Paraguay launched a human papillomavirus (HPV) vaccination program in 2013, so virological surveillance is important for measuring the impact of HPV vaccines. This study aimed to estimate the type-specific HPV frequency in unvaccinated sexually active women aged 18–25 years in the metropolitan area of Asuncion as a baseline for monitoring the HPV vaccination program. This study included 208 women, attending the Central Laboratory of Public Health between May 2020 and December 2021, were invited for testing through social networks and flyers at local health centers and higher education institutes. All participants who agreed to contribute to the study signed a free, prior, and informed consent form and answered a questionnaire that included basic demographic data and determining factors of HPV infection. Human papillomavirus detection and genotyping were conducted using the CLART HPV2 test  that allows the individual identification of 35 genotypes. 54.8% women were positive for any HPV type, with 42.3% positive for high-risk HPV (HR-HPV) types. Several factors were associated with HPV detection including the number of sexual partners, new sexual partners, non-use of condoms, and history of other sexual infections. Moreover, multiple infections were identified in 43.0% of the young women. We detected 29 different viral types present in both single and multiple infections. HPV-58 was the most commonly detected HPV type (14.9%), followed by HPV-16, HPV-51, and HPV-66 (12.3%). We estimated the prevalence of bivalent (16/18), quadrivalent (6/11/16/18), and nonavalent (6/11/16/18/31/33/45/52/58) vaccine types to be 8.2%, 13%, and 38%, respectively. These results reinforce the importance of surveillance studies and provide the first data regarding circulating HPV genotypes in the unvaccinated population in Paraguay, thus generating a baseline to compare future changes in the overall and type-specific HPV prevalence after HPV vaccination. Human papilloma virus (HPV) is the most common sexually transmitted infection (STI) among sexually active young women, being acquired shortly after the first sexual intercourse, with the highest prevalence seen in women aged 25 years or younger , 2.Based on epidemiological and biological data HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 should be considered carcinogenic, or high-risk types (IARC Group 1); HPV26, 53, 66, 67, 68, 70, 73, and 82 probably/possibly carcinogenic or probable/possible high-risk types (IARC Groups 2A and 2B) and HPV 6 and 11 non-carcinogenic, or low-risk types (IARC Group 3) ; 20–21 , 22–23 , and 24–25  .A total of 29 different viral types were identified, present in both single and multiple infections among 208 studied women . The mosThe prevalence of HPV types preventable with currently available vaccines, was 8.2% for the bivalent vaccine (16/18) (95% CI 4.5–11.9), 13.0% for the quadrivalent vaccine (6/11/16/18) (95% CI 8.4–17.6), and 38.0% for the nonavalent vaccine (6/11/16/18/31/33/45/52/58) (95% CI 31.4–44.6%). Infection prevalence of HPV types against which vaccine cross-protection has been reported, such as 31/33/45, was 10.6% (95% CI 6.4–14.8) .The WHO global strategy for the elimination of cervical cancer as a public health problem proposes a multidisciplinary approach that includes community education, social mobilization, vaccination, detection, treatment, and palliative care.HPV vaccination in combination with timely detection and appropriate treatment of cervical cancer precursor lesions is considered to have the potential to achieve the elimination of this disease, defined by the WHO as fewer than 4 new cases per 100,000 women-years .With the introduction of the HPV vaccine, changes in the transmission dynamics of the virus are expected, making necessary to monitor changes in the prevalence of HPV and the patterns of HPV-associated diseases. Population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low- and middle-income countries, so cross-sectional surveys in sentinel populations could be a suitable alternative .Paraguay, like many other countries worldwide, has incorporated the HPV vaccine into its national immunization program, requiring a virological surveillance to measure its impact. It can provide an early measure of vaccine effectiveness by quantifying the magnitude of reduction in HPV type-specific prevalence, including possible evidence of herd and cross protection and provide timely assessment of vaccination strategy. The principle of HPV prevalence surveillance is to recruit samples in age groups at risk of HPV exposure and conduct repeat-cross sectional analyses .In this study, we analyzed a cohort of unvaccinated sexually active women between 18–25 years of age and found an overall HPV prevalence of 54.8%, comparable to others cross-sectional studies in South American as Colombia with a prevalence of 60.3% in a same age group , and ArgPrevious studies have established sexual contact as the main route of HPV transmission. Thus, the higher HPV prevalence in women ≤25 years of age could be explained by recent first sexual contact, a condition that increases the chance of exposure to the virus , 25.Although the demographic data and all risk factors for acquiring HPV showed that the participants were moderate risk e.g., only 24.0% used condom, 33% reported STI, 40.9% had reported 2 or more partners, the results reported high risk HPV types. At least one type of HR-HPV was found in 42.3% of young women, with HR-HPV prevalence ranging from 18.7%–45.7% among women under 25 years old worldwide –33. A siChlamydia trachomatis have been associated with cell inflammatory processes in the cervix, facilitating HPV entry and reducing its elimination Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: PartlyReviewer #4: PartlyReviewer #5: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: YesReviewer #5: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: NoReviewer #3: YesReviewer #4: NoReviewer #5: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: YesReviewer #3: YesReviewer #4: YesReviewer #5: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for including me in reviewing this manuscript. The article is that “HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY”. The sample size is small. The current study does not deliver anything significant new or outbreaking in this matter; at this point, all of this, justifies rejection of the manuscript.1. The introduction is long. Authors should abbreviate some places by combining them. Otherwise, the interest in the subject and the flow of the subject are scattered. For example, sections on vaccines and HPV can be shortened. Places that indicate the purpose of the subject can be highlighted.2. There are minimal grammer errors. I recommend that a native English speaker review the article or use a professional language editing service.3. ”National primary screening is mainly aimedat women aged 30 years and above; younger women does not frequently attend health servicesfor screening tests.” What is this program like, how is it done?4. You mentioned the vaccination program that started in 2013. What is this program?5. “which was similar to that reported in other…” Is the age group of all the other studies mentioned in this paragraph the same?6. As you know, HPV can be eliminated after a certain period of time. There may be elimination, especially under the age of 25. What do the authors think about this and about screening in the early age group?Reviewer #2: The authors conducted an HPV prevalence and genotyping study among unvaccinated females using screening samples in women aged 18-25 years. The data are important because they establish a baseline for monitoring impact of Paraguay’s HPV vaccination program. The study appears to be internally valid and most of the analyses seem to be appropriate. Some edits could improve the readability and further focus the paper on the important information.Although the manuscript is generally well-written, additional attention to English grammar is warranted.Abstract:First sentence stating “several young women remain unvaccinated” is quite confusing. Isn’t it true that at the time of this study, women in the age group included were never vaccine eligible? Quadrivalent vaccine should be moved from second sentence to first . Methods in abstract are almost non-existent – please include some information on the recruitment procedures, including how epidemiologic data and specimen were collected and some basics of the typing assay, such as number of types detected.Introduction:The introduction is quite long and could be trimmed considerably to focus more on essential information needed to understand the context of the paper. Some examples are listed below.The first two sentences of the paper suggest the main reason young women are susceptible to STIs is immaturity of genital tract. While this may be a contributor, the initiation of sexual activity and non-monogamous relationships should not be ignored. Those first two sentences could be deleted – “HPV is the most common STI” is a better start. The second paragraph is largely extraneous and most if not all of it could be deleted. Line 53 introduces the term “high-risk types,” which needs to be defined, as it was not introduced in the previous paragraph about the categories of HPV types.The introduction really needs a more complete description of the vaccination program. There is a long paragraph on page 4 beginning with line 64 describing all the internationally approved vaccines, and easily lost at the end of this paragraph is one sentence about Paraguay: “In Paraguay, the quadrivalent Gardasil vaccine was introduced to 10-year-old girls in 2013.” The international information could be shortened, and more information on the Paraguay program added. How many doses of Gardasil do girls receive? In what