The tumor microenvironment (TME) of HNSCC is heterogeneous and complex, and it plays a crucial role in achieving effective cancer therapy. To develop effective cancer therapies, it is important to understand the crosstalk between cancer inflammation and immunity, as well as oncogenes and tumor suppressor genes. In this study, we aimed to gain a more comprehensive understanding of the transcriptomes of the TME in HPV-associated and HPV-non-associated TSCC by studying the gene expression profiles of 168 genes linked to various cellular mediators and factors involved in inflammation, immune crosstalk, transcription, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis. We found that the TME of HPV-associated and HPV-non-associated TSCC exhibited a remarkable heterogeneity of gene expression associated with cellular mediators and factors involved in inflammation, immune crosstalk, transcription factors, immune signaling pathways, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis.Head and neck squamous cell carcinomas (HNSCCs) are a common type of cancer, ranking as the sixth most prevalent cancer worldwide and having a high morbidity and mortality rate. Among oropharyngeal squamous cell carcinoma (OPSCC) cancers, tonsillar squamous cell carcinoma (TSCC) is the most prevalent and has a particularly aggressive clinical course with poor disease outcomes. The tumor microenvironment (TME) of HNSCC is complex and heterogeneous, playing a crucial role in effective cancer therapy. Understanding the interaction between cancer inflammation, immunity, oncogenes, and tumor suppressor genes is essential for developing effective cancer treatments. This study aimed to gain a comprehensive understanding of the transcriptomes of the TME in TSCC, both associated with human papillomavirus (HPV) and not associated with HPV. The gene expression profiles of 168 genes linked to various cellular mediators and factors involved in inflammation, immunity crosstalk, transcription, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis were analyzed. We identified 40 differentially expressed genes related to the communication between tumor cells and the cellular mediators of inflammation and immunity crosstalk. In HPV-positive TSCC patients, 33 genes were over-expressed with a fold change greater than 1.5, and 26 of these genes were unique to this group. In contrast, HPV-negative TSCC patients had 11 up-regulated genes. The results further showed that 48 gene transcripts related to oncogenesis, tumor suppression, angiogenesis, and apoptosis were up-regulated in both HPV-positive and HPV-negative TSCC patients. Among the HPV-positive TSCC patients, 37 genes were over-expressed, while the HPV-negative TSCC patients had 11 up-regulated genes. The tumor microenvironment (TME) of HPV-associated and HPV-non-associated TSCC exhibited distinct characteristics, including the dysregulation of various genes involved in cellular mediators, inflammation, immunity crosstalk, transcription factors, immune signaling pathways, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis. Additionally, we detected six Hr-HPV genotypes in 81% of the TSCC patients, with HPV-16 and HPV-35 being the most common types, followed by HPV-45 and HPV-18. HPV-39 and 31 were also identified. The presence of Hr-HPV genotypes in TSCC patients varied from single to multiple infections. In conclusion, we observed distinct heterogeneity in the transcriptome of the microenvironment in HPV-associated and non-associated TSCC. Further in vitro and in vivo studies are needed to investigate the functional implications of the identified over-expressed genes. Also, deeper molecular pathways and immunological studies on the TME are required to determine the potential of targeting genes for cancer therapy. Head and neck squamous cell carcinomas (HNSCCs) have a high morbidity and mortality rate and rank as the sixth most common cancer globally, with approximately 900,000 new cases and more than 450,000 deaths in the last two years ,2. By 20TSCC has an aggressive clinical course and poor disease outcomes . Around The tumor microenvironment (TME) of HNSCC is a crucial factor in achieving effective cancer therapy ,20,21. TSixteen formalin-fixed paraffin-embedded tissue samples of TSCC were collected from four different hospitals, King Saud Medical City, King Fahad Medical City, King Faisal Specialist Hospital & Research Center and Dr. Sulaiman Al-Habib Medical Group, between 2014 and 2021. TSCC was confirmed by two pathologists. The preliminary inclusion criteria used in this study were (1) both males and females aged above 18 years; (2) the presence of invasive TSCC; (3) sufficient material for HPV detection and genotyping; and (4) an available block of formalin-fixed, paraffin-embedded tissue for the transcriptome of the invasive carcinoma. Patients who received chemotherapy or radiotherapy for any cancer, pregnant women, immunocompromised patients, patients with immune-mediated diseases, those treated with immunotherapy, or those with incomplete or missing records or data, as well as those under the age of 18, were excluded from the study. A series of 10 μm-thick tissue sections were cut from each paraffin block. To ensure the accuracy of the results, standard measures were taken to avoid cross-contamination during tissue sectioning and processing.The study protocol was reviewed and approved by the Institutional Review Board (IRB) committee at King Saud University, College of Medicine (IRB number E-22-6932).Viral DNA total RNA was extracted using the AllPrep DNA/RNA FFPE Kit from Qiagen  following the manufacturer’s protocol. The concentration (ng/μL) and purity of the extracted nucleic acid were assessed using a NanoDrop2000 . cDNA synthesis and genomic DNA elimination were performed using an RT2 first-strand synthesis kit  with 1.5 µL of RNA, following the manufacturer’s instructions. The integrity of the extracted nucleic acid and the absence of inhibitors were assessed using two internal housekeeping genes (GAPDH and HMBS). We have restricted measurements for handling the total RNA, such as working in a biological safety cabinet and in RNase-free environments.® Green Master mix, 1 µL of forward primer, 1 µL of reverse primer, 2 µL of cDNA, and 6 µL of RNase-free water. The cycling program was as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles; denaturation at 95 °C for 15 s; annealing at 57 °C and extension at 72 °C for 45 s using the ABI-7500 Fsat . The list of specific primers can be found in For HPV detection and genotyping, we used qPCR using a SYBR Green/ROX PCR Master Mix according to the manufacturer’s protocol. The extracted total RNA was converted into cDNA using the RT2 first-strand synthesis kit  based on the manufacturer’s recommendations to detect HPV E6/E7 mRNA expression. The reaction contained 10 µL of 2× RT2 SYBR2 profiler PCR arrays were used for the expression of 168 genes that are linked to communication between tumor cells and the cellular mediators of inflammation and immunity, oncogenesis promotion, tumor suppressor genes, transcription factors, angiogenesis, and apoptosis. The detailed list of all genes in both RT2 Profiler PCR Array Kits can be checked on the manufacturer’s website . Real-time PCR was performed using the RT2 Profiler PCR Array Kit  in a mixture with the RT2 SYBR Green/ROX qPCR Master Mix (Qiagen), as previously described [® Green Master mix, 102 µL of cDNA, and 1248 µL of RNase-free water. Subsequently, 25 µL of the qPCR component mix was added to each well of the PCR array, and then the 96-well array was placed in the ABI-7500 Fast  using 40 cycles at 95 °C, with 10 min per cycle, followed by 95 °C for 15 s and 60 °C for 1 min for annealing, extension, and fluorescence data collection. The online RT2-PCR Profiler data PCR array analysis software was used for relative gene expression results . Gene expression was quantified as ΔΔCt, and calculations were normalized to the five housekeeping genes.For mRNA expression, cDNA synthesis and genomic DNA elimination were performed using an RT2 first-strand synthesis kit  following the manufacturer’s instructions. The synthesized cDNA was used for an RT-PCR array of cancer inflammation and immunity crosstalk (PAHS-181Z), oncogenes, and tumor suppressor genes (PAHS-502ZC-12) . These two RTescribed . This ist-test. The correlations between all variables were assessed using Pearson’s correlation test. A p-value of < 0.05 was considered statistically significant. For gene expression and real-time PCR array data analysis, online RT2 Profiler Data Analysis software was used. The fold regulation values for each gene were determined using the relative quantification 2-ΔΔCt method. ΔCt values were normalized using the mean values of five housekeeping genes.GraphPad 10 software  was used for data analysis. A group comparison was performed using a A functional analysis of statistically significant over-expressed genes was used for the construction of the biological network using the Retrieval of Interacting Genes/Proteins database (STRING version 11.0) and Cytoscape software 3.10.1.The total FFPE samples from TSCC patients  and three individuals without TSCC were included in this study. The age range was 24–70 years. The median age was 57 ± 12. The complete demographics and clinical characteristics are shown in From the PEPP tissue-extracted DNA, HPV DNA was detected in TSCC patients. Thirteen (81.3%) were HPV-positive, while 18.8% had HPV-negative TSCC. We detected six Hr-HPV genotypes in 81% of the TSCC patients. The most common types were HPV-16 and HPV-35 (62%), followed by HPV-45 (25%) and HPV-18. HPV-39 and 31 (6%) were the least common types in this study. The presence of HPV in TSCC patients varied from single to multiple infections. Single HPV-16 and HPV-35 infections were detected in three patients (60%) and two patients, respectively. Whereas double HPV infections (25%) (HPV 16 + HPV 35) were detected in two TSCC patients . On the Cancer, inflammation, and immunity are closely interconnected, and their crosstalk plays a critical role in tumor development and progression. Inflammation can promote the initiation of cancer by inducing DNA damage and genetic mutations. It can also create a microenvironment that supports tumor growth by enhancing angiogenesis and suppressing immune surveillance. Therefore, we studied the mRNA expression profile of immune genes involved in crosstalk between the cancer, inflammation, and immunity pathways using the Human Cancer Inflammation and Immunity Crosstalk RT2 Profiler PCR Array in HPV-positive TSCC and HPV-negative patients. A total of 84 key genes involved in mediating the communication between tumor cells and the cellular mediators of inflammation and immunity crosstalk were included in this qPCR array. Based on the internal positive controls, genomic DNA elimination control, and reverse transcription controls, this profile showed higher reproducibility and efficiency. We identified 40 genes related to communication between tumor cells and the cellular mediators of inflammation and immunity crosstalk that were differentially dysregulated in HPV-positive TSCC and HPV-negative patients . Thirty-2 Profiler™ PCR Array. We also wanted to investigate the interplay between them and inflammation-related genes. Our results showed that a total of 48 gene transcripts were up-regulated (fold change > 1.5) in the HPV-associated and the HPV-non-associated TSCC patients . The most up-regulated genes  were input into the gene list, and homo sapiens were selected. The medium confidence level was set to 0.400. Then, we hid the disconnected nodes of the network and adjusted the position of each node. The PPI contained 43 nodes, 372 edges, and an average node degree of 17.3, with a × 10−16. . The PPI× 10−16. . This PPThe TME plays a crucial role in the initiation and progression of several types of cancer, making it a significant area of research in recent years. A deeper and more complete understanding of the specific components of the TME, particularly the immune oncogenes and tumor suppressor gene components, remains a significant challenge. To gain a more comprehensive understanding of the gene expression in the TME of HPV-associated and non-associated TSCC, we focused on the gene expression