Despite the availability of effective and safe human papillomavirus (HPV) vaccines that reduce the incidence and impact of cervical cancer and other cancers, HPV vaccine coverage rates remain persistently low and the cervical cancer burden disproportionately high among Indigenous people globally. This study aimed to systematically identify, appraise, and summarize the literature on documented barriers and supports to HPV vaccination in Indigenous populations internationally. Forty-three studies were included and an inductive, qualitative, thematic synthesis was applied. We report on 10 barrier themes and 7 support themes to vaccine uptake, and provide a quantitative summary of metrics. Focusing on Indigenous perspectives reported in the literature, we propose recommendations on community-research collaboration, culturally safe intergenerational and gender-equitable community HPV vaccine education, as well as multi-level transparency to ensure informed consent is secured in the context of reciprocal relationships. Although the voices of key informant groups  are underrepresented in the literature, the identification of barriers and supports to HPV vaccination in a global Indigenous context might help inform researchers and health policy makers who aim to improve HPV vaccine uptake in Indigenous populations. Persistent infection with human papillomavirus (HPV) is responsible for the vast majority of cervical cancers, as well as causing oropharyngeal, vulvar, anal, and penile cancers , 2. ThisMany Indigenous people inherit and retain unique cultural ways of relating that are distinct from those of colonizing societies ; and desThe prevalence of HPV infection is significantly higher among Indigenous people than non-Indigenous people –20, withas reported by global Indigenous people themselves, recognizing that those most directly affected are often most contextually-informed on the social and structural determinants of their own circumstances.Enhancing HPV vaccination is an opportunity to reduce cancer risk among Indigenous people. However, it is critical to center Indigenous voices to understand the context in which HPV vaccine barriers exist and to develop and implement supportive programs. Therefore, we sought first to systematically identify barriers to and supports for HPV vaccination in Indigenous people worldwide as identified in the literature, regardless of who reported the information. Our secondary aim was to amplify the barriers and supports to HPV vaccination The land where the majority of our research team resides is today known as the western Canadian province of Alberta. It is home to a number of Indigenous people, including First Nations, Métis and Inuit people, who experience historical and ongoing harmful practices of colonialism and assimilation . As a laThe overarching research project in which this review is situated was a partnership project to explore the reasons behind disparities in HPV vaccine uptake in First Nations people in Alberta. The project’s researchers sought to establish culturally safe processes in our collaborative research partnership with First Nations representatives of a number of First Nations cancer prevention initiatives. By building on previous models of engagement, our team benefitted from best practices that recognize an empowered partnership approach, situating First Nations people, with both experiential and traditional knowledge systems, at the center of the work and creating a foundation for research engagement, implementation, and evaluation .This empowered partnership approach began with creation of a First Nations Elder Advisory Council to determine community interest in the project and to begin a dialogue on the unique needs and concerns of First Nations people regarding HPV and cervical cancer . MembersWe employed a broad search strategy, described previously in a published protocol , to captWe used descriptive statistics to summarize study characteristics and undertook iterative, inductive thematic synthesis on extractable barriers and supports, using the systematic review synthesis methods for qualitative research . We condIn our analysis and discussion, we emphasized the barriers and supports reported by Indigenous people, in order to amplify Indigenous voices and perspectives. We also identified studies that included evidence of community-based approaches, collaborative engagement in pilot studies, or explicit consultation with members of a relevant Indigenous community, so as to promote collective, community ownership of the research process and to highlight culturally-appropriate approaches.From 16,702 search-generated and de-duplicated records, 43 unique studies  met our inclusion criteria ; over 95From the 43 included studies, we identified 43 unique barrier codes and 35 unique support codes, which were grouped into 10 barrier themes and 7 support themes . We alsoFor each data point we identified who reported each barrier or support, and further classified each voice as belonging to the ‘Indigenous community’ , or non-Indigenous ‘colonized systems’ . Indigenous voices were represented through surveys, interviews, focus groups, and sharing circles, with 55% of the data points reflecting perspectives reported among Indigenous populations globally. Barriers and supports were reported by parents/caregivers (15 studies), health workers (4 studies), community members (4 studies), and vaccine-eligible youth (3 studies) and young adults (5 studies). Of 24 studies, six reflected Indigenous voices from more than one ‘reported by’ category. The reported barrier and support themes and codes are outlined in To determine which studies were conducted in a participatory manner through Indigenous community engagement, we incorporated ‘evidence of user involvement in design’ as a measure when using the QATSSD tool for quality appraisal. Indigenous people were, to some degree, engaged and/or involved in 47% of the studies identified in this review . HoweverBeliefs about sexual behavior were the most commonly reported barrier among those who identified as Indigenous, namely the perception that the vaccine might encourage sexual behavior among young people. These beliefs were particularly evident when concepts of cultural taboo were brought forth. For instance, despite overall support for vaccination in a community in Papua New Guinea, there were concerns expressed by community members about a perceived increase in promiscuity once disease protection was acquired from the HPV vaccine; some participants believed that there might be a community-wide increase in the sex trade . In settThe age appropriateness of the vaccine was a common area of inquiry in surveys about the barriers and supports to HPV vaccination among Indigenous people –38. In oThere is some evidence that vaccine-eligible Indigenous young adults viewed their risk of contracting an oncogenic strain of HPV as low, which further reduced their vaccine acceptance. In a focus group study of post-secondary students in the southwest United States , studentGiven that the HPV vaccine is offered predominately to youth under the age of 18, parents or caregivers might refuse to consent for their children to be vaccinated in school, or might influence the autonomous decision of their adolescent to seek the vaccine for themselves , 43, 44.Negative media reports can increase social stigma and fear of side-effects, and lead to community spread of misinformation regarding the HPV vaccine , 44, 46.During interviews with vaccine-eligible youth in Alaska , youngerConcerns about non-specific safety issues and unwanted side effects led Alaska Native parents to delay HPV immunization for their children . Many InConcerns about alleged side-effects unique to Indigenous bodies were raised by community members in various studies. In Victoria, Australia, one parent asked whether the clinical trials had been conducted with Indigenous women, stating “Our immune systems are set for things in the land…if this has not been tested on us, and the vaccine has things that are not from the land, it could make us very sick” . A vacciSome Indigenous people in the literature were uneasy about the possibility of infertility or sterilization. Community participants in Papua New Guinea feared that the HPV vaccine would result in the destruction of reproductive organs and trigger additional complications resulting in infertility . Other sDeep distrust of the institutions providing the HPV vaccine can be a barrier to uptake in Indigenous populations. Many communities engage in sharing stories and disseminating oral knowledge, including powerful stories of lived experiences of injurious or disabling interactions with the healthcare system. Social communication of experiences of perceived harm from vaccines and ill-intent of institutions can result in community-wide caution, distrust, and general apprehension towards HPV vaccination . InformaMistrust of any information stemming from pharmaceutical companies was highlighted by participants in Alaska . Fear ofIn multiple studies, vaccine-eligible young adults, parents/caregivers, and health workers alike expressed a desire for increased understanding of HPV and the vaccine. Participants asked for explicit details about HPV vaccines to allay concerns, to educate their family members, and to make informed decisions about their health, the health of their children and the health of their communities , 40, 44.Across the majority of studies reflecting Indigenous voices, there were low knowledge levels regarding both the HPV vaccine and the disease outcomes from HPV infection. Parents and caregivers stated that insufficient knowledge of the HPV vaccine was a barrier to providing consent for their children , 50, andDespite a pharmaceutical industry marketing campaign in Alaska prior to a study , vaccineInsufficient knowledge of the HPV vaccine was also an issue for the healthcare providers serving Indigenous communities, with one study identifying misunderstandings regarding vaccine eligibility requirements and beliefs about age-appropriateness among providers in Washington State . In a scLoring (2008) noted that Indigenous children from families with low awareness of the HPV vaccine are more likely to be overlooked in vaccination programs, especially when HPV and cervical cancer are not talked about within communities. In survey data, Indigenous people reported that one reason they had not consented to vaccination of their son or daughter was that they did not know about the vaccine , 41, 46.Some researchers reported knowledge disparities concerning gender equity in contracting HPV; in one study, parents were generally unaware that the HPV vaccine was recommended for males as well as females , and femA Canadian workshop with Indigenous vaccine recipients highlighted issues with age- and culturally-inappropriate health resources for the HPV vaccine . ParentsIf their healthcare provider did not recommend the HPV vaccine or provide education, then parents and caregivers were less likely to vaccinate their children , 35, 44.In some studies, there were reports of minimal efforts to ensure consent was fully informed and freely given before vaccinations were administered. Parents in Papua New Guinea said that they did not know what their children’s routine immunizations (outside of HPV) had been for, leaving them unaware as to what their children were protected against, often due to poor health messaging provided by health workers at the time of immunization . AdditioIn key informant interviews in New Zealand, a Maori health expert highlighted the importance of ensuring that existing colonial systems continue to support equitable HPV vaccine uptake for the Maori people . The MaoEven when the HPV vaccine was offered free of charge, a Peruvian study reported that the availability of medical staff, equipment, and facilities can be a barrier for access, especially in remote communities . For thoIndigenous people in rural or remote communities face additional barriers in terms of physical access to clinics and transportation issues , 43. PhyCommunicating the importance of receiving the HPV vaccine through the lens of ‘cancer prevention’ might be important to achieve optimal HPV vaccine uptake. Australian parents who were also health workers thought it was important to promote the