setting ? What has the coverage been? Doing the math, with the study conducted in 2020-21, the females included in the prevalence study had mainly not been vaccine eligible, is this correct? This needs to be spelled out for the reader. On first read, it seemed like the authors had screened out HPV-vaccinated women, which raised questions about vaccination coverage and known herd effects from HPV vaccination. This may still be an issue in the youngest women, and should be addressed in the discussion. It is not a real problem with internal validity, but it needs to be clear what exactly was done and why.Terminology with different meanings has been used interchangeably . Impact is the correct term for what this study provides a baseline to assess. Similarly, throughout the paper, the authors refer frequently to vaccines “containing” different HPV types; as there is no virus in the vaccines, it may be more appropriate to say the vaccines “target” different HPV types.Materials and methods:Study Population:Please provide more clarity regarding recruitment methods. Do women in this age group normally attend the LCSP for reasons other than study participation? Were women recruited from the LCSP or from other community sites? Was any incentive provided? Were women given results of the HPV testing, and if so, how were they counseled? Was the study actually conducted on “randomly selected samples”, as stated on page 5 in line 103, or were a convenience sample of women recruited to participate in this study? These details are important for replicating study methods in a future evaluation of impact.Statistical analysis:In the statistical analysis section on page 7 in lines 150-156, please describe the HPV type groupings, and how multiple types were accounted for. For example, if a woman had HPV 18 and HPV 58 detected, would she be included in the numerator of both types? In Figure 1, if a woman had both a HR and a LR type detected, where would she be represented?Figure 1 is potentially misleading because it shows proportion positive for the different type groups out of 100% -- this makes it appear that the prevalence of HR-HPV is higher in older age groups, but that may not be true, and the prevalence is not shown. Consider remaking this figure so that the height of the bars represents total prevalence in each age group.On page 7 in line 151, consider not using “qualitative variables” in the description of the methods. “Participant characteristics” would be a better descriptor.Some analyses presented in the results (e.g. odds ratios and 95% CIs) are not described in the stastistical analysis section.Results:Table 1 and the corresponding text description are inconsistent. For example, the text refers to percentage who were students without employment, but the table does not include employment status. The text refers to percentage with at least high school education, but the table combines primary and secondary. Table 1 does not show results by age; age is usually one of the strongest predictors of HPV prevalence, and this basic descriptive information needs to be provided.Consider not reporting odds ratios since this may not be the most appropriate measure of association for this study. Since HPV is not a rare outcome, odds ratios overestimate the strength of the association. Consider not reporting a measure of association at all  or consider using prevalence ratios instead. Regardless, the description of this measure of association should be included in the statistical analyses section.In table 3, please specify N. HPV6 prevalence appears to be off by .1.In the description of figure 2 on page 11 beginning with line 200, please be clear about how prevalence is determined for single and multiple infections. What is the denominator? Is it 208?In the legend of figure 2 on page 11 in line 210, HPV 72 LR is not mentioned.Discussion:Generally, the discussion could be trimmed to focus on the most salient information. For example, the paragraph on multiple types that starts on page 13 with line 245 may be extraneous. The last paragraph before the conclusion beginning on page 15 with line 295 is confusing, with discussion expanding to other STIs. This paragraph may not be needed as well. A very high number of references is cited (76), and the authors should focus on those that are most important to the topic of establishing a baseline HPV prevalence in Paraguay.On page 14 beginning with line 271, consider adding more context to the discussion of HPV 58. While HPV 58 has a high prevalence in Paraguay and in other countries worldwide, before suggesting it be given equal consideration to HPV-16, its degree of carcinogenicity should be considered. Most HPV-attributable cancers are caused by HPV-16 and 18. International studies found HPV-58 in 2.2% (de Sanjose JNCI Cancer Spectr 2018) and 4.4%  of invasive cervical cancers, far below the contributions of the high-risk types targeted by the quadrivalent vaccine.On page 15 in line 285, it is confusing to state that HPV 58 is not vaccine preventable (it is targeted by Gardasil-9); better to say it is not targeted by the vaccine currently in use in Paraguay’s vaccination program.The discussion does not appear to include a paragraph on limitations of the study. It is important to at least acknowledge limitations of the study population not being representative of the population of Paraguay or Asuncion and how recruitment of a comparable population for a follow-up study to measure vaccine impact would be accomplished.On page 16, in the conclusion in lines 308-309, consider deleting “Considering the high prevalence, multiple infections, and non-vaccine-type HPV infections“ from the second sentence. The data provide a baseline regardless of these considerations.Reviewer #3: 1) The title the authors picked is an interesting and pioneer base line focus of interest specific to Paraguay. Authors used simple random sampling to select 208 women aged 18-25. Do you have an explanation or a local reference for the statement on line 106 "younger women..."? Secondly, what is the conclusion you have drawn from this research? Furthermore, what are your recommendations?2) Although looking grossly sound, I have one questions regarding the result/analysis section. Why is the summation of those with History of STI and Negative test not 94; it is 91 in your case?Overall, it is a very well written manuscript discussing a hot issue mainly concerning developing countries like Paraguay. It is a good initiative but the conclusion and recommendation needed further elaboration than short statements.Reviewer #4: This study aimed to estimate the type-specific HPV frequency in unvaccinated women aged 18–25 years in the metropolitan area of Asuncion as a baseline for monitoring the HPV vaccination programI have reviewed the manuscript and would like to suggest the following changes.Discussion :The first paragraph should mention the noteworthy findings of the study. The authors should compare and contrast these findings with other studies in subsequent paragraphs.Also, author should add a paragraph regarding limitations and future implication of this study.the conclusion should be a separate paragraphthe references are very numerous, it may be necessary to reduce a littleReviewer #5: The paper was on the HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY. The results showed important information for public health.However, as the title was on prevalence, there were no mentioned on the estimation of sample size as N=208 was small. the demographic data showed that the participants were actually moderate risk eg. only 24.0 % used condom, 33% reported STI, 40.9% had reported 2 or more partners. These should add in the discussion, all these are risk factors for acquiring HPV. That's why the results reported quite high high risk HPV types.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Dr. Burak BayraktarReviewer #1: Reviewer #2: NoYes: Dr. Kaleab A. BetruReviewer #3: Reviewer #4: NoReviewer #5: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  23 Dec 2022Taking into account the important suggestions proposed by the reviewers and seeking to respond to each of them, all the points were answered in the letter to the reviewers, please refer to this attached file RESPONSE TO REVIEWERS.AttachmentResponse to Reviewers 23th Dec PONE-D-22-28769.docxSubmitted filename: Click here for additional data file. 25 Jan 2023
 PONE-D-22-28769R1

 HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY

 PLOS ONEDear Dr. Bobadilla,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by 10/02/2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at