profiling of 168 genes that are linked to the communication between tumor cells and the cellular mediators of inflammation and immunity, oncogenesis promotion, tumor suppressor genes, transcription factors, angiogenesis, and apoptosis. The TME of HNSCC is a crucial factor in achieving effective cancer therapy ,21. The The HPV-associated and non-associated TMEs are characterized by the dysregulation of immune-related genes, tumor suppressor genes, and oncogenes. This dysregulation may be attributed to the presence of HPV and its viral proteins, which can disrupt normal cellular processes and immune responses. Consequently, genes involved in immune surveillance and tumor suppression become dysregulated. Several studies have shown that the immune microenvironment plays a crucial role in the prognosis and targeted immune therapy of HNSCC patients ,36,37,38In this study, we found that several immune-related genes were up-regulated (CCL4 and CCL5) in most of our cohorts, whereas BCL2, CCR7, CSF3, CXCL9, CXCR2, and IL13 were down-regulated. The CCL4 and CCL5 chemokines play a vital role in various cancer types in terms of metastasis, invasion, and poor clinical disease outcomes. Several studies have revealed that the up-regulation of CCL4 and CCL5 in gastric carcinoma, tongue squamous cell carcinoma (TSCC), and colon cancer resulted in the initiation of invasion steps ,42,43. MTumor growth and metastasis in cancer have been linked to angiogenesis . SeveralCXCL9 is one of the immune activation chemokines; its expression plays a crucial role in anti-tumor immunity and is associated with improvements in different types of cancer patients’ survival and overall good clinical outcomes ,56. In tInterleukin-13 (IL-13) is a cytokine that plays a significant role in negatively regulating anti-tumor immunity. It has been found to have a pivotal role in promoting tumor growth and progression by enabling cancer cells to evade the immune system . HoweverThe immune microenvironments of HPV-associated and HPV-non-associated OPSCC were distinct. HPV-associated OPSCC is characterized by the up-regulation of numerous immunogens and immune signaling pathways, as well as immune cell infiltration ,66. Our Genetic stability is responsible for tumor initiation and progression. We aimed to study 84 genes related to oncogenesis promotion, tumor suppressor genes, transcription factors, angiogenesis, and apoptosis. Those genes were categorized into different clusters: the oncogene cluster; the tumor suppressor cluster; the transcription factors cluster; the angiogenesis cluster; the cell adhesion molecules cluster; and the cell cycle cluster. We found that 48 genes were different in their expression patterns. In the HPV-negative TSCC patients, only 11 genes were up-regulated. In head and neck cancer, several oncogene families have been shown to play an oncogenic role. It has been reported that oncogenes play an important role as prognostic and tumor-prognostic factors in many types of cancer . During In this study, many key transcription factor genes were over-expressed in patients with TSCC. Until now, there has been a gap in our understanding of the mechanisms and how transcription factor deregulation contributes to tumor development, progression, and metastasis, as well as treatment resistance and disease clinical outcomes. It is quite a surprising finding that in one patient, we observed a rather remarkable down-regulation of 168 genes. This could be indicative of various underlying conditions, such as a genetic mutations. However, further research is necessary to ascertain the root cause of this phenomenon. In any case, this discovery could have significant implications for the development of new treatments and therapies for a wide range of tumors and diseases that are related to gene expression and regulation. A complete and deeper molecular understanding of the exact role of those genes in HPV-associated and HPV-non-associated TSCC could lead to identifying a potential target for effective cancer treatment approaches.Globally, the most prevalent hr-HPV genotype detected in HNSCC is HPV-16, followed by HPV-31, HPV-18, HPV-56, HP-V52, HPV-33, and HPV-35, as well as other hr-HPV genotypes ,79,80,81The HPV genome codes for six oncogenes named E1, E2, E4, E5, E6, and E7 ,13. ThesLimitations should be acknowledged for this research. Firstly, this study is focused on a specific cohort of TSCC patients from a single hospital, which may limit the generalizability of the findings. Secondly, we used a relatively small sample size, which may affect the statistical power and generalizability of the results. Further research should be conducted with larger samples. Thirdly, the study did not investigate the functional implications of the identified over-expressed genes in the HPV-associated and HPV-non-associated TSCC patients, which could provide further insights into the underlying mechanisms.The TMEs of HPV-associated and HPV-non-associated TSCC have shown distinct and heterogeneous gene expression related to various cellular mediators and factors involved in inflammation, immunity crosstalk, transcription factors, immune signaling pathways, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis. Further in vitro and in vivo studies are needed to explore the functional implications of the identified over-expressed genes in HPV-associated and non-associated TSCC patients, which could provide more insights into the underlying mechanisms. These experiments can provide crucial insights into the biological effects of these genes and their relevance in different subtypes of TSCC, identify potential therapeutic targets, and pave the way for personalized treatment strategies. Additionally, more in-depth molecular pathways and immunological studies are required to assess the potential of targeting genes for cancer therapy.


Parental consent for adolescent human papillomavirus (HPV) vaccine uptake is important; however, refusal is prevalent. Therefore, this study aimed to understand factors associated with parental consent for their adolescent daughter’s HPV vaccination. A cross-sectional study was conducted in Lusaka, Zambia, between September and October 2021. We recruited parents from different social settings. The means and standard deviations or median and interquartile ranges were used as appropriate to summarise continuous variables. Simple and multiple logistic regression models were fitted with robust estimation of standard errors. The odds ratios are presented with 95% CI. Mediation analysis was conducted using a generalised structural equation model. The study enrolled 400 parents, mean age 45.7 years . Two hundred and fifteen (53.8%) parents reported consenting to their daughters’ HPV vaccination, and their daughters received it. None of the health belief model (HBM) construct scores showed an independent association with parental consent. Higher, compared to lower wealth index , knowing someone with genital warts , cervical cancer screening uptake  were associated with increased odds of parental consent. This study highlights factors influencing parental consent for their daughters’ HPV vaccination. Ongoing sensitisation programs are important to improve their decision-making. HPV is sexually transmitted, with a significant proportion of sexually active people getting infected in their lifetime . Infectie supply ,5. The ae supply . HPV vace supply .The HPV vaccine is safe and effective for the primary prevention of cervical cancer, which has high morbidity and mortality in sub-Saharan Africa (SSA) . StudiesZambia has one of the world’s highest cervical cancer incidence rates of 65.5 per 100,000 women and a mortality of 43.3 per 10,000 women, accounting for 23% of all new cancers in the country in 2020 ,12. WithTo complement the efforts made through cervical cancer screening, Zambia introduced a campaign-based free national HPV vaccination program in 2019 restricted to a single age cohort of 14-year-old girls with a two-dose regimen, 12 months apart . HoweverHPV vaccination in Zambia is non-mandatory; parental consent is required with or without the parent’s presence at the point of vaccination. WHO has guided the consent process for eligible adolescents to be reviewed according to country guidelines; hence informed parental consent may be written, verbal or implied . ParentaSince its national rollout, there has been a paucity of data on factors influencing parental HPV vaccination consent in the Zambian context. In addition, mechanisms that potentially drive parental HPV vaccination consent have not been explored. Previously, Lui et al.  reportedUnderstanding factors influencing parental consent to HPV vaccination, such as beliefs, knowledge, and attitudes, is critical in developing targeted strategies to increase HPV vaccine uptake among adolescent girls ,26. TherTo our knowledge, HBM has not been used previously in Zambia in the context of parental consent for HPV vaccination. Therefore, we hypothesised that parents with higher perceived severity, perceived benefits, perceived susceptibility, higher self-efficacy, more cues to action and lower perceived barriers would be more likely to consent to their daughters’ HPV vaccination . Further2 area and a population density of 4181/km2 [We conducted a cross-sectional study between September and October 2021, in Lusaka district, the capital city of Zambia, with a population of 1,747,152 million people, 417.9 km4181/km2 . The dis4181/km2 .The target number of adolescent girls for HPV vaccination is determined using school registers from the Ministry of General Education, headcount, and Central Statistical Office figures. Community health workers and civil societies additionally identify out-of-school girls. The HPV vaccination program is campaign based, conducted during the first round of child health week (CHWk1), lasting about six days from Monday to Saturday. The CHWk1 is a biannual event that aims to catch up on vaccinations for children under five years from health facilities and outreach/mobile sites. Additionally, for HPV vaccination, schools are used, making it a mixed approach to capture both in-school and out-of-school girls.The district health director and health facilities work closely with the Ministry of General Education and schools in planning. Vaccination dates are shared with the school by the district health team so that teachers can prepare for the activity and girls can inform their parents in addition to community and mass media messages. Information sharing with parents varies, ranging from flyers, educational materials, television adverts, radio programs and letters.The parental consent approach is opt-out, meaning consent is implied unless a girl categorically mentions that her guardians refused; most private schools, however, use the opt-in approach, which requires written parental consent before vaccination . HoweverBased on similar studies’ estimates , we assuA two-stage sampling technique was used to enroll respondents. In the first stage, we stratified Lusaka into six sub-districts. Then we randomly selected two zones in each sub-district using probability proportion to zone size. In the second stage, a random sample of 30 respondents per zone was drawn from various social settings, such as markets, saloons, and barber shops, until we reached 400 participants clustered within 12 zones. We inflated the final sample size to 400 participants after accounting for the assumed moderate design effect of 1.5 to counter the loss in precision caused by the clustering of respondents in the 12 zones.Face and content validity of the questionnaire was ascertained by Obstetrician/Gynaecology consultants through a consultative process. We developed an interviewer-administered questionnaire based on the literature ,38. The A screening question was asked if the potential participant had a daughter aged 15–18 years. If not, the interview was terminated. This age group was selected because the HPV vaccination program in Zambia targets 14-year-old adolescent girls for dose 1. Therefore, we anticipated that an age range of 15–18 years would give a sample of parents with daughters who would have been eligible to receive the first dose or completed the vaccination schedule during the HPV vaccination demonstration 