High-risk human papillomavirus (HR-HPV) testing has become an increasing important strategy in primary cervical cancer screening in recent years. It warrants the evaluation of molecular-based HPV tests for accuracy and efficacy of screening. The performance of Roche Cobas 4800 HPV test was validated and compared with Digene Hybrid Capture 2 (HC2) high-risk HPV DNA test for primary screening in a large Chinese screening cohort. Of 6345 women screened, overall agreement between Cobas and HC2 was 92.23% (95% CI: 91.57–92.89). The inter-assay agreement was correlated with the severity of underlying biology, with an increasing concordance found in samples with more severe abnormalities. Most of the discordant samples had the test signal strength closer to the test limits of the detection than concordant samples, reflecting a low viral load and infection of a cluster of low-risk HPV in these samples. The Cobas test demonstrated significantly higher specificity in identifying CIN2+/CIN3+ cases than HC2 test , with comparable sensitivity in clinical evaluation. Increased specificity of Cobas test would accent women having the highest risk of developing CIN2+, with the potential to reduce unnecessary colposcopy referral in a screening population. Cervical cancer is a preventable disease and its incidence and associated mortality rate have substantially declined since the implementation of massive screening programs . As HPV Currently, several HR-HPV assays have been validated and approved by United States Food and Drug Administration (FDA) . Roche CThe study population was 6345 women who had both HC2 and Cobas HPV tests done were selected from a recent prospectively randomized controlled trial (RCT) in Hong Kong . BrieflyCervical specimens were collected in PreservCyt® for liquid-based cytology, then followed by HC2 test. The HC2 test is a signal amplification assay that utilizes microplate chemiluminescent to detect 13 HR-HPVs. The cutoff (CO) values for each run were calculated based on the relative light units (RLUs) of the positive and negative controls and the results were reported as relative light unit to cutoff (RLU/CO) ratios.The Cobas test is a real-time PCR based assay targeting HR-HPV L1 gene and human β-globin gene as an internal control. It provides HPV16 and HPV18 genotyping and reports 12 other HR-HPVs as a pooled result. It is a fully automated system from DNA extraction to PCR amplification of HR-HPV DNA and carried out according to manufacturer’s instructions.The Linear Array HPV genotyping test (LA) generates individual qualitative results for 37 HPV genotypes, including the same 14 HR-HPV genotypes as Cobas test and 23 additional HPV genotypes which are defined as low risk-HPV (LR-HPV) . LA testThe results of HC2 HPV test were used for subject management in RCT, whereas the results of Cobas and LA tests were used for comparison analysis only.All statistical analyses were conducted using computer software SPSS  / GraphPad Prism (version 6) / R . Overall agreement, positive and negative agreements and corresponding Kappa coefficients with 95% confidence intervals were calculated to estimate levels of agreement between HPV tests. Kappa values from 0 to 0.20, 0.21 to 0.40, 0.41 to 0.60, 0.61 to 0.80 and above 0.81 indicate poor, fair, moderate, good and excellent strength of agreement, respectively. The Median score and Mann-Whitney U tests were used to calculate p values for the median cycle threshold (Ct) values of Cobas and RLU/CO (a relative light unit/cutoff) ratio of HC2 test for concordant versus discordant HR-HPV positive cases.Specimens that tested positive by Cobas and LA tests for HR-HPV were further analyzed by comparing genotyping results in the following categories: HPV16, HPV18, and other HR HPV types (OHR), as well as coinfections involving the three categories. Samples that were positive for 12 OHR types by Cobas test were considered concordant if the LA provided a HR-HPV genotype (other than HPV 16 and HPV 18), regardless of the number of genotypes detected.The clinical performance of both Cobas and HC2 tests were analyzed by comparing the sensitivity, specificity, positive and negative predictive values of the test for the detection of CIN2+ and CIN3+, respectively. The unconditional logistic regression model was used to calculate the odds ratio(s) (OR) and 95% confidence interval (s) (CI) for the detection rate of the co-testing group compared to cytology alone group. P values less than 0.05 (two‐sided) were considered statistically significant.The present study included 6345 women with valid cytology and HPV (including HC2 and Cobas tests) results. The mean age was 49.33 years (SD 7.78). A total of 6259 (98.64%) women had normal cytology, 48 (0.76%) had ASCUS and 36 (0.57%) and 2 (0.03%) had low-grade and high-grade intraepithelial lesions (LSIL and HSIL). Two hundred and sixty-one and 241 women were referred to colposcopy from intervention and control groups based on the referral criteria of the corresponding groups.p = 0.031), which indicated a moderate agreement. In addition, the Cobas genotyping demonstrated 1.15% (73 of 6345) of HPV16, 0.61% (39 of 6345) of HPV18 and 6.98% (443 of 6345) of 12 OHR HPV positive.Among 6345 women enrolled, Cobas and HC2 tests identified 555 (8.75%) and 506 (7.97%) cervical samples as HR-HPV positive, respectively . Of the p = 1.0 and p = 0.375 in ASCUS and LSIL). Contrary, the significant discrepancy of the two test results was found in women with normal cytology (p = 0.02).The HPV prevalence in different cytology groups showed no significant discrepancy of the two tests in HR-HPV detection in disease groups (p<0.001). Similarly, Concordant Cobas+/HC2+ samples had significantly higher median RLU/CO ratio of HC2 than discordant Cobas-/HC2+ samples . The median Ct value showed the similar patterns when stratified by Cobas genotypes. In addition, the median Ct value of Cobas test in samples of disease groups were significantly lower than samples in normal cytology group , as well as when stratified by HPV16 and 12 OHR, except HPV18. Likewise, the median RLU/CO ratio of HC2 test was significantly higher in disease group than in normal cytology group .The HPV viral load signal was compared in concordant and discordant cases by analysis of the Ct value of Cobas and the RLU/CO ratio of HC2 test . Concordp<0.001). LA test identified LR-HPV genotypes in 24.62% (32 of 130) Cobas-/HC2+ samples, which was significantly more than those detected in discordant Cobas+/HC2- samples . HPV53 and HPV62 were the most commonly found LR-HPV genotypes in discordant Cobas-/HC2+ cases (13 of 23) and Cobas+/HC2- cases (5 of 20), respectively.Linear Array HPV genotyping test was performed on 687 cases to confirm the results of Cobas and HC2 tests. HR-HPV positivity was confirmed in 92.14% (258 of 280) of concordant Cobas+/HC2+ cases . For thoOf the Cobas genotype identified, LA test detected 43.66% (31/71) of HPV16, 56.41% (22/39) of HPV18 and 64.24% (282/439) of 12 OHR HPV positive. The overall genotyping agreement between Cobas and LA were ranged from 73.36% of 12 OHR, 93.89% of HPV16+ to 96.94% of HPV18+. Only two CIN3 cases were found HPV16+ by both Cobas and LA tests, and HPV18 was negative in all CIN2+ cases.p<0.001), indicating a moderate agreement. There was no significant difference in HR-HPV detection between two tests in CIN2+ or CIN3+ samples (p = 0.250 and p = 0.5). The discrepancy of the two tests was found in women with normal histology or low-grade lesions. HC2 test showed more HR-HPV positive cases than Cobas test in these two groups .Totally 502 cases were referred to colposcopy and cervical and endocervical biopsies were taken for histological assessment. The clinical performance of the Cobas and HC2 tests was evaluated on these cases by comparing the abilities of the tests to detect CIN2+ and CIN3+. CIN2+ lesions were identified in 28 women, while CIN3+ lesions were found in 19 women, including one squamous cell carcinoma (SCC). p<0.001, p<0.001). Though HC2 test showed a better sensitivity than Cobas test (92.86% vs 82.14% and 89.47% vs 78.95%), it’s not statistically different (p = 0.083 and p = 0.157). The sensitivity of HPV16 in the detection of CIN2+ and CIN3+ were very low (7.14% and 10.53%) in our study, whereas its specificity was particularly high (96.41% and 96.48%).The agreements between positive Cobas test and CIN2+/CIN3+ were 67.33% and 65.94%, which were significantly better than the agreement between positive HC2 test and CIN2+/CIN3+  in the baseline round screening. On the other hand, the CIN2+ detection was less in co-testing group than in cytology alone group in the second round . Over the two rounds of screening, the total detection of CIN2+/CIN3+ was still higher in co-testing group , though not reaching to statistical significance (p = 0.139 and p = 0.119).The effectiveness of co-testing versus cytology alone in primary cervical cancer screening was analyzed based on 6345 cases in the present comparative study . Both thp<0.001; CIN3+: 0.62% vs 0.20%, OR 3.06, 95% CI 1.78–5.58, p<0.001), leading to the significantly reduction of the CIN2+/CIN3+ detection in subsequent round in the co-testing group . Over two rounds of screening, the cumulative CIN2+/CIN3+ detection was significantly higher in the co-testing groups compared to cytology alone group . Results of RCT study confirmed that the addition of a HR-HPV test to cytology for primary cervical cancer screening led to early detection of clinically significant preinvasive lesions, suggesting that co-testing may be more effective in reducing the risk of cervical cancer than cytology alone. Our result was in agreement with other studies showing that co-testing had a higher sensitivity than cytology alone [The present study was a sub-study of our previous RCT study with two rounds of screening to evaluate the effectiveness of co-testing and cytology alone in cervical cancer screening in a Chinese population cohort . In prevp = 0.083 and p = 0.157). Our results suggested that Cobas test demonstrated a comparable clinical performance to HC2 test in a screening. Similar findings were also observed in other studies [As HPV testing is an increasing important part of cervical cancer screening, effective implementation of HPV test requires comprehensively validations of the test. Present study compared the analytical and clinical performance of Cobas test to HC2 test, a standard HPV test, in a Chinese population-based