vaccine as a cancer prevention strategy , becauseParents and caregivers have a role to play in communicating the importance of the HPV vaccine , 51, 52,Teachers in New Zealand suggested involving youth as campaigners and role models to provide encouragement for their peers, as well as involving youth-friendly media in HPV vaccination campaigns . The socGreater knowledge of HPV or the HPV vaccine was found in multiple studies to increase parents’ and caregivers’ willingness to consent to HPV vaccination , 46, 59.Encouraging community-level discussion of the HPV vaccine and its protective effects was viewed as a viable method to support vaccination uptake in many Indigenous communities . SharingIt was suggested that educational efforts must discuss not only the protection afforded by the HPV vaccine, but also frank information about possible adverse effects. Young people, in particular, expressed the need in Papua New Guinea for information on the vaccine’s advantages, disadvantages, and side effects . ParentsFor parents in Victoria, Australia, community education was considered vital in the consent process. This education was also critical in unwinding any sexual implications that community members have attached to the HPV vaccine . EquitabHealthcare providers making a strong recommendation for HPV vaccination is seen to support Indigenous people in receiving the vaccine , 37. InfDirecting resources to create a position for a dedicated immunization nurse , or furtReminders and follow-up from healthcare providers might be helpful in encouraging parents to complete the consent form for HPV vaccination . The majPhysical access to the immunization clinic (or school) supported the receipt of HPV vaccination , as did Initiation of the vaccine series at a younger age has been a successful strategy to increase uptake in Indigenous populations. In the United States, Indigenous children who initiated the HPV vaccine series at age 9 or 10 were 2.5 times more likely to complete the series compared to those who started at the recommended age of 11 or 12 . The autFor Indigenous community members in the United States, participation in a traditional cultural practice was found to be significant. Those who participated in ceremony were two and a half times more likely to receive the HPV vaccine .In one study in Fiji, the majority of parent respondents included their daughter in the decision-making process , and in The primary aim of this systematic review was to identify barriers to and supports for HPV vaccination for global Indigenous people, as reported in the literature. In the results section, we have presented the findings as reported by both Indigenous and non-Indigenous study participants. In this discussion, we focus on our secondary aim, namely reflection on the barriers and supports specifically reported by Indigenous participants, including Indigenous parents/caregivers, health workers, community members, and vaccine-eligible youth. Our research team, which includes our First Nations research partners, offer this discussion to provide unique insight and give voice to Indigenous perspectives wherever possible. Given that the majority of our team (Indigenous and non-Indigenous) are based in Canada, we have included some additional reflections specific to the Canadian context in The studies included in this review illustrate the hesitancy of Indigenous parents and caregivers to vaccinate their children against HPV due to concerns about sexual health, uncertainty about vaccine safety, and general distrust of colonial systems. While HPV vaccination is not associated with an increase in sexual activity , 65, 66,Fear of sterilization or other feared vaccine injury was driven by lived or intergenerational experience with harmful practices whereby Indigenous people were used as nonconsensual research subjects . MembersInadequate awareness of community vaccination needs can contribute to barriers in vaccine service provision for Indigenous people . AlthougThe ability to address health issues within Indigenous communities is often dependent upon the capacity of the healthcare workers themselves. Responding to health crises can overshadow other concerns despite a recognized need to address them. In these instances, health researchers can play a key role by providing educational resources to healthcare workers to build capacity in the community and to ensure meaningful actions are taken that reflect a deep level of commitment to supporting the community. One example of such support can be found in actions that are taken to demonstrate a true sense of reciprocity. Reciprocity was exemplified by a recent series of Métis-led vaccination clinics against SARS-CoV-2 in Alberta, Canada, during the COVID-19 pandemic. The inclusion and broad support of Indigenous health workers by the provincial health authority, non-Indigenous healthcare workers, and leading researchers in the study resulted in a low-barrier, culturally safe immunization space for presenting vaccine recipients . Other eCommunity members expressed disappointment in the colonized approaches to HPV vaccine health education, acknowledging that the exclusion of Elders and the use of culturally inappropriate resources might contribute to lower vaccination rates for their communities. Enabling HPV vaccine health education for all community members was seen as a more culturally traditional way of sharing knowledge, with intergenerational health education working to normalize the HPV vaccine. Prior to colonization, Elders might have been traditionally involved in cross-generational health education, and with the right support, it is possible for Elders to reclaim these roles and to guide young people in decision-making that protects health . In provFor vaccine-eligible young adults over the age of 18, fears of vaccine injury and concerns about Indigenous women being ‘targeted’ and harmed by vaccination effectively limits HPV vaccine acceptance. The combination of a low-risk perception of HPV and a perceived risk avoidance through cancer screening presents difficulty in shifting the risk-reward beliefs of these young adults. The assessment lens is an example of something education can affect by encouraging young people to recognize the long incubation periods of HPV-related cancers and to understand the limitations inherent to screening or healthcare requiring detection to occur early.Only six studies investigated the perspectives of Indigenous young adults; more research on this topic is required. A lack of youth participation in the literature represents a clear and persistent obstacle to HPV vaccination strategy development. Knowledge translation strategies might not be effective if there is little understanding of the needs of the vaccine recipients. These factors have negative implications for both HPV vaccine strategy development and for HPV-related disease prevention. Research conducted by young adults with young adult participants might yield candid responses that could provide a helpful basis for such strategy development.Only three studies highlighted the voices of Indigenous youth under the age of 18 years. Despite this fact, we have made every attempt to highlight youth concerns because evidence suggests that tailoring vaccine strategies to the needs and preferences of the target adolescent population might increase uptake . IndigenThe perspectives of Indigenous youth for a service that is directed at this population are paramount given the barriers and supports identified. For example, support for HPV vaccine uptake included youth influence on parental consent for vaccination, as well as the role of youth in areas of social influence and decision-making. This finding mirrors vaccination preferences in non-Indigenous youth, where adolescents expressed the desire to be involved in the decision-making process more explicitly . FurtherOver 95% of the studies we identified in this review originated from North America and Oceania, where Indigenous people are more explicitly defined and maintain shared histories of colonization. This fact is notable, given the documented global existence of Indigenous people in at least 90 nations worldwide . This liWe also noted a general lack of embedded Indigenous consultation and involvement in the studies included in this review. This is concerning, given the well-documented importance of early and consistent community participation in improving the acceptability, applicability and success of Indigenous health research. Also, due to the challenges associated with recruiting minor-aged children, children who are eligible for HPV vaccination in many countries  have been excluded from participation in most research studies.Issues of racism and discrimination experienced by Indigenous people in accessing healthcare were limited in the findings of this systematic review. Given that the majority of the studies included did not situate the identity of the researchers (i.e. their Indigenous or non-Indigenous identities), we do not know if the authors of the studies included in this review approached their work with a lens geared towards identifying the multifaceted effects of colonization. This lack of deep engagement with Indigenous people’s experience of racism is an important gap in the current knowledge base which should be explored in future studies.More subtly, the legacy of systematic exclusion of Indigenous voices from healthcare decisions indirectly points to issues of racism as a barrier to HPV vaccine uptake for Indigenous communities globally. The barrier of deep distrust towards the institutions  recommending and providing the HPV vaccine is one example of intergenerational trauma caused by colonizing systems and oppressive practices, many of which continue today. Likewise, the lack of consistent, culturally-appropriate engagement by researchers is evidence of embedded colonizing practices at work. Although these issues were indirectly addressed in the literature, it is important to highlight that racism and discrimination prevent the full participation of Indigenous people in the health care system, and the insidious nature of structural racism—often subliminal and hard to identify—is a recognized barrier to health care access . It is eThe COVID-19 pandemic has further highlighted the need for adequate data and information on the health of Indigenous populations , 79. FurThere are a number of noteworthy limitations to our study. We experienced difficulty in identifying Indigenous populations within the literature and in how studies reported on the populations due to the varying ways these populations are defined . In addiIn presenting the barriers and supports for HPV vaccination separately for Indigenous and non-Indigenous people in the literature , we noteWe acknowledge that each community of people defined as ‘Indigenous’ is distinct, and the needs regarding HPV vaccine acceptance will vary due to cultural contexts situated within unique historical and current experiences of colonization. We acknowledge that the perspectives offered by our First Nations team members are not reflective of all Indigenous people within Canada or globally. Alternative interpretations might emerge if additional Indigenous voices were included in our team’s work.While there is no ‘one-size fits all’ solution to increasing HPV vaccine uptake among Indigenous people, we have systematically identified the extant barriers and supports to HPV vaccination for global Indigenous people as reported in the peer-reviewed and grey literature. Our focus on the Indigenous voices present in the literature highlights and distinguishes Indigenous perspectives from those voices of a Western background, and this work is critical to advancing HPV vaccine uptake. From its inception, our study team has been comprised of both Indigenous and non-Indigenous collaborative members; key understandings of the results presented here were co-led and co-interpreted. It is our hope that future HPV vaccine research involving Indigenous participants will center Indigenous voices and reflections in scholarly discussion and dialogue.The methodology of this research sought to bridge Indigenous ‘Ways of Knowing’ with Western scientific research processes. Although the methodology can refer to principles of community-based research for the benefit