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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Andrea GianniniAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments:Dear authors,the manuscript it has now been evaluated by our experts and they have recommended that minor changes be made to the submission.Please improving the manuscript with the reviewers' comments.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #4: All comments have been addressedReviewer #6: (No Response)Reviewer #7: (No Response)**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #4: PartlyReviewer #6: PartlyReviewer #7: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #4: NoReviewer #6: YesReviewer #7: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #4: YesReviewer #6: YesReviewer #7: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #4: YesReviewer #6: YesReviewer #7: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors made several corrections. But as I said in my first review:The current study does not deliver anything significant new or outbreaking in this matter.Reviewer #4: (No Response)Reviewer #6: I read with great interest the manuscript, which falls within the aim of this Journal. In my honest opinion, the topic is interesting enough to attract the readers’ attention. Nevertheless, the authors should clarify some points and improve the discussion, as suggested below.Authors should consider the following recommendations:- Manuscript should be further revised in order to correct some typos and improve style.- To date, several lines of evidence support the possibility to use specific biomarkers to identify early-stage cervical cancer and, in this way, offer a better prognosis to the patients. This point deserves to be discussed, referring to: PMID: 28918603; PMID: 35549629.- I would recommend to stress novel pieces of evidence about high-risk HPV-negative high-grade cervical dysplasia, which seems to have more favorable outcomes than patients with documented high-risk-HPV infection .Reviewer #7: I read with great interest the Manuscript titled "HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY " which falls within the aim of the Journal.In my honest opinion, the topic is interesting enough to attract the readers’ attention. Methodology is accurate and conclusions are supported by the data analysis. Nevertheless, authors should clarify some point and improve the discussion citing relevant and novel key articles about the topic.-The introduction should be extended and completed. I find interesting a reference to the efforts made for the prevention and early diagnosis of gynecological cancers (see PMID: 36141217).- Although it is a retrospective analysis, inclusion/exclusion criteria should be better clarified by extending their description.- Discussions can be expanded and improved by citing relevant articles (I suggest authors to read and insert in references the following article PMID: 35742340).Considered all this points, I think it could be of interest for the readers and, in my opinion, it deserves the priority to be published after minor revisions.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Burak BayraktarReviewer #1: Reviewer #4: NoReviewer #6: NoYes: Tullio Golia D'AugèReviewer #7: **********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  1 Mar 2023REVIEW COMMENTS TO THE AUTHOR1) REVIEWER #1: The authors made several corrections. But as I said in my first review: The current study does not deliver anything significant new or outbreaking in this matter. We respect your opinion, but as you know the incidence of cervical cancer in Paraguay is higher than other countries in the region and an important component of its prevention is the HPV vaccination. So, Paraguay, like many other countries worldwide, has incorporated the HPV vaccine into its national immunization program, requiring a virological surveillance to monitor changes in the virus transmission. We know that the population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low resources countries, thus cross-sectional surveys in sentinel populations could be a suitable alternative. For hence this study even its important limitations, such as the small sample size and the convenience recruitment strategy, the results are particularly important, providing information regarding HPV prevalence in groups that are no often included in national cervical cancer prevention programs. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the short-term estimation of the impact of the national HPV immunization program.  These explanations were added in the discussion.  Lines 342-351. We know that the population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low resources countries, thus cross-sectional surveys in sentinel populations could be a suitable alternative.For hence this study even its important limitations, such as the small sample size and the convenience recruitment strategy, the results are particularly important, providing information regarding HPV prevalence in groups that are no often included in national cervical cancer prevention programs. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the short-term estimation of the impact of the national HPV immunization program.2) REVIEWER #4: (No Response)3) REVIEWER #6: I read with great interest the manuscript, which falls within the aim of this Journal. In my honest opinion, the topic is interesting enough to attract the readers’ attention. Nevertheless, the authors should clarify some points and improve the discussion, as suggested below. Authors should consider the following recommendations: - Manuscript should be further revised in order to correct some typos and improve style. Thank you for your observation. The text was revised. - To date, several lines of evidence support the possibility to use specific biomarkers to identify early-stage cervical cancer and, in this way, offer a better prognosis to the patients. This point deserves to be discussed, referring to: PMID: 28918603; PMID: 35549629.  - I would recommend to stress novel pieces of evidence about high-risk HPV-negative high-grade cervical dysplasia, which seems to have more favorable outcomes than patients with documented high-risk-HPV infection . We appreciate your suggestions, we read the mentioned references and we added some important points in the introduction: Lines 63 – 69. Cervical cancer is a disease which reflects inequities among different populations depending on the availability of a national vaccination program and population-based cervical cancer screening, and access to quality treatment. In 2020, the World Health Organization (WHO) called for action to eliminate cervical cancer as a public health problem. With three key strategies and clear 2030 targets: 90% of girls fully vaccinated with HPV vaccine by 15 years of age, 70% of women screened using a high-performance test by 35 years of age and again by 45 years, and 90% of women identified with cervical disease treated [8].  Line 70. The primary prevention approach focuses on preventing disease before it develops. Lines 79 – 92. Secondary prevention, based on cervical screening program, is widely implemented to prevent. The adoption of Pap smear and HPV testing resulted in a significant increase in the diagnosis of cervical dysplasia and a significant decrease in cervical cancer, mainly in more developed countries. New screening strategies, such as HPV self-sampling, and the use of artificial intelligence in colposcopy assessment, must be disseminated in a near future [11].Some markers, including p16 ink4a, p16, E-cadherin, Ki67, pRb, and p53, have been found to be useful in identifying intra-epithelial lesions that are more likely to develop into invasive lesions. Others, such as CEA, SCC-Ag, CD44, were developed to detect invasive forms. These biomarkers represent a promising diagnostic, prognostic and dynamic tool and can also be easily performed in the management of cervical cancer [12]. And tertiary prevention in cervical cancer includes its treatment with surgery , radiotherapy, and systemic treatments  [11]. Minimally invasive surgery has been studied and associated with advantages in the treatment of various gynecological cancers [13].4)REVIEWER #7: I read with great interest the Manuscript titled "HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY " which falls within the aim of the Journal. In my honest opinion, the topic is interesting enough to attract the readers’ attention. Methodology is accurate and conclusions are supported by the data analysis. Nevertheless, authors should clarify some point and improve the discussion citing relevant and novel key articles about the topic. -The introduction should be extended and completed. I find interesting a reference to the efforts made for the prevention and early diagnosis of gynecological cancers (see PMID: 36141217). Based on the suggested reference, we have added information on new screening strategies and certain biological markers that may be useful in early diagnosis and prognosis of cervical dysplasia, and new approaches to the treatment pre-cancerous lesions and cervical cancer.  Lines 79 – 92. Secondary prevention, based on cervical screening program, is widely implemented. The adoption of Pap smear and HPV testing resulted in a significant increase in the diagnosis of cervical dysplasia and a significant decrease in cervical cancer, mainly in more developed countries. New screening strategies, such as HPV self-sampling, and the use of artificial intelligence in colposcopy assessment, must be disseminated in a near future [11].Some markers, including p16 ink4a, p16, E-cadherin, Ki67, pRb, and p53, have been found to be useful in identifying intra-epithelial lesions that are more likely to develop into invasive lesions. Others, such as CEA, SCC-Ag, CD44, were developed to detect invasive forms. These biomarkers represent a promising diagnostic, prognostic and dynamic tool and can also be easily performed in the management of cervical cancer [12]. And tertiary prevention in cervical cancer includes its treatment with surgery , radiotherapy, and systemic treatments  [11]. Minimally invasive surgery has been studied and associated with advantages in the treatment of various gynecological cancers [13]. - Although it is a retrospective analysis, inclusion/exclusion criteria should be better clarified by extending their description. We explained more clearly the inclusion/exclusion criteria. Lines 114 – 117. Sexually active women aged 18–25 years, who had not been vaccinated against HPV were invited for screening through social networks and flyers at local health centers and higher education institutes. Exclusion criteria were pregnancy, nonsexual onset at the time of the study and have been received HPV vaccine. - Discussions can be expanded and improved by citing relevant articles (I suggest authors to read and insert in references the following article PMID: 35742340).Considered all this points, I think it could be of interest for the readers and, in my