the panel members of the agency, which means that they were probably individuals with higher socio-economic statuses. Future research could more carefully examine socio-demographic differences among and within countries.Interest in insects as a potential source of food has grown considerably in recent years and consumer acceptance studies towards edible insects as food is crucial to support this emerging sector. The overall aim was to investigate the main difference in the acceptance of mealworms as food across five countries, i.e., Belgium, China, Italy, Mexico, and the US considering a sample representative for gender, age, and macro-region . Significant differences related to the entomophagy culture of each country were found, i.e., consumers in China and Mexico with a long-standing entomophagy tradition reported higher acceptance of whole and processed mealworms than the other countries. Across all countries, gender affected the willingness to consume mealworms. In this regard, men tended to reject mealworms less than women, confirming previous results. Regarding age, younger consumers were more open to accept mealworms in their diet. Considering data across all countries, the acceptance of processed mealworms was higher compared to whole mealworms suggesting that the focus of the food industry should be on processed mealworms rather than whole insects.Interestingly, from a marketing perspective, we observed how familiarity with edible insects as food in general increases acceptance of edible insect species not currently available on the market, . In countries with no entomophagy tradition, the introduction of (food products made with) mealworm should target first young males. These findings unlock business opportunities for insect producers to introduce new insect species (whole or processed) in food products in countries with entomophagy traditions.Locusta migratoria (migratory locust) and Acheta domesticus (house cricket). Future studies should also consider how the knowledge, previous experience towards entomophagy, and the drivers in each market  could influence acceptance to consume insects.Further research should investigate how consumers’ acceptance changes based on edible insect species, starting from the ones recently authorized in the EU, i.e., S1 Appendix(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


The aim of the present study is to summarize the current evidence published in the literature concerning the role of transoral robotic surgery in HPV+ oropharyngeal squamous cell carcinoma. Recently, in order to reduce the side-effects related to chemo-radiotherapy, the use of transoral robotic surgery has increased, especially for oropharyngeal tumors related to HPV. Our review highlights that we do not have enough evidence for specifically recommending TORS in HPV−driven oropharyngeal cancer. However, transoral robotic surgery shows good oncological and functional outcomes in general. Moreover, based on the current evidence, transoral robotic surgery could potentially represent a promising strategy for intensifying treatments in HPV−negative oropharyngeal cancer.Introduction: International guidelines include transoral robotic surgery (TORS) as an option for selected oropharyngeal squamous cell carcinomas (OPSCCs). In the perspective of treatment de-intensification, many surgeons have started recommending and performing TORS preferentially in p16- positive OPSCC in order to reduce the long-term morbidity related to chemoradiotherapy. The aim of the present review is to analyze the current evidence supporting the above-cited strategy. Materials and Methods: The study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Results: Twenty-two studies were included in this review, with a total of 3992 patients treated with primary TORS. The majority of patients were classified as HPV+ , and 8.2% (n = 327) as HPV−. The HPV status was unknown in only 10 (0.3%) patients. In particular, only five of the included studies compared survival outcomes of HPV−positive patients with HPV−negative ones treated with primary TORS, and only two of these found a significant improvement in survival in the HPV−driven cohort. Discussion: The current literature does not clarify whether HPV+ OPSCCs treated with TORS, alone or with adjuvant treatments, are associated with a better oncologic and/or functional outcome compared to those treated with radio- or chemoradiotherapy. However, TORS alone obtained good oncological outcomes in a high percentage of cases in the reviewed series. Recent data, on the other hand, suggest that TORS could represent a promising strategy for intensifying treatments in HPV− OPSCC. In recent decades, we have witnessed a dramatic increase in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) in Western countries, mostly due to the human papillomavirus (HPV) epidemic ,2,3. In HPV−driven OPSCC has been demonstrated to be a different entity compared to the “classical” smoking-associated tumors, as acknowledged by the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. Several studies have reported markedly better oncological outcomes for HPV+ OPSCC compared with their HPV− counterpart ,11,12,13In any case, the better survival for HPV+ OPSCC has led to an intense debate about the oncological safety of treatment de-intensification with the aim of reducing long-term morbidity ,20. The OSF .The present systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . This stBoth retrospective and prospective studies describing the clinical outcome of TORS in the treatment of OPSCC were included according to the PICOS system previously described. All studies had to report at least the HPV status, the adjuvant treatment performed, and overall survival. No restrictions on follow-up length were applied. All patients were included regardless of the adjuvant systemic or local treatment. All studies had to be published in peer-reviewed journals, while all abstracts were excluded. No language or publication date restrictions were applied.A comprehensive electronic search for relevant published studies was conducted on PubMed/MEDLINE, Google Scholar, and the Cochrane Library database. The searches were adjusted to fit the specific requirements for each database based on the following main keywords: transoral robotic surgery, oropharyngeal squamous cell carcinoma, and HPV. As an example, the search strategy used on the PubMed database was:  OR (OPSCC)) AND ((HPV) OR (human papillomavirus)) AND ((TORS) OR ). Then, a cross-reference search of the included studies was performed to minimize the risk of missing relevant data. The last search was run in August 2022, starting from January 2000.The National Institute for Health and Clinical Excellence (NICE) quality assessment tool was used to evaluate the quality of the included studies .The included studies were analyzed to extrapolate data about patients’ age, gender, HPV status, behavioral risk factors, stage, surgical techniques, margins, complications, adjuvant treatment, and outcomes. All data were summarized in tabular form. Formal meta-analyses could not be performed due to study heterogeneity. Dichotomous variables were reported as counts and percentages, and continuous variables as mean ± standard deviation or as median ± IQR (interquartile range), as reported by each study.n = 3655, 91.6%), while only 8.2% (n = 327) were HPV−. The HPV status was unknown in only ten (0.3%) patients. Overall tumor staging according to the AJCC was reported for 1299 patients (32.5%). Of those, the slight majority had an early-stage disease , while 49.5% were at an advanced stage .A flow chart of the study identification process is shown in n = 339, 8.5%) enrolled the patients prospectively, and only 9 studies  included an explicit statement that patients were recruited consecutively. Although 17 studies  reported stratified data, only 5 studies  stratified the outcomes on HPV status.The study quality assessment revealed an important heterogeneity between included studies, and several concerns were raised . In partOnly five studies compared the oncologic outcomes in HPV−negative and HPV−positive tumors treated with primary TORS. n = 45) and 80.6% (n = 25), respectively, for their entire cohort (n = 50), and 1-year and 2-year DFS of 97.8% (n = 45) and 92.6% (n = 25), respectively. Survival related to HPV status was not statistically different among the two cohorts according to OS  and DFS .Cohen et al.  reportedFurthermore, Blanco et al.  reportedp < 0.01).Meccariello et al.  reportedp < 0.001).Furthermore, based on a cohort of 120 patients undergoing TORS , O’Hara et al.  comparedp = 0.584).Finally, Sahovaler et al.  reportedThe above-mentioned studies highlight that it is not clear yet whether HPV−driven tumors have any survival benefit compared to HPV−negative ones when treated with primary TORS. Indeed, in summary, only two out of five studies found a significantly better survival in HPV−positive patients treated with primary TORS compared to