of enabling a holistic perspective of the project findings and outcomes, as previously noted, the collection, analysis and synthesis of outcomes relies on Indigenous interpretations and meanings to ensure the voices and perspectives of First Nations are given prominence in the findings , 83. TheThe perspectives of Indigenous vaccine-eligible children, youth, young adults, and Elders are underrepresented in the literature, creating a knowledge gap in understanding the barriers and supports to HPV in vaccination for Indigenous communities. Lack of formal investigation of child populations  should not preclude consideration of their input on HPV vaccine programs. The insights and preferences of this population should be captured and considered when developing and delivering local HPV vaccination programs within Indigenous communities. To appropriately inform vaccination policy worldwide, increased understanding of the perspectives of vaccine-eligible Indigenous children, youth and young adults should be prioritized, along with the perspectives of community Elders. In addition to exploring youth perspectives through an academic lens, potential approaches would be to involve Elders, parents, and grandparents  in collective efforts to inform communities about HPV-related cancers, HPV vaccination, and sexual health education. In many Indigenous cultures, Elders hold positions of influence and respect in communities as knowledge-holders and community guides , 85, andBased on findings from the literature, coupled with interpretation and dialogue from Indigenous team members, we have compiled the following general recommendations regarding HPV vaccination in Indigenous populations. We propose that it is essential for researchers, healthcare providers and policy-makers to:Prioritize collaboration with Indigenous leaders, Elders, children and youth.Ensure HPV vaccination is offered through multiple venues and various locations, allowing individuals to access HPV vaccines both within and outside of Indigenous communities.Focus on vaccine relevance, normalization, and ownership, and ensure Indigenous representation in media messaging .Address misconceptions about HPV vaccines and HPV disease risk perception through community-level discussion and educational support sessions.Foster intergenerational openness in community health education, with a focus on engaging Elders in knowledge gathering.Engage children and youth to receive their comments on what youth need regarding HPV education and knowledge to make informed familial decisions about the vaccine.Involve the men of the community; their knowledge and participation are an important component for communities when discussing sexual health promotion.Be honest and maintain full transparency at each stage of the knowledge-sharing process, working at the pace of the community, and ensuring fully informed consent prior to HPV vaccination.To enhance HPV vaccination for Indigenous populations globally, culturally-appropriate, collaborative, and inclusive health education for all community members might assist governments and vaccine providers to normalize the HPV vaccine, to address misconceptions about the HPV vaccine and to ensure fully informed consent. Future work to increase HPV vaccine uptake should be supportive of community-based research in partnership with Indigenous people, to ensure the richness of shared knowledge and to reflect accurately the holistic experiences of the young HPV vaccine recipients as well as their wider community.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


Black and Hispanic/Latinx individuals experience a greater burden of mental health symptoms as compared to White individuals in the general population. Examination of ethnoracial disparities and mechanisms explaining these disparities among veterans is still in its nascence. The current study examined perceived everyday discrimination and income as parallel mediators of the association between race/ethnicity and PTSD, depression, and general anxiety symptoms in a sample of White, Black, and Hispanic/Latinx veterans stratified by gender.A random sample of 3,060 veterans living across the U.S. (oversampled for veterans living in high crime communities) completed a mail-based survey. Veterans completed self-report measures of perceived discrimination via the Everyday Discrimination Scale, PTSD symptoms via the Posttraumatic Stress Disorder Checklist-5, depressive symptoms via the Patient Health Questionnaire, and anxiety symptoms via the Generalized Anxiety Disorder Questionnaire.Models comparing Black vs. White veterans found that the significant effect of race on PTSD, depression, and anxiety symptoms was mediated by both perceived discrimination and income for both male and female veterans. Results were less consistent in models comparing Hispanic/Latinx vs. White veterans. Income, but not perceived discrimination, mediated the relationship between ethnicity/race and depression and anxiety symptoms, but only among women.Results suggest that discrimination and socioeconomic status are important mechanisms through which marginalized social status negatively impacts mental health. Approximately 25% of veterans identify as a racial or ethnic minority, numbers which are projected to grow to almost 40% in the next three decades . As the Posttraumatic stress disorder (PTSD), major depressive disorder (MDD), and anxiety-related disorders are common mental health conditions experienced by veterans of the United States military, with current prevalence estimates for PTSD, MDD, and anxiety disorders ranging from 4.8–23% , and 3.7There is less research regarding ethnoracial disparities in depression among veterans. In the general population, Non-Hispanic/Latinx Black (hereafter referred to as Black) and Hispanic/Latinx individuals have been found to have higher rates of depression compared to White individuals –20 and aFindings on ethnoracial disparities in anxiety are also somewhat mixed in both civilian and veteran populations. Within the general population, White individuals have been found to have a higher 12-month and lifetime prevalence of anxiety disorders compared to Black, Asian, and Hispanic/Latinx individuals , 23. HowMinority stress theory, which in part posits that social and structural processes, including discrimination, are important mechanisms through which marginalized social status negatively impacts mental health  may explThe experience of systemic and chronic racism and discrimination by minority and marginalized groups has also been linked to poorer mental health outcomes . StudiesAlthough a large body of research documents both the negative impact of discriminatory experiences and lower access to economic resources on the mental and physical health of racial/ethnic minorities, relatively few studies have examined the extent to which discrimination and economic resources explain ethnoracial disparities in mental health of veterans. One study found that both discriminatory stress and PTSD severity were higher in Black and Hispanic/Latinx Veterans compared to White Veterans, and that discriminatory stress was associated with increased PTSD severity over time . This stExamination of mental health disparities often focus on single categories of identity . However, such approaches do not reflect the complexity of people’s identities and the fact that many individuals have multiple and intersecting identities that interact to define each person’s unique experience . FurtherIn the current study we examined both perceived discrimination from multiple potential sources and income as parallel mediators of the relationship between race/ethnicity and PTSD, depression, and general anxiety symptoms in a sample of White, Black, and Hispanic/Latinx veterans. To further understand differences between race/ethnicity within gender, analytic models were stratified by gender.Data for this study comes from the Longitudinal Investigation of Gender, Health, and Trauma (LIGHT) study, a prospective study examining the impact of exposure to ongoing trauma and violence on veterans’ mental health, functioning, and reproductive health. In order to address the primary aims of the larger investigation, female veterans and veterans living in high crime communities were oversampled. The VA/DoD Identity Repository (VADIR), a VA-managed dataset of all separated service members, was used to identify eligible veterans. High crime communities  were determined by zipcode using ArcGIS. A random sample of veterans between the ages of 18 and 50 were selected such that 19,441 (69.4%) veterans were living within high crime zipcodes and 8,559 (30.6%) veterans were living within not high crime zip codes . Female and male veterans were sampled in equal numbers from both zipcode groups. A total of 17,178 veterans were invited to participate  after accounting for duplicate and non-deliverable addresses . Of those invited, 3,544 veterans enrolled in the study (21% response rate), which included 2,047 (57.8%) veterans residing in high crime communities and 1,838 (51.9%) women. Veterans living in not high crime communities (22.0% vs. 19.7%), rural areas (21.5% vs. 20.4%), and/or were female (24.5% vs. 17.6%) were more likely to enroll in the study. For the current analysis, a total of 3,060 veterans who identified as Black, White, and/or Hispanic/Latinx were included.Data was collected via a mail-based survey administered using a modified Dillman mail survey approach . VeteranRace and ethnicity were ascertained using the clinical research reporting policies outlined by the National Institutes of Health , which dHousehold income was self-reported by asking the veteran to estimate their household’s yearly gross income. They were asked to include all sources of income from all earners in their household and to choose an income range from “no income” to “$150,000 or more per year” . Other demographic information, including gender, educational attainment, rank, and deployment status were also collected via self-reported.never) to 5  with scores ranging from 0–45. Participants also report what they believe are the main reasons for these experiences . The EDS has shown good internal consistency and test-retest reliability [α = .91).The Everyday Discrimination Scale (EDS)  is a widiability . Internathink about things that may have happened to you throughout your life that are unusually or especially frightening, horrible, or traumatic. If you have had one of these experiences, which experience causes you the most distress?” Then, they noted which type of event it was . Time since trauma was on average 15.4 years (SD = 10.1) with little variation between race/gender groups. Participants then rated the extent to which each PTSD symptom  bothers them in the past month from 0  to 4 (extremely) with scores ranging from 0–80. The PCL-5 has demonstrated sound psychometric properties among both civilians [α = .97).The Posttraumatic Stress Disorder Checklist-5 (PCL-5)  is a 20-ivilians  and veteivilians  with a civilians . Internanot at all) to 3 (nearly every day) with scores ranging from 0–27 and scores of ≥ 10 indicating moderate to severe depressive symptoms. The PHQ-9 has demonstrated excellent internal reliability, test-retest reliability, and strong criterion and construct validity [α = .93).The Patient Health Questionnaire (PHQ-9)  is a 9-ivalidity . Internanot at all) to 3 (nearly every day) with scores ranging from 0–21 and scores of ≥ 10 indicating moderate to severe anxiety symptoms. The GAD-7 has demonstrated excellent validity, internal consistency, and test-retest reliability [α = .95).The Generalized Anxiety Disorder Questionnaire (GAD-7)  is a 7-iiability . InternaAnalyses were conducted using SPSS version 24 and PROCESS version 3.5 . Parallen = 1,539) female and mostly ranged in age between 34 to 49-years-old . Participants were 62%  White, 24% (n = 734) Black, and 13% (n = 420) Hispanic/Latinx. As compared to White veterans, Black veterans were more likely to be older and female and were less likely to have been deployed. Hispanic/Latinx veterans were more likely to be younger than both White and Black veterans, and more likely to be male as compared to Black veterans. White veterans were the most likely to have a college degree and higher income, followed by Hispanic/Latinx veterans