2013–2017) [13–2017  or natioThe outcome variable was parental consent for the HPV vaccine for daughters measured on a binary scale . The primary exposure variable was the HBM constructs scores. The total scores for the HBM constructs were calculated using the following steps: For each item within the HBM constructs, the numeric values chosen by each respondent, with a five-point Likert scale, were added. Responses were coded such that higher scores indicate a greater level of the relevant construct  and the reverse for perceived barriers. The total possible scores per construct were as follows; (a) perceived susceptibility (3 items = 15), (b) perceived severity (4 items = 20), (c) perceived benefits (4 items = 20), (d) perceived barriers (8 items = 40), (e) self-efficacy (4 items = 20), and (f) cues to action (2 items = 10). Knowledge of HPV (18 items) and HPV vaccine (24 items) scale had three options . We assigned a zero for “false/don’t know” and a one for “true” options.In addition, the study measured HPV and HIV-related parental characteristics, including ever screening for cervical cancer  , parental HIV status , the daughter’s HIV status , and knowing someone with cervical cancer or other HPV-related cancers , Socio-demographics included age (continuous), sex , marital status  employment and socio-economic status .The socio-economic status variables used for calculating the wealth index included; source of drinking water, type of toilet, house ownership, employing anyone, use of electricity, type of cooking fuel used and ownership of any of the following; fridge, laptop, television, motorcycle, scooter, mobile phone, watch, livestock, land phone, car, agricultural land, washing machine .https://getodk.org/). ODK is a free mobile-based platform with four tools serving different functions, namely, Aggregate, Build, Collect and Voice [Data collectors were trained over five days to understand the questionnaire, the study protocol and the use and configuration of the Open Data Kit (ODK) for data collection . All analyses accounted for the clustering of respondents within the zones using robust estimation of standard errors . In addition, the analysis accounted for the stratification by sub-districts within Lusaka province.The item scores were summed to create a score for six HBM constructs scales and one knowledge scale. The means and standard deviations or median and interquartile ranges were used as appropriate to summarise continuous variables  and report whether the respondent consented to vaccinate their daughter.Simple logistic regression models were fitted with robust estimation of standard errors with “consent to vaccinate daughter” as the response variable and one of the predictor variables at a time, to assess for any association between the predictor variable and consent to vaccinate daughter. Afterwards, a multivariable logistic regression model was fitted with six HBM construct scores (priori), and other variables found significant at a 20% level from the univariable logistic regression model. Variables were dropped from the multivariable model sequentially until only important variables remained. Finally, interactions between six HBM construct scores and modifying variables that remained in the final model were considered individually. The Hosmer Lemeshow test was done to assess the goodness of the model fit. The odds ratios are presented with 95% confidence intervals.Using principal component analysis, we used household assets and other related characteristic variables to calculate the wealth index as a proxy for socio economic status . The weaFurther, generalised structural equation modelling was conducted to understand the interrelationships between variables and assess mechanisms of association . Direct,The writing of this manuscript was guided by the STROBE statement checklist .We enrolled 400 participants; 215 (53.8%) reported consenting to the HPV vaccine for their daughters, and all reported that their daughters received it. The demographic characteristics and total knowledge scores are summarised in Health Belief Model constructs scores according to parental consent to vaccinate their daughter.A higher proportion of female participants who consented to vaccinate their daughter had screened for cervical cancer compared to non-consenting parents. Participants who knew their HIV status as positive, knew someone with cervical or HPV-related cancers or someone who has had genital warts were more likely to consent to daughters’ vaccination In the univariable logistic regression model, we found that wealth index, knowledge score, knowing someone with cervical cancer or other HPV-related cancers, knowing someone with genital warts and living with HIV were associated with providing consent to vaccinate the daughter. When adjusting for variables that were significant at a 20% level in the univariable model, the multivariable logistic regression showed that HPV/HPV vaccine knowledge, ever knowing someone with HPV-related cancer and none of the HBM construct scores were independently associated with consent to vaccinate the daughter .Mediation analysis was performed to explore and assess if HBM constructs are a mechanism through which knowledge levels affect parental consent choices. Knowledge levels were significantly associated with parental consent as a direct effect; however, the indirect effects of knowledge (mediated through HBM constructs) were not significant . All othThis study found that over half of our sample participants provided parental consent for the daughters’ HPV vaccination. We framed our study within the HBM based on its usefulness in exploring vaccination behaviour. However, we did not find any significant associations between HBM constructs and parental consent for the daughter’s HPV vaccine uptake, associated factors included; having screened for cervical cancer, knowing someone with HPV-related conditions, socio-economic status and marginally for those living with HIV.Parental consent to daughters’ HPV vaccination was 53.8%, and their daughters vaccinated, giving an uptake of 53.8% amongst our sample. This uptake is higher than the 39%  coverageParental consent plays a crucial role in HPV vaccination as daughters believe and act upon what their parents tell them. An earlier hypothetical study done in Lusaka, Zambia, before the HPV vaccination demonstration project (2013–2017) and national rollout of the HPV vaccination 2019) showed that 100% of the parents intended to vaccinate their daughters or themselves . On the  showed tWe did not find any differences in HBM construct scores between consenting and non-consenting parents, as earlier hypothesized. Similarly, Vermandere and others found no differences regardless of the daughter’s vaccination in a selected cohort of parents in Kenya . HoweverThe plausible explanation could be that we had low awareness of HPV and HPV vaccine in our cohort, and people are unlikely to commit to or have firm perceptions/beliefs about what they do not know. Therefore, people empowered with knowledge may be able to give a well-informed position. Further, inherent within the HBM framework and previous literature, the constructs’ relationship was assumed linear without variable ordering, which is considered a more complex approach .Combined high knowledge of HPV and HPV vaccine was associated with vaccination in univariate analysis. However, this was not significant after adjusting for possible confounders. Similarly, in mediation analysis, knowledge was a statistically significant direct factor associated with parental consent for daughters, but not indirectly through the HBM constructs. Knowledge plays a key role, as low levels may predispose one to hold fast to myths and misinformation. In other studies, parental knowledge is highly associated with the willingness to vaccinate daughters ,35,50. LThe possible explanation is that the HPV vaccine is relatively new in Zambia, despite an earlier demonstration project; hence some parents may not be knowledgeable about it. Further, the HPV vaccination program is campaign-based, and mass media messages and other activities to raise awareness only occur briefly before and during the activity. Therefore, it is important to consider ongoing messaging that can be reinforced during vaccination .This study revealed that the rich were more likely to consent to the HPV vaccine than the poor. Similarly, a study in Uganda based on demographic health data found that those in the middle wealth quintile were more likely to vaccinate their daughters . AnotherWe found that HIV-positive parents are more likely to vaccinate their daughters in univariable and marginally in multivariable analyses. There has been an emphasis on cervical screening WLHIV, especially in high-burdened regions like Zambia, to reduce the burden of cervical cancer . As suchTherefore, it is not surprising that WLHIV and those who have screened for cervical cancer are more likely to consent to their daughter’s vaccination based on their exposure to information on HPV and the HPV vaccine’s benefits. Wigfall et al.  reportedThis is one of the first studies in Zambia regarding parental consent for the HPV vaccine since the national rollout, framed in a well-recognised model. Even though the data is regional and collected from the capital city, the study can serve as a pilot, which can be scaled up to get a nationally representative sample. Using the health belief model is important as understanding results framed in theories is easier.The HBM has been widely used as a cognitive theory; hence this study adds evidence to the existing literature on circumstances where the behaviour of interest, as in this case, may not be directly influenced by the HBM constructs . FurtherThis study is without limitations; firstly, it was done in the Lusaka district hence the results may not represent the rest of the country, affecting the external validity. Secondly, recall bias could exist as some adolescents’ vaccination period was as far back as two years. However, parents usually have a good memory for a child’s health-related issues. Thirdly, girls could have received the vaccine without parents knowing since the current approach is opt-out, and the girl’s desire is considered primarily. Therefore, these girls may have been reported as not-vaccinated by parents when the converse was true. Fourth, non-consenting parents may have reported consenting for their daughters due to social desirability since the questionnaire was interviewer-administered.Fifth, this data was collected during the COVID-19 pandemic when there was a lot of mistrust around the COVID-19 vaccine, which could have significantly heightened the barriers in both groups during our data collection. Finally, the data was collected when the behaviour of interest, HPV vaccination, had already occurred. Therefore, the hypothesis can only be adequately tested where beliefs are known to have existed before the behaviour they are to determine; otherwise, individuals’ perceptions could be modified in areas relevant to HPV vaccination. There is the potential that vaccination status may have led to selective exposure or recall bias, thus suggesting reverse causality.This study has highlighted modifiable correlates of parental consent for adolescent HPV vaccination, which include socio-economic status, cervical cancer screening uptake and knowledge. Equity in vaccination programs should be prioritised as the rich are more likely to consent and get the vaccine and more likely to have screened for cervical cancer.Focus on raising community awareness on the role of HPV vaccination in preventing cervical cancer, especially among those from low socio-economic status, should be a priority to improve future programming and uptake. Equitable access to the provision of the HPV vaccine is essential to reduce the gap between the rich and the poor. Further, education on the relationship between HPV and cervical cancer and the preventive role of HPV vaccination should be integrated into the screening programs. This approach may improve knowledge levels among parents. Additionally, at the policy level, the feasibility of having ongoing HPV vaccination should be explored as it may improve access; Zambia can take a leaf from countries like Senegal .