cohort. Overall, two tests were found comparable with 92.23% (95% CI: 91.57–92.89) agreement. There was no significant difference in identifying HR-HPV in cases associated with diseases between two tests, indicating that both tests were equally good in detecting cervical disease. In addition, the analysis of clinical performance showed that Cobas test had a significantly higher test specificity and better agreement with CIN2+/CIN3+ than HC2 test , while t studies . It is iWe noticed that Cobas and HC2 tests only moderately agreed with each other (Ƙ = 0.493) in our study. There were some factors in the study would interfere the comparison analysis. First, our study was performed in a large population-based screening cohort and the majority of the smear samples were cytological negative (98.64%), which strongly influencing Ƙ-coefficient in statistical analysis , 26. SecIn addition, a higher positive rate of HR-HPV detection was observed in Cobas test than HC2 test (8.75% vs 7.97%) in present study. Our HPV positivity rates in the total study population were similar to the results of the recent Canadian FOCAL study , where HThe added advantage of integrated genotyping of HPV16 and HPV18 in Cobas test has been demonstrated to be able to identify women at high risk of developing high-grade cervical lesions and cancer , 37, 38.The strength of our study is its large sample size, which allowed a statistically powerful and detailed analysis of Cobas performance in comparison to HC2 test. However, our study was limited to a large convenient sample from women initially enrolled in an RCT study, using the leftover ThinPrep specimens collected after cytology and HC2 tests for Cobas and LA tests. As Cobas test was not used for patient management in previous RTC study, the missing histological data in discordant Cobas+/HC2- cases may have impact on the assessment of the sensitivity and specificity of the Cobas test. Another limitation of the present study was inherited from the previous RCT study, the study cohort was a highly screened population, leading to a low incidence of high-grade lesions. This may also constrain the present comparison study.Our large comparison study demonstrated that addition of a HR-HPV test to cytology for primary cervical screening may be more effective in reducing CIN2+ risk than cytology alone. The Cobas test demonstrated significantly higher specificity in identifying CIN2+/CIN3+ cases than HC2 test with comparable sensitivity of both tests in clinical evaluation. Due to the study limitations, the added benefit of HPV16 and HPV18 genotyping of Cobas test in clinical performance has not be acknowledged and need to be further evaluated in a screening cohort with increasing number of high-grade lesions.S1 Checklist(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.


Lactobacillus abundance: Lactobacilli-dominated (LD) and non-Lactobacilli-dominated (NLD) groups. The LD group had significantly less bacterial diversity than the NLD group. In addition, the variety of bacteria was contingent on the prevalence of HPV infection. Among distinct histological groups, an abundance of L. iners  was discovered in both groups. A few fungi, e.g., C. albicans, were identified in the fungal community. The viral community analysis revealed that the presence of HPV considerably reduced the diversity of human viruses. Taken together, when we analyzed all our results collectively, we discovered that HPV infection was a significant determinant in the diversity of bacteria and human viruses in the cervix.Human papillomavirus type 16 (HPV16) and/or high-risk (Hr-) HPV are the main causes of cervical cancer. Another element that may contribute to the development of cervical cancer is the microbiota. To date, no study has investigated the entire cervical microbiome, which consists of bacteria, fungi, and viruses. In this study, cervical samples with different histopathology , with or without HPV16 and Hr-HPVs infection, were enrolled. From bacterial community analysis, 115 bacterial species were found and separated into 2 distinct categories based on  Cervical cancer is one of the most prevalent malignancies in women worldwide and the second most prevalent disease in emerging nations . More thPapillomaviridae family, which is currently split into 16 genera, of which 5 genera contain members that infect humans, namely Alpha-, Beta-, Gamma-, Mupa-, and Nupapapillomavirus [HPV is a member of the omavirus . Based oomavirus , while Lomavirus ; neverthomavirus ,11. Lactobacilli are believed to have a crucial function in vaginal health [Lactobacillus are often the most common microorganisms found in a healthy vagina [Lactobacillus spp., namely CST I (dominated by L. crispatus), CST II (dominated by L. gasseri), CST III (dominated by L. iners), CST IV -related bacteria), and CST V (dominated by L. jensenii) [Lactobacillus spp. promote a protective environment in the vagina by producing metabolites, bacteriocins, hydrogen peroxide (H2O2), and lactic acid, and by lowering the pH by producing hydrogen peroxide [Candida albicans. Typically, this C. albicans acts as an opportunistic pathogen [Although HPV infection is the primary cause of cervical cancer, environmental factors may also play a significant part in the disease’s progression. The International Agency for Research on Cancer (IARC) identified smoking as a co-factor of cervical cancer in 2004 ,13. Othel health . One or y vagina ,17. The ensenii) . These Lperoxide ,19,20,21pathogen . Neverthpathogen ,23,24.Malassezia sp. [Papillomaviridae, Anelloviridae, Genomoviridae, and Herpesviridae families [A few studies have detected, in addition to vaginal microbiota, the microbiota in the cervix where HPV infection occurs. Klein et al.  demonstrezia sp. . Virusesezia sp. . Severalezia sp. . Moreovefamilies . IntriguThe specimens were taken by colposcopy from patients who visited the Department of Gynecological Outpatient at King Chulalongkorn Memorial Hospital in Bangkok, Thailand. During a colposcopy, a gentle swab at the posterior fornix of the cervix using an Ayre Spatula was performed by a gynecologist. The swab was resuspended in lysis buffer and the sample was then rapidly stored at −80 °C until use. The Cobas 4800 high-risk HPV test identified the presence of HPV16, 18, and Hr-HPVs DNA in all samples at the Virology Unit, Department of Microbiology, King Chulalongkorn Memorial Hospital in Bangkok, Thailand . Blood-free samples with either HPV16 or Hr-HPVs positivity and reproductive-age women (21–50 years old) with histopathology results were the inclusion criteria for the selected samples . Basically, the classification of cervical intraepithelial neoplasia (CIN) ranges from 1 to 3. CIN1 refers to abnormal cells that compose around one-third of the epithelium’s thickness. Meanwhile, about two-thirds and more than two-thirds of the epithelium thickness are abnormal cells, known as CIN2 and CIN3, respectively. Exclusion criteria were menstruation at the time of specimen collection, antibiotic or vaginal antimicrobial usage during the previous month, current microbiological infection, HIV infection, and pregnancy. Only 43 of the remaining HPV-positive specimens met these requirements. Moreover, five CIN1 with HPV-negative samples were selected as a control group.The COBAS 4800 instrument automatically extracted total nucleic acid from the remaining cervical specimens . Prior to DNA extraction, the sample was beaten at 30 Hz for 10 min using Pathogen Lysis Tubes L  and TissueLyser LT  [The Swift amplicon 16S + ITS panel amplified the 16S rRNA of bacteria and ITS gene of fungus . This kit included a single pool of primers for all variable sections of the 16S rRNA gene (V1–V9) plus the fungal ITS1 and ITS2 genes. Based on a library size of 475 bp, the libraries were quantified using the KAPA library quantification kit .Before sequencing, libraries were denatured and diluted in accordance with the instructions for the MiSeq System . The library was adjusted to 11 pM by HT1 and placed onto the MiSq Reagent Kit v2 reagent cartridge . On an Illumina MiSeq platform, sequencing was then performed to generate two 151 bp reads.The Illumina Miseq FASTQ data were exported for the bacterial analysis. The Microbial Genomics Module of QIAGEN CLC Genomics Workbench 20 (version 20.0.4) was used to examine the sequences. The database utilized was Greengenes v13.8. At least 10 distinct reads were valid. In the case of any atypical microorganism, the sequence reads were reanalyzed by BLAST against the NCBI taxonomy database using 16S ribosomal RNA sequences (Bacteria and Archea) as the database. At least 97% and 99% sequence identity were acceptable for genus- and species-level identifications, respectively . The datThe cDNA and the second strand were generated from the extracted total nucleic acid to recover RNA viruses from samples. cDNA was produced using Roche Evoscript Universal cDNA Master and random hexamers from an RNA template . Second-strand buffer (10× NEB Buffer 2), 0.7 mM dNTP mix, 0.18 U/L RNaseH, 0.7 U/L DNA polymerase I, and 1.8 mM DTT were then utilized to synthesize the second strand of cDNA .Slight modifications were made to the SeqCap EZ Hypercap Workflow Version 2.3  recommendations to prepare the gDNA collection. Each reagent was acquired from Roche . gDNA was sheared enzymatically with the KAPA HyperPlus Library Preparation Kit. In brief, the gDNA was combined with KAPA Frag Buffer/Enzyme and incubated at 37 °C for 15 min to obtain an average fragment size of 200 bp. NanoDrop  was used to ensure the quality and yield of the above libraries. The Fragment Analyzer 3700 with PRO Size 3.0 software  was utilized to examine the size distribution, which must be between 150 and 500 bp with a peak of 320 bp.The samples were hybridized with the SeqCap EZ probe pool (VirCapSeq-VERT Capture Panel), which contains 2.1 million probes (varying lengths of 50–105 mers) encompassing the genomes of 207 viral species . NanoDroThe libraries were quantitated by the KAPA library quantification kit. The sample preparation was carried out by using the denature and dilute libraries guide for the MiSeq System  using the MiSeq Reagent Kit v3 . The procedure followed the reagent leaflet. The library was adjusted to 18 pM by HT1 and loaded onto the reagent cartridge for sequencing using an Illumina MiSeq platform to generate 2 × 300 bp reads.ftp://ftp.ncbi.nih.gov/refseq/release/viral/ (accessed on 11 October 