and then Black veterans.Full sample demographics are presented in M = 12.48) and Hispanic/Latinx veterans (M = 10.86) compared to White veterans (M = 9.49). Moreover, Black veterans had a significantly higher EDS mean score compared to Hispanic/Latinx veterans. Of the 10 potential reasons for perceived discrimination, the most frequently rated reason for discrimination for Black male veterans was race (64.75%) followed by gender (24.46%). For Black female veterans, it was race (65.06%) followed by gender (53.10%). The most frequently rated reason for discrimination for Hispanic/Latinx male veterans was race (42.50%) followed by ancestry (31.50%). For Hispanic/Latinx female veterans, it was gender (49.53%) followed by race (35.38%). For White male veterans, appearance (25.23%) and age (15.55%) were rated as the most frequent perceived reasons for discrimination, while gender (52.77%) and age (22.60%) were rated as the most frequent reasons for White female veterans. See EDS scores were significantly higher for both Black veterans , depression (40.44%), and anxiety (36.73%) based on established clinical cut-offs as compared to both White  and Hispanic/Latinx veterans .A series of 12 models were run to examine whether EDS and/or income mediated the effect of race/ethnicity on mental health symptoms within each gender, controlling for age and education level. The bootstrap confidence intervals of these models are included in For models comparing Black vs. White veterans the significant effect of race on PCL-5, PHQ-9, and GAD-7 was mediated by both EDS and income for both male and female veterans. Pairwise contrast testing revealed that EDS and income were not significantly different in strength as mediators across these models. Results varied in models comparing Hispanic/Latinx vs. White veterans. Hispanic/Latinx male vs. White male veterans showed no significant mediation effects of EDS or income on any of the mental health symptoms. However, Hispanic/Latinx female vs. White female veterans revealed that income, but not EDS, mediated the relationship between ethnicity and PHQ-9 and GAD-7. There were no mediation effects for Hispanic/Latinx female vs. White female veterans on the PCL-5. See Consistent with previous literature , 14, 20,Our findings highlight the ways in which minoritized individuals bear a disproportionate burden of these social stressors. Although all groups reported perceived everyday discrimination, the sources of discrimination differ across groups. Our data suggests that perceived discrimination based on race has particularly insidious effects on mental health. Discrimination and low income are chronic stressors that may persist throughout a person’s lifespan . ChronicWe did not observe gender differences in the models comparing Black vs. White participants, suggesting that both perceived everyday discrimination and income similarly explain the mental health disparity among both male and female Black veterans. Alternatively, results varied by gender when examining these mechanisms among Hispanic/Latinx veterans. Specifically, neither income nor perceived everyday discrimination mediated the association between race/ethnicity and mental health symptoms for male veterans. However, for Hispanic/Latinx female veterans, income explained a portion of the association for the increased risk of depression and anxiety, but not PTSD, symptoms. This suggests that the mechanisms explaining disparities between Hispanic/Latinx and White veterans in mental health symptoms varies by gender. These findings highlight the importance of examining disparities through an intersectional lens, as they illustrate that there is a unique experience of Hispanic/Latinx women as it relates to income that cannot be explained by gender or ethnicity alone. Hispanic/Latinx women may experience income-related stressors that are unique from those experienced by Hispanic/Latinx men and White women. As an example of the intersection of ethnicity and gender with respect to economic disparities, while the unemployment rate has narrowed between Hispanic/Latinx men and all other men (4.7% vs. 4.4% unemployed), the unemployment rate for Hispanic/Latinx women continues to remain higher compared to the rate for all other women (5.7% vs. 4.3%) . FurtherThis study was not without limitations. Unfortunately, there was not enough representation in other Non-Hispanic/Latinx racial groups  to allow for group comparisons. Second, the response rate in this study was relatively low  although consistent with other studies that have been conducted among veterans  , 70, andIn sum, the current study provides further evidence of the role of perceived everyday discrimination and income in mediating racial/ethnic differences in mental health symptoms. Assessing and recognizing the potency of perceived discrimination is critical in informing our interventions for mental health disorders, particularly for vulnerable subgroups who report higher rates of perceived discrimination.


Objectives: To study the prevalence and the association of HPV infection in retinoblastoma and to determine the most common genotype presented in RB.Methods: Following the PRIMSA guideline, 14 studies reporting HPV infection in RB acquired from six databases were included.Results: The prevalence of HPV from 941 RB samples was 15.6% [95% confidence interval (CI): 7.3–30]. Mexico followed by India and Brazil had the highest HPV prevalence in RB samples, 61.7% (95% CI: 17–93), 22.5% (95% CI: 9–47), and 12.1% (95% CI: 2–52), in order. HPV 16 was the most common genotype presented in RB samples 23% (95% CI: 9–47), followed by HPV 18 10% (95% CI: 3–30) and the combined HPV 16–18 6% (95% CI: 0–50). We did not find a significant association between HPV and RB . However, after removing the largest-weighted study, a significant association between HPV and RB was observed .Conclusion: HPV prevalence in RB samples was 15% and HPV 16 was the most presented genotype in RB samples. There may be an association between HPV and RB that is needed to be confirmed by high quality future studies. Preventive and treatment measures against HPV infection are essential for the prevention of any possible consequences, in particular, RB. Among all ocular tumors, retinoblastoma (RB) is known to be the major cause of intraocular tumor presented in the childhood period. The incidence of RB varies from one country to another and is estimated to affect 1 patient/20,000 individuals , 4. TherIn recent years, Human papillomavirus (HPV) has become an important driver of the non-inheritable proportion of many cancers . Such hiOur systematic review and meta-analysis was developed by following the guidelines of Liberati et al.  which arInclusion criteria: All papers that reported HPV infection in RB samples were included, whether reported alone or compared to a control population. We made no restrictions against the event of HPV (studies with 0 events were included), language of studies, and the publication year.Exclusion criteria: We excluded conference papers, previous systematic reviews, books, and duplicate papers including the same patients, which were checked by reviewing the country, recruitment year, and the hospital of the included patients along with the similarity of the authors of the suspected duplicated papers.After the search term development, one author retrieved all records from the six databases including title, abstract, link, DOI, publication year, and author list of each individual study. The process was performed by Endnote software, and then all records were transported to an Excel sheet for the screening process. Each record was screened from the excel sheet by both authors and a third reviewer when disagreement was presented. Then another step of full text screening was done by both authors for including the best matched studies according to our eligibility criteria. Two experienced members conducted a manual search to retrieve the missed relevant papers by searching the references of any previously published systematic reviews and a hand search in Google Scholar database.All the necessary data for the characteristics of the included studies and our outcomes were extracted through an Excel sheet. Our outcomes were: the prevalence of HPV in RB, the common genotype of HPV in RB, and the association between HPV and RB. The characteristics of the included studies were: study ID, male prevalence, country of patients, study design, control definition, age, and the diagnostic method of HPV. Two authors extracted the data and reviewed it for any potential errors. The online tool of the cross-sectional and the cohort studies of the National Institute of Health were retrieved and all studies were assessed for quality into good, fair, or poor domains .p values that resulted from Egger’s test were used to report the publication bias in outcomes that had ten or more included studies [The software used for the analysis was comprehensive meta-analysis software (CMA 3.0). Two effect sizes were pooled, the event rate to analyze the prevalence of HPV in RB, and the odds ratio (OR) to analyze the association between HPV in RB compared to controls along with their 95% confidence interval (CI). Due to the significant heterogeneity we found, we used random effect model in all the analyzed outcomes . Moreove studies , 17.From the six databases, 3,264 records were retrieved and only 2,459 records were screened using title and abstract screening after excluding duplicate records. We screened 16 full texts for potential eligibility in our study. Finally, we included 14 studies that included three studies from manual search methods  7–12, 1–25.in situ hybridization in formalin-fixed paraffin-embedded RB tissues. All studies were of fair criterion  .We performed subgroup analysis according to the country of each study. Mexico followed by India and Brazil had the highest prevalence of HPV in RB samples, 61.7% (95% CI: 17–93), 22.5% (95% CI: 9–47) and 12.1% (95% CI: 2–52), in order . Moreovep = 0.02)  .Five studies compared HPV prevalence in RB samples to controls , 20, 24.p = 0.09) (p < 0.001)  . However< 0.001) .HPV 16 was the most common genotype presented in RB samples 23% (95% CI: 9–47), followed by HPV 18 10% (95% CI: 3–30) and the combined HPV 16–18 6% (95% CI: 0–50) .We found that the prevalence of HPV in RB was 15.6% and was the highest in Mexico followed by India. Among all strains of HPV, HPV 16 had the highest prevalence in RB. We did not find an association between HPV and RB; however, the association was prevalent after performing a sensitivity analysis.Various studies had explored the prevalence of HPV in RB samples and a huge variability in the prevalence among all studies was found. Fuentes et al.  indicateIn our analysis, HPV 16 was the most common genotype presented in RB tissues. The results were consistent with many studies reported in literature , 22, 25.We did not find a significant association between HPV and RB; however the association was presented when we excluded the study of Antoneli et al. . In addiIntensive efforts have been devoted to exploring the possible method of HPV infection in the eye before inducing RB. Different routes of transmission were identified including transmission during pregnancy, sexual contact, autoinoculation transmission, and transmission through fomites –34. MoreTo our knowledge, HPV plays an important role in the deactivation of tumor suppressor genes, in particular pRB and p53 through the E7 and E6 proteins . HoweverOur study had many limitations therefore our results should be taken with caution when interpreted by future studies. Firstly, all studies were cross-sectional in nature, which cannot determine the exact relationship between HPV and RB, therefore more prospective studies with long follow up periods are needed to confirm this association. Secondly, we found a significant heterogeneity between the included studies. Thirdly, many studies did not report the socioeconomic profile of the included patients which may have a significant effect of the association between RB and HPV. Fourthly, the selection of the control samples  is of extreme importance for measuring the true association between HPV and RB. Fifthly, all studies were of fair criterion, therefore caution should be considered when interpreting our results.