HPV 16 was involved in 55.2% of high-grade lesions and in situ carcinoma and 76.3% of invasive carcinomas. In severe lesions, HPV 16 participation was predominant, whereas diverse genotypes were seen in low-grade lesions. Importantly, we observed that high-risk genotypes, for example HPV 52, can emerge for a few years then decrease even without vaccine pressure.Human papillomavirus is a predominant sexually transmitted viral pathogen. Our objective was to analyze the relative distribution of genotypes over time and to determine the genotypes associated with adverse clinical lesions. The study was based on data from adult women with cytological abnormalities from whom histological samples were obtained from 2005 to 2021. HPV genotyping was performed using PCR and INNO-LiPA assay (Fujirebio). Among the 1,017 HPV-positive biopsies, 732 (72%) were infected with a single HPV genotype and 285 (28%) were infected with several HPV genotypes. Most of the infections involved the high-risk genotypes 16, 31, and 52. Throughout the study period, HPV 16 was the most encountered genotype , while HPV 18 was rather under-represented , especially in invasive cervical carcinoma. HVP52  was detected mainly from 2008 to 2014, and its distribution reached 19.7% in 2011. Such epidemiological data underlines the possibility of an emergence of a high-risk genotype. The most detected low-risk HPV in combination with high-risk HPV was HPV 54 in 6.5% of samples. Monoinfection by HPV 16 led statistically more often to severe lesions than multi-infection involving HPV 16 ( Papillomaviridae family, is a pathogen that infects the epithelial tissue. It is the main risk factor for cervical cancer and is thus highly clinically relevant (The human papillomavirus (HPV), which belongs to the relevant . HPV is relevant , 3. The relevant , 5. Somerelevant , 7. Papirelevant . Persistrelevant .In 2018, there were nearly 3,000 new cases of invasive cervical cancer in France and more than 1,000 deaths . MoreoveA vaccination program was launched in France in 2007 with the quadrivalent Gardasil vaccine that targets HPV 6, 11, 16, and 18. In December 2019, a new vaccine, Gardasil 9, was implemented, targeting HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58. The low-risk (LR) genotypes, HPV 6 and 11, are responsible for 90% of condyloma acuminate cases. The HR genotypes targeted by the Gardasil 9 vaccine are involved in 80% of CIN2+ lesions and 90% of cervical cancers. Unfortunately, the poor perception of the HPV vaccine has hampered its implementation in France , 15, andThis retrospective cohort study was conducted at the Dijon University Hospital, France. The samples were collected for diagnostic purposes in the Medical Gynecology department, from September 2005 to December 2021. No additional samples were collected for the purpose of this study. Patient non-opposition was confirmed. All the women had cervical abnormalities, at least a pap smear with LSIL. Samples were mainly conizations, cervical, endocervical or endometrial biopsies, as well as some paraffin blocks from the pathological anatomy laboratory. All samples were placed in tubes with a transport medium. Briefly, biopsies were dilacerated with a scalpel, paraffin embedded samples were shaved to obtain slices, and all samples were incubated in a proteinase K solution at 56°C with stirring for at least 2 h up to 16 h maximum. Proteinase K was inactivated at 95°C for 10 min. At this stage, samples could be frozen at −20°C prior to analysis.in situ (AIS), and squamous cell carcinoma (SCC); and a third group included invasive cervical carcinoma (ICC).Classification of lesions was as follows: group 0, included samples close to normal; group 1, called low-grade lesions, included cervical intraepithelial neoplasia (CIN)1 and flat condyloma; group 2, named high-grade lesions group, included CIN2, CIN3, adenocarcinoma in vitro diagnostic use. It was designed for the identification of HPV genotypes by detection of specific sequences in the L1 region of the HPV amplification products that are subsequently hybridized using a single typing strip on which sequence-specific DNA probe lines and control lines are fixed. Classification of genotypes were indicated in the manufacturer’s instructions, according to Munoz et al.  that can determine the presence of the 28 genotypes (version EXTRA from 2005 to 2014) or 32 genotypes (version EXTRA II from 2015 to 2021). The INNO-LiPA HPV Genotyping is a line probe assay for z et al.  and IARCz et al. :HR means high-risk, LR means low-risk, and p, probable. The underlined genotypes were detected only in the EXTRA II version, while those in italic were no longer detected. When HPV 52 and a weak signal for HPV 31 were detected, only 52 was recorded seeing as non-specific activity was attributed to HPV 31, as indicated by the manufacturer.p <0.05 was considered statistically significant. STATA v15  was used for the statistical analyses.Continuous variables were expressed as means ± standard deviation (SD), categorical variables were expressed as percentages, and 95% confidence intervals (CI) were indicated. Categorical variables were compared using the Chi-square test and continuous variables were compared using one-way analysis test or Kruskal–Wallis test, as appropriate. A value of n = 1,017) were collected from female patients who were HPV-positive and analyzed using the INNO-LiPA hybridization test. Women were aged 18 to 92 . Samples were obtained in the cervix , endometrium , vagina , vulva , or anus . As expected for this cohort, most of the women had high-grade lesions. Women with an HR HPV infection represented 97.8% (96.7–98.6%) of patients (n = 995), while only 29 women 2.9% (1.9–4.1%) were found to have either only a LR (n = 22) or an indeterminate genotype (n = 7). Moreover, 10 of these 29 samples were extra-cervical.We analyzed the relative distribution of genotypes in histological specimens collected over a 16-year period, from 2005 to 2021. The samples  with a tendency to decrease over the years 2007–2016 when HPV 52 emerged  constituted 67.3% of the genotype cases .n = 285), women had an infection with 2 to 6 different HPV genotypes, dual infections were the most frequent , 61 samples included three different genotypes, 27 included four genotypes, 16 included five genotypes, and three samples had six genotypes, which led to the determination of 1,477 genotype results in total. HPV 16 was most frequently found as a single genotype (77.8% of HPV 16) whereas other HR types of HPV were often detected as coinfecting viruses ], including seven positive samples with indeterminate genotypes, and the remaining 285 samples were multi-infected [28% (25.3%–30.9%)]. When multi-infected . Of these, only eight involved an LR HPV as a coinfectant (9.5%). We then focused on preferential associations for prevalent genotypes . For thep < 0.05). HPV 16 was most frequent in women 26–45 years (p < 0.001). Although not statistically significant, the distribution of HPV 52, 31, and 33 also appeared to be higher from 26 to 45 years old (The mean age of the cohort was 38.9 years. The mean age of patients with monoinfection was 39.3 years while multi-infected women had a mean age of 37.9 years (ears old .p < 0.001)  of low-grade lesions were HPV-16-positive, 55.2% (51.9%–58.5%) of high-grade lesions and 76.3% (59.6%–88.6%) of ICC (< 0.001) . HPV 52 p < 001). Associations between age and any other genotypes could not be calculated because of the large diversity of genotypes in group 1 and the limited number of cases in group 3.We also explored the age of women with single-type HPV 16 infections in the different lesion classification groups. Women with HPV 16 in group 3 (mean 44.6 years old) were older than those in group 2 (mean 37.3 years old) while in group 1 (low-grade lesions), the mean age was 33.0 years old  to protect against HPV 31, 33, 45, 52, and 58 were added to these four genotypes to produce the updated Gardasil 9 vaccine. Gardasil 9 was implemented in December 2019 in France, when the women included in our study were too old to be eligible for vaccination. Therefore, the observed emergence and decline of HPV 52 cannot be attributed to any vaccine pressure against this genotype. It is worth noting, however, that vaccination may facilitate an increase in HR HPV types not targeted by the vaccine . Rather in situ and ICC , in accordance with previous reports (63%–65%) , 39, 40.Cervical cancer accounts for 83% of HPV-attributable cancer, two-thirds of which occur in less developed countries. HPV-DNA integration into cellular chromatin is a necessary event contributing to carcinogenesis. The persistence over time of a HR HPV infection , 48 and In conclusion, this study underscores the prevalence of HR HPV 16 over time in Burgundy in women undergoing biopsies or resections after abnormal colposcopies. The strong presence of HPV 16 has been consolidated over the years despite some vaccine pressure. In our population, which was not eligible for vaccination with Gardasil 9, we evidenced a spontaneous fluctuation of some genotypes over the 16 years. Such fluctuations could bias the results of vaccine policy evaluations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.J-BB, SD, and CM: study concept and design. SD, CA, J-BB, and CM: acquisition, analysis and interpretation of data. OC: laboratory detection. CM: writing. J-BB, SD, CA, and OC: critical revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Primary human papillomaviruses (HPV) cervical cancer screening can be strengthened by offering home-collection of biological specimen as a valuable option to increase screening coverage. As recommended by World Health Organization (WHO), screening programs should consider whether the inclusion of HPV self-sampling as a complementary option within their existing screening algorithms could address the gaps in current coverage. However, few HPV screening tests are validated for self-sampling according to international guidelines. This study aimed to test a self-sampling-based screening strategy, complementary to the main screening program based on clinician-collected cervical samples. The study took place in Trieste, Italy, and it aimed to evaluate the feasibility of self-testing at home under an opt-in system during COVID-19 pandemic in order to exploit self-sampling to reduce the screening delay generated by the lockdown.