Abnormal vessel patterns are specific signs in patients with early cervical abnormality and cervical cancer(CC) by colposcopy, but the impact of human papillomavirus (HPV) infections on abnormal vessel patterns remains unknown.A total of 6716 female patients with HPV infections or cytological abnormalities who underwent a colposcopy following abnormal CC screening results were included in the study. The final pathological diagnosis was confirmed to be the most severe pathological grade across cervical biopsy, endocervical canal curettage (ECC) and conization. Univariate and multivariate logistic regression analyses were used to investigate the association between HPV infections and abnormal vessel patterns, adjusting for age, gravidity and parity.There were 6124 normal vascular cases by colposcopy and 592 cases with cervical vascular abnormality. The prevalence of HPV infections was 4284 (70%) in normal patients, and the prevalence of HPV infections was 479 (80%) in cervical vascular abnormality patients. HPV high-risk type 16 infection alone increased the risk of cervical heteromorphic blood vessels . HPV 16 and 33 alone (other than the commonly recognized subtype of 18) or coinfection of these two genotypes could increase the risk of cervical punctate vascular and cervical vascular mosaic features and abnormal cervical blood vessels. An increased risk of abnormal cervical lesions was observed for HPV 16 and 33 alone or combined in coinfection compared to the negative group. The risk of cervical vascular abnormality was increased 10-fold by coinfection with HPV 16 and 33 . HPV 16, 33 alone or combined in coinfection were associated with an increased risk of lesions more advanced than high-grade squamous intraepithelial lesion (HSIL) when compared to the negative group. The risk of lesions more advanced than HSIL was up to 26-fold higher in the coinfection with HPV 16 and 33 group than in the negative group .HPV16 and 33 are the most dangerous HPV genotypes correlated with abnormal vascular patterns. Combined HPV16 and HPV33 infection increases the risk of abnormal vascular patterns. Combined HPV16 and HPV33 infection increases the risk of developing HSIL+. HPV iEarly-stage CC or high-grade CIN is always curable. However, recurrent or metastatic CC with the feature of tumour angiogenesis has a relatively poor prognosis with a 5-year survival rate of less than 20% . PersistThe impact of different genotypes of HPV infections on cervical vascular lesions in different races and regions remains unclear. There is currently no consensus on the topic of HPV infections in cervical angiogenesis and CC. Importantly, it is urgent to study the relationship between the subtype of HR-HPV and cervical vascular lesions based on large real-world sample data. We can determine the type of HPV infection in patients through HPV testing. We found that changes in cervical vascular morphology can be observed in colposcopy, and these changes were correlated with CIN and CC. Therefore, we intend to study the relationship between HPV infection and cervical blood vessel features in a large sample from southeastern China to promote progress in CC prevention, diagnosis and treatment.22.1in situ (AIS), squamous cell carcinoma (SCC) and adenocarcinoma. In all cases, the time between CC screening and histological examinations was less than 3 months. The inclusion and exclusion criteria are detailed in the flowchart  and/or HPV genotyping. In accordance with the system in the 2001 Bethesda system, the following TCT results were defined as abnormal conditions: atypical squamous cells of unknown significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), HSIL, atypical glandular cells (AGC), endocervical adenocarcinoma lowchart Figure 1The study was performed according to the Declaration of Helsinki (as revised in 2013) after approval was granted by the Ethics Committee of Fujian Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University (2022YJ002). The study was conducted without informed consent because it was a retrospective study.2.2PCR-RDB HPV genotyping (Yaneng Biotech) was used to distinguish 18 types of HR-HPV  and 5 types of low-risk HPV (LR-HPV) .2.2All patients in this study were referred for colposcopy and underwent colposcopy cervical biopsy according to the indication of the ASCCP guidelines. ECC should be carried out simultaneously when patients have the following conditions: HPV16/18 infections, AGC/AIS/HSIL cytology, and type 3 cervical transformation zone. If the results of liquid-based cytology were HSIL, AGC-FN , or AIS, or if cervical pathological biopsy/ECC results indicated CIN2-3 , cervical cone resection was also performed. Pathological evaluation of cervical biopsies/ECC and conization tissues were independently analysed by two blinded senior pathologists. The most advanced lesions among the cervical biopsies, ECC and conization tissues were taken as the final pathological diagnosis. According to the 2014 WHO classification of tumours of the female reproductive organs (4th Edition) , the his2.3in situ (CIS) with either well-differentiated, moderately differentiated or poorly differentiated squamous cell carcinoma or other variants, if applicable.Cervical vascular abnormality referred to lesions that were demonstrated as punctate vascular, cervical vascular mosaic, cervical heteromorphic blood vessel Figure 22.4We converted all of the numerical variables into categorical variables before analysis. Gravidity and parity were divided into two groups: less than three and greater than or equal to three. The categorical variables were expressed as frequencies (percentages) for description and analysis by SPSS 23.0 . The associations between age, cervical histopathology, HPV genotype, gravidity, parity and HPV infection patterns were analysed by using the chi-squared test. Univariate and multivariate logistic regression analyses were performed to examine the relationship between multiple HPV infections and cervical lesions, adjusting for age, HPV genotype, and pregnancy. Two-tailed P values less than 0.05.33.1A total of 6716 female patients with a definite pathological diagnosis were finally included in the analysis. Among them, there were 6124 cases with cervical vascular normality and 592 cases with cervical vascular abnormality. The mean age in 6124 normal cervical vascular patients was significantly higher than that in cervical vascular abnormality patients . The prevalence of HPV infections was 4284 (70%) in normal cervical vascular patients, while the prevalence of HPV infections was 479 (80%) in cervical vascular abnormality patients. The prevalence of high-grade lesions was 666 (10.9%) in normal patients, while the prevalence of high-grade lesions was 249 (42.1%) in patients with cervical vascular abnormality (P<0.001). The prevalence of cancer was 56(0.9%) in normal cervical vascular patients, while the prevalence of cancer was 72 (12.1%) in patients with cervical vascular abnormality. The prevalence of cervical vascular abnormality across different ages, gravidity, parity, HPV genotype, and histology is shown in 3.2The relationships between different HPV genotypes and punctate vascular features are shown in 3.3As shown in 3.43.5The correlations between different HPV genotypes and abnormal cervical blood vessel features are shown in 3.6The univariate and multivariate logistic regression analyses of multiple HPV infections and abnormal cervical lesions are shown in 3.7The univariate and multivariate logistic regression analyses of multiple HPV infections and abnormal lesions above HSIL are shown in 3.8To control for multipie tests, we additional use the Benjamini-Hochberg procedure for controlling the false discovery rate. Although p-values of HPV 52+, HPV 53+, HPV 81+ and HPV 43+ became non-statistically significant, for our primary focus on HPV 16+ or HPV 33+ results, the adjusted p-values remained statistically significant (Data not shown).In addition, the relationship between cervical vascular lesions and cervical lesion grades was shown in 4Metastasis and recurrence in patients with angiogenesis-associated CC carry a poor prognosis. Persistent HR-HPV infection leads to angiogenesis; however, how to determine the clinical meaning of local cervical vascular abnormality by colposcopy and its association with HR-HPV remain unknown. The present study assessed the prevalence of the subtype or coinfection of HPV and its correlation with cervical vascular abnormality within a retrospective cross-sectional cohort of 6716 outpatients in Fujian Province in southeastern China. The most prevalent genotype correlated with cervical vascular abnormality was HPV16, followed by HPV33, other than the commonly recognized subtype of 18. Moreover, HPV16 and HPV33 will increase the danger of cervical vascular abnormality and increase the danger of HSIL. Although other types of HPV coinfection also increased the risk of HSIL+, there were not as high as the HPV16 or HPV33 infection. While few studies have found increased risks with coinfection, we found that HPV33 infection increases the risk of cervical lesions above HSIL by 8 times. This is the first study to demonstrate the role of the nine-valent vaccine in preventing cervical lesions caused by HPV33.Colposcopy is a key instrument for early recognition of cervical lesions, especially vascular abnormalities in the cervix. HR-HPV is the definitive cause of high-grade CIN and CC. However, very few studies have focused on vascular abnormality and local