HPV−negative ones. Still, the cohorts of these two studies were made up of few patients, while larger cohorts of patients may be more appropriate for identifying real differences in survival. This lack of difference in survival according to HPV−status in patients undergoing TORS differs from the well-known better survival of HPV−positive patients already described by many studies regardless of the type of treatment, which could indicate that HPV−negative patients may benefit from primary TORS. Certainly, this is only a hypothesis, and larger studies are necessary to better define these aspects.A recurrent bias of clinical studies dealing with HPV−driven oropharyngeal carcinogenesis is the diagnostic tool used . Even ifAmong the studies included in the present review, HPV status was never assessed by mRNA E6 and E7 detection, but most often using assays with proven specificity issues, such as HPV DNA detection with PCR amplification or p16 IHC. Such sensitivity issues are evident also in the study by Moore et al. , which uThe use of a low specificity assay in defining HPV−driven carcinogenesis has been demonstrated to improve prognostic prediction between the 7th and 8th editions of the AJCC classification . HoweverAnother critical question is whether HPV−driven carcinogenesis is associated with higher radio-sensitivity, or if it is a pure prognostic marker regardless of the primary treatment . In the second case, which seems to be confirmed by some surgical series ,63,64, dSurgical studies stratifying patients according to HPV status generally show less evident differences between HPV− and HPV+ patients, as regards survival endpoints ,32,37, cSuch considerations acquire further value given the results of the ORATOR2 trial , compariCurrently, there are no intrinsic reasons why TORS should show more benefits in HPV−negative OPSCC patients compared to HPV−positive ones. In fact, TORS does not intervene on any particular molecular pathway, and is therefore effective both on HPV+ and HPV− tumors. On the contrary, evidence showing better oncological outcomes of HPV+ patients compared to HPV− ones are mainly based on cohorts treated with CRT ,10,65. IAnother fundamental assumption of the advocates of TORS for de-intensification is that upfront surgery in HPV+ OPSCC can be associated with a significant reduction in long-term morbidity compared with non-surgical approaches . This woAlthough TORS is often considered the preferred treatment for T1-T2 N0-N1 HPV+ OPSCC, our systematic review raises several concerns about the validity of this assumption. Even if a great number of OPSCCs were treated with TORS, only a minority of studies reported a stratification of the data based on HPV status. Moreover, they were all characterized by a small sample and retrospective data acquisition, with clear limitations for drawing firm conclusions. Furthermore, several studies used the detection of p16 for tumor stratification, and the oncological safety and the functional advantages of “surgical deintensification” are far from proven.On the other hand, TORS alone obtained good oncological outcomes in a high percentage of cases in the reviewed series, apparently allowing a reduction of surgical sequelae regardless of HPV status. In addition, the application of surgery in OPSCC provides additional information through the pathological examination of the sample, allowing for accurate staging and more tailored therapy based on tumor extension. Therefore, TORS remains a potentially useful tool in the hands of the head and neck oncologic surgeon. Furthermore, recent data open new perspectives for the use of TORS on the “opposite front”, in HPV−negative OPSCC . TORS co


This study revealed a low level of public awareness of HPV vaccination as a cancer prevention method in Poland.Human papillomavirus (HPV) infection is a risk factor for head and neck cancers (HNC). HPV-related head and neck cancers are preventable through vaccination. This study aimed to assess the attitudes towards HPV vaccination among adults in Poland, with particular emphasis on preventing HPV-related HNC, as well as identifying factors associated with a willingness to vaccinate children against HPV. This cross-sectional survey was carried out in November 2021 on a nationwide, representative sample of 1082 adults in Poland. The computer-assisted web interview (CAWI) technique was used. Only 42.5% of respondents were aware that HPV infection is a sexually transmitted disease. Less than one fourth of respondents (23.8%) indicated vaccination as an HPV infection prevention method and 51.9% of respondents correctly indicated HPV vaccine-eligible populations. Only 48.1% of respondents declared positive attitudes towards HPV vaccinations and declared that they would vaccinate their child against HPV. Males (OR: 1.43, 95% CI: 1.11–1.85;  Human papillomavirus (HPV) is a common viral infection ,2. The rMost HPV infections are asymptomatic and clear spontaneously ,2,7. HowHuman papillomavirus (HPV) causes more than 5% of cancers worldwide, including all cervical cancers and an increasing proportion of oropharyngeal cancers . The HPVEpidemiological data showed that the incidence of HPV-related head and neck cancer (HNC) is increasing ,13,14,15Vaccination is the most effective strategy for preventing HPV infection . In 2006HPV vaccination and HPV-based screening are cost-effective strategies for preventing cancer worldwide ,28. TherPoland is a country with one of the lowest HPV-vaccination coverage rates among the EU member states . Since 2In Poland, HPV vaccination is voluntary. There is an extra charge (cost of the vaccine—approximately 30 EUR) for prophylactic vaccination against HPV in primary healthcare settings  ,28,30. HData on public awareness of HPV vaccination and HPV infection prevention methods in Poland are very limited ,33,34,35We aimed to assess the attitudes towards HPV vaccinations among adults in Poland, with a particular emphasis on preventing head and neck cancers through HPV vaccination, as well as to identify factors associated with a willingness to vaccinate children against HPV.This cross-sectional survey was carried out in November 2021 on a nationwide, representative sample of 1082 individuals aged 18 years and over in Poland. The computer-assisted web interview (CAWI) technique was used . Data weRespondents were selected from the Nationwide Research Panel Ariadna out of >100,000 registered and verified individuals aged 18 and over. The Nationwide Research Panel Ariadna is the biggest independent nationwide research panel in Poland, widely used in nationwide, representative cross-sectional surveys ,39,40.A non-probability quota sampling technique was used . ParticiThe research tool was a questionnaire developed for the purpose of this study. Questions on head and neck cancer, HPV infection, and attitudes towards the HPV vaccine were addressed. Questions also addressed personal characteristics, including gender, age, marital status, having children, educational level, size of place of residence, occupational status, and financial status. Self-reported knowledge about HPV infection. Respondents were asked about their awareness of HPV transmission, using the question: “How can you get infected with human papilloma virus (HPV)? Please select all that apply” with 6, mutually nonexclusive answers: by a kiss; by touching; by having sex; during natural childbirth; by having contact with the blood of an infected person; and difficult to tell/do not know. Moreover, respondents were asked about HPV infection prevention methods, using the question: “What do you think are the human papilloma virus (HPV) infection prevention methods? Please select all that apply” with 6, mutually nonexclusive answers: vaccination before the initiation of sexual contact; condom use; limiting the number of sexual partners and avoiding risky sexual behavior; other forms; infection with HPV cannot be prevented; and difficult to tell/do not know. Moreover, respondents were asked about the oncogenic potential of HPV, using the question: “What do you think are the risk factors for head and neck cancer?” with 10, mutually nonexclusive answers (including HPV infection as one of the answers ).Self-reported knowledge about HPV vaccination. Respondents were asked about their knowledge of HPV vaccination recommendations (eligible groups), using the question: “In your opinion, who should be vaccinated against human papilloma virus (HPV)?” with four possible answers: girls before the initiation of sexual contact; boys before the initiation of sexual contact; boys and girls before the initiation of sexual contact; difficult to tell/do not know. Respondents who indicated “boys and girls before the initiation of sexual contact” were classified as those who were aware of the population eligible for the HPV vaccine.Willingness to vaccinate children against HPV was defined using the question: “Would you like to vaccinate your child against human papilloma virus (HPV)?”