opinion, it deserves the priority to be published after minor revisions. Discussions have been expanded and improved as suggested, with the follow changes:  Lines 235 – 253. The WHO global strategy for the elimination of cervical cancer as a public health problem proposes a multidisciplinary approach that includes community education, social mobilization, vaccination, detection, treatment, and palliative care. HPV vaccination in combination with timely detection and appropriate treatment of cervical cancer precursor lesions is considered to have the potential to achieve the elimination of this disease, defined by the WHO as fewer than 4 new cases per 100,000 women-years [8].With the introduction of the HPV vaccine, changes in the transmission dynamics of the virus are expected, making necessary to monitor changes in the prevalence of HPV and the patterns of HPV-associated diseases. Population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low- and middle-income countries, so cross-sectional surveys in sentinel populations could be a suitable alternative [8].Paraguay, like many other countries worldwide, has incorporated the HPV vaccine into its national immunization program, requiring a virological surveillance to measure its impact. It can provide an early measure of vaccine effectiveness by quantifying the magnitude of reduction in HPV type-specific prevalence, including possible evidence of herd and cross protection and provide timely assessment of vaccination strategy. The principle of HPV prevalence surveillance is to recruit samples in age groups at risk of HPV exposure and conduct repeat-cross sectional analyses [16]. Lines 326 – 329. Have been reported women immunized with quadrivalent vaccine at young age who still developed cervical intraepithelial neoplasia with evidence that these lesions are commonly associated with HPV types not targeted by this vaccine, but some of the HPV types associated with these lesions in vaccinated women are targeted by the Gardasil-9 [59]. Lines 332 – 335. Therefore, the high prevalence of HPV types targeted by nonavalent HPV vaccines could support its introduction in the national immunization program to achieve greater coverage, considering that the preventable proportion of cervical cancers is estimated to increase to 90% [60]. Lines 342 – 351. We know that the population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low resources countries, thus cross-sectional surveys in sentinel populations could be a suitable alternative.For hence this study even its important limitations, such as the small sample size and the convenience recruitment strategy, the results are particularly important, providing information regarding HPV prevalence in groups that are no often included in national cervical cancer prevention programs. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the short-term estimation of the impact of the national HPV immunization program. Lines 356 – 365. This research could provide key information to healthcare policy makers about the importance of increasing HPV vaccination coverage in national immunization programs. The results should be combined with other studies in cohorts of vaccinated women to investigate the reduction of types 16 and 18, as well as their possible replacement by other genotypes, thus evaluating the impact of the implementation of HPV vaccination in Paraguay and compare the results with other countries.In addition, these data can improve the acceptance of the HPV vaccine in the Paraguayan population, highlighting the high prevalence of HPV, especially the high-risk types, observed in unvaccinated young women. Increased public awareness of the benefits of HPV vaccination for cervical cancer prevention could increase public support for immunization. Lines 370 – 373. Surveillance and monitoring of changes in viral transmission that lead to modification of HPV type-specific prevalence and the patters of HPV-associated disease are crucial to progress toward the global elimination of cervical cancer.************************************************************************************************************************AttachmentResponse to Reviewers 28th Feb PONE-D-22-28769R1.docxSubmitted filename: Click here for additional data file. 12 Mar 2023"High prevalence and co-infection of high-risk Human Papillomavirus genotypes among unvaccinated young women from Paraguay”PONE-D-22-28769R2Dear Dr. Bobadilla,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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TLR4  and TLR9  gene polymorphisms with cervical persistent high-risk HPV (HR-HPV) infection, as well as multiple HR-HPV infections.Toll-like receptors (TLRs) may be involved in the natural history of human papillomavirus (HPV) infection. In our study, we aimed to investigate the association of TLR4/TLR9 gene polymorphisms between HR-HPV transient and persistent infections, as well as between HR-HPV single and multiple infections. HR-HPV genotypes were detected using Real-time polymerase chain reaction (RT-PCR). PCR-restriction fragment length polymorphism (PCR–RFLP) was used to determine TLR4 and TLR9 gene polymorphisms. Analyses of the different outcome variables (HR-HPV infection status and time for HR-HPV clearance) with respect to TLR4/TLR9 polymorphisms were carried out. Logistic regression analysis was used to determine the association of TLR4/TLR9 genotypes and alleles with HR-HPV infection status. The Kaplan–Meier method with the log-rank test was used to analyze the relationship between TLR4/TLR9 genotypes and the time for HR-HPV clearance.A total of 269 study subjects were enrolled and grouped by retrospectively analyzing the HR-HPV testing results and other clinical data of 2647 gynecological outpatients from Jingzhou Hospital Affiliated to Yangtze University. We conducted a case–control study to compare the role of TLR9 rs187084 and rs352140 were associated with persistent  and multiple   HR-HPV infection. However, no association was found between TLR4 polymorphisms and HR-HPV infection status. Kaplan–Meier time to HR-HPV clearance analysis demonstrated that women carrying rs187084 and rs352140 mutant genotypes take longer duration to clear HR-HPV infection compared with wild-type genotype carriers .The mutant genotypes of TLR9 polymorphisms, but not TLR4, were associated with cervical persistent and multiple HR-HPV infections, which could be useful as a potential predictor of HR-HPV infection status.Our results suggested that The online version contains supplementary material available at 10.1186/s12879-023-08116-z. Human papillomaviruses (HPV) are the most common sexually transmitted pathogens worldwide. More than 200 HPV genotypes have been identified so far , 2. HighToll-like receptors (TLRs) are belong to the family of pattern recognition receptors (PRRs), of which TLR9 is located intracellularly and recognizes unmethylated cytosine-phosphate-guanine (CPG) DNA, TLR4 is located at the cell membrane and mainly recognizes lipopolysaccharide (LPS) from bacteria and certain viral proteins –10. TLRsTLR4 rs10759931 and rs10116253 could be a binding site for GATA2 and NFATC2, respectively [TLR9 influenced the immune response to CpG, their further research found TLR9 rs352140 TT genotype enhanced gene expression and was associated with higher frequency of intracellular IgM + B cells. Therefore, TLRs gene mutations may alter the expression or function of the corresponding encoded proteins, and affect the individual immune responses. The majority of the previous studies have focused on the association between TLR4/TLR9 single nucleotide polymorphisms (SNPs) and HPV susceptibility [TLRs SNPs in the history of HPV infection were poor reported. In the Ludwig–McGill Cohort Study, Oliveira et al. [TLR9 rs5743836 in clearance/persistence of type-specific HPV infection and no association was found. There only one study conducted in India reported TLR4 10759931 AG/GG genotype showed protective effect against the risk of acquiring multiple HR-HPV infections [Functional analyses have shown that ectively , 21. In ectively  observedtibility –26. The tibility , 24. Howa et al.  investigfections .TLR4  and TLR9  gene polymorphisms with cervical HR-HPV infection status.Based on the important role of TLRs in antiviral immune response, the aim of this study was to investigate the association of In this case–control study, subjects were collected from 2647 women who tested for HR-HPV at the gynecological outpatient clinic of Jingzhou Hospital Affiliated to Yangtze university from Sep. 2019 to May. 2022, whose genomic DNA was extracted in our previous study. The HR-HPV testing results and clinical data for these 2647 patients were obtained from laboratory information system (LIS) and outpatient electronic medical records (EMRs), respectively. According to the inclusion and exclusion criteria, there were 593 patients were enrolled in the retrospective cohort to estimate individual HR-HPV infection status. Inclusion criteria: women who were HR-HPV positive for the initial testing and retested regularly within the following 36 months. Exclusion criteria: age < 18; pregnancy; history of total hysterectomy or cervical resection; tested for HR-HPV less than 3 times. Ultimately, 269 patients were enrolled in the study and grouped according to the definition of HR-HPV infection status, as transient HR-HPV infection, persistent HR-HPV infection, single HR-HPV infection, and multiple HR-HPV infection. The flow chart for study subject collection was shown in Fig. DNA was extracted from exfoliated cervical cells according to the instruction of the nucleic acid extracting kit  and stored at − 80 °C for use, as our previous studies described in detail –31. ThisThere were wide variations in the definition of HPV persistence used in literatures. In most studies, HPV