Cervical cancer (CC) is a prevalent malignancy affecting women globally. The primary causative factor of CC is the high-risk oncogenic human papillomavirus (HR-HPV). However, it is noteworthy that not all women infected with HR-HPV develop cancer, indicating the potential involvement of genetic predisposition in the development of CC. This study aims to identify genetic risks and their distribution in groups of women with different epidemiological features of HR-HPV.rs55986091 in HLA-DQB1, rs138446575 in TTC34, rs1048943 in CYP1A1, and rs2910164 in miRNA-146a.A comparison was conducted among four groups of women, comprising 218 HPV-negative women, 120 HPV-positive women, 191 women diagnosed with cervical intraepithelial neoplasia (CIN) grade 2 or 3, and 124 women diagnosed with CC. The analysis focused on four single nucleotide polymorphisms (SNPs): The rs55986091-A allele exhibited a protective effect within the “CC” group when compared to the “HPV-Negative” group  using a log-additive model. Additionally, similar protective effects were observed in the “CIN 2/3” group compared to the “HPV-Negative” group .The data obtained emphasize the importance of developing PCR-based diagnostic kits for the identification of SNP alleles, particularly for rs55986091, among HR-HPV-positive women within the Russian population. In 2020, approximately 604,000 new cases of CC were reported, resulting in over 340,000 fatalities and contributing to 7.7% of all cancer-related deaths . The incAccording to Russian cancer statistics from 2019, the existing preventive measures for cervical precancerous conditions have demonstrated limited effectiveness, particularly concerning vaccination programs. Furthermore, there is a lack of awareness among the population regarding the significance and accessibility of CC screening and a lack of motivation for undergoing preventive examinations. Consequently, the mortality rate for CC remains largely unchanged (Infection with high-risk oncogenic human papillomavirus (HR-HPV) has been established as a confirmed carcinogenic factor for CC. Within this study, 14 HPV types were identified as highly carcinogenic, namely, types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68  alleles associated with diseases enables the detection of potential inherited predispositions to the development of pathological conditions during the asymptomatic phase. This aids in the timely implementation of diagnostic and treatment measures .Given the lengthy asymptomatic period, sexual transmission, and the impact on women of reproductive age, assessing the genetic risk of developing CC holds significant clinical importance. This is particularly relevant for high-risk groups, including women infected with HIV .rs55986091 in HLA-DQB1, rs138446575 in TTC34, rs1048943 in CYP1A1, and rs2910164 in miRNA-146a.In our previous study, we conducted an analysis of SNPs associated with CC risk derived from genome-wide association studies (GWAS) and case-control studies, including a meta-analysis . For thirs55986091 SNP  was examined in a single GWAS  or invasive CC  was also examined in the same GWAS study . The finrs1048943 SNP  has been investigated in meta-analyses, revealing significant associations with CC. Among women from India, the meta-analysis showed an OR of 2.34, with a 95% CI of 1.37-3.99  has been examined in previous articles  kits. The investigation of HPV types was conducted using the AmpliSens® HR HPV screen-titre-14-FL (Registration Certificate RZN 2017/5387 of 22/2/2019) and AmpliSens® HR HPV geno-type-titre-FL (Registration Certificate RZN 2017/6533 of 27/3/2019) kits. These kits enabled HR-HPV genotyping for 14 specific types, namely, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.DNA extraction from whole blood samples and cervical scrapings (exocervix and endocervix) collected in the BD SurePath transport medium  was performed using the RIBO-prep (Registration Certificate FSR 2008/03147 of 15/9/2008) and AmpliSensThe PCR reactions were performed using a reaction mix of 25 μl, which included primers, probes, and dNTP (0.44 mM) in a volume of 10 μl. Additionally, 0.5 μl of TaqF polymerase and 4.5 μl of RT-PCR-mix-2 FEP/FRT reagents were added. The extracted DNA (10 μl) was also included in the reaction mix. Real-time PCR was carried out using the Rotor-Gene Q cycler  and DTprime  instruments. The following cycling conditions were employed: an initial denaturation step at 95°C for 15 minutes (1 cycle), followed by 45 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 10 seconds. During the amplification process, a fluorescent signal was detected at 60°C. The amplification results were analyzed by setting the threshold at 10% of the highest value of the fluorescent signal for each channel. All the reagents and kits used for PCR and DNA extraction were manufactured by AmpliSens, Russia, and provided by the Central Research Institute of Epidemiology of Rospotrebnadzor.The PCR products were sequenced using the PyroMark Q24  and 3500xL  instruments, and reagent kits were recommended by the equipment manufacturers.2.3A retrospective study was conducted on biological samples collected between 2017 and 2021 at the Central Institute of Epidemiology. Informed consent was obtained from all patients regarding the use of their DNA samples, and samples without informed consent were excluded from the study. The collected samples were categorized into five distinct groups:The “Population Control” group consisted of randomly selected whole-blood samples obtained from donors whose sex, age, and HPV infection status were unknown.The “HPV-Negative” group included women who tested negative for high-risk human papillomavirus (HR-HPV) and had negative results for intraepithelial lesion of malignancy (NILM) during cervical screening over a 5-year monitoring period. The cervical scrapings were used for this assessment.The “HPV-Positive” group consisted of women who tested positive for high-risk human papillomavirus (HR-HPV) and had negative results for intraepithelial lesion of malignancy (NILM) during cervical screening over a 5-year monitoring period. Within this group, 29% of women showed elimination of the virus, while 71% demonstrated persistent HR-HPV infection.The “CIN 2/3” group consisted of women who tested positive for high-risk human papillomavirus (HR-HPV) and had cytologically and/or histologically confirmed CIN grade 2 or 3.in situ or squamous cell carcinoma of the cervix.The “CC” group comprised women who tested positive for high-risk human papillomavirus (HR-HPV) and had histologically confirmed carcinoma Cytological and histological evaluations, as well as the diagnosis of carcinoma, were conducted based on investigations of cervical scrapings. All the samples included in the study were collected from residents of Moscow, Russia. Samples from the “HPV-Negative,” “HPV-Positive,” “CIN 2/3,” and “CC” groups were recruited between 2017 and 2021. Samples from the “Population Control” group were collected between 2018 and 2020.Detailed information about the samples is given in 2.4The obtained allele data were compared with the allele frequencies from the Ensembl database for Europeans and validated using the Hardy-Weinberg equilibrium and Pearson’s χ2 test. The associations of the SNPs were assessed using logistic regression and expressed as OR with 95% CI for standard genetic models in case-control studies . The Aka3The results of genotyping of the samples are given in In the initial stage of the study, we conducted genotyping of the Population Control group for four markers and compared the allele frequencies with the data obtained from the Ensembl database . To deters138446575, and rs1048943 between the groups. However, significant differences were observed for the marker rs55986091 in HLA-DQB1 across all four inheritance models. The codominant and recessive models specifically showed the lowest OR of 0.16. The calculation of OR for the five genetic models can be found in Based on the analysis, no significant differences were found for the markers rs2910164, The analysis of the AICs and BICs indicated that the log-additive model was the most likely inheritance model for the rs55986091 genetic variant. This suggests that carrying the A allele may provide a protective effect against CC development. On the other hand, the G allele was found to increase the risk of CC .Specifically, in the Dominant model, individuals carrying the GG and GA genotypes had an increased risk of CC development with an OR of 6.13 (95% CI=2.13-17.65). In the “CC” group, there were 120 individuals with the GG and GA genotypes compared to 4 individuals with the AA genotype. In the “HPV-Negative” group, there were 181 individuals with the GG and GA genotypes compared to 37 individuals with the AA genotype. These findings suggest a significant association between the GG and GA genotypes and an increased risk of CC.The frequencies of observed genotypes deviated from the Hardy-Weinberg equilibrium .rs55986091 showed a consistent association, with the dominant model having the lowest OR of 0.47. After adjusting for multiple comparisons using the Bonferroni correction, the statistically significant differences remained significant for the dominant and log-additive genetic models.In order to compare the HPV-Negative and CIN 2/3 groups, we examined four genetic markers. Among these markers, For further details and specific OR values, refer to The analysis using the Akaike information criterion (AIC) and Bayesian information criterion (BIC) reaffirmed that the log-additive inheritance model is the most likely model for this particular locus.Furthermore, we compared the HPV-Negative and HPV-Positive groups using four genetic markers. While some statistical differences were observed in the recessive model for rs55986091, these differences were considered insignificant after applying the Bonferroni correction to account for multiple comparisons. For more comprehensive information, please refer to The analysis of allele frequencies revealed that the “Population Control” group had a slightly higher frequency of the A allele compared to women without HPV, but this difference was not statistically significant . However, the frequency of the A allele in women with CIN 2/3 was similar to that in the “HPV-positive” group and significantly different from the “HPV-Negative” group . For a more detailed overview of the allele frequencies, please refer to The dominant HPV types in the HPV-infected group were 68, 16, and 31 . In the CIN 