® 4800 HPV .500 women, who should have received the screening call in 2020, were asked, via phone call, to participate in the study. To whom agreed, a home-collection kit, including a vaginal dry swab for specimen collection, was sent. The recipients performed the sample self-collection and sent back the swab through traditional mail using a prepaid envelope. Once received by the hospital, the samples were analyzed with HPV Selfy , a CE-IVD HPV screening test specifically validated for self-collection. Results were further compared using cobas80% women sent back their swab, showing one of the highest return rate obtained in comparable studies. 34 HPV-positive women were followed up and underwent the Pap test, that revealed 8 low squamous intraepithelial lesions (LSIL) cases, later triaged to colposcopy. HPV Selfy was confirmed to be an adequate test for self-sampling-based screening.This study further confirmed the feasibility of self-test at home screening strategy based on self-sampling with an opt-in system as a support method to enhance cervical cancer screening coverage in Italy. Enrolled women showed a high appreciation for this approach. HPV Selfy test demonstrated to be a valuable assay for cervical cancer screening based on home self-collection.Trial registration: ASUGI Trieste n. 16008/2018 and amendment 02-11/09/2020.The online version contains supplementary material available at 10.1186/s40001-023-01263-8. Cervical cancer is the fourth most common female cancer worldwide with 604,237 new cases and 341,843 deaths in 2020 [The validation of human papillomavirus virus (HPV) molecular tests on self-collected samples offers the opportunity to adopt this collection method for primary screening purposes with the final aim to increase cervical cancer screening coverage. In particular, by means of self-sampling WHO aims to reach a global target of 70% screening coverage by 2030 . This imIn particular, it has been shown that offering to the screening population the possibility to perform a vaginal self-collection specimen at home significantly increases the response rate compared to traditional screening methods based on clinician-collected biological sampling –20.These aspects have been documented through a plethora of studies, reaching participation rates, intended as the percentage of women who returned back the self-collected sample, ranging from 10 to 64% , 21–27.Surveys evaluating women’s experiences with self-sampling showed that most non-attendees would prefer self-sampling to clinician-based sampling in the next screening round –30.Notwithstanding, in 2020, no assays were clinically validated for primary screening purpose specifically on self-collected samples according to the VALHUDES international guidelines .At the time of the present study, only two CE-IVD assays reported self-sampling in their intended use: (a) HPV Selfy, validated for self-collection since May 2019 on a large population of more than 1,000 women , 33; datHowever, the introduction of HPV self-test at home based on self-sampling, both as an opt-in system  or an opt-out system , could be a valuable and effective strategy to increase primary cervical cancer screening coverage. Indeed, different studies showed how self-sampling can increase the participation in cervical cancer screening both in developed and developing countries , 38–42. In addition, this could be of particular interest in the post-COVID-19 era, where self-sampling could represent a timely, accessible, safe and cost-effective method to efficiently screen women while keeping social distance –50. ImpoThis study was started as a first experimental application of HPV self-test at home based on self-sampling offered to screening population in Trieste  during the interruption of the primary screening due to COVID-19 pandemic. In order to guarantee trustable results, a CE-IVD marked test specifically validated for self-collected samples (HPV Selfy) was selected for this study , 33.We applied an opt-in system, since according to some studies, it is more cost-effective on average , and we The women, during the call, received by the midwives an exhaustive explanation of the initiative, with particular reference to the self-sampling process, sample shipment and to the management of the informed consent form included in the kit. Moreover, the women were provided with a customer care telephone contact dedicated to the project for any further need. The demographic and clinical data of each patient enrolled were collected in a proper database with a case-report form that was filled during the enrollment phone call. Pregnant women and women with current diagnosis of uterine, endometrial, vaginal, vulvar or ovarian cancers were not included in the study.® validated for self-collection  with manufacturer’s instructions of use, general instructions of the self-sampling procedure, the informed consent form to participate to the study, a satisfaction questionnaire containing three questions about their opinion on the study procedure, and a prepaid and pre-addressed envelope to return the sample and the documents by standard postal mailbox to the laboratory of Anatomia e Istologia Patologica, Cattinara Hospital, Trieste, Italy.The self-sampling kits were manufactured by Ulisse BioMed Fig.  and wereIf women did not sent samples back, one or two reminders by phone call would have occurred.As previously mentioned, for this study, a vaginal swab FLOQSwabs®  was selected as a self-sampling device. The FLOQSwab® is a dry, sterile, flocked swab contained in a tube used to store the sample after collection to transport to the laboratory. The swab shaft has a red mark to indicate where to hold the swab during collection following a pictorial self-collection guide collection guide. The FLOQSwabs® has been validated for both point of care and home self-collection for the detection of HPV.Once the kits were received at their homes, women performed vaginal sample self-collection using sterile dry flocked swab contained in the kit. According to the manufacturer’s instructions, after collection, the swabs are stored dry in their own plastic tube, placed in a plastic bag, packaged and mailed to the hospital together with filled and signed consent documents and a satisfaction questionnaire. Upon receiving the samples envelopes, the hospital laboratory staff registered the arrival date and time and status of the samples.To quantify the time between the sample self-collection and arrival to the laboratory, the signature date written in the consent form has been taken into consideration as “collection date”.Vaginal self-collected samples were analyzed by HPV Selfy assay coupled with Ulisse Faster DNA reagent according kit’s instructions for use , that allows to perform direct PCR analysis, thus skipping DNA extraction steps.To verify the reproducibility over different PCR instruments, the samples were tested twice with HPV Selfy, once with Light Cycler 480  and once with Quant Studio 5 Real-Time PCR  instruments.® 4800 HPV (Roche Molecular Systems) and by genotyping test EasyPGX® ready HPV test .A portion of all the samples were also analyzed by cobas® 4800 HPV, used in Trieste for primary cervical cancer screening purposes, and by Pap test. If an abnormal Pap test result was observed during the follow-up, colposcopy was executed.Negative test results were communicated by phone. In case of positive HPV test result, the midwives called the positive women and booked a follow-up visit aimed to perform a traditional clinician-collected cervical sampling successively analyzed by cobasHPV Selfy is a CE-IVD full genotyping Real-Time PCR-based HPV screening test capable to detect and perform single genotyping of 14 High-Risk HPV types , thanks to the Ulisse BioMed SAGITTA patented technology, that allows to perform precise genotyping by means of melting curve analysis within the same Real-Time PCR run. The test is at date validated for cervical cancer primary screening, not only on clinician-collected samples, according to Meijer’s international guidelines, but also on self-collected samples, according to the VALHUDES protocol , 33.Prior to perform the analysis with HPV Selfy, the dry swab biological samples were resuspended in 2 ml RNAse- and DNAse-free sterile water, and 80 μl were pre-treated with Ulisse Faster DNA , a pre-treatment reagent that allows to skip DNA extraction and purification, in order to perform the so-called direct PCR, thus saving time and money. HPV Selfy test includes a human DNA amplification control (Hemoglobin subunit beta) to evaluate biological sample quality, thereby reducing the risk of false-negative results . Analysi® 4800 HPV is a CE-IVD and FDA-approved fully automated HPV screening test based on Real-Time PCR amplification for the detection of 14 High-Risk HPV types  and partial genotyping of HPV16 and HPV18; the assay is validated for clinician-collected cervical brush samples stored in ThinPrep PreservCyt Solution .CobasSamples were also analyzed with EasyPGX HPV kit , for the detection and genotyping of 14 High-Risk HPV types  through the Real-Time PCR amplification of the oncogenes E6 and E7. For EasyPGX analysis, DNA extraction of the samples was performed using the MagCore HF16 instrument , an automated nucleic acid extractor.Cervical smear slides were Pap-stained, and histo-technicians interpreted the results following the Bethesda 2001 classification .Simple questionnaire items assessed participants’ attitudes regarding the offered screening procedure. Attitudes toward the self-collection experience were assessed by evaluating the process with three questions through a satisfaction scale from 1 (very low) to 10 (very high). Questions were adapted from the questionnaire of the VALHUDES study . An openP values of < 0.05 were taken as statistically significant. All statistical analyses were performed using the Statistical Software R .The agreement between the collection methods were calculated using concordance and discordance rates and the Cohen’s kappa statistic. The kappa statistic was calculated to determine the level of chance-agreement between two methods with a kappa value of 0 indicating no agreement better than chance, a value of 1 indicating perfect agreement better than chance, and intermediate values of 0.00–0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80 and > 0.81 indicating poor, fair, moderate, substantial and excellent agreement, respectively . The datOut of 1500 women aged 31–66 contacted by midwives by phone, 500 agreed to participate in the study and were enrolled. Over the 500 swabs sent to the enrolled women, 400 swabs returned to the laboratory (400/500 = 80%) . 