. Respondents who indicated positive attitudes toward HPV vaccination by choosing the answers: “Definitely yes” or “Rather yes” were classified as those who declared a willingness to vaccinate their children against HPV.The data were analyzed with SPSS v. 28 . The distribution of categorical variables was shown by frequencies and proportions. Statistical testing to compare categorical variables was completed using the independent samples chi-square (χ2) test.Associations between sociodemographic factors such as gender, age, marital status, having children, place of residence, educational level, occupational status , self-reported financial situation as well as self-reported knowledge about the oncogenic potential of HPV (head and neck cancer risk factor), and awareness of HPV infection prevention methods were analyzed using the logistic regression analyses. Vaccination before the initiation of sexual contact, condom use, and limiting the number of sexual partners and avoiding risky sexual behavior were considered separately as dependent variables in the model. These variables were selected because they are some of the most common HPV infection prevention methods that are well described in the literature.Additionally, similar logistic regression analyses were carried out for the associations between sociodemographic factors and attitudes towards HPV vaccination (“positive attitudes towards HPV vaccination”), as well as knowledge about HPV vaccination recommendations (“awareness of population eligible for the HPV vaccine”). In univariate logistic regression analyses, all variables were considered separately. In multivariate logistic regression analyses, all sociodemographic variables were included. The strength of association was measured by the odds ratio (OR) and 95% confidence intervals (95% CI). The level of statistical significance was set at alpha 0.05. This study was performed in line with the principles expressed in the Declaration of Helsinki. The study protocol was revised and approved by the Ethics Review Board at the Central Clinical Hospital of the Ministry of the Interior and Administration in Warsaw, Warsaw, Poland . Informed consent was obtained from all the subjects involved in the study.Completed questionnaires were received from 1082 individuals , aged 18–86 years (mean age 44.5 ± 16.1). Detailed characteristics of the study population is presented in Only one third (32.5%) of respondents were aware of the oncogenic potential of HPV and indicated HPV infection as a risk factor for head and neck cancer . Most ofp < 0.001) or limiting the number of sexual partners and avoiding risky sexual behavior  as a method of preventing HPV infection. The percentage of respondents who indicated vaccination before sexual initiation as a method of preventing HPV infection was significantly higher among those who lived in larger cities compared to those who lived in small cities  or rural areas  or limiting the number of sexual partners and avoiding risky sexual behavior  are the HPV infection prevention methods. Respondents aged 30–44 had higher odds of indicating that condom use is an HPV infection prevention method . Those respondents who lived in cities with more than 500,000 residents had higher odds of indicating that vaccination before the initiation of sexual contact  or condom use  are HPV infection prevention methods compared to those who lived in rural areas. Respondents who had a higher education had greater odds of being aware that vaccination before the initiation of sexual contact is an HPV infection prevention method . Respondents who were aware of the oncogenic potential of HPV (head and neck cancer risk factor) had higher odds of being aware of HPV infection prevention methods. Details are presented in The results of the univariate and multivariate regression analyses are presented in p < 0.05) as well as indicating positive attitudes towards HPV vaccination . Respondents who self-declared a rather good, good, or very good financial situation  or moderate financial situation  had lower odds of being aware of the population eligible for the HPV vaccine (p < 0.01) as well as those respondents who did not have children  had higher odds of indicating positive attitudes towards HPV vaccination. Respondents who lived in cities had higher odds of indicating positive attitudes towards HPV vaccination compared to those who lived in rural areas (p < 0.001) as well as indicating positive attitudes towards HPV vaccination . Details are presented in Respondents who had received a higher education had higher odds of being aware of the population eligible for the HPV vaccine  correctly indicated sexual contact as a route of HPV transmission. We observed a relatively high percentage of respondents who incorrectly believed that HPV may be transmitted through blood (42.5%), by kiss (20.5%), or by touch (7.4%). A similar phenomenon was observed by Smolarczyk et al. in a group of parents in Poland . In 2018In this study, limiting the number of sexual partners and avoiding risky sexual behavior as well as the use of condoms (44.4%) were the most recognized HPV infection prevention methods, wherein the percentage of respondents who were aware of these methods was significantly higher among females. In the study by Smolarczyk et al., 63.3% of parents in Warsaw, Poland indicated the use of condoms as an HPV infection prevention method and 70.6% indicated limiting the number of sexual partners as an HPV prevention method . The difIn this study, only one quarter of respondents indicated vaccinations as an HPV prevention method. Respondents with a higher education, those who lived in cities with more than 500,000 residents, as well as those who were aware of the oncogenic potential of HPV (head and neck cancer risk factor), were more aware of the HPV vaccine as an infection prevention method. Our findings indicate that there is a need to increase the level of knowledge about HPV vaccinations among Poles with a lower level of education, as well as those who live in rural areas. Local governments, as well as local non-governmental organizations and associations , can be involved in education on HPV vaccinations. According to the Polish Preventive Vaccination Plan, HPV vaccination is recommended for girls and boys before sexual initiation . In thisRegular monitoring of public awareness towards the HPV vaccine is an essential part of vaccination policy. Identification of public concerns and hesitant attitudes allows for the promotion of educational campaigns that build vaccine confidence. Gańczak et al. showed that between 2013–2014, 85.1% of parents of high school students from three selected high schools in Zgorzelec, Poland, declared a willingness to vaccinate their children against HPV . In 2018This study has practical implications for vaccination policies. The proportion of respondents who declared their willingness to vaccinate their children against HPV was five times lower than the HPV vaccination coverage rate among teenagers in Poland, which may indicate systemic barriers to receiving HPV vaccinations. Further activities are needed to ensure easy access to HPV vaccines for those individuals who intend to vaccinate their children. Organizational barriers, as well as financial barriers, are considered the most important factors ,34. PublOur findings suggest that there is an urgent need to conduct a nationwide information campaign on HPV infection prevention methods and the health risk of HPV-related diseases. In Poland, most educational campaigns on HPV infections were led by obstetricians or gynecologists and were addressed to females. Further educational campaigns should target all genders. This study had several potential limitations. This study was carried out using the CAWI research method, which excludes the possibility of interaction with the respondents. Therefore, the ability to assess the competencies of the respondents, for example, whether they sufficiently understand the questions asked, is impossible. Second, we cannot exclude non-response bias. However, similar methods were used in previously published papers ,39,40. MThis study demonstrated the low public awareness of HPV infection prevention methods among adults in Poland. Less than one quarter of adults in Poland were aware that HPV infection can be prevented through vaccination, and only half of the Poles declared positive attitudes towards HPV vaccinations. Male gender, living in cities, and having a higher education were the most important factors associated with positive attitudes towards HPV vaccinations. The current study indicated significant gaps in the recognition of HPV vaccine-eligible populations. The presented data underscore the importance of adopting an educational campaign on HPV infection prevention methods, with particular emphasis on vaccinations. Otolaryngologists should be actively involved in the educational campaign on HPV vaccines, due to the significant burden of HPV-related head and neck cancers in Poland.