2/3 group, 16, 31, and 33 HPV types were most often detected . In the group of women with CC, the HPV types often identified were 16, 18, and 33 . Detailed information about the percentage of people infected with different types of HPV in three groups is provided in 4The rs55986091-A allele exhibited a protective effect in the “HPV-Negative” group compared to the “CC” group. The rs55986091 is located in the regulatory region between the HLA-DQA1 and HLA-DQB1 genes . In partWe propose two hypotheses regarding the influence of this SNP on the development of CC in women:There is a genetic linkage between the observed SNP and a specific allele of the human leukocyte antigen (HLA) gene. This HLA allele is believed to enhance the effectiveness and/or prolong the duration of the immune response in individuals who have received the HPV vaccine.This SNP affects the expression of the HLA-DQB1 gene, whose product may be associated with a protective effect against cancer.The first hypothesis is supported by several pieces of evidence. Previous studies have demonstrated the protective effects of certain HLA alleles, such as HLA-DQB1*02, HLA-DQB1*0603, DQB1*03, DQB1*0604, DQB1*0501, and DQB1*0603, in CC and other precancerous lesions . CarrierOne limitation of the present study is the lack of information regarding HPV vaccination in the study group. Adjusting for vaccination status could provide a better understanding of the observed effects. In future research, this limitation should be addressed by incorporating data on HPV vaccination and employing bioinformatics tools to investigate the linkage disequilibrium of this SNP with HLA alleles DQB1 and DQA1.of HLA-DQB1, it is possible that it is associated with alterations in gene expression. Reduced or impaired expression of HLA molecules has been linked to the development of CIN and CC , indicating a departure from the expected genotype frequencies. Specifically, the frequency of observed heterozygotes was lower than expected, while the frequency of observed homozygotes in both genotypes was slightly higher than expected. Further analysis of the super-enhancer region (NC_000006.12:c32651217-32660516) revealed the presence of at least 100 SNPs with similar genotype distributions.Based on the findings, it is plausible to suggest that homozygous SNP variants in this region exhibit a higher affinity for recognition by RNA polymerase, resulting in enhanced gene expression and a more robust immune response. Additionally, there may be a genetic linkage between rs55986091 and one of the SNPs situated within the exon, influencing the encoding of the peptide allele .Graffelman et al. previously observed a deficiency of heterozygotes between genes as well as an excess within the genes for HLA class II, which they attributed to the presence of null alleles and sequence errors . HoweverAdditionally, the association of the HPV type with one of the host genotype variants was considered. We did not find a statistically significant association for any of the types in each group. We assume that with an increase in samples, it will become more realistic to establish a relationship between this SNP and the type of HPV. The realization of the carcinogenic potential of the virus is influenced by many factors , including the presence of a genetic predisposition, which is realized by a combination of unfavorable combinations of risk alleles in SNP.One limitation of this study is the absence of adjustment for demographic factors such as age, smoking status, occupation, vaccination history, and specific HPV types. These factors can potentially influence the development of CC and could have confounding effects on the observed associations. Additionally, the lack of clinical data on patients further limits the comprehensive understanding of the relationships between genetic variants and disease outcomes. Future studies should consider incorporating these demographic factors and clinical data to obtain a more comprehensive analysis.This study focused exclusively on CC and did not investigate other HPV-associated cancers. Therefore, it is important to replicate the findings in samples of patients with head and neck cancer, including male patients, to determine the association of this SNP with HPV persistence in different cancer types.The identified associations between specific SNPs and the risk of developing CC have significant implications for the development of screening programs aimed at identifying individuals at higher risk. Additionally, these findings play a crucial role in personalized management approaches for patients infected with high-risk HPV, particularly for individuals with a positive HIV status . The per5In the Russian population, the presence of the rs55986091 variant was observed in AA genotype carriers with an OR of 0.16, and in GG and GA genotype carriers with an OR of 6.13. Identifying both individual and population risks of CC development will improve the monitoring of individuals infected with high-risk HPV (HR-HPV). This will increase their motivation for vaccination and cervical examinations, thereby making a significant contribution to reducing CC-related mortality rates.The findings highlight the importance of developing and conducting clinical trials for an allele discrimination assay targeting rs55986091 in HR-HPV-positive women. Future research plans involve investigating the underlying mechanisms by measuring the expression level of HLA-DQB1 in individuals with different genotype variants. Additionally, testing other SNPs that have shown statistically significant results in previous studies will be pursued (The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Council on Bioethics of the Central Research Institute of Epidemiology. The patients/participants provided their written informed consent to participate in this study.MV: Writing - Original Draft, Formal analysis, Investigation, Visualization. KM: Methodology, Data Curation, Writing - Review and Editing, Investigation. ED: Conceptualization, Methodology, Writing - Review and Editing, Data Curation. TR: Conceptualization, Investigation, Writing - Review and Editing. AP: Conceptualization, Data Curation, Writing - Review and Editing. VA: Supervision, Resources, Project administration, Writing - Review and Editing. All authors contributed to the article and approved the submitted version.


S-adenosylmethionine-dependent methyltransferases (SAM-MTs) of HtmA2(-module 6)-MT domain and HtmB2(-module 18)-MT domain are responsible for the installation of methyl group at C-45 and N-12, respectively, whereas the FK506 methyltransferase (FKMT) type O-methyltransferase of HtmB1(-module 16)-MT domain take care of the methylation at O-21 of HTM. We also reported the antibacterial activities of HTM in this study, and found that it showed activities against M. luteus, B. thuringiensis and A. baumannii with MIC of 4 μg/mL, 4 μg/mL, and 64 μg/mL, respectively.About two-thirds of small molecule drugs contain methyl group and it plays a very important role in the drug development. So, methyltransferases catalyzing the methylation have always attracted great attention. Hangtaimycin (HTM) is a potent hepatoprotective agent. Previous study showed that its biosynthetic gene cluster contained three methyltransferase domains, but their characteristics in HTM biosynthetic pathway has not been revealed. In this study, we clarified multi-methylations in HTM biosynthesis in vivo. It showed that the two  The abiechanism . The majechanism .M. luteus, B. thuringiensis and A. baumannii.Hangtaimycin (HTM), a product of a hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), containing a rare unsaturated hexalactone ring embedded in a conjugated dodecanoic acid and a terminal diketpiperazine units . Accompa22.1E. coli and Streptomyces. All chemical reagents and antibiotics were obtained from Sigma-Aldrich. Primers were synthesized by Tsingke . The seed culture was obtained in TSBY liquid medium  at 28 °C with shaking of 220 rpm for 20 h. And then, the seed culture was inoculated to the modified SFM medium  and fermented for another 4 days at 28 °C and 220 rpm. E. coli strains were cultured in 2 × TY  or on 2 × TY agar medium at 37 °C with the appropriate antibiotic at a concentration of 100 μg/mL ampicillin, 35 μg/mL apramycin, or 30 μg/mL nalidixic acid for selection.The wild-type and mutants of 2.3S. spectabilis CCTCC M2017417 by conjugation using donor strain ET12567/pUZ8002 on ABB13 plates. After incubation at 28 °C for 12 h, the plate was overlaid with the final concentration of 35 μg/mL apramycin and 30 μg/mL nalidixic acid. Exconjugants were selected on ABB13 plates supplied with 35 μg/mL apramycin and 30 μg/mL nalidixic acid to confirm their antibiotic resistance. Then single colonies were patched onto ABB13 plates containing 35 μg/mL apramycin and onto ABB13 plates without antibiotic, respectively, to screen for the double crossover mutant. The correct phenotype (AprR) candidate mutants were further verified by PCR and sequencing.The constructs used for gene disruption were introduced into 2.4To obtain the HtmB2(-module 18)-MT domain in-frame deletion mutant, two homologous recombination fragments were amplified by PCR using primer pairs of OMT-L-up and OMT-L-re, OMT-R-up and OMT-R-re, respectively  eluting with a linear gradient elution system of CH3CN/H2O  at a flow rate of 1 mL/min, on an equipment of Shimadzu LC-20A. Meanwhile LC-ESI-HRMS was carried out on a Thermo Electron LTQ-Orbitrap XL mass spectrometer in a positive ionization mode with 35% relative collision energy, equipted with a linear gradient elution system of CH3CN/H2O  at a flow rate of 1 mL/min.After fermentation in modified SFM medium  for 4 da2.8Micrococcus luteus ATCC 10240, Bacillus thuringiensis BT01, Bacillus subtilis CGMCC 1.3358, Staphylococcus aureus ATCC 29213, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 47085 were added to each cell with a final concentration of 106 CFU/mL, respectively. After incubation at 37 °C for 16–20 h, the compound's concentration in cells with no turbid were defined as the MIC of the tested compounds, respectively. Experiments are triplicated for three times. Apramycin and kanamycin served as the positive controls, while DMSO served as the negative control.HTM was further tested for its antibacterial activities under the method described before . Briefly33.1222 and a larger lactone-polyene peptide fragment [S. spectabilis is tryptophan-dehydrobutyrine diketopiperazine (TDD), which had been isolated for