2020).The NGS raw FASTQ files were explored for the presence of viruses in each sample. The Virus Identification Pipeline (VIP) was run on Docker and Ubuntu 20.04 LTS and was performed by using sense mode . In the t-test (Mann–Whitney test) was performed, whereas PERMANOVA was used for beta diversity analysis. A Chi-square test was used to calculate the similarity of percent ubiquity among samples.The hierarchical clustering tree was generated using UPGMA and Euclidean as the algorithm and similarity index, respectively, via Qiagen CLC Genomics Workbench 20 (Version 20.0.4) using the Microbial Genomics Module  and Past 4.03 software . The heat map was constructed by CLC software  and GraphPad Prism 8 software . The alpha  and beta diversity  based on Bray–Curtis dissimilarities of the microbial community structure) were calculated by CLC , ITS Metagenomics Module , and Past 4.03 software , whereas the boxplots, rarefaction curve, bar chart, and pie chart were constructed by GraphPad Prism 8 software. To observe the statistical difference between the alpha diversity and richness, a non-parametric unpaired p = 0.1688, CIN1 HPV-negative vs. CIN1; p = 0.2701, CIN1 HPV-negative vs. CIN2/3; p = 0.5187, CIN1 vs. CIN2/3; Mann–Whitney U-test). The peripheral clinical data of the patients are shown in A total of 48 (43 HPV-positive and 5 HPV-negative) leftover cervical samples were collected. They were classified according on the results of histology, i.e., cervical intraepithelial neoplasia (CIN) type 1 and CIN2/3 . Due to Lactobacillus spp., Gardnerella sp., Bombiscardovia sp., Prevotella sp., and Shuttleworthia sp. were the five taxa that predominated in the cervical samples (n = 27) and non-lactobacilli-dominated  cervical microbiota groups were identified based on the 60% abundance of Lactobacillus spp. [Lactobacillus spp. was 97% and 6%, respectively  high-quality reads were retrieved with an average of 80,723 per sample. One hundred and fifteen species of bacteria were found in cervical samples.  samples . Lactobalus spp. . In the ectively A. The LDrevalent B. In bot for NLD . The alp for NLD . Similar for NLD E.In this investigation, samples were separated into three categories: CIN1 HPV-negative, CIN1, and CIN2/3. CIN1 HPV-negative (40.00%) and CIN1 (40.091%) had comparable percentages of NLD samples, however CIN2/3 had a slightly greater percentage of NLD samples (45%) A. The alAtopobium sp., Bifidobacterium sp., Bombiscadovia sp., Gardnerella sp., Prevotella sp., Sneathia sp., and Lactobacillus spp.  high-quality reads were retrieved. Using VIP, 1.5 M  viral reads were subtracted [Lactobacillus spp. dominance and the viral community, alpha and beta diversity were computed. The viral diversity between LD and NLD groups did not differ significantly  were shared by all histological categories E, howeveL. iners was the most prevalent bacterial taxon in all groups (L. iners was discovered in the CIN1 HPV-negative group (100%), its relative abundance was higher in CIN1 (43.78%) and CIN2/3 (30.54%) than in CIN1 HPV-negative (21.33%). Gardnerella sp. was the second most common bacterial taxa in all groups. C. albicans was absent in the CIN1 HPV-negative group, although it was present in the CIN1 and CIN2/3 groups. As expected, the HPV16 abundance was the most predominant virus in all groups. However, the relative abundance of HPV16 in CIN1 HPV-negative samples was low (1.93%) compared to HPV-positive samples . Besides HPV16, other viruses were discovered. Notably, MCV was found in all samples -associated bacterial species [Lactobacillus spp. acidic pH generation hindered the colonization of harmful bacterial species, such as Chlamydia trachomatis and Gardnerella vaginalis [Despite that the vaginal microbiome has been demonstrated for several years, the microbial composition of the vagina has been found to differ between ethnic groups ,23,24. B species . Lactobaaginalis ,40. Thisaginalis . Lactobacillus spp., the presence of HPV also affected bacterial diversity , which is known to have antibacterial and antiviral activities [L. iners, had a less acidic pH, but Lactobacillus spp. in CSTs I, II, and V were able to produce more acidic pH, which significantly decreased the BV rate [L. iners in illness progression remains unclear, as it was detected in both normal and pathological settings, as demonstrated by the LD group   J,K. Parval flora . To datebacteria . This su with BV . TogetheC. albicans, an opportunistic fungal pathogen, colonized 20% of women without causing any clinical manifestations in many healthy women [Saccharomyces sp. infection caused vaginitis, but most of the cases were caused by Saccharomyces cerevisiae [Saccharomyces cariocanus and Densospora sp. have never been isolated from the vagina or cervix. The role of Saccharomyces cariocanus in vaginitis should be further explored.For fungal community, a previous study from Spain demonstrated that the Shannon diversity of fungi in the cervicovaginal area was statistically significantly higher in atypical squamous cell undetermined significant (ASCUS) when compared to the negative for squamous intraepithelial lesion (NSIL) and high-grade squamous intraepithelial lesion (HGSIL) . Howeverhy women . Saccharrevisiae . InteresLactobacillus spp., have a great potential to control cervical illnesses, further research is required [Lactobacillus spp. did not influence viral diversity  and Human parainfluenza virus 1, were found in all histological groups E. We hyp illness . Recentl illness . In addi illness . A membepatients .Surprisingly, a few HPV16 reads (relative abundance < 0.1%) were found in the CIN1 HPV-negative group F. At theLactobacillus spp. played a significant role in bacterial diversity, while HPV infection played a crucial role in both bacterial and human viral diversity. The correlation of several microorganisms with HPV infection and the severity of dysplasia may be advantageous for the use of microbial communities as diagnostic tools. However, the mode of transmission and function in pathogenesis of many microorganisms, particularly viruses, remain obscure and require further investigation. In addition, our research revealed the presence of non-human viruses in the cervix. Although a small sample size of the CIN1 HPV-negative group was recruited, the statistical analysis of the Shannon index (p > 0.05), representing a normal population. Thus, all statistically significant analyses in the comparative analysis of the microbiome were reliable. However, according to the limitation of the small sample size, further research is required to confirm and validate these results. More clinical samples should be necessary for further analysis. Additionally, samples of cervical cancer must be included.Our study shed new light on the modifications of the cervical bacterial, fungal, and viral communities in HPV-infected patients with varying degrees of dysplasia severity. We demonstrated that on index  obtained


Ethical discourse on prognosis disclosure is not yet well established. The core of the problem continues to be the dilemma between the right of self-determination and non-maleficence of patients. The prognosis disclosure policy based on Kantian autonomy provides a good solution for the problem. The policy includes demand for strict truth telling and its compatibility with patients’ best interest. However, there remains a discrepancy between theory and practice, especially when prognosis is disclosed just prior to their death. Kantian theory of prognosis is supplemented by a moralistic perspective. The moralistic perspective places high importance on temporality and relationships with others, which all human beings inherently possess. From the moralistic viewpoint, decisions about prognosis disclosure at the final stages of life must be individualized in order to be authentically autonomous. The decision to disclose a prognosis or not can only be determined by the relationships fostered over time with patients. One of the classic conundrums in biomedical ethics remains truth telling in the medical setting. Although ethical positions on diagnosis disclosure have converged somewhat, the literature on practices around disclosure of prognosis in cancer patients documents a variety of approaches, highlighting continued ethical questions ,2,3,4. SA notable gap exists between patient attitudes regarding prognosis disclosure and actual prognosis disclosure . On the The depth of the ethical discourse on prognosis disclosure remains insufficient . OncologNotably, prognosis disclosure opens up the possibility that a patient will experience psychological distress. Studies have identified cultural differences in prognosis disclosure, particularly with regard to how much consideration is given to the principle of non-maleficence when physicians withhold information. For example, O’Kelly found that physicians in the Middle East and Asia tend to withhold diagnoses and prognoses, in accordance with the ‘do no harm’ consideration toward patients . Thus, wThe argument for nondisclosure on the basis of respecting the right of self-determination and avoiding non-maleficence in prognosis disclosure contains a dilemma. First, if patients desire prognosis disclosure that would likely cause psychological pain, this is problematic; a typical example of this is when the physician’s prognosis is less optimistic than the patient’s expectations. Second, when patients refuse prognosis disclosure based on their right not to know, this can be problematic as well, as prognosis disclosure is often required for good outcomes in clinical practice. This is true for cases in which disclosing the prognosis leads to the provision of appropriate palliative care. Unfortunately, prognosis disclosure strategies based solely on self-decision making can neither address nor resolve these two challenges.Stahl and Tomlinson (2017) characterize respect for autonomy as “the capacity of people to make considered choices in light of the facts in order to achieve their values and goals, not merely their expression of a preference .” PatienThe Kantian approach to autonomy resolves the perceived conflict between respect for the right of self-determination (the right to refuse information) and the principle of non-maleficence (the obligation to avoid harm in the disclosure). Stahl and Tomlinson (2017) further argue that the Kantian approach to autonomy resolves the conflict between the right of self-determination (the right to refuse