persistence was defined as the detection of the same HPV genotype or the same type-group in two consecutive visits , 27, 32.HPV testing was performed using the HR‐HPV typing test kit  according to the manufacturer’s instructions in an AGS 4800 real-time PCR System. Four fluorescent channels of different reaction tubes correspond to specific HPV types and internal control (IC) . The positive results were with a cycle threshold value  ≤ 38 and with a typical "S-shape" amplification curve.TLR4 and TLR9 gene polymorphisms. We designed new primers with the software Primer Premier 6 and synthesized by Sangon Biotech Co., Ltd. The primer sequences as followed, rs10116253 F: 5′-TGTGATGATTAGGGCTGAA-3′, rs10116253 R: 5′-GTGGACTGGGCACAAACT-3′; rs1927911 F: 5′-CATGTGCCTCTGAACTTA-3′, rs1927911 R: 5′-CATGCACTCTAAAGATTTC-3′; rs10759931 F: 5′-ACATTGGTAGCACCAGAGTC-3′, rs10759931 R: 5′-ATTTCCCTTACTTCCTCATT-3′; rs187084 F: 5′-TCCCAGCAGCAACAATTCATTA-3′, rs187084 R: 5′-CTGCTTGCAGTTGACTGTGT-3′; rs352140 F: 5′-CCAGGTAATTGTCACGGAGA-3′, rs352140 R: 5′-TCTCGCAGGCAGTCAATG- 3′. The details of primers and specific restriction enzymes were shown in Additional file 2O, 2.5 μl 10 × buffer (Mg2+) (Takara), 2.0 μl dNTPs (Takara), 1.25 μl forward and reverse primers (20uM) (Sangon Biotech), 0.125 μl Taq DNA polymerase (5 U/L) (Takara), and 2 μl DNA extraction. PCR under the following conditions: initial denaturation of 95 °C for 5 min, 35 cycles of 98 °C for 10 s, 55 °C or 60 °C for 45 s, and 72 °C for 1 min. The PCR products were digested by specific restriction enzymes, and then analyzed on 3% agarose gel electrophoresis stained by GelRed nucleic acid dye (Biotium).Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) was performed to identify the 2 test. The distribution of HR-HPV infection status and time for HR-HPV clearance with respect to the different TLR4/TLR9 genotypes and alleles were furtherly analyzed. Logistic regression analysis was used to determine the association of TLR4/TLR9 genotypes and alleles with HR-HPV infection status by computing the adjusted odds ratios (ORs) and 95% confidence intervals (CI). Hardy–Weinberg equilibrium (HWE) and frequencies of haplotype were determined by SNPstats program (http://bioinfo.iconcologia.net/SNPstats). The time for HR-HPV clearance was plotted by the Kaplan–Meier method and compared by log-rank test to analyze its relationship with TLR4/TLR9 genotypes (the time for HR-HPV clearance was defined as the time span between the first HR-HPV positive testing and the subsequent negative or different genotypes testing). A two-tailed P < 0.05 was considered statistically significant.All statistical analyses were performed with SPSS software version 25.0. The continuous variables compared between case and control group using Student’s t-test. Comparison between categorical variables was performed by the χP < 0.001), as well as multiple HR-HPV infection (46.62 ± 11.02) was higher than single infection (43.64 ± 8.25) (P = 0.018). Among single HR-HPV infection, the most prevalent genotypes were HPV52 (51/160 31.9%), HPV16 (21/160 13.1%) and HPV58 (17/160 10.6%). Among multiple HR-HPV infection, the most prevalent genotypes were HPV52 (50/109 45.9%), HPV58 (47/109 43.1%) and HPV16 (28/109 25.7%) . The average age (mean ± SD) of patients with persistent HR-HPV infection (46.89 ± 9.72) was higher than that of patients with transient infection (41.55 ± 8.65) (P > 0.05). Two SNPs, rs187084 and rs352140 of TLR9, were in significant association with persistent and multiple HR-HPV infections after they were adjusted by age and HR-HPV genotypes. Compared with wild-type genotype, TLR9 rs187084 heterozygous and mutant genotypes increase the risk of persistent  and multiple  HR-HPV infections. TLR9 rs352140 heterozygosity and mutant genotype also significantly increased the risk of HR-HPV persistent infection . Without adjusted by age, TLR9 rs352140 mutant genotype was associated with multiple HR-HPV infections in the dominant model . The mutant allele of TLR9 rs187084 and rs352140 increase the risk of persistent  and multiple  HR-HPV infections.The frequencies of genotype and allele and their association with persistent and multiple HR-HPV infections were shown in Tables TLR4 SNPs  genotype and allele frequencies were found between the persistent and transient HR-HPV infection groups, as well as between the single and multiple HR-HPV infection groups .However, no significant differences in TLR4 and TLR9 SNPs candidate loci were in strong linkage disequilibrium (LD), respectively. The most frequencies of haplotypes T-C and C-T of TLR9(rs187084-rs352140) in the study population were 63.93% and 34.00%, respectively, and the most frequencies of haplotypes T-C-A and C-T-G of TLR4 (rs10116253-rs1927911-rs10759931) were 58.73% and 39.58%, respectively. Compared with the TLR9 haplotypes T-C, carriers with the C-T haplotypes were more susceptible to persistent and multiple HR-HPV infections  . There were 162 (60.22%) patients to be HR-HPV negative or tested with different HR-HPV genotypes than the initial test at the end of our follow-up. The median HR-HPV clearance time was 10.5 months in those 162 patients   and rs352140 (located in the exon) are 2 common SNPs. It was reported that rs187084 TT genotype and T allele were associated with HPV infection in Mexican women [TLR9 gene and its association with the risk of acquisition and clearance of type-specific HPV infection using a case–control sampling strategy within the Ludwig–McGill Cohort Study, whose data showed no consistently significant associations between TLR9 rs5743836 polymorphism and HPV clearance or persistence, probably due to the limited sample sizes. In other studies, the association of rs187084 with HBV infection and rs352140 with HCV spontaneous clearance were also reported to be absent [an women . In addian women . Above san women  investige absent –35. Howee absent  stimulatTLR4 and TLR9 polymorphisms were associated with HR-HPV spontaneous clearance. Similar to other studies [TLR9 rs187084 and rs352140 presented a significantly longer duration time for HR-HPV clearance compared to wild-type patients, which was consistent with the conclusions from our case–control studies. Our findings suggest that women with different TLR9 SNPs genotypes might undergo different time for HR-HPV infection, and a personalized screening strategy might be a more efficient and economical choice.As the definition of persistent HPV infection is ambiguous, there may be selection bias in our subgroups. Kaplan–Meier time to clearance analysis better reflects the natural history of HR-HPV. We performed Kaplan–Meier analysis to clarify whether  studies , 39, ourTLR9 rs187084 and rs352140 increase the risk of HR-HPV multiple infections as well. It indicated that TLR9 SNPs rs187084 and rs352140 may play the same roles in persistent and multiple HR-HPV infections. However, Pandey et al. [TLR9 rs187084 and rs352140 polymorphisms and multiple HR-HPV infections in India. The reason for this may be the influence of different genetic background or other environmental factors, such as smoking, sexual behavior, and pathogens infection.The prevalence and distribution of single and multiple HPV infections vary widely worldwide and are affected by diverse factors , 40, 41.y et al.  never obTLR4 rs1927911 increased the risk of cervical cancer and HPV16/18 infection, while the role of SNP rs10116253 and rs10759931 in the HPV infection was not clear. In our study, we found that TLR4 polymorphism was in lack of association with HR-HPV persistent and multiple infections. TLR4 could recognize lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, as well as some envelope proteins from the virus [TLR4 polymorphisms were significantly associated with the susceptibility to Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) infection [The chronic inflammation mediated by TLR4/iNOS and TLR4/MYD88/NF-κB signaling pathway was associated with HPV-related cervical cancer , 43. It he virus , whetherhe virus . Moreovenfection –48. HencTLR4/TLR9 polymorphisms. Only the superficial correlation between TLR4/TLR9 polymorphisms and HR-HPV infection status was investigated in our research. Thus, we will expand our study populations for subgroup analysis and explore underlying mechanisms in further research.The main strengths of this study were that the subject was collected from a retrospective cohort that included patients followed-up for a long period of time (about 36 months), and HR-HPV infection status was confirmed with consecutive visits at least three times. Several limitations in our study should be considered as well. We were unable to collect more information of patients, such as smoking, use of condoms, and the number of sexual partners, which potentially have an impact on HR-HPV infection. The sample size was limited to some extent, we failed to analyze type-specific HR-HPV infection and its association with TLR4 and TLR9 gene polymorphisms and HR-HPV infection status in Chinese Han population. Our results suggested that the heterozygous genotypes and mutant alleles of TLR9 rs187084 and rs352140 were associated with persistent and multiple HR-HPV infections, which maybe provide important information for further investigation of risk factors for HR-HPV infection and assessment of the HR-HPV infection status.In conclusion, this study initially investigated the relationship between Additional file 1: Fig S1. Kaplan–Meier curve for time to HR-HPV clearance in patients with different TLR4 SNPs genotypes . The differences were determined by the log-rank test. The number of patients at risk was listed below each curve.Additional file 2: Table S1. Definition of persistent and transient HR-HPV infections.Additional file 3: Table S2. Primer sequences and restriction enzymes used for genotyping the studied SNPs.