several decades ahead of HTM, but its absolute configuration was confused [S configuration for the natural product TDD from S. spectabilis by comparing with synthetic standard, which is likely reflected in the corresponding portion of HTM, for the reason of absence of E domain in HTM biosynthesis [222 could be part of the cause of the degradation of HTM  and N-methylase (E × G × G × G was mutated to E × R × R × R) in HTM biosynthetic gene cluster were performed, respectively (222) [222 and demethyl-TDD, which are corresponding to the fragment of C-demethyl-HTM and N-demethyl-HTM were found from mutants of CMT and NTM, respectively   Fig. 2)O-methyltly (222)  would faectively . These r3.3C- and N-methylation at C-45 and N-12 in HTM, the remaining methylation at O-21 in HTM may be catalyzed by the FKMT type O-methyltransferase domain in module 16 of NRPS HtmB1. It is interesting that a FkbM family methyltransferase motif of “DxGxNxG” corresponding to the conserved SAM-MT motif of “E/DxGxGxG” was identified -MT domain. Results showed that the biosynthesis of HTM and relevant TDD and HTM222 were also abolished  and NTM, respectively, however no detectable demethyl-HTM was observed in ΔHtmB1(-module 16)-MT domain. Possibly, it is the difference substrate tolerance of HtmA2(-module 6)-MT domain, HtmB1(-module 16)-MT domain, HtmB2(-module 18)-MT domain in HTM biosynthesis. Although we haven't obtained the intermediates from the mutants, but thanks to cometabolites of HTM222 and TDD in HTM biosynthesis containing a C and N-methyl group that of HTM, respectively, we could detect the demethyl-HTM222 and demethyl-TDD in mutants of CMT and NTM, respectively, and finally determine the action sites of three methyltransferases in HTM. Although, the possibilities that the in-frame deletion may lead to the inactivation of the modular type protein (NRPS) could not be excluded, however, we still could conclude that HtmB1(-module 16)-MT domain is responsible for the installation of O-methylation at O-21 according to the results of CMT and NTM. Through this study, we found that HTM could gradually decreased with time, while the content of HTM222 and TDD increased accordingly, and HTM222 and TDD may be produced by degradation of HTM. Due to the unique structure and good hepatoprotective bioactivities of HTM, and the abilities of methylation to optimize properties of candidate drugs, the understanding of the methylation in HTM biosynthesis seems very important.In HTM biosynthesis, three methyltransferases existed as the following order of HtmA2(-module 6)-MT domain, HtmB1(-module 16)-MT domain, HtmB2(-module 18)-MT domain. So, we conclude that the Bacillus subtilis and Candida albicans [M. luteus, B. thuringiensis and A. baumannii with MIC of 4 μg/mL, 4 μg/mL, and 64 μg/mL in this study, respectively, as to the other strains HTM showed no antibacterial activities against to them at a concentration of 64 μg/mL, which are consisted with the antibacterial activities results reported by Zuo et al. [Meanwhile, we reported the antibacterial activities of HTM in this study. HTM was reported previously to have weak antibacterial activities against albicans , howeveralbicans , but also et al. . The fur222 and TDD increase accordingly, suggesting they may be partly produced by degradation of HTM. Further antibacterial activities test unravel that HTM have antibacterial activities against M. luteus, B. thuringiensis and A. baumannii. The findings in this study have given us a deeper understanding of HTM's biosynthesis and bioactivities.In conclusion, we unveil that HtmA2(-module 6)-MT domain, HtmB1(-module 16)-MT domain and HtmB2(-module 18)-MT domain are responsible for the installation of methyl group at C-45, O-21, N-12 of HTM respectively in vivo in this study, and observe that HTM could gradually decrease with time, and HTMNational Key R&D Program of China (2018YFA0903200), General Program of National Natural Science Foundation of China (32370083), Natural Science Foundation of Chongqing CSTB (CSTB2022NSCQ-MSX0995), and Graduate Innovation Program (GZLCX20232114)This work was supported by The authors declare no conflict of interest.Minghe Luo: performed the genetic experiments and LC-ESI-HRMS analysis. Yulu Dong: performed the genetic experiments and LC-ESI-HRMS analysis. Zixin Deng: analyzed the data and revised the manuscript, is an editorial board member for Synthetic and Systems Biotechnology and was not involved in the editorial review or the decision to publish this article. Yuhui Sun: conceived the overall project, analyzed the data and wrote the manuscript.


The incidence of high-grade anal intraepithelial lesions (HSILs) has increased in recent years among men who have sex with men with human immunodeficiency virus (HIV). This work evaluated the validity of the human papilloma virus viral load (HPV-VL) versus cytological and qualitative HPV results to detect HSILs.κ) were also calculated.From May 2017 to January 2020, 93 men who have sex with men and HIV were included in an anal cancer screening program from the Infectious Diseases Unit at a tertiary-care hospital in Alicante (Spain). The gold-standard for the screening of anal HSILs is the anal biopsy using high-resolution anoscopy. The diagnostic methods compared against gold-standard were HPV-16-VL, HPV-18-VL, and HPV-16-18-VL co-testing, anal cytology, and qualitative HPV detection. The receiver operating characteristic (ROC) curve and cut-off points for HPV-VL were calculated. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen’s Kappa coefficient (n = 18) HSIL, 29.1% (n = 27) LSIL, and 51.6% (n = 48) negative. An HPV-16-VL >6.2 copies/cell was detected in the HSIL biopsy samples (p = 0.007), showing a sensitivity of 100% and a specificity of 46.2%. HPV-18-VL and HPV16-18-VL co-testing showed a sensitivity of 75% and 76.9% and a specificity of 72.7% and 61.3%, respectively. The highest PPV was 50% obtained with the cytology and HPV-18-VL. The HPV-16-VL showed a NPV of 100%, followed by 88.9% in the HPV-18-VL and 87% in the abnormal cytology. Cohen’s Kappa coefficient were: HPV-18-VL (κ = 0.412), abnormal cytology (κ = 0.353) and HPV-16-VL (κ = 0.338).The mean patient age was 44.6 ± 9.5 years. All of them received antiretroviral treatment, 96.8% had an HIV viral load of <50 copies/mL and 17.2% had a previous diagnosis of AIDS. The diagnosis of the anal biopsies were: 19.4% (HPV-VL testing improved the detection sensitivity but not the specificity for HSIL biopsies compared to anal cytology and the qualitative detection of HPV. In men who have sex with men and HIV the HPV-VL could be an useful tool for diagnosis of HSILs in anal cancer screening programs. Further studies will be needed to evaluate the clinical implications of HPV-VL in these programs. Anal cancer is a rare digestive tumour type whose incidence has increased in recent years , especiaAnal biopsy using high-resolution anoscopy (HRA) is currently considered the gold standard for the diagnosis and management of HSIL . HoweverThe validity of these methods for the detection of anal precursor lesions or anal cancer in people with HIV and MSM was evaluated in a meta-analysis . For anaGiven the wide variability in the results published on the sensitivity and specificity of the different screening tests, new biomarkers such as HPV E6/E7 mRNA, p16, and Ki67 have been explored that could help in the screening of HSILs. The sensitivity of these tests for the detection of HSILs is also variable; for E6/E7 the sensitivity ranged from 69.6% to 71% and the specificity from 56.1% to 55.6%, while for p16 or p16/ki67, a sensitivity of 38.1% to 90% and a specificity of 50.5% to 87.9% was obtained . As we cDetermination of the HPV viral load (HPV-VL) is another biomarker directly associated with the severity of cervical intraepithelial lesions in cervical cancer screening , with thTo the best of our knowledge, HPV-VL has not been studied in the population of MSM with HIV in Spain. Therefore, considering the good predictive results of the majority of published international studies, we decided to study the ability of the HPV-VL to detect high-grade anal lesions in a population of MSM with an HIV infection who continued clinical monitoring at our clinic. The objective of this study was to evaluate the validity of using the HPV-VL to detect HSILs in men with HIV who have sex with men compared to the efficacy of qualitative and cytological HPV test results.This was a cross-sectional study of a diagnostic test evaluation.Eligible candidates for anal cancer screening were included from the Infectious Diseases Unit at a tertiary-care hospital in Alicante (Spain). Men who attended an anal cancer screening test between May 2017 and January 2020 were consecutively selected to participate in this study. All of them met the following inclusion criteria: males aged 18 years or older, a confirmed HIV infection, an HIV transmission mechanism of MSM sexual relations, and participating in the anal cancer screening program. The only exclusion criterion was having been previously vaccinated against HPV.ad hoc structured data collection questionnaire was prepared for data collection. Sociodemographic variables (sex and age), HIV-related clinical variables , undetectable HIV viral load [HIV-RNA <50 copies/mL], CD4+ nadir, current CD4+ cell value/µL, CD4/CD8 ratio and presence of genital warts), toxic habits , and variables related to sexual habits  were collected.An Anal cytology was performed on all the participants using the specific cytobrush material which was later transferred to ThinPrep sample preservation jars®. This technique involves introducing a brush into the anal canal using enveloping movements from the perianal skin up to four cm above the anal margin. Subsequently, this brush was introduced into a liquid medium for a few seconds  for preservation and analysis. Analysis of the anal cytology results was first performed by a single pathologist specialising in the diagnosis of anal dysplasia. The Bethesda classification was used  with theThe anal cytology was subsequently sent to the clinical microbiology service to determine the viral load and qualitative detection of HPV. The viral load of HPV-16 and HPV-18 was determined and the viral load co-testing of HPV-16-18 was performed taking the highest quantified value of HPV-16 or HPV-18 as the reference. Detection of human genomic DNA in the HPV viral load was performed using TaqMan™ Control Genomic DNA . The results were normalised and expressed as viral copies/human