HPV infection. In the 6716 patients included in the analysis, the prevalence of HPV infections was 70% (4284/6716) in normal vascular patients and 80% (479/6716) in patients with cervical vascular abnormality. To the best of our knowledge, the results provide initial direct data on local HPV infections and vascular abnormality by colposcopy. Furthermore, colposcopic vascular abnormality predicted higher cervical lesions, which was consistent with other studies  may affect the type distribution of HPV infections in the long run (20 years) . This phIn the previous study, female patients who underwent colposcopies/conization following abnormal CC screening results were included, and it’s found that multiple HR-HPV infections do not increase the risk of HSIL+, compared to single HR-HPV infection . The preThe findings are useful for developing HPV vaccination strategies and assessing future HPV vaccine impacts. According to this study, HPV16 and 33 were the most dangerous genotypes that introduced cervical vascular abnormalities. In addition, HPV 16 and HPV33 co-infection may cause more severe cervical vascular lesions than HPV 18. While the 9-valent HPV vaccine is the only currently approved vaccine offering protection against HPV33 in China. Therefore, the 9-valent HPV vaccine could provide the most effective protection against cervical vascular abnormalities.The major limitation of this study was its retrospective design. It was also limited in that the data collected were from a single institution. The study was a cross-sectional study performed in a single centre and could be affected by selection bias. Moreover, residual confounding, including the number of sexual partners, duration of HPV infection and vaginal microbiota, may continue to influence the result after adjustment for potential confounders. Thus, further large-scale and multicentre prospective cohort studies should be conducted to confirm the results. At last, this study is a clinical study, which the internal mechanism has not been further explored, and we will be further explored in the future.5HPV16 and HPV33 are the most dangerous HPV genotypes correlated with abnormal vascular patterns. Combined HPV16 and HPV33 infection increases the risk of abnormal vascular patterns. Combined HPV16 and HPV33 infection increases the risk of developing HSIL+.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of Fujian Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University (2022YJ002).YZ and HL were involved in the design of the study. ZL, XL, QY, YS, YC, and JC carried out data collection and analysis. YZ and HL assisted in drafting the manuscript. YS, HY, and XZ revised the manuscript. All authors contributed to the article and approved the submitted version.


The HPV vaccine is used as one of the main prevention tools for HPV-related cancers globally, yet it is not part of the Indian National Immunization program. In light of the introduction of the indigenous vaccine, we examine the effectiveness of health education about uptake, acceptance, and awareness.Research was performed in the following databases: PubMed, CINAHL, Scopus, and Embase to identify studies between 2008 and 2022. Studies were included if: they were conducted in India including primary data research and health education intervention, and participants were between 9 and 29 years old.Out of the 10.952 results, 7 studies were included. Four studies focused on adolescent girls, aged from 9 to 20 years old, and 3 on university students aged from 17 to 26 years. Five studies were implemented in urban areas and 2 in rural areas. Health education interventions proved to be effective in increasing uptake, awareness, and acceptance of the HPV vaccine. The barriers included among others: cost, lack of awareness, and cultural barriers.Observations from this study outline immediate action for policymakers to educate and encourage the young population toward HPV vaccination. Future programs should be aimed at different population groups and be adjusted according to their special characteristics and needs. Attention should be given to the male population and marginalized groups. The involvement of various stakeholders proved to be beneficial, and it is highly recommended. Infection with the Human Papillomavirus (HPV) is the most common sexually transmitted infection (STI) of the reproductive tract with the majority of the sexually active population estimated to acquire the virus at some point in their lifetime , 2. The Globally, it is estimated that 570,000 cancer cases in women and 60,000 cases in men per year are related to HPV. The majority of them occurring in low and middle income countries. More specifically, 275,429 and 29,324, almost half of the global HPV-related cancer cases, occurin Asia in women and men respectively . MoreoveNumerous efforts have been undertaken in India to control cervical cancer. In 1975 the government of India launched the first National Cancer Control Program aiming to provide cytology-based exams for women in premier cancer institutions . In 2010However, the development of HPV vaccines has offered new opportunities for early prevention. Approximately 80% of cancers caused by HPV can be prevented by administering the HPV vaccine . ThroughNevertheless, the landscape of availability and acceptance of the HPV vaccination in India is promising. Since 2016, cost-effective vaccination programs against HPV have been successfully implemented in different states, achieving high coverage . In JulyThe role of health education appears central to the successful implementation of the HPV vaccination strategy, as shown by the problems due to misingormationthat arose during the first rollout of a vaccination program. Health education has been widely used to increase health literacy, especially among young people, helping them to adopt healthy habits in a variety of fields , 26. HeaTo date, there is ample questionnaire-based evidence assessing the knowledge and the acceptance of the HPV vaccine among the youth in India. However, the existing research is solely based on extracting information regarding these sectors without applying any intervention to improve them. Moreover, the lack of systematic review on the impact of health education regarding uptake, knowledge, acceptance, and willingness to get vaccinated represents a significant gap of knowledge –33. NotaIn this systematic review, we examine the effectiveness of health education among people from 9 years old, which is the minimum age to receive the HPV vaccination. Our upper limit is 29 years old as, 90% of the Indian population has their first sexual intercourse before the age of 30. The current vaccine guidelines suggest that the first dose should be administrated before the first sexual contact .In June via discussion and with the aid of a third reviewer who has expertise in the sexual and reproductive health field. Qualitative, quantitative, and mixed methods studies reporting primary data in English language published in peer-reviewed journals or grey literature between 2008 and 13th July 2022 were included. Studies were excluded if: they reported primary data from non-Indian residents, the age group was outside the age limits of 9–29 years, and if the participants have already been diagnosed with an HPV attributable cancer.Two researchers independently screened the results (titles and abstracts) resulting from the electronic searches and evaluated the full-text articles. Discrepancies were solved The PRISMA flowchart  summarizSeven eligible studies based on the inclusion and exclusion criteria, with the earliest reported in 2013, were included in our systematic review . Four stDifferent types of health education interventions were reported yet some of the studies did not feature analytical descriptions of the methods , 4. The Out of 7 studies, the participants had the opportunity to receive the HPV vaccine following the health education intervention in only 3 studies. In the state of Sikkim and rural West Bengal, the uptake among adolescent girls was reported to be above 95%. In a study implemented in a college in Bhubaneswar although 86% of the girls agreed to receive the vaccine, only 58.33% received their first dose. In 3 studies it was reported that some of the participants had already taken the vaccine, but the percentage was low  , 37, 41.Four studies examined the awareness of the participants regarding HPV, cervical cancer, HPV vaccine, screening methods, and symptoms of cervical cancer two of which reported poor awareness , 38–40. Similarly, all the studies that reflected on the knowledge of the participants after the intervention reported an improvement in the participant's knowledge , 38, 41.Four studies examined the acceptance and willingness to get vaccinated. Although acceptance and willingness appear distinct terms they were similarly used in the included studies , 40, 41.The reasons underlying the HPV vaccination hesitancy were manifold . The repThis review systematically collected reports from primary studies done in India on the effectiveness of health education interventions in improving the uptake, awareness, and acceptance of HPV vaccination among people aged 9–29 years and provides an evidence base for further research studies and policy. Our study's results highlight the effectiveness of health education in improving all the sectors that have been studied. Poor awareness was reported to be common prior to interventions, especially among the general population. In 