2 women declared that they sent their samples back despite they were never received by the laboratory (2/100 = 2.0%).Additional data regarding the women whose swab was received by the laboratory are described in the Table Data regarding the lead time of the swabs are presented in Table We analyzed the 400 returned swabs with Quant Studio 5, following manufacturer’s instructions; 2 samples were invalid due to low or null control gene amplification (2/400 = 0.5%). Out of 398 valid samples, 364 samples were HPV-negative (364/398 = 91.4%), whereas 34 samples were HPV-positive with at least one type of High-Risk HPV (8.5%), being HPV prevalence similar to that reported in previous literature . We founTo evaluate the assay inter-device reproducibility, we performed a second run of the HPV Selfy test on another instrument, the Light Cycler PCR machine: 3 invalid samples were found (3/400 = 0.75%), of which two were the same invalid samples detected by Quant Studio 5. Therefore, 397 samples were considered for the comparison. Agreement between the paired results obtained over the two machines was 99.5% , demonstrating high grade of reproducibility of the assay , of which 1 was found invalid also by HPV Selfy performed on Quant Studio 5. cobas® 4800 HPV detected 372 negative cases (372/396 = 93.9%) and 24 HPV-positive cases (24/396 = 6%), 10 fewer than HPV Selfy.The 400 swabs were also analyzed by cobas® 4800 HPV results, we considered 395 samples that were valid for both assays, showing a total agreement of 95.9%  , the clinician-collected cervical specimen were retested with EasyPGX® ready HPV test, according to its intended use, and 6 out of 9 retested samples resulted HPV-positive confirming the result previously obtained with HPV Selfy on self-samples .Consequently, the number of discordant samples between cobas® 4800 HPV assay on clinician-collected samples.On the same 33 samples we performed also cytology analysis through Pap test: we found 8 Low-grade Squamous Intraepithelial Lesions (LSIL) cases , all detected as HPV-positive with HPV Selfy on self-collected samples as well as with cobas® 4800 HPV assay performed on self-collected samples, out of its intended use, thus further confirming the importance to use tests clinically validated on self-collected samples in order to perform screening self-test at home  were negative with cobas% were neAll the LSIL-positive women were subjected to colposcopy obtained a negative result, thus a 1-year follow-up was scheduled for them..Enclosed within the kit, a survey form with three simple questions regarding the screening project based on self-collection was administered to the participants. To each question, the women could provide a satisfaction indicator, ranging from very low (1) to very high (10). Overall women appreciated the project proposal very much, with 9.01 average Fig. . The ans2HPV testing on home self-collected vaginal specimens is an effective primary cervical cancer screening method, valuable both to recruit non-responders and to screen the entire relevant screening population. HPV screening based on self-collection of dry swabs indeed could be advantageous since (i) the self-collection-based screening does not require specialized personnel for the collection step, thus simplifying the organization and lowering the costs; (ii) it could reach women who do access the gynecological visit by cultural, religious, psychological or other social barriers; (iii) it ensures to achieve equivalent performance compared to cervical collection, if the HPV test method is specifically validated for this purpose. In addition, recent studies showed that more extensive self-collection in the anogenital area  could even allow detection of HPV reservoirs that could have an impact on cervical and not-cervical HPV-cancer development –58.We showed that HPV Selfy, a test validated for primary screening purposes according to Meijer’s guidelines and specifically validated also on self-collected samples according to VALHUDES protocol , 33, has® 4800 HPV assay), possibly because HPV Selfy assay was specifically optimized for self-collected samples and had this claim in its intended use, while Cobas had not [Moreover, HPV Selfy generated a lower number of inadequate samples  up to very high return rate , show that there is not a significant difference among invitation methods, although it has to be underlined that the studies were different in the setup , in the sampling devices , in the sociodemographic and economical background of the investigated population and, most importantly, in the communication strategy  , 61. A rA possible bias of this result could be due to the fact that we selected women who had at least a screening test in their life (so we focused on a more responsive population by excluding the non-attendee’s population), however, it has been previously shown that also screening attendees, expressed their preference for self-sampling compared to clinician-based sampling , alike mOther possible factors leading to this result include the fact that we provided a user-friendly home-collection kit, with clear instructions for use, and the entire study was done with a simple returning method . Finally, we also provided a dedicated customer care telephone contact to the women, to provide them any required assistance.Next step will be to enlarge and further validate the established opt-in protocol also to non-responders’ population, aiming to increase the screening participation even among those hard-to-reach women.In this clinical study performed on 500 enrolled women, we verified that HPV Selfy test is a suitable assay for primary cervical cancer screening programs based on self-collection performed at home. Thus, we confirmed that the clinical performance of HPV Selfy executed on self-collected vaginal samples is higher than the performance, on the same self-collected samples, of assays that do not have self-collection in their intended use, therefore, confirming that the HPV Selfy test is suitable to be used in primary cervical cancer screening programs based on self-sampling.We also demonstrated that the opt-in system we set up was effective and provided a very high return rate, confirming that this system could be used to increase non-responders’ participation rate to screening, and that by the time could be used as the main primary cervical cancer screening collection method.Additional file 1: Table S1. Intralaboratory reproducibility of HPV Selfy using two different Real Time PCR machines. HPV Selfy assay was performed twice on 397 samples using two different Real Time PCR instruments: Quant Studio 5, indicated by the manufacturer’s protocol, and Light Cycler. Overall concordance observed was 99.5% .


Polyomaviruses have both structural and functional similarities with papillomaviruses. Accordingly, their role in human papillomavirus (HPV) associated malignancies has been studied with conflicting results. Our goal was to disclose any association between BK (BKPyV) and/or JC (JCPyV) polyomavirus serology and HPV data derived from Finnish women (327) in a 6-year prospective follow-up.Glutathione S-transferase fusion-protein-capture (ELISA) in combination with fluorescent bead technology was used to analyze antibodies to BKPyV and JCPyV. In the longitudinal setting, BKPyV or JCPyV serostatus was related to i) oral- and ii) genital low (LR)- and high risk (HR) HPV DNA detection, iii) HPV16 persistence at both these sites, iv) results of the Pap (Papanicolaou) smear taken at baseline, and v) development of incident CIN  during the follow-up.Being BKPyV or JCPyV seropositive was not significantly associated with HPV seropositivity to either LR- or HR-genotypes, genital- or oral HPV DNA positivity, persistence of genital- or oral HPV16 infection, grade of Pap smear, or development of incident CIN.Thus, the present study could not provide any confirmation to the concept that co-infections by HPyV and HPV have interactions that impact on the clinical manifestations or outcomes of HPV infections either in the genital tract or in the oral mucosa. HPyVs are non-enveloped DNA viruses with circular double-stranded genome varying from 5100 to 5400 base pairs . CurrentAlso HPVs are small, non-enveloped double-stranded DNA viruses of which currently more than 200 genotypes have been identified. Based on their different risk to induce precancer lesions and cancers, HPV genotypes are further categorized as low- (LR) or high-risk (HR) HPV genotypes . Both HPin vitro studies on the mutual interactions between polyoma- and papillomaviruses are scanty. However, some clues on the possible interaction between these two viruses are