IL17RB and IL17REL are associated with a higher viral load and memory B and activated NK cell TS, as well as improved prognosis in HPV-infected HNSCC patients. The transcriptional parameters that we describe may be optimized to improve prognosis and risk stratification in the clinic and provide insights into gene and cellular targets that may potentially enhance anti-tumor immunity mediated by NK cells and memory B cells in HPV-infected HNSCC patients.Changes in the cellular secretome are implicated in virus infection, malignancy, and anti-tumor immunity. We analyzed the association between transcriptional signatures (TS) from 24 different immune and stromal cell types on the prognosis of HPV-infected and HPV-free head and neck squamous carcinoma (HNSCC) patients from The Cancer Genome Atlas (TCGA) cohort. We found that HPV-positive HNSCC patients have tumors with elevated immune cell TS and improved prognosis, which was specifically associated with an increased tumor abundance of memory B and activated natural killer (NK) cell TS, compared to HPV-free HNSCC patients. HPV-infected patients upregulated many transcripts encoding secreted factors, such as growth factors, hormones, chemokines and cytokines, and their cognate receptors. Analysis of secretome transcripts and cognate receptors revealed that tumor expression of  Head and neck squamous cell carcinomas (HNSCC) are the most common type of malignancies that arise in the head and neck. Over 90% of head and neck cancers are HNSCC that develop from the mucosal epithelium in the oronasal cavity, pharynx, and larynx. In the past two decades, accumulating studies have revealed the link between prior infection with oncogenic human papillomavirus (HPV) strains and tumors that develop in the oropharynx. An estimated 30,000 oropharyngeal cancers were caused by HPV infection worldwide each year, and HPV has been detected in ~25% of all HNSCC patients . DespiteIL17A to IL17F (encoded by IL17A–IL17F). Five relevant IL17 receptor (IL17R) proteins have also been described as IL17RA through to IL17RE . The IL17 family has pivotal roles in inflammation, autoimmune disease, and cancer. IL17A (IL17) is the prototypic member produced by RAR-related orphan receptor gamma t (RORγt)-expressing cells and predominantly expressed by TH17 cells [IL17A binding and activation of the IL17RA/IL17RC heterodimer signaling complex induces the expression of cytokines and chemokines such as tumor necrosis factor (TNF), CXC-chemokine ligand 1 (CXCL1), CXCL2 and CXCL5, from macrophages and stromal cells that are critical for defense against extracellular pathogens [IL17A expression is implicated in inflammatory disorders, such as psoriasis, ankylosing spondylitis and psoriatic arthritis [IL17A in cancer also remains controversial [IL17A usually indirectly shapes immune suppression and helps tumor cell proliferation during the early stages of cancer by upregulating phosphorylated ERK1/2, angiogenesis and self-renewal [The interleukin 17 (IL17) cytokine family contains six structurally related members from 17 cells . IL17A bathogens . In contrthritis . The roloversial . IL17A u-renewal ,11,12, b-renewal ,13,14.IL17A has been well established, the immunological roles of other IL17 family members are less well known. Human IL17B is a monomer that only shares 21.3% homology with human IL17A in the amino acid sequence [IL17RB homodimer, which is expressed in the epithelial cells of various organs [IL17RB has also been found in TH cells and innate lymphocytes [IL17RB can combine with IL17RA to form a heterodimeric complex that recognizes IL17E and prevents the binding of IL17B to IL17RB. In inflammatory diseases, the IL17B/IL17RB pathway is protective and might restrict pro-inflammatory by the IL17E/IL17RA-IL17RB signaling complex [While the knowledge of sequence . In contsequence ,17,18. Is organs . The expphocytes ,21. IL17 complex .IL17RE has also been found to be expressed on TH17 cells in addition to stromal cells and IL17RE signaling can amplify TH17 cell responses in autoimmune disease [IL17RE stimulated TH17 cells to produce pre-tumorigenic cytokines and deficiency of IL17RE dramatically decreased intestinal tumor growth [Several reports have shown the association between the IL17B/IL17RB signaling pathway and tumor development in breast, gastric, lung, pancreas, prostate, brain and blood cancers, however the precise mechanisms involved remain unclear ,24,25,26 disease . In inter growth . CollectIL17REL with minor allele frequency (MAF) of less than 0.01 have been associated with inflammatory bowel disease (IBD), ulcerative colitis (UC) and gout. However, the role of IL17REL in malignancies remains uncertain [Following the discovery of the IL17 cytokine family, a gene encoding a novel IL17RE-like protein (IL17REL) was defined by similarity searches of amino acid sequences ,33. Rarencertain ,35.IL17A and IL17B were both expressed in the GC microenvironment, dedicated to B cell recruitment and antibody production [Accumulating evidence has revealed the role of the IL17 cytokine family members in regulating the migration and functions of germinal center (GC)-derived B cells ,36,37,38oduction ,36,37.NK cells are innate lymphocytes with cytotoxic and cytokine-secreting functions that comprise approximately 10–15% of total lymphocytes in human peripheral blood. They are known to initiate potent anti-tumor immunity regulated by “missing-self” and “induced-self” recognition ,43,44. N+ T cells, which are a well-established lymphocyte subset associated with improved clinical outcomes [Tumor immune infiltration is largely linked to the survival of a variety of HPV-related cancers. It has been demonstrated that high volumes of tumor-infiltrating lymphocytes (TILs) is associated with good prognosis in HPV-related cancer ,53. In aoutcomes ,55, B ceoutcomes  and HPV-outcomes . NK celloutcomes . HoweverIL17RB and IL17REL, are associated with improved prognosis and may modulate the anti-tumor activity of memory B, NK cells, and T cell subsets in HPV-infected HNSCC patients.In this study, we set out to test the prognostic values of HPV infection and the expression of transcripts encoding components of the cellular secretome, such as growth factors, cytokines, chemokines, hormones, and their cognate receptors in HNSCC patients. We have applied a 24-cell-type TS and provided an overview of the TIL profiles in HPV-negative and -positive HNSCC patients as well as their prognostic associations. We find that a high abundance of activated NK cell or memory B cell TS are associated with good prognosis in HPV-infected HNSC patient tumors. Moreover, we also find that two genes encoding IL17 receptor family members, We first collected RNA-seq data and clinical results for 500 tumor biopsies in the GDC Data Portal . AccordiThe processes of the generation and benchmark of 24 cell type transcriptional signatures were described in our former publications ,62. BrieRaw read counts from TCGA-HNSCC RNA-seq data were scaled with the trimmed mean of M values (TMM) method  to compeIL17RB and IL17REL. Four open-access HNSCC single-cell RNA sequencing datasets from GEO: GSE139324 [Single-cell RNA-seq (scRNA-seq) analysis has also been performed to understand the cell type expressing SE139324 , GSE1033SE139324 , GSE1641SE139324  and GSE1SE139324 ,70,71,72p-values.We estimated the cell type relative fractions for each biological replicate with our reference RNA-seq-derived transcriptional signature and the RNA-seq data from TCGA-HNSCC based on CIBERSORT . Then, KData analysis and visualization were performed using the R environment in RStudio. Packages include tidybulk , tidyHeaA previous study has shown that HNSCC patients have significantly lower rates of