3 studies where the participants were provided the opportunity for vaccination, the uptake was between 58,33% and more than 95%. Overall, female participants demonstrated greater acceptance and awareness compared to men. A variety of health education methods were identified. Additionally, our study provides information on the underlying causes of HPV vaccine hesitancy with cost and lack of awareness being the major reasons among others. Our results align with the results of the systematic review conducted by Thulaseedharan et al. in 2019 . ThulaseThe authorization of the indigenous HPV vaccine to enter the market in July paves the way for achieving the Cervical Cancer Elimination goal of 90% vaccination of girls up to 15 years old by 2030 . FurtherFurthermore, policymakers could consider best practices from successful vaccination efforts implemented in India and overseas. Our research highlights the successful approach of the state of Sikkim where more than 95% of the target population received both doses . The sucOur research identified a lack of HPV vaccine health education-related studies conducted in neighbouring countries such as Sri Lanka, Nepal, and Bangladesh which highlights the need for global evidence-based strategies in South Asia . HoweverConsidering the findings of our study in which the main population group constists of students, HPV vaccine information is essential to be part of the curriculum of health education in schools. Moreover, since awareness appeared to be inadequate among university students health education and opportunistic vaccination could be part of the university's curriculum by organizing workshops, thematic days, etc . HoweverThe HPV vaccination strategies could be integrated into already existing screening efforts for cervical cancer prevention in India. Despite the existing guidelines, screening programs are mostly based on opportunistic screening or after the appearance of symptoms . The curTherefore, to achieve the goals of cervical cancer elimination strategy for 90% HPV vaccination coverage and 70% screening by 2030 there is an urgent need for amelioration and a combination of existing strategies . Future In rural areas, where cancer prevalence is alarming due to a variety of factors such as illiteracy, poor hygiene, and early marriage the role of Accredited Social Health Activist (ASHA) workers could be expanded by adding HPV vaccination counselling . ASHA woLastly, efforts out to be made to increase the vaccination rates among girls who do not attend school. In 2021, Holroyd et al. published research on designing a pro-equity HPV vaccine delivery program for girls who dropped out of school in a community set-up in India . Their fThe main limitation of our study lies in the inclusion of a diverse body of literature including qualitative, quantitative and mixed-methods studies. There was therefore no scope for the conduction of a meta-analysis on the present data due to the lack of consistent measures of outcomes and large heterogeneity of the studies included. In addition, the true effect of health education on HPV uptake in India may not be ascertained based on the findings of the current paper, due to the lack of studies based on experimental and randomized control trials. These study designs may have helped understanding the true effect of intervention. Furthermore, the results of this present paper may be generalised to India as a whole. In fact, the number of the studies included remains relatively small and the samples not representative. Lastly, the lack of official statistics and surveillance obtained from government sources  and the absence of previous studies in this area is a matter of consideration.Considering the development of the indigenous HPV vaccine, India has the capacity to achieve success in the Cervical Cancer Elimination goal for 90% of coverage among girls up to 15 years old . With th


To observe the effects of high-risk human papillomavirus (HR-HPV) infection on P53, pRb, and survivin in lung adenocarcinoma (LUAD).The cancerous and cancer-adjacent tissues of 102 patients with LUAD from January 2020 to April 2022 were selected for the study. HR-HPV infection was detected by flow fluorescence method, and P53, pRb, and survivin protein expression was detected by immunohistochemical staining method. Statistical analysis was performed to determine the differences in the HR-HPV infection and the expression of P53, pRb, and survivin proteins between LUAD tissues and cancer-adjacent tissues; the correlation between HR-HPV infection and P53, pRb, and survivin protein expression in cancer tissues; and the correlation between HR-HPV infection and clinicopathological features of LUAD.P < 0.05). The positive rates of P53 and survivin protein were higher in the LUAD group  than in the cancer-adjacent group , and the difference was statistically significant (P < 0.05). The positive rate of pRb protein was lower in the LUAD group (58.82%) than in the cancer-adjacent group (92.14%), and the difference was statistically significant (P < 0.05). The positive rates of P53 and survivin proteins were significantly higher in the HR-HPV LUAD group  than in the non-HR-HPV LUAD group , and the difference was statistically significant (P < 0.05). The expression rate of pRb protein was significantly lower in the HR-HPV LUAD group (37.93%) than in the non-HR-HPV LUAD group (67.12%), and the difference was statistically significant (P < 0.05). The expression of p53 and survivin protein was positively correlated with HR-HPV infection , whereas the expression of pRb protein was negatively correlated with HR-HPV infection . HR-HPV infection was not associated with gender, age, and smoking in patients with LUAD (P > 0.05). HR-HPV infection was associated with lymph node metastasis and clinical stage of LUAD (P < 0.05).The infection rate of HR-HPV was higher in the LUAD tissues (28.43%) than in cancer-adjacent tissues (7.84%), and the difference was statistically significant (HR-HPV infection was associated with lymph node metastasis and clinical stage of LUAD, which may be achieved by up-regulating p53 and survivin protein expression and down-regulating pRb protein expression. Human papilloma virus (HPV) is a known cause of human cancer, and has been shown to be associated with multiple sites of human cancer, such as cervix, reproductive tract, skin, head and neck, oropharynx and other sites . The latThe paraffin specimens of cancerous and cancer-adjacent tissues of 102 patients with invasive LUAD admitted at the Second Affiliated Hospital of Shandong First Medical University from January 2021 to April 2022 were collected for the study. The study was approved by the ethics committee of The Second Affiliated Hospital of Shandong First Medical University (ID: 2021-8). Inclusion and exclusion criteria: (1) Inclusion criteria: The diagnosis of LUAD was in accordance with WHO pathological diagnosis norms; patients were untreated and had complete clinical data. (2) Exclusion criteria: Patients with other malignant tumors and past history of invasive LUAD were excluded. All specimens were used with written consent from participants or family members.Basic information of patients: 40 males and 62 females; age: 25 patients < 60 years old, 77 patients ≥60 years old, with an average age of 63.02 ± 8.08 years; smokers: 28 patients, non-smokers: 74 patients; lymph node metastasis: 11 patients, no metastasis: 91 patients; tumor diameter < 3 cm: 87 patients, ≥3 cm: 15 patients; clinical stage I+II: 93 patients, III+IV: nine patients.Two 7 µm slices of tissue wax blocks were used to extract DNA, and 5 µl of DNA template was added to each 15 µl PCR reaction system to carry out PCR amplification reaction; 3 µl of amplification product and 22 µl of microsphere hybridization solution were added to each well of the microwell hybridization plate for denaturation hybridization .Then, 75 µl of phycoerythrin (SA-PE) was added to each well to continue hybridization , and the HPV infection data were read on the Luminex 200 platform. A Globin value ≥150 was considered as a successful test, and positive high-risk types were counted in the results . The kit was purchased from Shanghai Toujing Life Technology Co., LTD.2O2 was added to eliminate endogenous peroxidase. Then, the samples were rinsed for threetimes with PBS. Primary antibodies were added . The samples were incubated for 90min at room temperature and then rinsed for threetimes with PBS. Secondary antibody (ready-to-use Quick MaxVisionTM-HRP kit (mouse/rabbit)) was added dropwise and incubated for 15 min at room temperature. Then, it was rinsed with PBS for threetimes and stained with DAB for 3–5 min. Hematoxylin was used for staining cell nuclei, and then conventional dehydration, transparency, and sealing of the film.Tissue paraffin blocks were sliced at 3 µm, incubated at 75 °C for 60 min, routinely deparaffinized to water, and rinsed for threetimes with phosphate buffer solution (PBS). Antigen retrieval was performed by using pH9.0 EDTA solution at high temperature and high pressure for 2 min. The samples were rinsed with PBS for threetimes, and 3% HThe results were interpreted by two senior pathologists in a double-blind method. Judgment criteria: ① P53-positive location was in the nucleus, which was diffusely positive (positive rate ≥70%) or completely negative (positive rate 10%). ② pRb-positive localization was in the nucleus. pRb staining intensity  and the range of positive expression were recorded at