provided by i) the older in vitro studies on SV-40 performed with HPV-positive cell lines, e.g. HeLa (HPV18-positive) and Caski (HPV16-positive) or ii) Cos-1 cell line, the latter containing one copy of SV40. Of particular interest is the study by Wildeman and coworkers (1989) on large T-antigen, which is required to start viral replication (It has been suggested that during their simultaneous infection (either at same location or different), polyomaviruses could potentiate the transforming properties of HPVs . Howeverlication . The autlication .Until now, the simultaneous presence of both BKPyV or JCPyV and HPV has been analyzed in genital, colorectal, head and neck, or lung lesions . The resThe Finnish Family HPV Study has been ongoing since 1998 in Turku University, Finland, focusing on HPV dynamics related to oral and genital infection and immunology in a longitudinal study setting within regular Finnish families . In the In the present study, we analyzed the association between BKPyV and/or JCPyV serostatus (positive/negative) and several key HPV-related covariates in women participants from the longitudinal Finnish Family HPV Study. These HPV-covariates of interest include HPV serology and DNA status for both LR- and HR-HPV types, persistence of HPV16 infection in oral- and genital mucosa, Pap smear abnormalities, and incident CIN during the six-year follow-up.22.1In total, 327 of the 329 women enrolled originally to the Finnish Family HPV Study, were eligible in this study. The participants were pregnant in their third trimester when enrolled at the Maternity Unit of the Turku University Hospital, Turku, Finland . The par2.22.2.1Blood samples from these mothers were collected at baseline (n=327) and at 12(n=278)-, 24(n=220)-, and 36(n=261) months - visits as described earlier . The sam2.2.2A routine Pap (Papanicolaou) smear was taken at the baseline visit and at 12-month, 24-month, 36-month, and 6-year follow-up visits with the conventional 3-sample technique with wooden spatulas and a brush  as described earlier . The sli2.2.3Scrapings for HPV DNA testing were taken from the cervical and oral mucosa by using a brush  as described earlier . Oral br2.2.4The samples were analysed for antibodies to VP1 of BKPyV and JCPyV. The antibody-detection method was based on glutathione S-transferase fusion-protein (GST)-capture ELISA  in combi2.2.5The samples were analyzed for antibodies to the major capsid protein L1 of LR-HPV types 6, 11 and HR-HPV types 16, 18, and 45 by multiplex HPV serology based also on glutathione S-transferase fusion-protein capture on fluorescent beads . Sera we2.2.6®  which detects 24 LR- and HR-HPV-genotypes as follows: LR-HPV6, 11, 42, 43, 44, and 70; and HR-HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 as described earlier  were first identified as described earlier . Among t2.2.8Following two cervical smears taken at 6–12 month intervals containing abnormal atypical squamous cells of undetermined significance or worse (ASCUS+), women were referred for colposcopy and biopsies to confirm the cytological findings as described earlier . All bio2.3All statistical analyses were run by using the SPSS for Windows  software package. Conventional 2x2 tables were used to analyze categorical variables, tested using the likelihood-ratio test or Fisher’s exact. Differences in the means of continuous variables were analyzed by ANOVA or Mann–Whitney/Kruskal–Wallis test for two and multiple independent samples, respectively. Univariate logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (CI) between two binary variables. All statistical tests performed were two-sided and declared to be significant when p<0.05.3The median and mean IgG-antibody levels of BKPyV in serum of 327 pregnant women were 8640 MFI with a range from 72 to 20773 MFI and 8497 ± 416 MFI, respectively. The corresponding IgG-antibody levels of JCPyV were lower, median being 765 MFI with a range from 0 to 15007 MFI and mean ± SD 2340 ± 2901MFI. The mean IgG antibody levels of BKPyV were nearly four-fold higher than those of JCPyV. Also the standard deviation for BKPyV IgG antibodies is nearly seven times lower than that for JCPyV.3.1Of the HR-HPV seropositive women , 97.3% and 67.6% were also BKPyV- and JCPyV seropositive (MFI 400). The BKPyV and JCPyV seroprevalence (MFI 400) was practically identical among LR-HPV seropositive women (IgG-antibodies to HPV6 and/or HPV11 L1), 93.5% and 64.1%, respectively. Next we used the very stringent MFI values for BKPyV seropositivity (MFI 5000) and for JCPyV (MFI 2000) 3.2BKPyV and/or JCPyV serostatus was stratified by HR- and LR-HPV DNA status (at baseline) as well as HPV16 persistence in genital or oral mucosa 2. NearlNo statistically significant differences in BKPyV seroprevalence were observed among women with and without persistent genital- or oral HPV16-infection, and the same was true with JCPyV serostatus as well. No statistically significant differences between BKPyV and JCPyV serology and HPV DNA data were found even the stringent cut-off for BKPyV and JCPyV seropositivity was used Table 2.3.3Of the 327 women, 35 had cytological abnormalities  in their Pap smear taken at the entry to the study when pregnant. ASC-US and low SIL was detected in 33 and 21 BKPyV- and JCPyV-seropositive women, respectively. However, as nearly all women with normal cytology were also seropositive to BKPyV (95%) and 59% were JCPyV seropositive, the differences did not reach statistical significance (p=0.094). During the follow-up, 12 women developed an incident CIN. Similarly as found with cytology most of these women were seropositive to BKPyV and 58.7% were JCPyV seropositive Table 1.In assessing the risk estimates (OR) of being BKPyV or JCPyV seropositive, none of the HPV-related covariates proved to be significant predictors Table 3.4Papillomaviruses were originally grouped in the family Papovaviridae, but since 2002, papillomaviruses were designated as a distinct family, Papillomaviridae, in the 7th Report of the ICTV . BecauseSimultaneous presence of both BKPyV or JCPyV and HPV has been analyzed in genital, colorectal, head and neck, lung, and prostate lesions . AccordiContrary to our findings in the uterine cervix, JCPyV might be more common in head and neck carcinomas than BKPyV . JCPyV DIn the absence of longitudinal studies on this topic, our results are of particular interest. In this setting among healthy women, it was shown that being BKPyV and/or JCPyV seropositive was not associated with any of the HPV-related variables, including HPV-seropositivity to LR- or HR-HPVs, LR- or HR-HPV DNA detection, HPV16 persistence in the oral or genital mucosa, Pap smear abnormalities or incident CIN in the biopsy samples. When stratified by these HPV-related covariates, in practically all categories, over 90% of the women were BKPyV seropositive while seropositivity to JCPyV ranged from 52% to 68%, with no significant differences between strata.It is known that HPyVs, especially BKPyV and JCPyV are ubiquitous in populations. In healthy adult population, BKPyV seroprevalence is around 90% while that of JCPyV is lower, around 50-70% . In our Some previous studies have focused on etiological association between HPyVs and HPV in cancer patients. However, the results are mostly in line with those observed in the present study, showing no associations between these two virus groups even in cancer patients. Martinez-Fierro and coworkers analyzed the presence of DNA of BKPyV, JCPyV, and HPV in 130 subjects: 55 prostate cancer patients and 75 controls. HPV sequences were detected in 11 (20%) of the cases and 4 (5%) controls. BKPyV, JCPyV, and SV40 sequences were not detected in any of the samples examined . Llewellin vivo that the first HPV16 to come established the persistent infection but the next HPV16 with slight genomic changes was able to cause only a transient HPV infection  did not associate with CIN/CIS or cervical cancer . In contnfection . Thus, bIn vitro models are not available as there are neither BKPyV nor JCPyV positive epithelial cell lines and HPV can not be cultured. The ultimate strength of the study is that the study design is novel and innovative which in the best case scenario would encourage other research groups to study the current issue within their own cohorts and especially utilizing in situ hybridization methods to localize these viruses in same cells progressing toward malignancy.One limitation of our study is that HPyV DNA analysis was not performed in the samples used for HPV-genotyping . In addition, the number of these samples is low. In the clinical diagnostics, e.g. HPV serology has limited value and HPV seropositivity is usually regarded as a sign of past history of HPV infection. However, serological analysis is the only current method to study the possible association between HPV and HPyV types. In conclusion, BKPyV and/or JCPyV seropositivity had no significant associations with any of the HPV-related covariates included in the present analysis. Thus, the present study could not provide any additional support to the concept that co-infections by HPyV and HPV have interactions that affect the clinical manifestations or outcomes of HPV infections either in the genital tract or in the oral mucosa.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Research Ethics Committee of Turku University Hospital. The patients/participants provided their written informed consent to participate in this study.HL, TW, KS, SG, KL, and SS contributed to the study conception and SS created the study design. Material preparation and data collection were performed by SS and TW. The analysis was performed by HL, SS, and KS. The first draft of the manuscript was written by HL and SS. All authors commented on previous versions of the manuscript and read and approved the final manuscript. All authors contributed to the article and approved the submitted version.