information) and the principle of beneficence (promotion of patient well-being) . The rigAccording to this theory, prognosis disclosure is desirable because it helps patients make deliberative choices about their own treatment, which promotes the right of self-determination. This type of rational choice also seems to accommodate the best interest of patients. In fact, healthcare professionals failing to share prognoses with their patients can result in continuation of futile treatment and delays in receiving palliative care , and endRealizing the value of the Stahl and Tomlinson’s (2017) ideological and intellectualistic analysis of the prognostic disclosure policy based on Kantian autonomy requires a consideration of temporality and the patient’s relationships with others. Even if the prognostic disclosure policy based on Kantian autonomy could theoretically avoid the two dilemmas, it creates another dilemma, i.e., that few people can actually implement it. This new dilemma is revealed in prognosis disclosure just before death.The Kantian autonomy-based prognosis disclosure policy places a great deal of value on loyalty to truth. Accordingly, even if a patient likely has only 24 h left to live, this policy would have physicians communicating this to their patients once they recognize it. Admittedly, many patients would have already lost consciousness 24 h from death, but this is not the case for all, and some patients remain conscious until the moment of death. Vince and Petros (2006) use the example of an end-stage pediatric patient whose death prognosis is given after the patient required ventilation and sedation due to severe lung disease . Some ofAs conceptualized, the prognosis disclosure policy based on Kantian autonomy lacks consideration of both temporality and relationships. With regard to prognosis disclosure, consideration of human temporality according to disease progression is essential. For example, for a patient with recurrent cancer, the timing of prognosis disclosure can be divided into three stages depending on the clinical turning point. The first stage is when cancer recurrence is diagnosed and treatment is initiated (prognosis in years), the second is when active treatment methods have been exhausted (prognosis in months), and the third is when death is imminent (prognosis in days). We suggest the prognosis disclosure policy based on Kantian autonomy is effective only up to the second stage; by the third stage, the dilemma of prognosis disclosure becomes apparent. One could argue the Kantian justification for prognosis disclosure is no longer useful when there are no viable treatment options and death is imminent. A reason is needed in order for the prognosis to be disclosed in the third stage . One reason for prognosis disclosure just before death is that the patient wants to know. Other justification may be possible, e.g., if disclosure contributes to the achievement of expressed goals of the patient or is in concordance with the patient’s values. Regardless of whether a patient has a few days left in their life, or even 24 h left, they may be able to accomplish matters of importance in the time they have remaining. For example, patients may be able to convey important information to family members, as a will of sorts. They may also be able to express appreciation to those who have taken care of them in their lives.The third stage  is more complicated. Family members and healthcare professionals must interpret patient statements in the context of the patient’s whole life. Although it conflicts with the prognosis disclosure policy based on Kantian autonomy, it may be acceptable to withhold prognosis disclosure during this stage, as was decided in the pediatric case presented by Vince and Petros (2006) . We exteTo complement the prognosis disclosure policy based on Kantian autonomy, we present the moralistic policy for prognosis disclosure. We suggest it as fundamental principles of prognosis disclosure. The moralistic policy for prognosis disclosure is compatible with prognosis disclosure based on Kantian autonomy up through the second stage. However, in the third stage, we would argue that a moralistic policy for prognosis disclosure replaces Kantian autonomy-based prognosis disclosure. To add to the plurality of fundamental principles, we maintain that there is no sole fundamental principle in prognosis disclosure, but rather a combination of competing principles, which may or may not coincide at times along the clinical timescales. The situation changes every moment, depending on the timescale. Depending on the combination of psychosocial and contextual factors in a real-world ethical situation, multiple principles may exert different moral weights. Prognosis disclosure based on Kantian autonomy is a theory that is too universal and intellectualistic to be of practical use, especially in the third stage. Some humans are ambivalent about knowing when they will die. Some believe a person can live a more meaningful life by adjusting the purpose of their life and the means of achieving it if they know the approximate time of their inevitable death. Some fear facing death and superimpose the possibility of achieving one’s life purposes on the uncertainty of the future. The ambivalence and variety of perspective are crucial details about human nature that must be acknowledged in order to have a meaningful discussion about prognosis disclosure in the medical setting. Prognosis disclosure based on Kantian autonomy misunderstands or overlooks this aspect of human nature.In contrast, moralistic policy for prognosis disclosure is situation-dependent and individualistic. François de La Rochefoucauld, a French moralist, is famous for his maxim, “neither the sun nor death can be looked at steadily” . The morFor the community of patients, family members, and physicians affected by this, it may be cruel to inform patients explicitly that they will die in the next 24 h. It has traditionally been explained that prognosis disclosure just before death is unlikely to increase significantly the patient’s remaining quality of life; it also seems cruel because it may cause pain to patients with insufficient time to mitigate that pain. The prognosis disclosure policy based on Kantian autonomy values the truth that lies beyond that pain. However, the moralistic point of view that we present in this paper is different from both the Kantian account and the traditional account; in the latter, voluntary self-decision making potentially conflicts with non-maleficence toward patients. Truth is certainly important; however, in prognosis disclosure during the third stage (just before death), the value of truth telling must be congruent with a firm consideration of the patient’s values and humanity. It does not mean only considering the psychological pain (of the patients themselves). Rather, the root of the cruelty in prognosis disclosure lies in the fact that patients themselves may be alienated from the network of relationships, such as those with family members and healthcare professionals. Relationships and historicity are essential to the person, and prognosis disclosure must accommodate the individual patient; decisions must be informed by a patient-specific narrative. The 24 h prognosis disclosure may disrupt these relationship and historicity, yielding a feeling of cruelty. If the historicity of this network of relationships is not taken into account and prognosis disclosure is based on mere (Kantianistic) ideology, it would mean the collapse of this network of relationships. Historicity in this context is the accumulation of time shared by the patient, the family, and the health care professionals who have provided care, and the narratives developed over that time. Historicity shapes the patient’s personality in the network of relationships. Without respect for this historicity, any decision about the patient is meaningless. To disrupt this network of relationships and send the patient off into another world alone would cause the patient emotional distress, but more than that, it would be cruel in the sense that the patient’s historic personality would be disregarded. In order to avoid this cruelty, it would be necessary to adopt a flexible approach that emphasizes the values of patients at the forefront.The ethical discourse on prognosis disclosure is not yet well established. Although the West and East show cultural differences in prognosis disclosure, particularly with regard to patient awareness, they share in common the acknowledgment that prognoses are not actually disclosed enough in advance to satisfy patient wishes. The prognosis disclosure policy based on Kantian autonomy demands truth telling under all circumstances. However, the application of the Kantian point of view must take into account the clinical timescale specific to the patient. Certainly, the introduction of the Kantian point of view is advantageous when the prognosis is expected to remain somewhat predictable (first and second stage). However, we must hesitate to introduce the Kantian point of view in the just before death (third stage). That is because the intellectualistic view of humans that underlies this policy impairs the social relationships patients have with those around them. All human beings inherently possess temporality and relationships with others. Relationships between patients, family members, and healthcare professionals, which have been formed uniquely and concretely until the patient approaches their end of life, share this temporality, and the patient’s values are formed by such relationships. We call this the moralistic perspective. Prognosis disclosure must always be unique in order for the patients living in the final stages of their life to be authentically autonomous. In other words, the decision to disclose a prognosis or not can only be determined by the relationships fostered over time with the patients. One significance of the present study is its demonstration of how to balance a theory and a practice pertaining to the ethical discourse on prognosis disclosure. The policy based on Kantian autonomy presented by Stahl and Tomlinson (2017) represents groundbreaking ethical discourse on prognosis disclosure. This paper supplements their theory by adding a moralistic perspective that accommodates the clinical stage of the patient prior to death, allowing the ethical discourse on prognosis disclosure to be taken a step further.