the same time . The staining intensity score was multiplied by the positive expression range of pRb to obtain a composite expression score (CES). According to CES, it was divided into RB1 negative  and positive . ③ Survivin-positive location was in the cytoplasm/nucleus, and the positive localization area was stained with brown-yellow particles under the microscope. Fivefields of high magnification (×200) were randomly selected. According to the coloring intensity score, 0 was no coloring, 1 was light yellow, 2 was yellow, and 3 was brown yellow. Meanwhile, according to the percentage of positive cells in the same type of cells, 0 was negative, 1 indicated < 10% positive cells, 2 indicated 10%–50% positive cells, 3 indicated 50%–75% positive cells,and 4 indicated >75% positive cells. The sum of the two scores was taken as the total score, in which 0–3 was negative and>3 was positive.The samples were divided into the LUAD group and cancer-adjacent group. The LUAD group was further divided into HR-HPV LUAD group and non HR-HPV LUAD group according to the presence or absence of HR-HPV infection. Our study obtained the consent of the patients and their families, and was approved by the hospital ethics committee, the ethics approval number is 2021-8.2 test or Fisher’s exact probability method was used between groups. Measurement data were expressed as . The count data were expressed as the number of cases or percentage, and the XP < 0.05)  .P < 0.05). HR-HPV infection was not associated with gender, age, and smoking, and the difference was not statistically significant (P > 0.05)  .P < 0.05). The positive rate of pRb was lower in the LUAD group than in the cancer-adjacent group, and the difference was statistically significant (P < 0.05)  .P < 0.05). The positive rate of pRb was lower in the HR-HPV LUAD group than in the non-HR-HPV LUAD group, and the difference was statistically significant (P < 0.05). The expression of p53 and survivin was positively correlated with HR-HPV infection, whereas the expression of pRb was negatively correlated with HR-HPV infection  in the cell ubiquitin ligase E6AP, and then E6/E6AP heterodimer degrades p53 lazily. Degradation of p53 is a marker activity of HPV E6 protein . P53 genPRb is the protein of tumor suppressor that was first discovered and determined by human beings. Its family members, pRb/p105, p107 and pRb2/p130, are one of the most important regulators of G1/S transition in the cell cycle. The point mutation or inactivation deletion of Rb gene leads to the loss of protein regulation ability, which is related to the occurrence and development of many tumors . At the Survivin, a member of the inhibitor of apoptosis protein family, is composed of 142 amino acids. Its multiple residues can bind with caspase, directly inhibit its activity, accelerate cell proliferation, and resist apoptosis. Survivin directly regulates apoptosis and mitosis of tumor cells during tumor occurrence and metastasis. Survivin is highly expressed in cancer tissues of patients with malignant tumors, and is the strongest inhibitor of apoptosis protein found so far . SurviviIn the correlation analysis between the clinicopathological characteristics of LUAD patients and HR-HPV, it was found that HR-HPV infection was related to the clinical stage of LUAD and whether lymph nodes were metastatic. The proportion of HR-HPV infected LUAD with high clinical stage and the proportion of lymph node metastasis were higher than those of HR-HPV negative LUAD, suggesting that HR-HPV infection is a high risk factor for LUAD, and its mechanism may be involved in the occurrence and development of LUAD by regulating the expression of P53, pRb, and survivin. P53 mutation plays an important role in the therapeutic drug resistance of cancer, and p53 related signal pathway may reverse the process of cisplatin resistance . PRb defIt is worth mentioning that the number of cases in the middle and late stages in this study is relatively small, which may be related to the widespread application of screening methods such as electronic computer tomography and the early detection of LA . This is10.7717/peerj.15570/supp-1Supplemental Information 1Click here for additional data file.


Different types of HPV have been associated with cancer in humans, but the role of HPV in esophageal cancer (EC) is controversial. The purpose of this study was to evaluate the correlation between HPV infection and EC in the Chinese population and to provide the scientific basis for the future prevention, control, early diagnosis, and treatment strategies of EC in China.PCR detected HPV infection in 1112 esophageal cancer tissue samples, and 89 HPV-positive samples were detected by genotyping. Proximity ligation assays (PLAs) and immunohistochemistry were used to detect the expression of HPV E6 and E7 proteins. Real-time fluorescent quantitative PCR was used to detect the integration of HPV16 E6. The level of HPV-specific antibody IgG in serum was detected by ELISA and PLA.The positive rates of HPV L1, HPV16, HPV18, hpv16 + 18 E6 and hpv16/18 E6 in 1,112 EC tissue samples were 77.6%, 41.4%, 27.2%, 14.2% and 55.4% respectively. Multiple HPV subtypes were detected in HPV-positive EC samples. PLA showed that E6 and E7 were expressed in EC109 and formed complexes with p53 and pRb, respectively. Immunohistochemistry showed that the positive rates of hpv16 + 18 E6 and E7 in HPV-positive EC samples were 56.4% and 37.0%, respectively. HPV-DNA integration rate in HPV-positive EC tissues (88.79%) was higher than that in adjacent tissues (54.17%). HPV antibody was found in the serum of EC patients by a serological test.The study suggests that HPV, especially HPV16 and HPV18, the infection may be a risk factor for EC in the Chinese population and that the E6 protein may play a key role in HPV-associated malignancies. These results may be important for the prevention and treatment of HPV-positive EC in China.The online version contains supplementary material available at 10.1007/s00432-022-04322-5. Cancers of the esophagus account for over 500,000 cancer deaths annually, representing 5.3% of all global cancer deaths. However, the worldwide regional distribution varies substantially  and HPV (31.1%)  tissue specimens were taken from 10 high-risk area pathology departments in China from 2009 to 2012 . 1,112 histologically confirmed ESCC specimens (FFPE tissue blocks) were sectioned and DNA was extracted. The specific process refers to our previous research .To identify HPV types and any sequence variations, PCR products from all samples were sequenced and analyzed using the T7 Sequence Version 2.0 DNA PCR product sequencing Kit . The nucleotide sequence was then determined using BLAST  was used for rapid and accurate HPV genotyping. It can identify 21 HPV including 15 high-risk types  and 6 low-risk types .Cell culture and preparation of centrifuge cell smear. In the experimental group, EC109 cell lines infected with HPV18 were suspended in PBS at medium density, then coated on glass slides, dried at room temperature and fixed with poly-oxy-methylene (4%). The control group was treated with normal HEK 293 cells in the same way.In situ PLA (Proximity ligation assay). In the experiment, each protein molecule was detected by a monoclonal antibody for p53, pRb, HPV18 E6 and E7. HPV18 E6-p53 protein complex was detected by Rabbit anti HPV18 E6 antibody and mouse anti p53 antibody, and HPV18 E7-pRb protein complex was detected by Rabbit anti HPV18 E7 antibody and mouse anti p53 antibody. The PLA test kit was purchased from Olink Bioscience  and the specific operation was carried out by the manufacturer's operating procedures. Then the results were observed by laser confocal microscope.HPV16+18 E6 and HPV16+18 E7 monoclonal antibodies were used to detect cancer tissues. The antibodies used to detect HPV-18 and 16 are mouse monoclonal antibodies. We used the same procedure previously used in the laboratory  in the RotorGene fluorescent PCR system.One hundred and sixteen cancer tissues and 72 adjacent tissues were randomly selected for real-time fluorescent quantitative PCR to detect the integration of HPV DNA in esophageal cancer. The research is based on the following assumptions: the special region of the E2 open reading frame (ORF), which is most often missing during HPV integration, such as HPV exists in the form of free type, and the proportion of E2 and E6 copies is equal. If HPV is integrated into the host chromosome, E2 is deleted, and the copy number ratio of E2 to E6 is zero; HPV exists in a mixed form of free and integrated, and the copy number of E2 is less than E6. Refer to our previous article  were used to detect HPV-related antibodies in serum. HPV18-infected EC109 cells were detected by nasopharyngeal carcinoma detection method and PLA technology. Normal HEK293 cells are used as the negative control. Please refer to the equipment manual for detailed operation.HPV antibody was detected by ELISA. The human papillomavirus IgG antibody detection kit  was used to detect human papillomavirus-related IgG antibody levels in serum. Please refer to the instructions for specific operations.P value of the tests had to be < 0.05 to be statistically significant. The correlation coefficient of the lollipop chart and multi-type pie chart by the free online data analysis and visualization platform https://www.bioinformatics.com.cn.SPSS23.0 statistical software was used for statistical analysis of the results, and the correlation between HPV-related