cell (copies/cell). The qualitative detection of HPV was carried out with the Cobas® 4800 HPV-Test. The results obtained were for HPV-16 and HPV-18, as well as a combined method for 12 high-risk human papillomavirus genotypes (HR-HPV), including 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.The HRA was performed on all the participants by the same team which comprised a proctologist and a nurse, both trained in performing this technique. The anoscopy was carried out in an operating room set up for this purpose. First, a digital rectal examination and visual examination were performed to detect macroscopic changes. Second, a transparent plastic anoscope (THD® N-Ano) was introduced into the anal canal for visualisation purposes. Using a colposcope with a light source and high magnification binocular vision, the anal tissue was then visualised. Afterwards, a swab wrapped in gauze and impregnated with 3% acetic acid (Lugol’s stain) was introduced for 2 min and was withdrawn; the anoscope was then reintroduced to visualise the anal tissue. After staining, areas likely to be preneoplastic—acetowhite positive—were sought. When necessary, a further 5% Lugol staining was performed with a swab to improve visualisation of the possible lesions. Third, an anal biopsy was taken from areas suspected of a lesion using Baby Tischler forceps and was then sent to the pathology department for analysis. When a patient had multiple areas biopsied on the same day, the area with the highest degree of dysplasia was used for analysis. The histological classification of the anal biopsy for its diagnosis was based on the Lower Anogenital Squamous Terminology Project , with thAll the participants were informed of the reason for the screening and signed an informed consent form for their participation in the study and for data collection. Participants were informed during HIV medical follow-up visits of the simultaneous performance of: anal cytology, qualitative HPV detection and high-resolution anuscopy with anal biopsy. These results were analysed by the Pathological Anatomy Service. The Clinical Microbiology Service subsequently analysed the viral load and carried out the qualitative detection of HPV.SD) and of the categorical variables using the absolute frequencies and percentages. Anal biopsy using HRA was used as the gold standard method of screening for HSILs. In the statistical analysis of the association and comparison of the different diagnostic tests, the histological result of HSIL versus non-HSIL was used. The latter included negative anal biopsy, LSIL, and anal warts. Thus, the continuous variables were analysed using Student t-tests or Mann–Whitney U tests, depending on the normality of the variables . Categorical variables were analysed using chi-squared tests and Fisher tests for association studies. In all the analyses, the value of p < 0.05 was considered statistically significant.A descriptive analysis of the continuous variables was performed using the mean and standard deviation (κ) were calculated to compare the usefulness of the different screening methods: HPV-16, HPV-18, HPV-16-18 co-testing, anal cytology, and the qualitative determination of HPV. Taking into account the high prevalence of anal HSILs data found in other studies  curves, together with the area under the curves (AUCs), were calculated to detect the cut-off point of the viral load of HPV-16, HPV-18, and the HPV-16-18 co-test to differentiate between the diagnosis of HSILs and non-HSILs by HRA biopsy. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen’s Kappa coefficient . All the participants were informed about the study and authorisation was requested by written informed consent before their participation. The confidentiality and anonymity of the data was ensured in accordance with current legislation on the protection of personal data and guaranteed rights (LOPD 03/2018 of December 5) and EU Regulation 2016/679 of the European Parliament.From May 2017 to January 2020, a total of 103 patients were seen at a tertiary-care hospital anal cancer screening clinic: 93 men and 10 women. A total of 93 participants who were MSM with an HIV infection were included in this work. Sociodemographic variables, clinical variables related to HIV, toxic habits, and sexual habits were collected of all the participants . The mean = 18) presented a HSIL, 29.1% (n = 27) a LSIL, and 51.6% (n = 48) a negative result. The quantification of the viral load in the HPV-16-18 co-test was higher for the total number of participants compared to HPV-16 and HPV-18 (41 copies/cells vs. 28.9 copies/cells vs. 2.16 copies/cells). In the anal cytology, 17.2% (n = 16) of the participants had a cytological alteration  while 81.7% (n = 76) obtained a negative result. There was one patient whose cytological result could not be obtained because insufficient material had been obtained. The highest prevalence of HPV detected was HR-HPV accounting for 83.7% (n = 77), followed by HPV-16 at 40.2% (n = 37), and finally HPV-18 with 17.4% (n = 16). In the comparisons made between participants with HSIL versus non-HSIL anal biopsy results, there were significant differences in HPV-16-VL  and the HPV-16-18 co-test viral load . In the rest of the variables studied, significant differences were found in abnormal anal cytology (p = 0.001), qualitative HPV-16 (p = 0.044), and qualitative HR-HPV (p = 0.037).A descriptive analysis of the different diagnostic tests studied for anal cancer screening was performed using HSIL and non-HSIL anal HRA biopsy for comparison . In the κ = 0.412) showed the highest Cohen’s Kappa coefficient followed by abnormal cytology (κ = 0.353) and HPV-16-VL (κ = 0.338). However, the poorest results were in the qualitative HR-HPV (κ = 0.037) and the qualitative HPV-18 (κ = 0.063).The results regarding the validity of HPV-VL, anal cytology, and qualitative HPV detection for the diagnosis of anal HSIL in participating HIV positive MSM were compared . The besThe aim of this study was to evaluate the validity of HPV-VL determination, compared to cytological and qualitative HPV results, for the diagnosis of HSILs using the histological results of anal biopsies as the gold standard. HPV-16-VL showed the highest sensitivity for the diagnosis of HSILs in anal cancer screening, although with a low specificity. HPV-18-VL showed the most balanced results between sensitivity and specificity with the drawback that only a few participants were studied. However, HPV-16-18-VL co-testing produced the most similar sensitivity and specificity results compared to conventional methods of anal cytology and qualitative HPV detection.As already observed in previous studies in HIV-infected men , the anaIn contrast, in our sample there was a high prevalence of qualitative HPV. The prevalence of HPV-16 was around 40%, similar to that found in other studies , which wvs. 100% vs. 76.9%, respectively) but high specificity . A meta-analysis reported a detection sensitivity of 85.2% and a specificity of 52.8% for anal cytology of HSILs , although the specificity was still improved (61.3% vs. 46.2%). It is possible that including HPV-18-VL with HPV-16-VL to obtain a combined result will improve the diagnostic validity of viral load in the diagnosis of HSILs. In addition, HPV-18-VL produced results with a lower discrepancy between sensitivity and specificity, at 75% and 72.7%, respectively. However, these data must be interpreted with caution because very few cases with HPV-18-VL and HSILs have been studied, and so these results are difficult to extrapolate. Notwithstanding, other studies have suggested that HPV-18 could also be a useful biomarker for the detection of anal lesions (Nonetheless, the sensitivity of the HPV-16-18-VL co-test was lower than that of the HPV-16-VL (76.9%  lesions .Of note, this current work had several limitations. Firstly, there could have been a participant selection bias because all the individuals who attended the anal cancer screening consultation were included consecutively. The fact that these patients were attending this consultation was conditioned by the fact that they had a greater interest in undergoing screening for possible anal symptoms suggestive of disease. Secondly, the low number of participants with qualitative HPV-18 could have influenced the interpretation of the results because these data cannot be extrapolated to other contexts and so more patients must be studied in future work.However, despite the limitations of this study, this work is the first of its type to be carried out at a national level in Spain to evaluate and compare the validity of HPV-VL to detect HSILs to that of conventional techniques in anal cancer screening in a population with HIV. While other studies have screened participants with a prior pathological result in anal cytology , with poGiven the wide variability in the results obtained for the predictive capacity of conventional methods such as anal cytology and the qualitative detection of HPV, studying new methods such as HPV-VL quantification can help in the detection of HSILs. This technique is a minimally invasive, well-tolerated test that also eliminates inter-observer variability in the interpretation of anal cytology. Given the overall high sensitivity of viral load quantification reported in this current work, perhaps in the future this technique could be considered a good biomarker for anal cancer screening.versus non-HSILs. All of this reinforces the idea that HPV is implicated in anal cancer and can be used for screening and detection of high-grade lesions. Nonetheless, further studies with more participants will be needed to evaluate the clinical implications that HPV-VL quantification may have in anal cancer screening. Indeed, it would be interesting to conduct longitudinal studies that evaluate HPV-VL at different screening time points in the same patients in order to study the factors that interfere with the progression of the HSILs or the clearance of HPV itself.In conclusion, in our work, quantification of HPV with cut-off values for HPV-16, HPV-18, and HPV-16-18 co-testing of 6.2, 59.8, and 41.7 copies/cell, respectively, improved the sensitivity for the detection of HSILs compared to anal cytology or the qualitative detection of HPV, although it did not improve the specificity. In addition, the HPV-16-VL and HPV-16-18-VL co-testing results were statistically significant for the diagnosis of HSILs 10.7717/peerj.15878/supp-1Supplemental Information 1Click here for additional data file.