Many of the United States’ more than 18 million veterans obtain healthcare through the Department of Veterans Affairs’ (VA) Veterans Health Administration system; however, recent legislative changes have expanded veterans’ access to non-VA care in their communities, particularly for those who do not live near VA medical centers. Veterans are seen by physicians in outpatient practice across the United States and are admitted to non-VA hospitals; this is particularly salient for older veterans, who may require a more frequent and high level of care. We present a review of characteristics of U.S. veterans from two conflicts: World War II (WWII) and the Korean War. While non-VA clinicians are well equipped to provide care for patients of all different ages, veterans of armed conflicts have a unique constellation of exposures and cultural considerations that must be accounted for when providing them care. In this review, we describe characteristics of the generations of American veterans who served in WWII and the Korean War conflicts in a brief historical context. We then note conflict-specific exposures and potential long-term sequelae to watch for during physical examinations and to monitor thereafter, age-specific health and emotional concerns, and best practices for providing care to this cohort of veterans. There were approximately 18.5 million veterans in the United States in 2021 .Veterans of armed conflicts often have experiences and exposures that are different from non-veterans and that impact their health and function in the long term. Non-VA primary care clinicians are well equipped to care for patients of all different ages, including older adults with significant comorbidities. However, veterans' unique exposures  and cultural norms that must be considered when providing care have an additive effect on their health and quality of life. While most veterans appreciate the specialized care that is available through the VA, they are often able to access care more conveniently with less travel at non-VA community facilities.Because of increases in non-VA healthcare utilization following enactment of the VA MISSION legislation , as wellWorld War II (1941-1945): the “GI” generationThere were an estimated 182,603 U.S. veterans of WWII as of 2021 . MembersWhile most WWII veterans are men, women were instrumental in war efforts. Millions took wartime jobs, and more than 350,000 served in the Pacific, European, and Asian theaters, representing 2% of uniformed personnel. Though women were excluded from combat, many cared for men who were injured in battle as nurses. Women were not immune to wartime suffering: more than 400 lost their lives during the war.Members of the GI generation tend to be team-oriented, patriotic, and attentive to rules .The Korean War (1950-1953): the silent generationAs of 2021, there were approximately 800,000 U.S. veterans of the Korean War .While a few “silent generation” (born 1925 to 1942) members fought in WWII, most were too young. Those who joined the military were much more likely to serve in the Korean War . These individuals faced numerous global adverse experiences in their childhoods, such as the Great Depression and WWII, in a context where “children should be seen and not heard” and authoritarian parenting approaches were dominant. Members of this generation often became “rebels without a cause,” starting adulthood as conformists who then began to challenge conventional culture. This generation produced America’s leading civil rights activists, its first “rock and roll stars,” antiwar leaders, and feminists.MethodsTo understand the needs of these special populations, we conducted a literature review using the National Library of Medicine’s PubMed database and an environmental scan of government materials  using Google searches. Our initial search identified 328 articles. We performed title and abstract review to determine which papers were relevant and reviewed PubMed’s “Similar Articles” and “Cited By” features of highly relevant papers, resulting in a set of 164 potentially relevant articles. We selected the 57 most salient resources for inclusion.Unique health risks for WWII and Korean war veteran populationsResearch has demonstrated that veterans of WWII and the Korean War have poorer self-rated health than non-veterans with similar sociodemographic characteristics . Though All veterans should be assessed for exposure- and conflict-specific risks. Below, we detail areas of exposures, conflict-related risks, and related-health conditions, along with recommendations about how to solicit and utilize this information.Physical TraumasThough many decades have passed since any veterans incurred battle wounds during these conflicts, the effect of combat injuries - lost limbs (and traumatic amputations), head and brain injuries, gunshot and shrapnel wounds, and wounds that limit range of motion, among others ,9 - can Dermatologic complaints were a primary cause for concern in the European theater, where 15%-45% of American sick call visits were for skin complaints . VeteranPractice recommendations: Ask the veteran about any war injuries that limit their functional abilities. Examples may include neuralgia, which can be well managed with medications such as gabapentin, or paresthesia/reduced sensation, which can raise the risk of falls, but can be countered with fall prevention measures. Perform a thorough evaluation of the skin, assessing for skin cancers and residual lesions from frostbite/cold injury. Assess for neurologic injury, focusing on symptoms such as bouts of pain in the extremities, and hot or cold and/or tingling sensations.Affected veterans may need physical therapy to improve physical symptoms and/or occupation therapy consults to help them establish a safer home environment and task completion skills.Psychological InjuriesPosttraumatic stress disorder (PTSD) develops after someone experiences or witnesses a life-threatening event. PTSD may present differently in older than younger veterans; the condition has been more extensively studied among veterans of the Vietnam War and later conflicts. Referred to as “combat fatigue,” “war neuroses,” or “shell shock” during WWII and the Korean War, PTSD was considered an unfortunate but unavoidable consequence of war. We thank the reviewer for this question and have amended the sentence to read: "Korean War veterans in particular are more likely to experience lifelong poorer mental health than veterans of other conflicts; this may be due to the greater prevalence of direct combat exposure or the closeness in time of this conflict to WWII, the largest conflict in human history” .Studies of Vietnam and subsequent conflicts have demonstrated that veterans with PTSD have a higher risk of suicide, and those with comorbid PTSD and depression have higher risk than those with either condition alone ,17. PTSDThere is some evidence that PTSD can be exacerbated among older veterans by traumatic and stressful life experiences ,26 and tVeterans who were prisoners of war have unique vulnerabilities ,29,30. TPractice recommendations: It is important to recognize when a veteran may be suffering from PTSD and refer them to mental health specialists who are trained in caring for patients with PTSD.Assess if any veteran is experiencing symptoms of PTSD, which include flashbacks, nightmares, and severe anxiety, as well as uncontrollable thoughts about the event(s). PTSD symptoms may emerge years later. PTSD is more common after certain traumas, such as combat and sexual assault. Ask about recent exposures that could trigger PTSD symptoms.Reassure veterans who are experiencing PTSD symptoms that there are many different treatment options available for PTSD, such as trauma-focused psychotherapy, including group and individual psychotherapy  -35, mindAssess veterans with PTSD for both depression and metabolic syndrome, as an association between the two has been demonstrated . MonitorExposure to Nuclear MaterialsBetween 1945 and 1962, the United States conducted atmospheric and underground nuclear tests in Pacific islands and in Nevada; service members during this time could be exposed before, during, and after such testing. Veterans were exposed to nuclear materials during the occupation of Hiroshima and Nagasaki, Japan  following the United States’ detonation of two nuclear weapons, as were Americans who were prisoners of war in Japan in areas close to the two cities and who participated in the cleanup of contaminated areas.The long-term impacts of radiation exposure are highly dependent on the dose received and its duration. While elevated rates of leukemia, solid cancers, and myelodysplastic syndromes (characterized by an increased risk of developing acute myeloid leukemia) have been observed in heavily exposed populations ,39, the The VA recognizes a specific set of conditions as potentially associated with radiation exposure during military service, such as all forms of leukemia, except chronic lymphocytic leukemia; cancer of the thyroid, breast, pharynx, esophagus, stomach, small intestine, pancreas, bile ducts, gall bladder, salivary gland, urinary tract, brain, bone, lung, colon, or ovary; bronchioloalveolar carcinoma; multiple myeloma; lymphomas, other than Hodgkin’s disease; and primary liver cancer, except if there are indications of cirrhosis or hepatitis B . RadiatiPractice recommendations: Ensure that patients have age-appropriate screenings for cancer, and ask about their radiation exposure and service history. Pay close attention to vision problems due to lens opacities, evaluate for cataracts if there are clinical concerns, and be mindful of the fact that veterans exposed to radiation can have an increased likelihood of thyroid problems and cardiovascular disease and consider screening them for thyroid and cardiovascular disease.WWII Mustard Gas ExposuresSulfur mustard  is a vesicant and blistering agent used during World War I as a chemical weapon ,45. DuriMustard gas causes blistering to the skin, eyes, and lungs between 2 and 24 hours after contact . ExtensiPractice recommendations: Assess veterans with this exposure (and others) for chronic respiratory disease and lung scarring. Consider screening for exposure-related cancers such as cancer of the lungs, nasopharynx, or larynx, or skin.NoiseHearing loss and tinnitus due to occupational noise damage/injury are prevalent among older adults and veterans, who were often exposed to loud noise during operations, training, and combat . HearingPractice recommendations: Evaluate the hearing of older adults and consider additional supports (such as using secure text messaging or resources such as TTY-based Telecommunications Relay Services) to establish and maintain connections with patients with hearing impairments. Additionally, make sure that their physical address is current; sensory deficits can make it more difficult to reach these individuals by telephone, and they may be more likely to miss appointments and follow-up.Because individuals in this age group are more likely than younger people to have hearing impairments (whether service-related or not), it is important to minimize background noise and to always face the patient and speak clearly. The same is true for visual impairments; therefore, ensure that materials are available in large print versions as well.Other Occupational Health ExposuresBoth combat and non-combatant veterans may have been exposed to numerous dangerous substances . IllnessPractice recommendations: Take a thorough occupational exposure history, especially for veterans who served in the above-mentioned professions. Asbestos-related diseases, such as shortness of breath, coughing, and chest pain, often do not appear until 20 to 50 years post-exposure.Infectious DiseasesDespite the discovery of sulfa drugs and effective antibiotics before and during WWII, many American veterans suffered from infectious diseases. Nearly one million cases of tropical infections, mainly intestinal protozoa (mostly Entamoeba histolytica), helminths (hookworm infection), or malaria, were diagnosed during or shortly after WWII in U.S. troops, especially in the Pacific theater . DysenteAt least 4,000 U.S. troops were infected with Plasmodium vivax during the Korean War, and many others were infected with Plasmodium falciparum ,52. ManyPractice recommendations: Veterans of the WWII Pacific theater and the Korean conflict may have experienced infections by capsulated organisms due to splenectomies. Patients without spleens may be more likely to experience overwhelming infections and greater mortality than individuals with functioning spleens. Keep this vulnerability in mind when patients have any signs of infection, particularly if they are unvaccinated for capsulated organisms.Back to Today’s Encounter: Integrating Exposure Histories and Age-Specific Health and Emotional ConcernsAll WWII veterans and most Korean War veterans are now age 80 or older ,6, and mVeterans in this age group are generally proud of their service to our country and enjoy talking about their years in the armed forces . Asking During this stage of life, many may suffer from isolation as a result of loss of family members and friends, which has been associated with poorer overall health ,57. EvalPractice recommendations: Track changes in address/residence and confirm that you can reach these patients after the encounter, as (1) frequent visits for ongoing concerns may necessitate close follow-up and (2) members of these populations may move into residential care or with family members for assistance. Many veterans in this age group tire easily; when providers need to instruct them on treatment or prognoses, it is best to teach while the veteran is at peak energy and to communicate information in short segments. Avoid rushing and allow additional time for making decisions and responding to questions.Every healthcare provider outside of the VA Healthcare system will likely eventually encounter a veteran patient, and it is important for physicians to be familiar with the challenges that veterans may face due to their service history and exposures. The service of American veterans of WWII and the Korean War should be recognized and honored, and their health and wellness protected as they proceed through their golden years.