proteins and esophageal cancer was analyzed. The R=0.701, P=0.024 and R=0.848, P=0.26, respectively). HPV16/18 showed the strongest correlation with EC , followed by Linzhou 93.4%) and (Puyang 92.1%), and Jieyang had the lowest rate (40.9%). The positive rate of HPV16 was higher in Cixian, Linzhou, and Huaihe River Basin, with positive rates ranging from 16% to 86%. The positive rates of HPV18 in Huaihe River Basin, Tangshan, and Puyang were 50.0%, 47.47%, and 34.2% respectively. The positive rate of HPV16 E6 and HPV18 E6 was 39.4% in Huaihe River Basin, 28.9% in Puyang, and 27.8% in Tangshan. The positive rate of HPV16/18 was the highest in Cixian (86.0%), followed by Linzhou (68.0%), and Huaihe River Basin (62.1%). The HPV prevalence rates in ten examined high-risk areas of China and the details of patients with EC positive for different HPV types are shown in Figs. .4% and , and the positive detection rate of HPV16 and HPV18 was high. HPV51 was also widely distributed in EC tissues (positive rate: 15.7%). HPV45, 33, 52, 53, 58, 45, 31, 66, 68, and other types were also found  was performed in 275 EC cases positive for HPV DNA. HPV-specific proteins, including monoclonal antibodies against HPV16+18 E6 and HPV16+18 E7, were used to detect cancer tissues. Representative IHC images are shown in Fig. Of 116 HPV16 E6-positive EC specimens, 103 (88.8%) were positive for E2 gene integration, whereas, among 72 adjacent tissue specimens, 39 54.2%) were positive for E2 gene integration   antibodies labeled with single-stranded DNA. Only when a pair of probes are close enough (<40 nm), a pair of probes labeled with antibodies will combine to form a ring, which is reflected by an increase in fluorescence intensity. We observed that E7–pRb and E6–p53 protein complexes were highly prevalent in EC109 cells. This not only directly proves the interaction between the two proteins, but also indicates that the cells expressing HPV E6 and E7 proteins are carcinogenic. Therefore, we believe that HPV-positive cells or tissues play an important role in carcinogenesis. We also found that the expression of E6 was stronger than E7 after HPV18 infected EC109 cells Below is the link to the electronic supplementary material.


Anal cancer, mainly attributed to human papillomavirus (HPV) infection, is rising in prevalence among the general population in Pakistan. This study aimed to examine the knowledge, attitudes, and practices (KAP) towards anal cancer screening and HPV of the general population in Pakistan.We surveyed anal cancer KAP using social media and snowball sampling from December 2022 to May 2023. The questionnaire had 16 knowledge, 12 attitudes, 6 practice questions, and socio-demographic variables. We applied validity criteria for inclusion and exclusion and used cutoffs ≥50% for each KAP category. We analyzed data in R with Guttman’s λ2 for reliability, did univariate and bivariate analysis, and reported frequencies, percentages, p-values, coefficients, odds ratios, and 95% confidence intervals.We surveyed 1620 people and discovered low awareness of HPV and anal cancer causes prevention, and screening (11%-24%), high stigma and embarrassment for screening (54%-70%), strong moral beliefs (89%), condom nonuse (91%), and low engagement in health services and programs (9.1%-14%). Knowledge  was shaped by socio-demographic factors, attitude, and practice, with higher education enhancing knowledge . Attitude  was influenced by socio-demographic factors, practice, and knowledge as well. Younger females, single, unemployed, students, living with more family members, earning more income, and residing in Islamabad had a more positive attitude , while religion did not affect attitude (p = 0.51). Practice  was determined by socio-demographic factors, knowledge, and attitude. Older males, employed teachers, living with more family members, earning less income, and residing in Islamabad had better practice , but marital status and religion did not influence practice (p > 0.05).Pakistani young adults need more education, awareness, health services, and programs on HPV and anal cancer, as they have low awareness, high stigma, and socio-cultural challenges. In addition, it is recommended for more research and policy initiatives are needed to address socio-cultural factors and increase anal Pap to overcome anal cancer. HoweverPakistan is a country where Islam shapes society’s moral and social fabric. Any sexual activity outside the marital bond, especially homosexual sex, is considered a grave sin and a violation of Islamic law. Many Pakistanis also assume that HIV cannot affect them, as they believe Muslims are immune to this disease associated with illicit sex , 19. HowGiven the increasing burden of sexually transmitted diseases in Pakistan, it is imperative to investigate the basic knowledge and awareness of the population about anal cancer. However, there is a dearth of research on this topic in Pakistan, and the level of awareness and prevention of anal cancer among the public is unknown. Therefore, this study is the first of its kind in Pakistan, aiming to explore the knowledge, attitudes, and practices (KAP) regarding HPV-associated anal cancer in the community.22.1We used an anonymous online questionnaire to collect the data for this study, which evaluated the public’s knowledge, attitudes, and practices concerning anal cancer. We employed a snowball sampling technique to distribute the questionnaire link through various social media platforms, such as WhatsApp, Twitter, Facebook, Messenger, Gmail, and Instagram. The respondents had to answer every item on the questionnaire, which was available in both English and their native language. The data collection period for this study spanned seven months, from December 2022 to May 2023. The participants completed the questionnaire voluntarily after receiving information about the nature of the study and assurance of anonymity and confidentiality of responses. The study encompassed all the regions of Pakistan, including Khyber Pakhtunkhwa (KPK), Punjab, Balochistan, Sindh, Gilgit-Baltistan (GB), Azad Jammu Kashmir (AJK), and Islamabad Capital Territory (ICT), as illustrated in 2.2This study aimed to assess the Pakistani population’s KAP parameters on a large scale, regardless of gender and residence. The study included respondents who met the following criteria: (i) they were 10 years old or above, and (ii) they were residents of Pakistan. These criteria were consistent throughout the study. To ensure the validity and relevance of our study, we excluded respondents who met any of the following criteria: (i) had a history of allergy to the HPV vaccine and could not receive immunization, (ii) the respondent was a foreign citizen studying or living in Pakistan, and (iii) had HPV-associated malignancy, as their personal experience might bias opinions.2.3The questionnaire was developed by adapting previously validated questionnaires to the study’s context –31. We mFurthermore, we investigated the attitudes toward HPV using 12 questions about individual beliefs, perceptions about anal screening, and willingness toward HPV vaccination. The responses were categorized as “Yes,” “No,” and “Not Sure.” Likewise, the overall attitude was divided into positive and negative scales. The attitude score was obtained by giving one point to “Yes” as the positive attitude and zero points to “No” and “Not Sure” as the negative attitude. A cutoff of six was applied to differentiate positive and negative attitudes, with equal or higher scores indicating a positive attitude and lower indicating a negative attitude.Additionally, we appraised the practices of participants using 6 questions about screening for anal cancer, participation in HPV awareness campaigns, and safe sex practices. Respondents were asked to answer using the “Yes” and “No” scales, and one point was awarded for good practices. A cutoff of six was applied to classify “good practice” and “poor/bad practices”, where scores equal or above the cutoff indicated good practice while low scores indicated poor/bad practices. Finally, we collected a broad range of socio-demographic variables, including gender, age, marital status, employment status, occupation, number of family members, level of education, religion, monthly household income, and place of residence to categorize their knowledge, attitudes, and practices in different variables, as illustrated in 2.4Ethical approval was obtained from the Departmental Bioethical Committee of The University of Haripur (UOH-RC-EA-142). Informed consent was taken from all participants before they participated in the study. In the case of participants under 18 years old, the participant’s legal guardian provided written informed consent to participate in this study.2.5λ2  and percentages (%) for each category of the variables under consideration  to clean and screen the data and to estimate the reliability of the test items; in addition, we adopted Guttman’s λ2  instead 33.13.2The respondents had varying levels of knowledge about HPV and anal cancer, as shown in 3.3The survey results revealed the respondents’ attitudes towards HPV and anal cancer, as shown in 3.4The survey results revealed the respondents’ practices related to HPV and anal cancer, as shown in 3.5We used binary logistic regression analysis to explore the association between the KAP of anal cancer and various socio-demographic factors. The dependent variables were knowledge, attitude, and practice grading, while the