The twisted bundled-tube locomotive device is an elongated soft robot that moves inside a pipe in a helical bending motion. This motion mimics the behavior of microorganisms called spirochetes. This device is inexpensive and easy to miniaturize because of its simple structure, which consists of three inflatable tubes twisted together. It can move in pipes of various diameters without a change in design. Therefore, it has a high capacity for water pipe inspection. However, it has not yet been shown to pass through step parts wherein the diameter of the pipes decreases. In this study, we developed a device that was deformed into a tapered shape by changing the pitch of the spirals at each location. The prototype device was able to move from a pipe with an inside diameter of 52.9 mm to a pipe with an inside diameter of 21.6 mm for horizontally fixed pipes, and from a pipe with an inside diameter of 41.6 mm to a pipe with an inside diameter of 21.6 mm for vertically fixed pipes. Although snakes have a simple structure with a cord-like body, they can move on uneven ground. They also sometimes use their bodies dexterously to perform various actions such as climbing trees. Therefore, despite their simple structure, many robots that mimic snake behavior have been studied due to the fact that various movements are possible ,4,5,6,7.In-pipe robots that are usually proposed can be categorized into wheel, screw, worm-like, and snake-like robots, depending on their movement method. Their characteristics are summarized in Wheel-type robots have the advantages of high traveling accuracy and speed . HoweverTo overcome these problems, we propose a twisted bundled-tube locomotive device. As shown in Because a non-stretchable axis exists in the center of the device, it can be equipped with electric cables. Thus, a camera can be attached to function as an endoscope. Moreover, this device has the same cross-section as the axis. Therefore, mass production by extrusion is possible . It has r, and the angle of the spiral of the silicone tube around the body axis is r and Consider a device model with one helix cycle that focuses on the body axis and one of the three silicone tubes that helically wrap around the body axis, as shown in Based on this relationship, the device model is then developed into a plane in d is the diameter of the silicone tube, and D is the diameter of the helix in the center of the silicone tube located outside after deformation. From r and The deformation of the device can be analyzed based on geometric relationships. Initially, Equation  is validEquation . EquatioEquation .(3)L1=PD yields Equations ..D yieldsThe radial expansion of the silicone tube is considered here. The radial expansion rate is Equation .(10)A=DThe relationship between Equation  25].. Fi. Fiα = 1To pass through the step part, we propose a device that deforms into a tapered shape that is thicker at the root and thinner toward the tip when air pressure is applied, as shown in In Japan, the 20A pipe is widely used for water pipes, with the designation A being the nominal diameter of a water pipe, as defined by the Japanese Industrial Standard (JIS). The inside diameter of a 20A pipe is 21.6 mm. Considering the deformation of the device, the device was designed such that its diameter is approximately half of the inside diameter of a 20A pipe. Therefore, silicone tubes with an outer diameter of 6 mm were used to fabricate it. A commercial silicone tube is not designed to be inflated and can only be inflated at high pressure. In addition, it does not return to its original size. Therefore, soft silicone tubes have been fabricated. Molds and caps were used, as shown in For comparison, an experiment was conducted using a device with a constant initial pitch. From the analysis results, the relationship between quations –11), a , a α andThe device was driven by applying air pressure using three solenoid valves  driven by a USB I/O Terminal . The period, duty ratio, and strength of the air pressure were controlled. The period spans the time from the start of pressurizing a tube to the start of pressurizing it again after depressurizing it (indicated by T), as shown in Next, the period was verified by simultaneously changing the duty ratio and the period such that the time during which the two tubes were simultaneously pressurized was within this range. If the period is more than 700ms with a duty ratio of 0.47, the period is sufficiently long to expand the silicone tube because there is a moment when the tube is sufficiently inflated and the device stops moving, as shown in Finally, air pressure was confirmed. The expansion rate of the tube depends on the strength of the air pressure. The constructed device was gradually pressurized, and when the pressure reached 0.14 MPa, the device was deformed and acquired the same outer diameter as the inner diameter of the 50A pipe, as shown in A device was designed with the proposed tip geometry to pass from a 50A to a 20A. As before, the initial pitch at the root was designed to be 320 mm, assuming that it was driven with an Experiments were performed using the pipe used in the previous experiment. The pipe was fixed horizontally. The device fabricated in The pipe used in the previous experiment was fixed vertically. The device used in the previous experiment was driven within a period of 700 ms and at a duty ratio of 0.47. When the device was driven by an air pressure of 0.14 MPa, the device remained stationary inside a pipe, as shown in A transparent pipe with the same inner diameter as that of the 40A was manufactured and connected to a pipe with the same inner diameter as that of the 20A, such that their centers coincided and were fixed vertically. When the device was driven by an air pressure of 0.14 MPa, it remained stationary inside the pipe, as shown in First, the minimum and maximum pipe diameter within which the device can move at an air pressure of 0.14 MPa is discussed. When pressure is applied to the silicone tube, the device expands both axially and radially. From the relationship between Equation , the bodIn an experiment using a horizontally fixed pipe, the device moved when driven at 0.14 MPa, but not at 0.10 MPa and 0.15 MPa. Based on these results, the effect of air pressure strength on the device is discussed. We also discuss the velocity of the device. From the experimental video shown in In an experiment using a vertically fixed pipe, the device that was meant to move within the 50A pipe failed to achieve this. Because the device could remain stationary in the pipe, it was deformed and acquired a diameter of 52.9 mm, as designed. The slippage and fall during the drive can be attributed to the length of the device. The device has a short contact length with the inside of the pipe owing to the small diameter of the tip, which results in a low gripping force on the pipe. Increasing the length of the device and the length that contributes to its sticking allows it to move through the pipe without slipping.Finally, we verified whether it was possible for the device to move inside a vertically fixed pipe that had the same diameter as that of the 50A pipe by using a device with a uniform initial pitch of 320 mm. When the device was driven by an air pressure of 0.17 MPa and 0.18 MPa, it sometimes moved upward in the pipe. However, it slid inside the pipe, as shown in The purpose of this study was to develop a twisted bundled-tube locomotive device capable of moving over a step. A new tip shape and method for gradually changing the initial pitch were proposed. In the experiments using a horizontally fixed pipe, the device moved from 50A to 20A when driven at 0.14 MPa. The same result was obtained even after the experiment was repeated. In the experiments using a vertically fixed pipe, the device moved from 40A to 20A when driven at 0.15 MPa. However, the device could not move inside the 50A even when a uniform device whose gripping force was larger than that of tapered devices was used. In the future, we will attempt to move this device through a pipe with a larger diameter difference. Therefore, we will improve the tubes that can be applied with greater pressure and make a device with a larger initial pitch in order to achieve a more considerable deformation. Moreover, we will try to make the device with an extrusion method in order to enable mass production. The proposed method required a different pitch at the front tip. It is necessary to develop methods that can make different pitch areas by extrusion.