During the Vietnam War, several unknown chemicals, such as Agent Orange, were used in Vietnam by the military. Therefore, there have been continuous health concerns among the Vietnamese population and veterans exposed to these hazardous chemicals. This study aimed to investigate the risk of all cancers and also organ-specific cancers among Korean veterans of the Vietnam War.This study used a national representative cohort that included all Korean Vietnam War veterans as the interest group, with 1:4 age-sex-region-matched general Korean citizens as the reference group, from 2002 to 2018. Age-standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) were calculated for all cancers and for 31 organ-specific cancer categories based on the medical facility visit data.An increased SIR of 1.07  was observed for all cancers among the veterans. There was a significantly increased risk of cancer among 22/31 organspecific cancers, with 18 cancer categories showing a significantly higher risk than all cancers. The highest risk was observed for “malignant neoplasms of other parts of the central nervous system” .This study evaluated the risk of cancer among Korean Vietnam War veterans. Further studies are warranted to investigate various health determinants in the veterans as well as the Vietnamese population. There iThere have been concerns regarding the adverse health effects from the various unknown chemicals used in the war, including cancer. The association between dioxin and some cancer types, such as soft tissue sarcoma, lymphomas, respiratory tract cancer, and prostate cancer, has been reported previously; however, there are limited or inconsistent results for some other cancer types. In Korea, Yi et al. reported cancer incidence among Korean VW veterans . From 19This study aimed to investigate and compare the risk for, and status of, cancer incidence between Korean VW veterans and Korean citizens using the newly opened national representative cohort data of the Korean Vietnam War Veterans’ Health Study Cohort (KOVECO) .22.1https://nhiss.nhis.or.kr/bd/ab/bdaba000eng.do) and the Ministry of Patriots and Veterans Affairs  of Korea. NHISS collects, stores, and manages data obtained from mandatory public health insurance services for all Korean citizens, which has been available to researchers for policy and academic research purposes since 2002. The NHISS database comprises data regarding hospital facility visits based on the standardized protocol of the Korea Classification of Diseases and Causes of Death, 4th edition, which corresponds to the International Classification of Diseases (ICD), 10th revision. All diagnoses were described based on ICD codes, with individual codes, dates, and regions  and its subsequent amendments. Data from the NHISS were collected after obtaining written informed consent from all the participants, and the information was anonymized. This study was approved by the Institutional Review Board of the Gachon University Gil Medical Center (IRB No. GCIRB2019-076).2.2The VW veteran group was defined based on VW-related information from the MPVA. The reference group was matched with the VW veterans group based on the 5-year-interval age groups, sex, and residence in 16 metropolitan cities in Korea in a 1:4 ratio.Cancers were defined based on ICD diagnosis codes for malignant neoplasms for all cancers C00–C97) and organ-specific categories according to the human organ system  and 95% confidence intervals (CIs) were calculated based on the 5-year-interval age groups. SIR was estimated using age-specific cancer incidence rates and the number of person-years in each age group of the reference group (non-VW veterans), indicating the age-specific expected incidence among VW veterans. The sum of the expected cases was compared with the total observed cases of the VW veterans, and the ratio was calculated. If the SIR and the lower limit of the 95% CI were both >1, the cancer risk was considered significantly higher in the VW veterans’ group than in the reference group. The 95% CIs were estimated using Poisson distribution. All analyses were performed using SAS, version 9.4 .3KOVECO comprised 250,842 (19.2%) VW veterans Table 1.The SIRs and 95% CIs according to neoplasm sites are shown in 4This study found an increased risk of all cancers among Korean VW veterans as compared to the general population. There was a significantly increased risk of cancer in 22/31 organ-specific categories, of which 18 categories showed a significantly higher risk than that of all cancers. Although the highest risk was observed for “other malignant neoplasms of the female genital organs” among VW veterans, the difference was not statistically significant. VW veterans showed a significantly increased risk for “malignant neoplasms of other parts of central nervous system.”TCDD is a known human carcinogen , 11. ManIn this study, the incidence of several cancers was significantly higher in the VW veterans than in the non-veterans. According to reviews conducted by the National Academies of Sciences, Engineering, and Medicine , there iWe found a significantly higher incidence of malignant neoplasms of the central nervous system . An association between agricultural exposure to herbicides or pesticides and tumors of the central nervous system has been reported . PesatorCancers of the head and neck, including malignant neoplasms of the eye, adnexa, lip, oral cavity, and pharynx also showed significantly elevated SIRs. Several studies have reported the possible mechanisms of carcinogenicity with regard to Ah receptor actions –27 as weThis study has several strengths. First, we used nationally representative data from the NHISS, which includes all Korean VW veterans and is the largest, most reliable, and objective data currently found in the world. The follow-up period was sufficiently long to observe the incidence of cancer, which may have been higher in older patients. Second, this was the first study to include female VW veterans. Although the number of female participants was much lower than that of male participants, investigating health risks in female veterans should be encouraged. Third, we found a significantly increased incidence of malignant neoplasms of the central nervous system, malignant neoplasms of the eye and adnexa, and other head and neck cancers. The risk of these cancers has not been conclusive, owing to the small number of cases. We believe that the elevated risk of these cancers observed in this study is actual, rather than coincidental.However, this study has some limitations. It only demonstrated the risk of cancer in VW veterans who were alive during the baseline data collection of KOVECO in 2002. The VW ended in the mid-1970s, and early onset or high-mortality cancers occurring before the start year of the cohort study may not have been included because of lack of information from those periods in Korea. This is a type of left censoring cohort bias. However, the standards for the VW troop’s conscriptions were reasonably high at that time, and only the healthy population may have been dispatched to the VW. Thus, the current results may have clinical implications for chronic non-severe cancer epidemiology in VW veterans. Some Korean VW veterans may also have served in the Korean Civil War from 1950 to 1953 or in dioxin-contaminated areas in Korea from the late 1960s to the early 1970s. However, the number of those veterans is believed to be small. Most importantly, Korean veterans who served in the VW were exposed to AO, although the exposure levels may differ. Therefore, whether the veterans had served in both wars did not seem to affect the exposure profile and their cancer incidence rates significantly. Furthermore, our study relied on hospital visit information to assess cancer diagnoses, and did not obtain pathological confirmation. If patients had ICD codes for neoplasms for diagnosis before confirmation, they might not have been actual cancer cases. However, this error has no direction with regard to the VW veteran group or the non-VW veteran group in the age-sex-region matched cohort; hence, non-differential misclassification may have occurred. Consequently, the risk of cancer may instead have a forward-to-null relationship. We could not demonstrate cancer- related factors, such as screening results, familial history, or other medical conditions, due to the nature of the dataset. Further studies on cancer-related factors are warranted. Finally, other conditions, such as the areas where the veterans served, and environmental conditions, may have affected exposure and health outcomes. Unfortunately, we could not obtain information about other environmental conditions besides the variables of veterans dispatched to the VW included in the manuscript owing to security reasons and lack of data. However, herbicides were sprayed in almost all areas of Vietnam during the war, even outside the official operations field. Therefore, all Korean veterans who serve in the VW should be considered exposed to AO, although the exposure levels may differ. A comparison between different situations or times requires a cautious approach.The risk of certain cancer types in Korean VW veterans was higher than expected. Although the toxicological effect of various chemicals used in the VW such as AO or dioxin could explain the large body of biological mechanisms, not all the oncologic effects on the VW veterans can be explained by current analysis. Further studies are warranted to investigate the various health determinants in veterans.The datasets presented in this article are not readily available because they belong to the National Health Insurance System of Korea, and access requires certification. Requests to access the datasets should be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Institutional Review Board of Gachon University Gil Medical Center (IRB No. GCIRB2019-076). The patients/participants provided their written informed consent to participate in this study.S-KK and W-JC conceived and supervised the project. WL, SP, J-HY and W-JC conducted data curation, analysis, and prepared the data visualization. WL, J-HY and W-JC contributed to the validation of results and data. WL, SP, S-KK, SH, J-HY and W-JC conducted the study and drafted the manuscript. All authors contributed to the article and approved the submitted version.