metastases with HPV infections, suggesting that viral infection may enhance cancer immune surveillance and influence HNSCC patient prognosis . To undePTPRC), were significantly upregulated in HPV-infected compared to HPV-free HNSCC tumors, respectively  with the aNK TS  and NKG2A (encoded by KLRC1) were significantly upregulated in HPV-infected patients /Helper T, γδ T cell (GD) T and monocyte TS; BMP3 (encoding bone morphogenetic protein 3)/fibroblast (Fibro) TS; IGFALS (encoding acid labile subunit)/T helper cell and GD T TS; CSPG5 (encoding chondroitin sulfate proteoglycan 5)/naive CD8 T and M2 macrophage TS; and cytokines/chemokines and receptors: IL17REL/Treg, T Helper, aNK, monocyte, mast cell and memory B TS; CCL25 (encoding chemokine C-C motif ligand 25)/Treg and GD T, CD8 Tem, IL2NK, monocyte, Mast cells and memory B cells TS; IL17RB/Treg, GD T, naïve CD8 T, CD4 Tem, aNK, monocyte, M2 macrophage and memory B TS; and hormones and receptors: ENHO (encoding adropin)/naïve CD8 T. CD8 Tcm, CD4 Tem, aNK, Macro M2 TS; NPPC (encoding natriuretic peptide precursor C)/Treg, GD T cell, aNK, monocytes and memory B TS; and TG (encoding thyroglobulin)/GD T cell, CD4 Tem, monocyte, mast cell and memory B TS in HPV-infected HNSCC tumors  are among the most common cancers worldwide, with over 870,000 new cases and 440,000 deaths in 2020 . SmokingPTPRC) encoding the common leukocyte antigen, CD45, were associated with improved prognosis in HPV-infected HNSCC patients compared to uninfected patients and positively correlated with HPV viral loads, suggesting HPV infection may enhance anti-tumor immunity in HNSCC patients. Analysis of the tumor abundance of 24 different cell-type-specific TS [HLA-A, -C, and -F and high tumor abundance of activated NK cells were associated with improved prognosis  [IL17RE [IL17REL was found to be highly associated with gout and ulcerative colitis in genome-wide association studies (GWAS) [IL17REL in different cell types remain largely unknown, especially in human cancers. In contrast, IL17RE is expressed on both epithelial and TH17 cells and recognizes IL17C as ligand. IL17C binding to IL17RE induces IL17 production from TH17 cells, which can enhance host innate immunity [IL17RB or IL17REL. However, in contrast to bulk RNA-seq data, scRNA-seq data had a lower resolution for detecting IL17RB or IL17REL (IL17RE and how these might influence prognosis in HPV-infected HNSCC patients. (SEFIR) . However [IL17RE . Though s (GWAS) ,35, the immunity . We cond IL17REL . Thus, iIL17RB, IL17REL and memory B cell and aNK TS expression (+ or CXCR5+ GC B cells were induced to migrate to CXCL12 or CXCL13 through the IL17RB signaling pathway [IL17RB and IL17REL may participate in the recruitment of memory B and activated NK cells in HPV-infected HNSCC patients.We have also found a positive correlation between pression D. It has pathway . Strikin pathway ,120,121.IL17RB with CD4 Tem, memory B, and activated NK TS and IL17REL with CD8 Tcm, memory B, M1 macrophages and T Helper cell TS and the improved prognosis of HPV-infected HNSCC patients. Our results have important consequences for anti-tumor immunity and reveal potential new biomarkers or targets for immunotherapy in HPV-infected HNSCC patients.To summarize, our study set out to uncover a possible role for genes encoding secretome factors or their receptors and their association with immune and stromal cell TS and HPV infection on the prognosis of HNSCC patients. We have uncovered a novel association between a higher tumor expression of


X2 = 2761.62, df = 2.96; RMSEA = 0.07 ; CFI = 0.72). An exploratory factor analysis was conducted to analyze how the data fit with our specific population, yielding 10 factors. Finally, a qualitative inter-item analysis of these factors was conducted, resulting in nine factors. Our findings suggest that measuring TIC attitudes and beliefs may vary according to field of practice and ethno-racially diverse workers. Further refinement of the ARTIC may be necessary for various services domains.Human service organizations (HSO) have increasingly recognized the value of employing trauma-informed care (TIC) in a variety of practice settings. Evidence suggests that effectively adopting TIC has shown client improvements. Organizational barriers to TIC implementation, however, exist. To improve TIC practice, the attitudes related to trauma-informed care (ARTIC) scale was developed to measure staff attitudes and beliefs towards TIC. The ARTIC has been widely adopted by researchers without evaluating its psychometric performance in diverse practice settings. The purpose of this study was to independently validate the ARTIC scale drawn from a sample of staff (n = 373) who provide services to substance-using parents. Psychometric tests were conducted to evaluate how the ARTIC performs with our HSO population. Results from a confirmatory factor analysis showed poor fit ( Experiences of trauma are common, impacting as many as 50–70% of people worldwide ,2,3. UndTrauma-informed care (TIC) is a widely accepted framework characterized by being mindful of traumatic experiences and their impact, creating safe environments, prioritizing clients’ voices to guide treatment, and flexibility ,9,10,11.Validated tools measuring attitudes and practices related to TIC are an important component of closing the implementation gap that currently exists across programs ,16,20,21A recent scoping review of TIC measures by Wathen and colleagues  showed tIt is unclear why the ARTIC has been rapidly utilized in human service organizations without additional testing in other settings and among different populations. Outside of the ARTIC’s creators, no other validation of the ARTIC-45 exists to the authors’ knowledge. The existing external validation ,23 articEffective TIC practices improve care by minimizing the re-traumatization of individuals within human service organizations ,25,26. TDespite the growth of TIC, multiple organizational barriers still impede effective TIC implementation ,34. BarrAs one of the first scales to measure attitudes and beliefs about TIC, the attitudes related to trauma-informed care (ARTIC) has been widely adopted by researchers and evaluators examining the implementation and effectiveness of TIC . The ARTThree versions of the ARTIC were created, the ARTIC-45, 35, and 10, with the number indicating how many items the scale included. Normative data established by the ARTIC’s creators show strong construct and criterion-related validity ,24. EachWhile this scale has provided valuable information about these processes, it is worthwhile noting that the sample utilized in establishing the psychometric properties of the ARTIC predominantly comprised white college-educated staff ,24. TherImplementing TIC is challenging, particularly due to its flexible nature in practice ,11, makiA total of 376 staff members from three human service organizations specializing in substance-use treatment and parent–infant mental health who participated in TIC trainings were invited to participate in this study, of whom 373 agreed. Demographic items were not initially included in the survey, but among 226 participants who provided demographics when they were added, 82% identified as female, 42% reported their race as non-white , and 20% identified as Hispanic, see . ParticiData were collected from frontline staff, supervisors, and administrators of three human service organizations located in the Northeastern United States. Participating organizations were part of a trauma-informed intervention, the attachment, regulation and competency (ARC) model, aimed at improving attachment experiences for individuals . ARC empThe ARTIC-45 measures staffs’ beliefs and attitudes about TIC. It contains