Human papillomavirus (HPV)-associated cancers, such as anal cancer, are a major risk among people living with HIV (PLWH). High-risk (HR) HPVs are the causal agent of anal precancer and cancer. However, there is a paucity of data regarding the HPV genotypes that cause especially anal cancers and pre-cancers in PLWH. In this study we characterize the specific HPV types in anal tissues, including benign, pre-cancer, and cancer samples from PLWH, and compare them to similar samples from HIV-negative individuals. The results from our study suggest that a broader range of HPV types may play a role in anal cancer in PLWH than in HIV-negative individuals. Proposed screening approaches that include HPV testing might need to differ by HIV status, with extended HPV genotyping included for PLWH.The incidence of anal cancer is increasing, especially in high-risk groups, such as PLWH. HPV 16, a high-risk (HR) HPV genotype, is the most common genotype in anal high-grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) in the general population. However, few studies have described the distribution of HR HPV genotypes other than HPV 16 in the anus of PLWH. HPV genotyping was performed by DNA amplification followed by dot-blot hybridization to identify the HR and low-risk (LR) genotypes in benign anal lesions (n = 34), HSIL (n = 30), and SCC (n = 51) of PLWH and HIV-negative individuals. HPV 16 was the most prominent HR HPV identified, but it was less common in HSIL and SCC from PLWH compared with HIV-negative individuals, and other non-HPV 16 HR HPV (non-16 HR HPV) types were more prevalent in samples from PLWH. A higher proportion of clinically normal tissues from PLWH were positive for one or more HPV genotypes. Multiple HPV infection was a hallmark feature for all tissues  of PLWH. These results indicate that the development of anal screening approaches based on HPV DNA testing need to include non-16 HR HPVs along with HPV 16, especially for PLWH. Along with anal cytology, these updated screening approaches may help to identify and prevent anal disease progression in PLWH. Human papillomaviruses (HPVs) comprise a diverse group of DNA viruses that have different epithelial tropisms. HPV primarily infects squamous epithelia, including keratinized and non-keratinized skin and mucosa of the anogenital region and oropharynx; some glandular epithelia can also be infected with HPV—e.g., endocervical mucosa . Most HPThe incidence of anal carcinoma in both men and women in the United States has been increasing over the years. The American Cancer Society estimated that in 2022, there would be 9440 new cases (3150 in men and 6290 in women) and 1670 deaths (930 in women and 740 in men)  (p = 0.0015) and in the presence of HIV , and that HIV infection did not elevate the risk of anal cancer. To assess the role of non-16 HR HPV types in HPV-16-associated anal disease (p < 0.0001)—slightly less strongly than infection with HPV 16 alone . The effect of co-infection with both high-risk types was intermediate between the solo effects . The risk of anal disease associated with HPV 16 was much stronger when adjusted for the influence of non-16 HR HPV types (Model 3) than its unadjusted effect (Model 1).The prevalence of anal disease (HSIL/SCC) is strongly associated with HPV 16  [risk ratio (RR) = 2.23   0.0001] , Model 1 0.0001] , Model 2 disease , Model 3 disease , Model 3The aim of this study was to perform a detailed analysis of HPV genotypes found in benign, anal HSIL, and anal SCC of both PLWH and HIV-negative individuals. Understanding the distribution of different HR HPV genotypes in the spectrum of anal disease, especially in PLWH, is imperative as they are at increased risk of anal cancer.Compared with samples collected from HIV-negative participants, four distinctive features were observed in samples collected from PLWH: (1) a lower proportion of anal HSIL and SCC that contain HPV 16 alone; (2) a higher proportion of clinically normal tissues containing HPV; (3) infection with a wider spectrum of HPV genotypes both HR and LR; and (4) an overall higher proportion of infection with multiple HPV typesThe frequency of multiple infections  was higher in benign, HSIL, and SCC samples from PLWH compared with samples from HIV-negative participants. Higher rate of infection with more than one HR type was also found in anal cancer samples from PLWH (19.2%) versus HIV-negative anal cancers (8%). The number of LR genotypes identified in combination with HR types in samples  from PLWH was at least 5-fold to 8-fold higher than samples from HIV-negative participants. A recent study of HPV genotype distribution in MSM obtained similar results, i.e., anal infection by multiple HR HPV genotypes was higher in HIV-infected participants compared to their HIV-uninfected peers . It has In this study, HPV 16 was the predominant HR HPV type found in anal HSIL and anal SCC irrespective of HIV status. Our results are similar to previous studies that have shown that HPV 16 is the most important HR type associated with anal carcinogenesis in both PLWH and HIV-negative individuals. Therefore, HPV 16 is a clear target for anal cancer prevention for both these groups ,27. HoweHPV 16 infection was found either as a single infection or in combination with other HR and LR types (multiple infection). However, the frequency of single infection with HPV 16 was higher in samples from HIV-negative individuals versus PLWH. A recent meta-analysis consisting of 95 studies showed that HPV 16 is more common in single infection in HIV-negative anal cancers and its prevalence is lower in HIV-positive anal cancers . SimilarConsistent with the above, we found that the frequency of non-16 HR HPV types were higher in HSIL and SCC from PLWH versus HIV-negative patients. Therefore, non-16 HR HPV genotypes are more strongly affected by immunological deficiency than HPV 16, as has been reported by others ,35.Due to the limited number of samples in our study, we analyzed the overall prevalence of anal disease (HSIL/SCC) between PLWH and HIV-negative participants. We found that HPV 16 was strongly associated with anal disease. This association of HPV 16 with anal disease was not affected by HIV serostatus. In fact, non-16 HR HPV in absence of HPV 16 was also associated with anal disease.The recent findings of the ANCHOR trial support the inclusion of routine screening in the detection and treatment of anal HSIL, as part of anal cancer prevention for PLWH . OptimalRecent studies suggest that HPV latency may be more common than previously thought ,37. We fOur study has some limitations. The sample size is small, which limits our ability to analyze the association between the risk factors HIV and HPV genotypes at the subgroup level  between PLWH and HIV-negative participants. Secondly, due to the cross-sectional study design, the causal relationship between the presence of a particular HPV genotype and the development of lesions cannot be established. Due to the small sample size, potentially important confounders, such as age and sex, were not adjusted for in the model and could impact the results. The results of this study need to be confirmed in a larger study. Despite these limitations, the current analyses provide valuable data regarding differences in HPV type distribution in the anus of PLWH and HIV-negative individuals.In summary, we have shown that HPV 16 is the most prevalent HPV genotype in both anal HSIL and SCC irrespective of HIV serostatus. Anal HSIL and SCC in PLWH was frequently associated with infection with multiple HPV types. The increased detection of non-16 HR HPV types in PLWH underscores the need to include these genotypes in anal screening programs. Combined with anal cytology, these approaches to anal HPV type detection will need to be validated in prospective studies of PLWH and HIV-negative individuals. The detection of HPV in a high proportion of benign tissues, particularly in PLWH, is consistent with a latency state for HPV, but the clinical significance of this finding also needs further study.


Dendritic cells (DCs) are specialized antigen-presenting cells that play a critical role in the immune response against human papillomavirus (HPV) infection, and represent a therapeutic target in cancer.To identify and quantify DCs in tonsillar squamous cell carcinoma (TSCC) under the influence of HPV infection.CD1a and CD83 antibodies were used to identify immature dendritic cells and mature dendritic cells by immunohistochemistry in 33 primary TSCC and 10 normal tonsils (NTs), respectively. For the TSCC samples, the number of DCs per area was evaluated in the intra- and peritumoral compartments. For the NTs, the quantification of DCs was evaluated in the intra- and peritonsillar compartments. HPV detection methods were determined according to the ASCO Clinical Practice Guidelines from the College of American Pathologists Guideline (2018).There were fewer intratumoral CD1a+ DCs in the HPV-positive and HPV-negative TSCC groups than in the NT group (p<0.05). In the peritumoral compartment, there were fewer CD83+ DCs in the HPV-positive and HPV-negative TSCC groups than in the NT group (p<0.001). The quantification of DCs subtypes showed no statistical differences between HPV-positive and HPV-negative TSCC groups (p>0.137). Patients with HPV-positive TSCC had significantly better overall survival rate than those with HPV-negative TSCC (p=0.004).Tumor activity contributes to DC depletion regardless of intralesional HPV positivity. An improved prognosis has been reported in patients with HPV-positive TSCC. In an immature state, DCs patrol tissue microenvironments and efficiently unleash various pathways when they encounter an antigen. DCs migrate to the draining lymph nodes through a maturation process that enables the presentation of pathogen-derived peptides to CD4+ and CD8+ T cells.2Dendritic cells (DCs) are antigen-presenting cells responsible for specific immune responses that capture, process, and present antigens to T lymphocytes. DCs can activate other cells, such as natural killer cells, B cells, macrophages, and eosinophils as well as generate immunological tolerance.4Epithelial DCs play a fundamental role in antiviral immunity since T cells depend on the presentation of viral antigens by DCs. The antigen presentation process conducted by DCs generates an effective immune defence against HPV. Epidemiological studies suggest that HPV takes advantage and interferes with the cell cycle to avoid being eliminated when the immune system is compromised or deficient, resulting in persistent infections and in the development of HPV-positive malignant lesions.6 This relationship of TSCC with HPV has been reported to have a better survival prognosis and less risk of recurrence than HPV-negative lesions.7 Some investigations have shown a correlationbetween DC subsets with distinct