AF to CIN2+ was predominated by HPV 16 with 56.2%, followed by HPV58 (12.0%), HPV52 (8.5%), HPV18 (4.3%), and HPV51 (2.9%). HPV52 and 58 in the prophylactic HPV vaccine would enhance the protection against CIN2+ by approximately 20%. Conclusions: Regarding multi-valent HPV vaccine development in China, the HPV types 16, 52, 58, and 18 should be given priority for their high prevalence at the population level, high AR, notable AF, and high relative risk to high-grade cervical lesions.Background: To investigate the human papillomavirus (HPV) genotype distribution among the general population and assess the attribution of HPV genotypes targeted by vaccines to protect against cervical lesions theoretically. Methods: Cervical samples were collected from women aged 21 to 64 years old from Inner Mongolia and Shanxi Province in China who had not been vaccinated against HPV. HPV type-specific absolute risk (AR) to classified cervical lesions was calculated and then the attributable fraction (AF) was estimated, together with the combined contributions of the HPV types, targeted by four available HPV vaccines and five HPV vaccines in clinical trials in China to protect against cervical lesions. Results: A total of 6286 women with an average age of 44.1 years ± 8.41 (range: 21–64) participated in the study. The age distribution of 14 HR-HPV and HPV16/18 all showed a ‘U’ shape, which peaked in the ≤25 year-group and >55 year-group. The five most common genotypes were HPV16 (4.3%), HPV52 (4.1%), HPV58 (2.1%), HPV51 (2.1%), and HPV66 (1.7%). The prevalence of HPV types 6 and 11 infections was 1.1% and observed with  A total of 570,000 new cases of cervical cancer worldwide was recently reported in 2018, ranking the fourth most prevalent disease in terms of both incidence and mortality among women . It is eHPV is the common sexually transmitted virus worldwide. Infection of certain types of HPV is regarded as a causal and necessary factor for cervical cancer . AccordiHPV-DNA-based screening and prophylactic vaccination are effective measures against cervical cancer ,11. The In this study, we looked at the Absolute Risk (AR), Attribute Fraction (AF), and Relative Risk (RR) of single HPV genotypes, as well as the HPV infection status of different HPV vaccine groups, including both market and clinical trial vaccines, using different cytology and pathology stratification among rural women who had not been vaccinated against HPV. This work can illuminate the risk of different HPV infections and the variation trends among different HPV groups and vaccines in different disease status and provide evidence for efficient HPV vaccine designation and HPV prevention in the future.The women from Inner Mongolia and Shanxi province in China who attended cervical cancer screening at local Women and Children’s hospitals were recruited from June 2016 to June 2017. Women were eligible if they (a) aged from 21 to 64 years; (b) had signed the informed consent form; (c) were not pregnant, or 8 weeks after the end of pregnancy; (d) had no history of hysterectomy; (e) were not menstruating when screening was done; (f) had not been vaccinated against HPV; and (g) had not undergone cervical cancer screening within three years. ® medium, and stored under −80 °C.The demographic information and risk factors were investigated by trained nurses with a structured questionnaire. The gynecologic examination was performed by experienced clinicians, and cervical samples were collected, kept in a PreservCytThe first aliquot samples in Inner Mongolia were tested by cobas 4800 , the samples from Shanxi Province were tested by Aptima , and the rest were used for liquid-based cytology (LBC). The second aliquot was used for the HPV genotyping test , which uses a PCR-based hybridization line probe assay to detect 28 different HPV types, including 14 HR-HPV: HPV16/18/31/33/35/39/45/51/52/56/58/59/66/68; and 14 LR HPV: HPV6/11/26/40/43/44/53/54/69/70/71/73/74/82.The LBC slides were reviewed by experienced cytologists at the National Cancer Center/Cancer Hospital Chinese Academy of Medical Sciences (CHCAMS), and the results were graded according to the Bethesda System : negativHPV16/18-positive (cobas or Aptima tests) or ASCUS+ were referred to colposcopy within 12 weeks. Biopsies were taken directly on visual lesions, and random biopsies were taken if no visual lesions were detected for the subjects with cytology LSIL+. Histological diagnoses were performed by two experienced pathologists from CHCAMS and the diagnoses were categorized as: negative; cervical intraepithelial neoplasia grade 1 (CIN1); cervical intraepithelial neoplasia grade 2 (CIN2); cervical intraepithelial neoplasia grade 3 (CIN3); squamous cell carcinoma (SCC); adenocarcinoma in situ (AIS); and adenocarcinoma (ADC).The final disease status for the women with no pathology results were classified in different pathology groups ,18: (1) 2 test was used to analyze the significance of data. Statistical significance was set at p < 0.05 and the AF was calculated using a mathematical algorithm in EXCEL.According to the HPV vaccines approved by NMPA or ongoing clinical trials in China, we categorized HPV infections into different groups: HPV6/11, HPV16/18, HPV16/18/58, HPV6/11/16/18, HPV16/18/52/58, other LR-HPV26/40/43/44/53/54/69/70/71/73/74/82, HPV 6/11/16/18/31/33/45/52/58, HPV6/11/16/18/31/33/45/52/58/59/68 and HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59. Means and standard deviations were used to describe the numeric variable. HPV type-specific AR and corresponding AF  to diffe2 = 5.20, p = 0.023; HR-HPV: χ2 = 4.83, p = 0.028). The HPV-positive rates were different among women using different contraceptive methods . The HPV-positive rates in women using condoms were significantly lower than women without any contraception methods  and women who used precautions other than condoms .In this study, 6241 women were screened and 6286 (97.9%) women had met the inclusion criteria. The age distribution of HPV infection by cytological and pathological grade is shown in The five most common genotypes were HPV 16, 52, 51, 58, and 6616 (4.3%), HPV52 (4.1%), HPV58 (2.1%), HPV51 (2.1%), and HPV66 (1.7%); In women with NILM: the top five types of HPV infections indicated by AR or AF were HPV52, 16, 51, 66, and 58. Among 223 LSIL women: HPV16, 52, 56, 31, and 58 were the most frequent HPV types by the order of AR, which is a little different from the AF, the infection rate of HPV 58 increased from the fifth to the third, and the HPV 56 infection decreased from the third to the fourth; For the RR, HPV52 was the highest, followed by HPV58, 66, 16, and 18. In 147 HSIL or above women, the AR of HPV16, 58, 52, 18, and 51 were higher than the other HPV types, which is the same for the AF ranks; The RR ranks, from high to low, were HPV16, 31, 58, 18, and 33, respectively .The HPV types with a higher AR in CIN1 were: HPV58, 16, 52, 18, 33 and 51, and the AF were HPV58, 16, 52, 33, 18, the order of HPV33 and HPV18 were changed; For the RR, the HPV58 was the highest, followed by HPV33, 16, 18, 52. By contrast, the 5 HPV types with a higher AR in CIN2+ were: HPV16, 52, 58, 18, 51, 35; For AF, the types were: HPV16, 58, 52, 18 and 51, the order of HPV52 and HPV 58 were changed; The RR ranks, from high to low, were HPV16, 58, 18, 33, 45, respectively .p < 0.001). The HPV prevalence in the groups of NILM and abnormalities were 49 (0.9%) and 19 (2.1%) for HPV6/11, and 72 (14.5) and 421 (48.1%) for 14 HR-HPV. The other LR-HPV were 300 (5.7%) and 118 (28.2%), HPV16/18 were 181 (3.4%) and 161 (47.1%), HPV6/11/16/18 were 229 (4.3%) and 176 (43.5%), HPV6/11/16/18/31/33/45/52/58 were 536 (10.1%) and 321 (38.2%), HPV16/18/58 were 238 (4.5%) and 223 (25.5%), HPV16/18/52/58 were 399 (7.5%), HPV6/11/16/18/31/33/45/52/58/59/68 were 607 (11.4%) and 350 (40.0%), and HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59 were 724 (13.6%) and 401 (45.8%), respectively.HPV prevalence was 24.2% for 28 HPV types, 19.3% for 14 HR-HPV, and 1.1% for HPV 6/11. The cytological diagnosis was as below: 5309 (84.5%) NILM, 876 (13.9%) abnormal cytology (ASCUS or worse), 101 (1.6%) unsatisfactory. The prevalence of different HPV infection groups in 6286 women, stratified by cytology, is shown in p < 0.001). In the group of HPV 6/11 and other LR-HPV, there were no obvious linear trends with the pathological grade (p > 0.005). In the pathological normal and CIN2+ cases, the prevalence in the HPV6/11 group was 61 (1.0%) and 2 (1.5%), 923 (15.7%) and 119 (90.2%) in the 14 HR-HPV group, 362 (6.2%) and 17 (12.9%) in the other LR-HPV group, 220 (3.8%) and 83 (62.3%) in HPV16/18, 279 (4.8%) and 83 (62.3%) in HPV6/11/16/18, and 639 (10.9%) and 114 (86.4%) in HPV6/11/16/18/31/33/45/52/58, 294 (5.0%) and 98 (74.2%) in HPV16/18/58, 480 (8.2%) and 108 (81.8%) in HPV16/18/52/58, 722 (12.3%) and 114 (86.4%) in HPV6/11/16/18/31/33/45/52/58/59/68, and 868 (14.8%) and 118 (89.4%) in HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59, respectively. From For all the pathology results: 5861 (93.2%) normal, 98 (1.6%) CIN1, 68 (1.1%) CIN2, 59 (0.9%) CIN3, 5  cancers, and 195 (3.1%) had no results. This cross-sectional study mainly discussed the HPV prevalence and genotype distribution in different age groups and cervical lesions. We focused on women without HPV vaccination to elaborate the level of HPV infection in China, evaluate the oncogenic potential of individual HPV genotypes, and estimate the population-based impact of 4 HPV vaccines approved by NMPA and 5 HPV vaccines in clinical trial in China.The prevalence of overall HPV infection and 14 HR-HPV infections had two peaks at age of <25 years old and at an age of >55 years old , similarHR-HPV prevalence is 19.3% and 5  cervical cancer cases were found in this screening program. Both the HR-HPV infection rate and cervical cancer prevalence were higher in women from Shanxi and Inner Mongolia (our data) than in Shanghai  , Gansu  The smoking rates among Chinese women are low. In our study, the smoking rate is too small to evaluate its role in HPV infection. (2) We only have the data about women in the Shanxi and Inner Mongolia provinces; two regions with high HPV prevalence areas. But all the women in our study came from the general population and didn’t undergo cervical cancer screening within recent three years, so it can truly reflect the prevalence of HPV infection in China. In addition, the cytology and pathology results were graded by senior doctors in CICAMS and reported an HPV prevalence of LR-HPV infection, which was seen in only a few studies.Our study suggested that the type-specific prevalence of HPV changes with age, and HPV16, 18, 52, and 58 had high prevalence, absolute risk, notable attributable fraction, and relative risk to high-grade cervical lesions among Chinese women. The priority should be given to HPV16, 52, 58, and 18 for multi-valent HPV vaccine development and HPV screening programs in China.