independent variables were all collected socio-demographic variables. However, odds ratios and the 95% confidence intervals indicate the direction and magnitude of the association between each predictor and the practice grading.3.5.1The dependent variable was the knowledge grading, categorized as knowledgeable (75.23%) or not-knowledgeable (24.77%) based on the scores obtained from the knowledge questions. As shown in 3.5.2The dependent variable was attitude grading, categorized as negative (21.55%) or positive (78.45%) based on the scores obtained from the attitude questions. Overall, age, gender, employment status, occupation, family members, income, resident province, practice, and knowledge were significant predictors of the attitude grading (p< 0.05). As shown in the 3.5.3The dependent variable was the practice grading, categorized as good (9.16%) or poor (90.84%) based on the scores obtained from the practice questions. Overall, age, gender, employment status, occupation, family members, income, resident province, knowledge, and attitude were significant predictors of the practice grading (p< 0.05). As shown in 3.6Moreover, females were more knowledgeable , positive , and good practicing  than males . Single and widowed respondents were more knowledgeable , positive  and good practicing  than married and divorced respondents . Business owners and teachers were more knowledgeable , positive  and good practicing  than other occupations . Respondents from ICT and Punjab were more knowledgeable , positive  and good practicing  than other regions .4The survey participants were 1620 individuals who answered questions about their knowledge, attitude, and practices regarding anal cancer and HPV infection in Pakistan. Most of the participants were young, female, single, unemployed, students, and had large families. Most of them did not disclose their income, had a university education, were Muslim, and lived in KPK province. Our results show that the respondents had varying knowledge about anal cancer and HPV infection but low awareness about the HPV vaccine and its availability in Pakistan. The respondents also had mixed attitudes toward HPV prevention, screening, and treatment. For example, 82% of the respondents knew that anal cancer is more common in men than in women, but only 39% had heard of the HPV vaccine, and only 57% knew that it could prevent penile cancer in males.Similarly, 17% of the respondents had heard of anal Pap screening, but only 31% knew medications could not cure HPV infections. These findings are consistent with some published studies on HPV KAP in Pakistan and other countries. For example, a study by Ejaz et al.  found thFurthermore, a study by Husnain et al.  found thCurrent study results revealed that respondents had varying awareness and opinions about HPV prevention, screening, and treatment. However, participants had a fair amount of knowledge and confidence regarding the signs, symptoms, diagnosis, and treatment of anal cancer. Most of them (67%) reported being well-informed, and 36% were aware of the signs and symptoms of anal cancer. In contrast, findings are inconsistent with previous studies that reported low levels of awareness and education about anal cancer among the general population . Our surFurthermore, as many as 66% of them agreed that adolescents should be encouraged to get HPV immunization, 14% agreed that HPV vaccination should be mandatory, 71% agreed that they would go for an anal Pap if they could collect a sample themselves, 84% agreed that they would like to learn more about the anal pap test, and 77% of participants would not recommend their family members for anal Pap. These findings suggest a positive attitude and readiness for HPV vaccination for adolescents but negative for anal cancer screening. In contrast, findings were reported in previous studies that have reported high levels of acceptance and willingness for HPV vaccination , 42 and The survey also showed a high level of awareness and resilience among the participants regarding the risk factors and barriers for HPV and anal cancer. As many as 64% agreed that multiple sex partners make an individual more likely to get anal cancer, 67% responded that social pressure and stigmatization would prevent them from anal Pap, and 71% responded that myths or religious beliefs would prevent HPV vaccination. This is consistent with previous studies that found that most men who have sex with men knew the link between HPV infection and anal cancer. Our findings are consistent with previous studies; some men who have sex with men may avoid HPV vaccination and anal cancer screening due to social pressure, stigma, myths, or religious beliefs , 48, 49,The current survey indicates a low level of participation and utilization of health services and programs related to HPV and anal cancer among the participants. Similarly, these findings are consistent with previous studies that have reported high levels of moral beliefs among the general population –58 and lThis is the first study in Pakistan to explore how young adults attending a university/college think and feel about HPV and anal cancer and what they do to prevent them. We found that males and females had similar knowledge, attitudes, and screening behaviors, but they were significantly low. Most participants did not know much about HPV and anal cancer, felt ashamed and embarrassed to talk about them, and faced many socio-cultural challenges to protect themselves. They also reported following moral beliefs and avoiding condoms for safe sex. However, they hardly ever participated in health education programs or screenings related to HPV and anal cancer. Only a few of them had ever learned about HPV, checked themselves for anal cancer, or got tested for anal cancer by a doctor. Even fewer were willing to get an anal Pap test if they noticed genital warts. These findings show an urgent need for more awareness campaigns and support services to help people understand and access health care for HPV and anal cancer and overcome the myths and barriers that may prevent them from getting timely and proper treatment. We also found that young adults in our study were not well informed or motivated to get vaccinated or screened for HPV and anal cancer, which means the existing prevention efforts have not reached them. Consequently, this shows the importance of training health professionals and educators to inform young adults about this issue.Our study has some limitations, such as being unable to represent all young adults in Pakistan because most participants were from the KPK and Punjab provinces. This study used a self-administered questionnaire as the data collection method, which may introduce misclassification bias due to the lack of validation of the participants’ responses. The anonymity of the questionnaire may not have eliminated the social desirability bias, as the topic’s controversial nature may have influenced the participants. Moreover, we lacked detailed data on their sexual behavior, HPV vaccination status, and anal cancer screening history, which could confound their disease risk. However, our study is among the pioneer studies to assess HPV and anal cancer awareness among young adults in Pakistan. Thus, it provides valuable insights for future research studies and public health interventions to enhance HPV and anal cancer screening and prevention.5This study demonstrated that young adults in Pakistan have inadequate awareness, elevated stigma and embarrassment, and multiple socio-cultural challenges regarding HPV and anal cancer. These factors may impede their engagement and utilization of health services and programs that can prevent and treat these diseases. Hence, there is a need for more education and awareness campaigns and more available and affordable health services and programs to address the gaps and barriers in the prevention and management of HPV and anal cancer among this population. Furthermore, there is a need for more research and policy initiatives to comprehend and address the socio-cultural factors that influence the knowledge, attitudes, and experiences of young adults in Pakistan regarding HPV and anal cancer. By doing so, the health outcomes and quality of life of this population can be enhanced.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Ethical approval was obtained from the Departmental Bioethical Committee of The University of Haripur (UOH-RC-EA-142). Informed consent was taken from all participants before they participated in the study. In the case of participants under 18 years old, the participant’s legal guardian provided written informed consent to participate in this study.UA: Conceptualization, Data curation, Methodology, Writing – original draft, Writing – review & editing. WN: Conceptualization, Data curation, Methodology, Writing – original draft. AK: Data curation, Investigation, Methodology, Writing – review & editing. TM: Formal Analysis, Methodology, Software, Validation, Visualization, Writing – original draft. ShK: Conceptualization, Data curation, Methodology, Writing – original draft. SuK: Data curation, Investigation, Methodology, Project administration, Formal Analysis, Writing – review & editing. XG: Conceptualization, Formal Analysis, Project administration, Supervision, Writing – review & editing. ZY: Funding acquisition, Project administration, Supervision, Writing – review & editing. JL: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. AN: Writing – review & editing.