clinical behaviors of squamous cell carcinoma of the oral cavity and larynx.11 Hence, this study aims to investigate the population of DCs in different stages of maturation in TSCC under the influence of HPV infection.Smoking, alcohol consumption, and oncogenic HPV infection have been found to be etiological factors for tonsillar squamous cell carcinoma (TSCC).in situ hybridization (ISH), 5 μm thick histological sections were obtained according to the manufacturer’s recommendations.This study was approved, according to the Helsinki statement, by the Ethics Committees of the Antônio Prudente Foundation - Hospital do Câncer - AC Camargo (CAAE: 00741212.8.0000.5432). Thirty-three tissue samples of primary TSCC and 10 of normal tonsils (NT) after tonsillectomy were collected between January 2015 and December 2020 from the Head and Neck Cancer Service at the above institution. Medical and anatomopathological reports of all patients were reviewed. The tissue samples were fixed in 10% formalin for 24 h, and four transverse sections were obtained. All fragments were embedded in paraffin blocks and 3 μm thick histological sections were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). For 12 (2018). Only samples with at least 70% nuclear and cytoplasmic expression values for p16 IHC, with a moderate to strong intensity, were considered as positive. ISH was performed in cases when the immunohistochemical analysis was discordant among the pathologists. For ISH, a wide spectrum probe  was used for genotypes 6, 11, 16, 18, 31, 33, 35, 45, 51, and 52. A 16/18-specific probe  was also used. Sections from a carcinoma of the uterine cervix were used as positive controls.HPV detection methods for the analyzed samples were based on the American Society of Clinical Oncology (ASCO) Clinical Practice Guidelines from the College of American Pathologists GuidelineINK4a antibodies . Subsequently, the slides were incubated for 60 min at room temperature with secondary antibodies using the EnVision/Dual-Link System-HRP , and developed with the chromogenic substrate . The slides were counterstained with Carazzi hematoxylin. For each reaction, negative controls were prepared by omitting the primary antibodies. Two blinded, calibrated examiners analyzed the slides.Immunohistochemistry reactions with primary monoclonal mouse antibodies against CD1a  and CD83  were used to stain immature and mature DCs, respectively. Additionally, the expression of HPV was evaluated using p162 of each compartment was determined using ImageJ 1.51k software .Ten fields of each slide were captured at 400× magnification with a binocular optical microscope  coupled to a digital image acquisition system  and a microcomputer , in which the images were stored. The number of DCs per area was evaluated in the intra- and peritumoral compartments in TSCC groups. For NTs, the quantification of DCs was evaluated in the intra- and peritonsillar compartments. The number of CD1a+ and CD83+ DCs per mmpost-hoc tests. The Mann-Whitney test was used to analyze the association between HPV status and the clinicopathologic findings or the number of intra-and peritumoral DCs in TSCC cases. The clinical and microscopic findings in the different groups with HPV-positive and HPV-negative TSCC were compared using the Mann-Whitney U, Fisher’s Exact, and Pearson chi-square tests. The log-rank test was performed to analyze the overall survival rate between HPV-positive and HPV-negative TSCC. Statistical significance was set at p<0.05. Statistical software SPSS version 23.0 was used for the analyses.Values are expressed as medians with interquartile ranges. Comparison of DCs between the groups was performed using Kruskal-Wallis and Dunn’s INK4a in the NT group. In the HPV-negative TSCC group, 24 patients were men (92.3%) and 2 were women (7.7%), with a mean age of 59.9 years . The HPV-positive TSCC group included 5 men (71.4%) and 2 women (28.6%), with a mean age of 48.5 years . The NT group had 6 men (60%) and 4 women (40%), with a mean age of 38.9 years . All patients were followed up for a mean of 70.34 months (ranging from 2-252 months). Of the 33 TSCC cases, 26 (78.8%) were HPV-negative and seven (21.2%) were HPV-positive. There was no detection of p16CD1a+ DCs showed multiple dendritic extensions, mainly in the intratumoral compartment. In contrast, CD83+ DCs had an ovoid morphology with scarce dendritic extensions in the same compartment . There wp<0.05). A lower number of intratumoral CD83+ DCs in the HPV-positive TSCC group was observed when compared with the NT group (p<0.05). In the peritumoral compartment, there were fewer CD83+ DCs in both the HPV-positive and HPV-negative TSCC groups compared to in the NT group (p<0.001)  .Survival rate of TSCC patients showed no improvement with increases or decreases in intra- or peritumoral CD1a+ and CD83+ DCs . HoweverDC populations are an essential group of cells that act as mediators between innate and adaptive immune responses. The inhibitory or stimulatory functions of DCs depend on their subsets and maturation state. Although DCs are important in regulating an antitumor immune response, our study revealed a depletion of mature and immature DCs in TSCCs, regardless of HPV infection.14 In this study, there was a significant decrease in CD1a+ and CD83+ DCs in HPV-positive and HPV-negative tonsillar tumors when compared with NTs. Tumor cells and the tumor microenvironment favor the release of factors that inhibit the function and maturation of DCs. Laguens, et al.15 (2002) showed that the densities of S100+ and CD1a+ DC in regional lymph nodes from cancer patients were significantly lower than those in the control lymph nodes. Another study16 showed a depletion of Langerhans cells (LC) and interdigitating cells in the epithelium of tonsils with recurrent tonsillitis, idiopathic tonsillar hyperplasia, and recurrent tonsillitis with persistent obstructive hyperplasia.Previous studies have shown that HPV may regulate the distribution, differentiation, and function of DCs, interfering with immune surveillance.17 (2007) showed a lower quantity of CD1a+ DCs in cervical squamous cell carcinoma than in normal ectocervix, and these findings could be associated with tumor progression and severity. Moreover, Kindt, et al.18 (2016) demonstrated that a high CD1a+ LC number is associated with longer recurrence-free survival in both intratumoral and stromal compartments and longer overall survival rate in the stromal compartments of the head and neck squamous cell carcinoma specimens. We found no difference between intra- and peritumoral DC population of patients in the early and advanced stages of TSCC  showed a depletion of CD1a+ and CD83+ DCs in lower lip squamous cell carcinoma when compared with the normal epithelium of patients, suggesting that an imbalance in cellular immunosurveillance would be a determining factor for the early development of lip cancer. In contrast, Costa, et al.9 (2016) reported a higher number of CD1a+ and CD83+ DCs in lip squamous cell carcinoma than in actinic cheilitis and in healthy labial mucosa. These divergences can be caused by other factors that contribute to the distribution and maturation of DCs, such as tobacco and ultraviolet radiation, which are strongly associated with lip cancer.Gomes, et al.21DC maturation is a continuous process that is induced and/or regulated by inflammatory cytokines. Tumor cells are known to produce several cytokines, such as TGF-β, VEGF, IL-6, and IL-10, which mediate the suppression of DC maturation and immune responses in the tumor microenvironment. Moreover, pathogen-related molecules and bacterial DNA impair DC maturation and interfere with the balance between pro- and anti-inflammatory signals in the local microenvironment.22 CD83+ cells in cervical lymph nodes and palatine tonsils were more reduced in AIDS patients than in non-AIDS patients, and a high expression of receptors and regulatory molecules has been associated with immunosuppression. Thus, the phenotype and amount of DCs may influence tumor growth and disease progression.23We have previously reported that conditions of immunosuppression interfere with the maturation of DCs and their functionality.24 These data corroborate our findings, in which 21.2% of tonsillar tumors were HPV positive. Previous clinical trials have shown that 50%-70% of oropharyngeal cancer cases are related to HPV.26 Variations in the proportion of HPV-associated squamous cell carcinoma (SCC) are associated with geographic differences and sexual behavior. In contrast, patients with HPV-negative head and neck tumors have a higher consumption of tobacco and alcohol.27Some investigations have estimated that HPV causes approximately 22% of oropharyngeal cancers.28 Fakhry, et al.28 (2008) found a 2-year overall survival rate of 95% in HPV-positive patients with SCC of the oropharynx or larynx, in contrast to the 62% survival rate of HPV-negative cases. Our findings are consistent with these studies, since we found a better survival rate in the HPV-positive TSCC (100%) group than in the HPV-negative TSCC cases (42%) (p=0.004). Several studies have pointed out that the survival improvement is due to a greater radiotherapy sensitivity or a stronger anti-tumor immune response in these tumors.30HPV-positive tumors have a better prognosis than patients with HPV-negative tumours.29 However, Jardim, et al.30 (2018) stated that decreased peritumoral CD1a+ cells predict a worse prognosis with shorter survival time span in patients with oral SCC. Our data showed no influence on tumor infiltration by mature (CD83+) and immature (CD1a+) DCs on the survival of patients with TSCC. Variations in survival rates amongst the studies may be due to the anatomical regions affected by the tumor and the stage of the disease in which the DCs were analyzed.TSCC patients with high expression of CD83+ DCs have been correlated with improved survival.Our study had some limitations. Future studies are needed with a larger sample of TSCCs associated with HPV, following the most recent ASCO Clinical Practice Guidelines from the College of American Pathologists. Furthermore, the study of other subtypes of DCs could be useful to better understand the innate and acquired immune responses against head and neck tumors.The DC networks have been shown to contribute to the pathophysiology of several human diseases involving immunological components. Hence, developing novel immunotherapies based on DCs may induce protection and help the immune system to recognize and respond to cancer antigens.We showed that the population of mature and immature DCs in the intratumoral and peritumoral compartments of TSCC was severely decreased regardless of HPV status. Although the CD1a+ and CD83+ DC subsets showed no association with prognosis, HPV-positive tumors contributed to the greater survival of patients with TSCC.