seven subscales, with the first five “core” subscales of (1) underlying causes of problem behavior and symptoms, (2) responses to problem behavior and symptoms, (3) on-the-job behavior, (4) self-efficacy at work, and (5) reactions to the work. The last two subscales are considered “supplementary” and include (6) personal support of TIC and (7) system-wide support for TIC . The fivTo assess the ARTIC-45’s psychometric properties with workers in substance-use disorder facilities that focus on parent–infant mental health, we first examined validity and reliability by conducting an item analysis, internal reliability checks, and confirmatory factor analysis (CFA). We tested for internal reliability by conducting an intra-item correlation and testing for Cronbach’s alpha. Modeled after Baker and colleagues’s work , we perfX2) test, the chi-square X2 to degrees of freedom ratio or (X2:df), the root mean square error of approximation (RMSEA), and the confirmatory factor index (CFI). While the chi-square X2 is often reported, it is generally not an acceptable model fit indicator as it is sensitive to sample size. Fit index values that indicate adequate CFA model fit are a nonsignificant chi-square X2 test, a chi-square to degrees of freedom ration (X2:df) ranging between 2 and 5, RMSEA less than 0.06 [A CFA is most appropriate for measures that are well-established; thus, our goal was to examine the validity of the ARTIC-45 with our specific sample . Fit indhan 0.06 , and CFIhan 0.06 .Based on the CFA results, we next performed an exploratory factor analysis (EFA) using maximum likelihood estimation to examine the ARTIC-45 factor structure in our sample ,45. An EAdditional sensitivity analyses were conducted. As the ARTIC-45 was created to measure seven independent constructs, a forced 7-factor EFA was conducted. Additionally, a CFA was performed on the ten items that make up the ARTIC-10 due to our quantitative results suggesting a unidimensional factor approach.Using the results from the original EFA, we conducted an inter-item qualitative analysis for each factor with items loading at 0.3 or greater to identify how the ARTIC’s items performed in a substance-use treatment setting . The goaA majority of the items (4 or more) within each of the five core subscales did not correlate with one another. When examining the two supplementary subscales “personal support of TIC” and “systems-wide support for TIC”, all items correlated with fellow subscale items at 0.3 or greater. Next, in an effort to replicate the original validation, all seven subscales were correlated with one another and the ARTIC composite score. Correlation values ranged from 0.56 to 0.85, or satisfactory to strongly satisfactory.The full ARTIC-45 scale’s internal consistency was strong in our data, (α = 0.98), suggesting that it could be shortened . All sevX2 = 2761.62, and a X2:df ratio = 2.96 (2761.62:930), suggesting a good model fit. Both the RMSEA  and the CFI (0.72) indicated a poor model fit  using maximum likelihood estimation was conducted to assess the number of distinct ARTIC-45 factors in our sample. Our sample produced 10 eigenvalues greater than one suggesting potentially 10 factors for our data see . Factor X2 = 185.41, and a X2:df ratio = 5.30 (185.41:35), suggesting a poor model fit. Both the RMSEA  and the CFI (0.86) indicated a poor model fit.A forced factor EFA was conducted to analyze whether the data, when forced, recreated the same spread of items on its original seven factors. Results from both EFAs showed that 40–43 of the 45 items loaded at 0.3 or greater onto factor 1, further suggesting a unidimensional factor structure. Results from the CFA using items that make up the ARTIC-10 showed poor fit. This CFA produced a chi-square of Initial qualitative analyses of the factors indicated that the tenth factor should be eliminated from the scale as no items had loadings of 0.3 or greater on this factor. The final nine factors included the following themes: organizational support for provision of tIc, trauma theory, reflective functioning on the job, worker efficacy, relationships with clients, efficacy of TIC, importance of consequences, beliefs about clients, and secondary traumatic stress see . ARTIC-4Building on existing work, our results indicate that additional refinement of the ARTIC is needed ,22,23,24Quantitative results indicated that the ARTIC-45’s original seven factors and corresponding items did not hold up similarly with our population of frontline workers who work with substance-using populations. EFA results suggest a 10-factor scale. Upon closer examination, with this working population, the ARTIC-45 may be a unidimensional scale as more than 40 of the 45 items loaded onto factor 1. The ARTIC-10, created at the same time as other versions of the ARTIC, does represent a unidimensional scale. However, our additional CFA on items representing the ARTIC-10 did not show good model fit with our population, suggesting that more inquiry is needed to understand why the ARTIC is not performing as strong.The ARTIC’s psychometric properties were originally examined using workers in community-based mental health and healthcare facilities and workers in education. It is possible that those working in a substance-use disorder treatment and parent–infant mental health (PIMH) support may understand and experience examples of trauma-informed care differently in these settings. Workers in these specific HSO settings are balancing multiple foci of care including substance-use recovery, individual trauma-focused work, and attempting to improve parent knowledge and their bond with their child. Often, workers in these HSOs specialize in one or two of those areas, but not always all three, possibly making it difficult to integrate a one-size-fits-all trauma-informed practice.These differences may be due to our diverse sample . The ARTIC was normed on a largely white, upper- and middle-class socio-economic population (91% white ), raisinAs with understandings of measuring psychological variables with diverse ethno-racial groups, it is possible that workers of color may interact with certain questions differently . Those fFollowing qualitative inter-item analysis, the nine subscales resulted in some similar and differing themes from the original ARTIC. While we do not suggest uptake of our nine-factor scale, we do suggest more inquiry into the qualitative nature of the ARTIC’s items. Aligned with previous work, we suggest that this scale being categorized as a bipolar Likert-type scale is potentially a misnomer and closer resemblance to a semantic differentiation scale ,23. TakeTo date, the ARTIC has been shown to perform differently with at least three varying worker populations . TheAs the field continues to evolve and new knowledge about TIC is developed, there is also a corresponding need to consider how this new knowledge should be captured in research. For example, it is clear that while TIC enjoys a broad level of endorsement and support from frontline social service providers and mental health workers, there is little shared understanding of how the core principles of TIC should be operationalized, which has resulted in widespread variation in implementation ,15,16.While the ARTIC has value as one of the first TIC scales utilized in HSO settings, it is imperative for the field to continue to advance in regard to measurement to achieve a robust and rigorous set of assessments about the provision of TIC in different social service settings with different client populations. More research is needed to identify the conditions under which the ARTIC should be used and how it may perform differently across different working populations and diverse ethno-racial groups.