High rates of new cervical cancer cases and deaths occur in low- and middle-income countries yearly, and one reason was found related to limitation of regular cervical cancer screening in local and low-resource settings. HPV has over 150 types, yet certain 14–20 high-risk and 13–14 low-risk types are common, and, thus, most conventional HPV nucleic acid assays, for examples, Cobas 4800 HPV test  and REBA HPV-ID  were developed to cover these types. We thereby utilized bioinformatics combined with recent isothermal amplification technique at 35–42 °C to firstly describe multiplex recombinase polymerase amplification assay that is specific to these common 20 high-risk and 14 low-risk types, and also L1 and E6/E7 genes that target different stages of cervical cancer development. Multiplex primer concentrations and reaction incubation conditions were optimized to allow simultaneous two gene detections at limit of detection of 1000 copies  for L1 and 100 copies (0.0125 fg) for E6/E7, respectively. The assay was validated against urogenital and other pathogens, normal flora, and human control. In 130 real clinical sample tests, the assay demonstrated 100% specificity, 78% diagnostic accuracy, and 75% sensitivity compared with REBA HPV-ID test, and is much more rapid (15–40 min), less expensive (~ 3–4 USD/reaction) and does not require instrumentation . Hence, the developed novel assay provides alternative screening tool for potential local screening. Furthermore, as this assay uses safe chemical reagents, it is safe for users. The reasons were found primarily related to a smaller rate of access to cervical cancer vaccination as a preventive venue and to regular cervical cancer screening programs  compared with high-income countries2. In Thailand and other Southeast Asia countries, cervical cancer represents the most common cancer among women. The Institute of Oncology/International Agency for Research on Cancer reported that ~ 9158 women were diagnosed with cervical cancer and 4705 died every year, and estimated approximately 3.4% of the Thai women population carried human papillomavirus (HPV) high-risk types 16 or 183. In addition, the cost of radiation therapy for cervical cancer and treatment of side effects, such as gastrointestinal or genitourinary side effects, is as high as ~ 63,103 USD per patient. The 2018 economic burden for merely radiation therapy in Thailand was ~ 131 million USD, and for the diagnosis of these advanced cervical cancer patients was ~ 129 million USD4. Subsequently, an effective, simple, easy-access, and low-price screening method for early prevention is important to minimize the number of cases and the economic burden of advanced cervical cancer patients.Cervical cancer is a major public health problem in developed and developing countries. According to the World Health Organization (WHO), an estimated 604,000 new cases and 342,000 deaths of cervical cancer occurred worldwide in 2020, and 90% of these cases were in low- and middle-income countries7. HPV has over 150 types and is classified into high-risk (HR) or low-risk (LR) for cervical cancer development. Common HPV HR types include 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 58, 59, 68, and 82, given types 16 and 18 generally account for 70% of cervical cancer. Common HPV LR types include 6, 11, 32, 40, 42, 43, 44, 54, 61, 70, 72, 81, 84, and 879.More than 95% of cervical cancer are caused by HPV. HPV is also associated with more than 60–90% of anal cancer, vaginal cancer, oropharyngeal cancer, and penile cancer11. For instance, Spence et al.12 reported a false negative rate of Pap smear to be 35.5% on average with the sensitivity ranging from 30 to 87%11. Furthermore, the Pap smear may require another HPV-specific nucleic acid test, such as Cobas 4800 HPV test  and REBA HPV-ID , as accompaniment and identification of HR types. Therefore, the HPV-specific tests were suggested as an alternative screening tool and some even reported that these tests could allow screening intervals to be extended to ≥ 5 years15. The Cobas 4800 HPV test  utilizes amplification of target DNA by polymerase chain reaction (PCR) and nucleic acid hybridization for detection of HPV HR types 16, 18, and a pool of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 6815. REBA HPV-ID  is a PCR-based reverse blot hybridization assay for 19 HPV HR probes  and 13 LR probes  for HR and LR genotyping16. Although the Cobas and REBA assays are available commercially and used widely in in vitro clinical laboratories, they require an expensive thermal cycler for PCR amplification of HPV DNA and a special instrument for product-probe detection16.Traditionally, a cytological examination by Papanicolaou test (Pap smear) is simple and established. However, the procedure is inconvenient, cannot be performed in local resource- and clinician-limited settings, and its sensitivity  and specificity to detect precancerous or cancerous changes in cells are relatively low compared with nucleic acid amplification test17. For HPV detection, Ma et al.18 developed RPA for HPV HR types 16 and 18, and Gong et al.19 developed RPA for 13 HPV HR types based on the L1 gene. We, thereby, further developed a multiplex recombinase polymerase amplification (mRPA) assay with attempt to cover broad HPV HR and LR types, as well as normal and precancerous stages of HPV developmental cycle in humans, as potential local use in a single tube for broad and precancerous screenings of cervical cancer. We determined the limit of detection, validated proper specificity on positive controls comprising some HR and LR types and negative controls comprising urogenital bacteria, other viruses, human and mouse, and evaluated the assay efficiency  on real 130 clinical cervical swab samples compared with the Cobas and REBA results. In HPV developmental cycle, a late gene L1 encodes for a major viral capsid protein, while early genes E6 and E7 encode for oncoproteins and their detection suggests an increased risk of cancer development . E6 degrades a human growth suppressor protein p53 and E7 degrades a human retinoblastoma tumor suppressor protein pRb, and, thus, the E6 and E7 induce cancer development by evasion of human growth suppressors, resisting cell death, sustaining proliferative signaling, enabling replicative immortality, angiogenesis induction, and activation of invasion and metastasis20. Consequently, this study designed multiplex degenerate primers based on HPV L1 and E6/E7 genes, covering up to 20 HR and 14 LR types, and the E6/E7 amplification product could suggest an increased risk of cancer.Recently, an isothermal amplification assay like recombinase polymerase amplification is emerged to solve this problem. The recombinase polymerase amplification utilizes 3 enzymes, a recombinase, a single-stranded DNA-binding protein, and strand-displacing polymerase, to allow isothermal amplification of specific primer-binding target(s) at 37–42 °C. This allows the reaction to be simply performed by a heat block or water bath, or even holding a tube in hands, supporting low cost, rapid and point-of-care molecular testswww.megasoftware.net/), and RPA degenerate primers were designed using Primer-BLAST21 or PrimedRPA22 and manual design (Table All GenBank available HPV type L1 (and E6/E7) sequences were aligned using ClustalW , urogenital normal flora (Staphylococcus aureus and Escherichia coli), skin normal flora (Staphylococcus epidermidis), viruses, and mammals ; and the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University , and the Institutional Biosafety Committee (IBC) of the Faculty of Medicine, Chulalongkorn University (MDCU-IBC016/2019), respectively. In brief, cervical swab samples were collected by genecologists from patients who visited the Buddhachinaraj Phitsanulok Hospital during 2013–2014 and the Department of Gynecological Out Patient, King Chulalongkorn Memorial Hospital, Bangkok, during 2019–2020. The samples were lysed in Pathogen Lysis Tubes L  using Tissue Lyser LT (Qiagen), and total nucleic acids were extracted automatically using the Cobas 4800 system (Roche Diagnostics). Then, these genomic DNA were HPV genotyped following the Cobas 4800 HPV test (Roche Diagnostics) and the REBA HPV-ID test (Molecules and