Studies on human papillomavirus (HPV) infection in oropharyngeal squamous papilloma (OPSP) are lacking, although HPV is a critical oncogenic virus for the development of oropharyngeal cancer. This study evaluated the prevalence and characteristics of HPV infection in OPSP and showed a 14.5% overall prevalence. High-risk HPV accounts for 75% of all HPV infections, with HPV16 being the most prevalent genotype, accounting for 58.3% of all HPV infections. There was a trend toward a higher prevalence of high-risk HPV infection in patients with OPSP aged ≤45 years, never-smokers, and those with multifocal diseases, which corresponded with the clinicodemographic profiles of HPV-associated oropharyngeal cancer. These findings could enhance our understanding of HPV infection in OPSP and could be used as valuable epidemiological data for the management of HPV-associated OPSP.Studies on human papillomavirus (HPV) infection in oropharyngeal squamous papilloma (OPSP) are lacking, although HPV infection has been recognized as the primary cause of oropharyngeal cancer for several decades. This study aimed to evaluate the prevalence and characteristics of HPV infections in patients with OPSP. We retrospectively enrolled patients with histologically confirmed OPSP in whom the presence of HPV infections and p16 expression were evaluated. The results of HPV infection in OPSP were analyzed according to the clinicodemographic profiles. Of the 83 patients included in this study, HPV test results were positive in 12 patients, with an overall prevalence of 14.5%. HPV genotypes involved low-risk and high-risk HPV types in three (3.6%) and nine (10.8%) patients, respectively. The most prevalent genotype was HPV16, accounting for 58.3% of all HPV infections. None of the OPSPs showed p16 IHC positivity. There were trends toward a higher prevalence of high-risk HPV infection in patients with OPSP aged ≤45 years, never-smokers, and those with multifocal diseases. These findings could enhance our understanding of HPV infection in OPSP and be used as valuable epidemiological data for the management of HPV-associated OPSP and regarding the possible efficacy of HPV vaccinations in OPSP. Head and neck squamous papilloma (SP) is a benign tumor histologically characterized by papillary epithelial growth with fibrovascular cores. It can occur in any part of the head and neck with squamous epithelium ,2. AlthoAlthough SP is a commonly encountered disease entity in actual clinical practice, its etiology is still not universally confirmed. Mechanical and chemical irritations have been considered the main causes of conventional SP ,2. SinceThough there are sufficient data on HPV infection in SPs of the paranasal sinus, larynx, and oral cavity, there are only a few studies on HPV infection in oropharyngeal SP (OPSP) with various geographic distribution. In two studies conducted in Japan involving 12 and 15 patients with OPSP each, HPV infection was not detected in any of the patients . In contThis study aimed to evaluate the prevalence and characteristics of HPV infection in OPSP to enhance our understanding of HPV infection as a causative factor of OPSP, ultimately providing clinically available epidemiological data for the management of HPV-associated OPSP.This was a retrospective cohort study conducted between April 2016 and June 2022. The study protocol was approved by our institutional review board, and the need for informed consent was waived owing to the retrospective nature of the study.The inclusion criteria were patients with SP, (1) which was pathologically confirmed after surgical treatment, and (2) that arose from the oropharynx. The exclusion criteria were patients (1) in whom evaluation of HPV status was unavailable, (2) who had a history of HPV vaccination, (3) who had a history of head and neck cancer, and (4) whose medical records were incomplete on review.The prevalence of HPV infection in OPSP has been reported to be 0–53.8% in six different studies ,14,15,19Detection and genotyping of HPV infection was performed using a CLART HPV2 kit  with formalin-fixed paraffin-embedded (FFPE) tissue samples of diagnosed OPSP . CLART H® p16 Histology  in all OPSPs. The intensity of staining was scored using a known p16 expression in HPV-positive OPSCC as a positive control. Overexpression of p16 was defined as homogeneous, strong nuclear, and cytoplasmic staining present in >70% of tumor cells [OPSP was diagnosed histopathologically by a specialized head and neck pathologist. Along with routine histopathological examination using hematoxylin and eosin staining, p16 expression reflecting the biological activity of HPV infection was analyzed using immunohistochemistry with CINtecor cells . All othThe primary outcome variables were defined as the overall prevalence and genotype of HPV infection in OPSPs. The secondary outcome variable was the prevalence of HPV infection as a function of clinicodemographic characteristics, such as age, sex, smoking status, tumor focality, and anatomical subsites of the oropharynx. The presence of dysplasia on histopathological examination, p16 IHC results, and recurrence after treatment were also evaluated.p-value < 0.05.Statistical analyses were performed using SPSS . Continuous data are presented as the mean ± standard deviation. Categorical variables are presented as number of patients (%). The associations between HPV infection and clinicopathological parameters were assessed using an independent two-sample t-test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables. Statistical significance was defined as a two-sided The HPV test was positive in 12 patients; thus, the overall prevalence of HPV infection in OPSP was 14.5% . HPV genp = 0.595); 13.0% and 17.2% in men and women, respectively (p = 0.412); 16.3% and 12.5% in never-smoker and ex- or current-smoker, respectively (p = 0.431); and 13.9% and 25.0% in unifocal and multifocal disease, respectively (p = 0.471). The prevalence of HPV infection according to oropharyngeal subsites, including the tonsil, tongue base, soft palate, and posterior wall, was 13.9%, 11.1%, 10.7%, and 33.3%, respectively (p = 0.517)  .p = 0.324); 9.3% and 13.8% in men and women, respectively (p = 0.386); 14.0% and 7.5% in never-smokers and ex- and current-smokers, respectively (p = 0.279); and 10.1% and 25.0% in unifocal and multifocal disease, respectively (p = 0.374). The prevalence of high-risk HPV infection according to oropharyngeal subsites, including the tonsil, tongue base, soft palate, and posterior wall, was 11.1%, 11.1%, 7.1%, and 16.7%, respectively (p = 0.895).The prevalence of high-risk HPV infection in OPSP was 14.8% and 8.9% in patients aged ≤45 and >45 years, respectively (p = 0.255); 71.4% and 80.0% in men and women, respectively (p = 0.636); 85.7% and 60.0% in never-smokers and ex- or current-smokers, respectively (p = 0.364); and 72.7% and 100.0% in unifocal and multifocal disease, respectively (p = 0.750). The proportion of high-risk genotypes in all HPV infections according to oropharyngeal subsites including the tonsil, tongue base, soft palate, and posterior wall was 80.0%, 100.0%, 66.7%, and 50.0%, respectively (p = 0.895).The proportion of high-risk genotypes in all HPV infections was 100.0% and 62.5% in patients aged ≤45 and >45 years, respectively , with HPV11, HPV16, HPV58, HPV39/66, and HPV84 genotypes. Data on HPV infection in OPSP are scarce because there have been no studies focusing on HPV infection of OPSP alone, but a few studies have included the oropharynx as an anatomical site for the evaluation of HPV infection in overall head and neck SPs ,12,14,15/83, withGiven the lack of sufficient studies on the prevalence of HPV infection, there is limited data on HPV genotypes in OPSP with large heterogeneity, even among the results of previous studies. Since 2010, only four studies have addressed specific HPV genotypes in OPSP, and HPV6, HPV11, HPV51, and HPV74 have been identified in different studies, showing a high predominance of low-risk HPV ,15. A stIn the present study, there were no differences in the prevalence of overall HPV infection according to clinicodemographical profiles, such as age, sex, smoking, tumor focality, and anatomical subsites. However, there were trends toward a higher prevalence of high-risk HPV infection in patients with OPSP aged ≤45 years, never-smokers, and multifocal diseases. Interestingly, these findings were consistent with the clinicodemographical profiles of HPV-associated oropharyngeal cancer, which include younger age, absence of traditional risk factors, such as alcohol and tobacco, and frequent metastatic disease ,18. Thisp = 0.008) [Recently, there has been growing interest and discussion about the effects of HPV vaccination to prevent and treat HPV infection or HPV-related diseases of the head and neck. In a cross-sectional study of the US population aged 18 to 33 years (N = 2627) within the National Health and Nutrition Examination Survey 2011 to 2014, HPV vaccination was associated with reduced vaccine-type oral HPV infection  . Further= 0.008) . Althoug= 0.008) ,37. Curr= 0.008) ,36,37. GThis study had several limitations. First, although this is the largest study on HPV infection in OPSP, the number of study subjects was still too small to represent the true prevalence of HPV infection in OPSP and to demonstrate statistical differences as a function of the clinicodemographic characteristics. Second, given that the prevalence and characteristics of HPV infection are highly affected by sexual and sociocultural behavior in different geographic regions, the results of the present study may not be universal in all geographic societies. Third, because we enrolled patients with pathologically confirmed OPSP after complete surgical resection, the natural course of HPV-associated OPSP could not be followed. Thus, it is still questionable whether current HPV infection in OPSP only acts as a causative factor for the development of OPSP or can progress to HPV-driven carcinogenesis. To overcome these limitations of the present study, a prospective case–control study involving a much larger number of patients with OPSP from various geographical regions should be performed.This study showed that the prevalence of overall HPV and high-risk HPV infections in OPSP were 14.5% and 10.8%, respectively. The most prevalent genotype was HPV16, accounting for 58.3% of all HPV infections. There were trends toward a higher prevalence of HR-HPV infection in patients with OPSP aged ≤45 years, never-smokers, and multifocal diseases. The findings of the present study could enhance our understanding of HPV infection in OPSP and could be used as valuable epidemiological data for the management of HPV-associated OPSP, expecting the possible efficacy of HPV vaccinations in OPSP.