It is unclear whether disparities in the care provided before lower extremity amputation (LEA) is driven by differences in receipt of diagnostic work-up versus revascularization attempts.We performed a national cohort study of Veterans who underwent LEA between March 2010 and February 2020 to assess receipt of vascular assessment with arterial imaging and/or revascularization in the year prior to LEA.Among 19,396 veterans , Black veterans had diagnostic procedures more often than White veterans (47.5% vs. 44.5%) and revascularization as often (25.8% vs. 24.5%).We must identify patient and facility-level factors associated with LEA as disparities do not appear related to differences in attempted revascularization. PAD can lead to significant morbidity and cardiovascular mortality.9It is estimated that nearly 10% of all veterans have PAD, and ∼1800 veterans undergo amputation each year within the Veterans Health Administration (VHA). Most amputations can be prevented with well-established diagnostic and therapeutic interventions; however, utilization of these strategies is variable.10 It is currently unknown why vascular assessment and attempts at revascularization are not more widely used before LEA.Several studies among nonveterans report significantly low rates of diagnostic vascular assessment as well as attempts at revascularization before LEA. Goodney et al. reported that 54% of Medicare beneficiaries do not have an attempt at revascularization before an amputation.It is also unclear what proportion of veterans undergo only diagnostic procedures without a subsequent attempt at revascularization before LEA, and whether this may be a potential explanation for observed disparities in LEA rates. As such, the objective of this study was to determine what proportion of veterans undergo diagnostic work-up with and without an attempt at revascularization in the 1 year before LEA, and to examine whether there are racial or regional differences.This study was approved by the Emory institutional review board and the Atlanta VA Health Care System research and development committee. All veterans over age 18 years who underwent LEA within the VHA between March 1, 2010, and February 28, 2020, were identified in the VA Corporate Data Warehouse. Current procedural terminology (CPT) codes for major LEA (defined as any amputation above the level of the ankle) were used to define the study population. We excluded any amputation revision and patients who received their care in the community (outside VHA).The primary outcome was the receipt of a vascular assessment in the 1 year before a major LEA. Vascular assessment was defined as any imaging study or diagnostic procedure performed on a veteran to either establish a diagnosis of arterial insufficiency (diagnostic only) or to improve lower extremity arterial flow (revascularization). We used CPT codes to determine which veterans underwent vascular assessment and whether the assessment was performed only for diagnostic purposes or was an attempt at revascularization. We excluded procedures performed for the indication of acute limb ischemia.Race was the primary exposure variable of interest. We first categorized race as American Indian/Alaskan Native, Asian, Black/African American, Native Hawaiian/Pacific Islander, White, and “unknown.” However, we found that >90% of patients were either Black or White and 6% were listed as “unknown.” Therefore, we only included Black and White veterans in our analysis to compare vascular assessment among groups. Hispanic veterans were not excluded, however, analyses were not stratified by ethnicity.2) or not obese (<30 kg/m2). Chronic kidney disease (CKD) was categorized as no CKD, CKD (stage III and above) not on dialysis, and end-stage renal disease.11 All other comorbidities were defined using the International Classification of Diseases, 9th and 10th revisions (ICD-9 and ICD-10) diagnosis codes.Region was the secondary exposure variable of interest and was categorized as northeast, midwest, west, southwest, and southeast. This variable identified the region of the country in which the veteran resided at the time of index amputation. Age was categorized as <60, 60–69, 70–79, or ≥80 years. Body mass index (BMI) was categorized as obese . All analyses were conducted using SAS, version 9.4 .n=12,782) were male. The mean age was 66.78 years  and 64.9%  were under the age of 70 years. The cohort included 1114 (5.7%) American Indian/Alaskan Natives, 37 (0.2%) Asians, 5167 (26.6%) Black/African Americans, 133 (0.7%) Native Hawaiian/Pacific Islanders, 12,782 (65.9%) Whites, and 1114 (5.7%) veterans of unknown race. Self-identification as Hispanic ethnicity comprised 6.0% (n=1171) of the cohort. Demographic data for the cohort are provided in In total, 19,396 veterans underwent major LEA during the study period. Among them, 98.5% . In addition, significantly more Black veterans had an attempt at revascularization than White veterans in the year before LEA .When stratified by region, the volume of major LEA did not change from year to year p=0.42; . A vascuThere were also significant regional differences noted when considering those who underwent only diagnostic procedures relative to those who underwent an attempt at revascularization . For exaPreviously published work has demonstrated striking racial disparities in PAD outcomes—specifically, Black patients are at higher risk of major LEA than any other racial group. Many believe that most amputations are preventable when guideline-directed medical therapy and vascular assessments are performed. However, there is notable variation in who gets a vascular assessment before an LEA. In this regard, our study presents novel findings that potentially shed new light on this specific issue: (1) vascular assessment is performed in over two-thirds of veterans who undergo major LEA; (2) as compared with White veterans, Black veterans are more likely to receive diagnostic studies, as well as to have an attempt at revascularization before LEA.3–6 There are several conflicting ideas as to why this may be the case, and very few proposed solutions to address or eliminate this disparity.12–14 Some have suggested Black patients may have a lower likelihood of being offered revascularization, which in turn leads to higher LEA rates.16 In our national cohort of veterans, Black patients represented over one-quarter of the study population (this over-represents this subgroup relative to the U.S. population and the veteran population receiving care at the VA).17Over the last few decades, Black patients have consistently been found to have higher LEA rates than White patients.Despite this over-representation, our work suggests Black veterans receive both diagnostic studies and attempts at revascularization more often than White veterans. As such, it does not appear that a lower likelihood of attempted revascularization is a major driver of racial disparities in LEA, at least among veterans receiving care at the VHA. Further evaluation of clinical indications for LEA, exploration of health care utilization among Veterans, and examination of prescription of guideline concordant care among Veteran providers is needed.19There are some limitations of note that should be considered. This was an observational study of a cohort of veterans who underwent major LEA. As such, selection bias cannot be entirely excluded as a reason for our study findings. As well, we did not perform a deep dive into veteran comorbidities in this study and their association with components of vascular assessment: diagnostic and therapeutic interventions. It is well known that diabetes mellitus and severe renal disease each are risk factors for PAD that work synergistically to elevate the risk for LEA.In addition, because the data were abstracted from the VA Corporate Data Warehouse, we did not include veterans who may have undergone an LEA outside of VHA. As such, we may have underestimated the proportion of veterans who receive vascular assessment before LEA, as we did not include data from care in the private sector. Finally, it is unclear how severe a patient's PAD was on presentation or whether some LEAs were performed in an emergent setting due to severe infection.Although racial disparities in major LEAs exist within the VHA, our findings suggest this does not appear to be due to racial differences in the receipt of diagnostic or revascularization procedures within the VHA. Future study should consider patient and facility factors that may be associated with receipt of vascular assessment before a major LEA.