Diagnostics); and were also the respective genomic DNA for our mRPA assay. All subjects provided written informed consent, and all data were fully anonymized before assessment. The study was performed in compliance with the Declaration of Helsinki.For HPV positive controls and clinical samples, the respective genomic DNA were provided by the Buddhachinaraj Phitsanulok Hospital, Phitsanulok; and the Faculty of Medicine, Chulalongkorn University, Bangkok® Basic reaction powder , 14.75 μL dehydration buffer (TwistDx), ~ 25 ng DNA (unless specified), and 1.25 μL of 280 mM magnesium acetate . The reaction was incubated at 39 °C (unless specified) for 40 min (unless specified). The optimization of the mRPA reaction condition included the determination of each minimal primer concentration that yielded both L1 and E6/E7 gene amplification products, and the shortest incubation time with the highest limit of detection (LOD) and correct specificity. The reaction was terminated by 80 °C heating for 2 min. The result was analyzed via 3% agarose gel electrophoresis: the amplicon sizes for L1 and E6/7 RPA were ~ 250 bp and ~ 170 bp, respectively.The mRPA reaction (25 μL) comprised 0.12–0.48 µM each of primers (unless specified) in Table ® GT PCR Master Mix , 0.12–0.48 µM each primer, and ~ 25 ng DNA (unless specified). The PCR conditions were 95 °C 5 min followed by 35 cycles of 95 °C 15 s, 56 °C 30 s, and 72 °C 30 s, with a final extension at 72 °C 5 min. The proper size amplicon product was analyzed by 3% agarose gel electrophoresis. The amplicon sizes for L1 and E6/7 PCR were 188–194 bp and 117–121 bp, respectively. Quantitation of the amplicon was additionally performed using Qubit™ dsDNA HS and BR Assay Kits . This kit quantifies the amount of amplicons  at high sensitivity.Single-gene PCR and L1-E6/E7 multiplex PCR (mPCR) were employed as the parallel nucleotide amplification experiments for our mRPA primers and LOD. The PCR reaction (25 µL) comprised 12.5 µL EmeraldAmp23) ÷ (PCR product length (bp) × 6.6 × 1011).For preparation of HPV L1 (and E6/E7) gene as representative HPV HR and LR DNA templates, the PCR primers /e2, given p at an average incidence of 75%, confidence level of 95%, and e of 7.5% for margin of error. This yielded an N of 129. Therefore, we tested 130 random HPV-positive and negative samples by our developed mRPA methods compared with the conventional Cobas and REBA results. The assay efficacy was calculated based on the following equations: sensitivity = true positive ÷ , specificity = true negative ÷ , false positive = 1 − specificity, false negative = 1 − sensitivity, and accuracy = (true positive + true negative) ÷ total samples.To determine the performances of the conventional (Cobas and REBA) and our developed methods, statistically required sample numbers (N)  were calculated according to the equation: N = (p (1-p) z29. Hence, the inclusion of E6/E7 in this study benefited the diagnosis in this HPV DNA integration subjects, and the mPCR and mRPA of the positive HPV HR samples that mostly no E6/E7 gene band appeared might reflect the non-precancerous state of the sample.As sequences of HPV L1 and E6/E7 are highly variable among types, a primer multiplex system containing 8 degenerate primers was required to cover broad HPV L1 and E6/E7 gene types, of 20 HR  and 14 LR  types. We performed bioinformatic analyses by BLASTN on each HPV primer sequence specificity against NCBI GenBank non-redundant database , and the primers were specific to HPV (Table 31. Noted that in our mPCR and mRPA displayed the < 100 bp bands (< ~ 200 bp for mRPA), which were primer dimers and non-specific amplification, and were found in some positive and negative clinical specificity samples, for examples, influenza B, C. trachomatis, N. gonorrhoaea, S. saprophyticus, S. aureaus, S. epidermidis, H. sapiens and M. musculus  in RPA) making the amplicons heavier and we performed a simple one-step clean-up the reaction for low-cost and rapid potential local use, in replace of an amplicon purification step using commercial kits. The shifts of RPA amplicon bands had been reported elsewhere32 to obtain equal amplification efficiency. In brief, each gene primer was individually checked for a single amplification fragment, given that synthetic PCR amplicons of HR-HPV types 18 and 33 and LR-HPV type 11 also supported the validation of the effectiveness of the primers . We found the minimal concentration of multiplex primers for L1 (0.1–0.2 µM each) and E6/E7 (0.2 µM each) RPA to be different . Individual L1 (and E6/E7) primers could successfully amplified the HPV templates Fig. A,C. Cons36. Meanwhile, the Cobas and REBA results exhibited 18.46% diagnostic disagreement between each other. Furthermore, the mRPA result analysis by agarose gel electrophoresis may be replaced by using fluorescent DNA dye, such as SYBR Green I , or DNA probe paper strip for an even more rapid total assay time. We are continuing to improve the mRPA limit of detection by reducing the number of primers to focus on the HPV HR types and the gene expression detection of E6/E7 as reverse transcription-mRPA37.The schematic illustration of RPA and multiplex 2-gene amplification concepts as local HR- and LR-HPV screening tool was summarized in Fig. Our developed HPV mRPA assay for broad-type HPV detection  is simple, rapid, inexpensive, specific, sensitive, and does not require any complicated instrumentation  Fig. . EvaluatSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Women who test positive for the human papillomavirus (HPV) but have normal cytology constitute the predominant subgroup of patients in the screening population in the post-vaccination era. The distribution of HPV genotypes changed dramatically, which was attributable to an increase in HPV vaccination coverage. These changes have created uncertainty about how to properly manage women with normal cytology, non-HPV16/18 infections, or persistent infections. Current recommendations include retesting and continued surveillance in the absence of HPV16/18 infection. However, these are not always applicable. The ability to implement genotyping or incorporate HPV16/18 with some additional high-risk HPV (HR-HPV) types for triage and management with the aim of identifying type-specific risks in this population could be acceptable. When the next set of guidelines is updated, generating potential triage strategies for detecting high-grade cervical lesions, such as the p16/Ki67 cytology assay and other alternatives that incorporate genotyping with newer tests, should be considered. Current clinical management is shifting to risk-based strategies; however, no specific risk threshold has been established in this population. Importantly, innovative triage testing should be evaluated in combination with primary screening and management. Furthermore, there is an untapped opportunity to coordinate HPV genotyping in combination with colposcopic characteristics to modify risk in this group. Hence, providing a more personalized schedule through the efficient application of risk stratification and improving the detection of pre-cancer and cancer is an option worth exploring. Human papillomavirus- (HPV-) based screening, either alone or in combination with cytology, is reported to be more sensitive than cytology alone in detecting high-grade squamous intraepithelial lesions (HSILs); this has reduced the number of subsequent screening rounds required and has been used in several settings –3. BecauCurrent guidelines recommend that women who are NILM be referred for colposcopy if HPV16/18 is positive, and that those who are negative for HPV16/18 be retested within 1 year , 6. On tOther triage options including incorporated cytology and HPV16/18/31/33/45 genotyping should be considered in countries with high rates of referral for colposcopy , and theIn this review, we examined the need for colposcopy referral in women who are NILM but positive for non-16/18 HR-HPVs by evaluating the risk for precursor lesions relative to these types. We also summarized the current management strategies  for this population with the aim of providing a valuable reference for policymakers to tailor further alternative management strategies or strategies complementary to colposcopy.Women who are NILM but positive for HR-HPV account for most of the subjects with positive screening results encountered in routine practice, ranging from 6.7 to 14.9% –18. In tSeveral longitudinal and cross-sectional studies have reported a relatively high prevalence of HSIL in women who were NILM but positive for HR-HPV. The proportion of HSIL or more advanced lesions ranges from 5.40 to 20.39% , 27–29. Human papillomavirus is a major carcinogen in the uterine cervix. Although most HPV infections tend to regress, there is a risk of preneoplasia and cancer, particularly in women who are NILM but have non-16/18 HR-HPV infections. The fact that HPV16/18 poses a considerable risk for developing HSIL in women who are NILM is well-established in the literature. Women with HPV16/18 infections have a higher risk for HSIL and more advanced lesions than those with non-16/18 HPV infections , 30. SevAccording to HPV genotypes, different age groups have different risks of progression over time . MoreoveThe different oncogenic potentials of genotypes confer disparate risks for cervical precancerous lesions in women who are concurrently NILM and HPV positive. However, an HPV infection must be persistent to cause precursor lesions and cancer. The natural history of HPV infection is well-documented. It has been demonstrated that women who are NILM but have HPV persistence after a baseline HPV-positive test have an increased risk of HSIL. The rate of oncogenic development in natural course models of precancerous lesions and cancer is a crucial parameter because it determines the age at which models predict that HSIL will be identified during screening . Women wHuman papillomavirus genotypes vary substantially in terms of cumulative risk and annual rate of progression to pre-cancer . Women wLactobacillus crispatus or Lactobacillus iners predominance at the time of HSIL diagnosis  and cancer, with preneoplastic lesions taking several years to develop . Howeveriagnosis .Therefore, recognizing the natural history of precancerous lesions, cancer, and other potential cofactors is of great clinical importance for determining specific risk thresholds and proper clinical screening intervals, particularly in women who are NILM positive for non-16/18 HR-HPVs. In addition, it has been observed that the maintenance of the vaginal microbiome and the improvement of vaginal microecology favor the regression of cervical lesions through a prebiotic effect . ElucidaAs discussed earlier, these data highlight that women who are NILM but positive for non-16/18 HR-HPVs are still at risk of developing precancerous lesions and cancer beyond 3 or 5 years. The differences in the likelihood of developing precursor lesions and cancer depend primarily on the carcinogenic potential of different genotypes and on geographic and ethnic variations.The current major guidelines for the management of women who are NILM but positive for non-16/18 HR-HPVs are reported in Partial HPV triage of normal cytologic findings offers an effective approach to discriminate between women with HPV16/18 infections and those with non-16/18 infections; however, the implementation of this strategy is limited by the higher distribution of non-16/18 HR-HPVs in women who are NILM. HPV16/18 genotyping is unlikely to be effective in triaging women who are NILM but positive for HR-HPV; it has a positive predictive value (PPV) of 11% and is not generalizable to younger women who have been vaccinated for HPV. Conversely, the HPV E6/E7 messenger ribonucleic acid (mRNA) assay may play a role in risk management in this population . The datAnother unresolved problem is the management of women who are NILM but have HPV persistence after two rounds of triage , includiThe role of colposcopy in predicting cervical disease and in cervical cancer screening remains controversial. Colposcopic impression of underlying disease appears to be less effective in the presence of non-16/18 HPVs and in those over the age of 50  suggesteAccording to the literature, vaccination will not fully avert an increase in colposcopy referrals and the detection of preneoplasia in the first 2–3 years of HPV-based screening. Given the herd effect of the HPV vaccine, the amount is thought to be reduced after 5–6 years, and then by approximately 20% , 58. It In screening management settings, women who are NILM may have a greater association with smaller and undetectable lesions than those who have cytological abnormalities . HPV16-aMorphological appearance is less sensitive and highly subjective and can therefore lead to false-negative results , 63. WomThe interpretation of cytomorphology is influenced by sampling, different cytologic presentations, and other factors . The paucity of abnormal cells is the major cause of cytological differences, and variations in interpretation are affected by opaque inflammation , 65. Furp = 0.04). The low risk for cervical preneoplasia in DS-negative women allows safe extension of follow-up intervals to 3 years. Moreover, Li et al.  cytology. Combining cytology and HPV testing is intended to improve the detection of cervical abnormalities in the future . The addi et al.  comparedCervical lesions linked to non-16/18 HR-HPV infections represent the predominant subgroup in the screening population due to the dramatic change in the distribution of HR-HPV genotypes among vaccinated women , 70–72. As HPV testing assays become more well-established, the genotype and duration of infection can allow for more accurate risk evaluations . HPV genThe data presented by Arbyn et al.  confirm Extended genotyping is an important part of precise risk evaluation and appropriate clinical management and offers the opportunity to apply risk discrimination and tailor management . Increasing evidence supports the concept that extended HPV genotyping is effective for risk stratification in women who are NILM . The stuMultiple studies have illustrated different approaches to triage for women who are NILM but positive for non-16/18 HR-HPVs, as presented in In addition, the DS cytology assay, which has been approved as a triage test by the FDA, is more accurate in detecting preneoplasia in HPV-positive women than cytology alone . This coSeveral innovative screening assays have been introduced, including HPV triage protocols (HPV mRNA and DH3), surrogates for cytology (automated DS cytology), biomarkers  methylation), and other technologies . For instance, in China, the DH3 HPV assay, a newly developed RNA–DNA hybrid capture-based technique similar to the Hybrid Capture 2 (HC2), has been utilized (in combination with HPV16/18 genotyping) for cervical cancer screening , 84, 85.The anticipation of these screening options will provide a promising insight into developing a proper screening program in women who are NILM in collaboration with cytology. Nevertheless, clinical evidence for these performances is still being studied, particularly in association with risk prediction during a follow-up.In conclusion, our review supports the combination of various HPV genotyping assays and recommends that HPV persistence and regional contexts be considered in overall assessment, follow-up, and management in women who are NILM, which will allow for the evaluation of genotype-specific test outcomes and will support risk discrimination or tiered risk stratification. It will also reduce the burden of colposcopy referral as clinicians will be able to better distinguish women with HPV persistence from those with incidentally detected HPV infection. It is noteworthy to consider extended genotyping based on different natural courses and risk profiling . MoreoveMW conducted the literature search and drafted the first manuscript. XM, HL, BL, CW, XF, and AF contributed significantly to writing and critically revising the review. FX conceived and designed the structure, framework of this review, and critically revised the work. All authors were involved in the study design and read and approved the final manuscript.This work was funded by the Tianjin Municipal Science and Technology Commission Special Foundation for Science and Technology Major Projects in Control and Prevention of Major Diseases (Grant No. 18ZXDBSY00200).The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.