HR-HPVs occurred in 24% of UC, 32% of Vself, 30% of V and 24% of P samples. Only 22.2% of all HR-HPV infections were shared by both partners . The type-specific HR-HPV concordance was significant between male to female OC-OC (κ 0.56 ± 0.17), V-VSelf (κ 0.70 ± 0.10), UC-V (κ 0.54 ± 0.13), UC-Vself (κ 0.51 ± 0.13) and UC-female AC (κ 0.42 ± 0.15). Conclusions: HPV infections are prevalent in HIV-positive couples in Rwanda but concordance within couples is low. Vaginal self-sampling for HPV is representative of cervical HPV status.Background: The prevalence of human papillomavirus (HPV) infections in other anatomical sites besides the uterine cervix is unknown in East Africa. Here, we assessed the prevalence and concordance of HPVs in different anatomical sites in HIV concordant couples in Rwanda. Methods: Fifty HIV-positive concordant male-female couples at the HIV clinic at the University Teaching Hospital of Kigali in Rwanda were interviewed, swabbed from the oral cavity (OC), oropharynx (OP), anal canal (AC), vagina (V), uterine cervix (UC) and penis. A pap smear test and a self-collected vaginal swab (Vself) were taken. Twelve high-risk (HR)-HPVs were analyzed. Results: HR-HPVs occurred in 10%/12% in OC, 10%/0% in OP and 2%/24% in AC ( Chronic infection by high-risk human papillomaviruses (HR-HPVs) of the uterine cervix is the most important risk factor associated with the development of high-grade squamous intraepithelial lesions (HSIL) and cervical cancer where HPV16 and HPV18 stand for most cervical cancer cases ,3. Co-inIn our previous study, we showed that 56% of all HR-HPV infections are chronic in Rwandan women living with HIV (WLWH) . While cIn this cross-sectional study, 50 HIV-positive concordant opposite-sex couples attending the HIV clinic at the University Teaching Hospital of Kigali (CHUK) in Kigali, Rwanda were recruited after informed consent. The couples lived together, and all participants were treated with antiretroviral therapy (ART). Inclusion criteria were literate individuals who were diagnosed with HIV and were 21 years old or older. Exclusion criteria were previous HPV vaccination, cervical lesions, women and men who have not had sexual intercourse or sexual activity with other individuals, and men and women for any reason considered not able to comply with the study protocol.The participants were given a structured questionnaire including questions covering medical history, sexual and obstetric history, previous sexually transmitted infections (STIs), and questions covering risk factors for contraction of HPV infection. This was followed by taking a swab sample for HPV using the Aptima Multitest Swab  from the buccal mucosa, tonsillar area, and anal canal. In men, swab samples were taken from the glans penis/opening of the urethra. In women, swab samples for HPV were taken from the vagina by the woman herself and another vaginal sample was taken by the physician. This was followed by an HPV swab sample and a pap smear sample taken with a wood spatula from the uterine cervix taken by the physician. A real-time PCR assay targeting type-specific segments of the E6/E7 region for 12 high-risk , two low-risk (HPV6 and 11) HPV types and beta-globin was used . NucleicCytology of pap smears was classified according to the 2014 Bethesda System and the following cytological diagnosis: Negative for squamous intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells cannot exclude HSIL (ASC-H), atypical glandular cells (AGC), endocervical adenocarcinoma in situ (AIS), high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC).p < 0.05.The SPSS version 24 (IBM) was used to assess the significance of differences detected in different main outcomes between and within groups. The chi-square test was used to compare categories. To compare the concordance of HR-HPV infections between different mucous membranes and between couples, Cohen’s kappa statistics were used. The Cohen’s kappa coefficient (κ) was estimated where κ < 0 = poor, 0–0.20 = slight, 0.21–0.40 = fair, 0.41–0.60 = moderate, 0.61–0.80 = substantial and 0.81–1.00 = almost perfect agreement. Bivariate logistic regression analysis was performed, and crude odds ratios (COR) and adjusted odds ratios (AOR) were calculated for explanatory variables correlating with being HR-HPV positive in any mucous membrane. Statistical significance was set to p < 0.0001 and p = 0.017, respectively). Male participants reported a higher number of previous sex partners and orogenital sex partners than their female partners . No participant reported same-sex sexual activity. Previous STIs were common among the participants, i.e., gonorrhea infection had occurred in 44% and 34%, syphilis in 18% and 24% and chlamydia in 12% and 12% of men and women, respectively. The proportion of men with CD4 counts lower than 200 cells/mL was significantly higher compared with women (p = 0.01), however, no difference was observed in HIV viral load between men and women. Ninety percent of men and women were in the agreement with the kind of contraceptive method used with their partner or in the agreement with not using a contraceptive method. Among 30 men reporting using contraceptive methods, 17% reported a different contraceptive method than their female partner or reported using a contraceptive method while their partner reported not using a contraceptive method.Demographic data are displayed in The prevalence of different HPV types in different mucous membranes in men and women is displayed in p = 1.00 and p = 0.06, respectively). Oral and oropharyngeal LR-HPV infections were not detected in any of the study participants. Anal HR-HPV infections were significantly more common in women than men . In contrast, anal LR-HPV infections were rare in women and occurred to a similar degree in men . Cervical HR-HPV and LR-HPV infections occurred in 24% and 6% of women, respectively. Vaginal HR-HPV and LR-HPV infections were detected with self-sampling in 32% and 4% of women, respectively, and with physician-taken sampling in 30% and 4% of women, respectively. A similar degree of genital HPV-positivity occurred also in men, i.e., penile HR-HPV and LR-HPV infections occurred in 24% and 4%, respectively. Co-infections of more than one HR-HPV type occurred in 1/5 male oral sample, 0/6 female oral sample, 2/5 male oropharyngeal sample, 0/1 male anal sample, 0/12 female anal sample, 1/12 cervical sample, 2/15 physician-taken vaginal sample, 2/16 vaginal self-sample and 5/12 penile sample. Low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL) in pap smears occurred in 8.7% and 4.3% of female genital HR-HPV-positive cases and in 11.1% and 0% of female genital HR-HPV-negative cases . Male-to-female oral HPV concordance was 90% . The concordance of type-specific HR-HPVs in vaginal samples taken by the physician and by the participant herself was 87% . The type-specific HR-HPV concordance between cervical samples and vaginal samples taken by the physician and the participant herself was 81%  and 80% , respectively. The type-specific HR-HPV concordance between cervical and female anal samples was 79% . The type-specific HR-HPV concordance between vaginal self-sample and physician-sample vs. female anal samples was 74% and 72% . Only 22.2% of HR-HPVs infections were shared by the couples and concordance was 35%  but not in multivariate analysis  and WLWH, but HR-HPV infections were discordant within couples. While couples displayed concordant oral to oral HR-HPV infections, oral to genital HR-HPV infections were discordant. A high concordance between vaginal self-collected, vaginal physician-collected and cervical HR-HPV samples was observed. Previous exposure to STIs, the number of sex partners and undergoing abortions were factors correlating with the risk of being infected by HR-HPV. Our study and previous studies have shown a high prevalence of HPV infections in Rwandan men and women, especially in MLWH and WLWH ,11,12,13In agreement with previous studies in sub-Saharan Africa ,25,26, wBesides a positive concordance between male and female oral HR-HPV strains, we observed a high discordance of type-specific sharing of HR-HPV infections between men and women. Our findings contrast with a larger study conducted in South Africa where HIV-concordant male-female partners shared HR-HPV strains . In our We confirm again the relationship between gonorrhea infection and not knowing if being previously infected with chlamydia with HR-HPV infections in women . To haveRwanda is at the top in Africa when it comes to the vaccination of schoolgirls, reaching 89% of the target population . Still, The likelihood of being cervical cancer screened is higher in WLWH than in HIV-negative women in East Africa . To noteThe strength of our study is that we were able to analyse the concordance of HPV infections in multiple mucous membranes and analyse concordance of HPVs among HIV-positive couples. As far as we know, no previous study in East Africa has studied HPV co-infections in mucous membranes among risk patients. The present study has, however, also limitations. First, the cohort assessed was small and this may explain why no variable was related to HPV-positivity in men. Secondly, in our questionnaire, we did not cover extramarital sexual behaviour so we can only present indirect evidence for extramarital sexual activity contributing to the high degree of HR-HPV-positivity in men. Thirdly, we had a high average age of the participants, which most likely led to a lower degree of HPV positivity than in a younger cohort of individuals.HPV infection is prevalent in mucous membranes among HIV-positive concordant couples in Rwanda. While oral to oral transmission was common in HIV-positive couples, genital infections were discordant. Vaginal HPV self-screening has, moreover, a high sensitivity to detect cervical HPVs. Our findings highlight the need for additional studies on HPV transmission in other mucous membranes than the uterine cervix in sub-Saharan Africa. In addition, the high prevalence of HPV concordance among HIV-positive concordant couples suggests that to further lower the HPV burden of disease in Africa, the focus should also be set on the vaccination of boys.