The coverage of the human papillomavirus (HPV) vaccine remains low worldwide. The Family Health Strategy (FHS) in Brazil has an important role in health promotion in communities. Given the FHS's close contact with assisted communities, the coverage of the HPV vaccine should be high in children. This study aims to investigate the acceptance of the HPV vaccine of parents or guardians of a peripheral community of the Brazilian Amazon region assisted by the FHS and influencing factors.A cross-sectional community-based study recruiting the residents of a subnormal agglomerate of Belém (Pará-Brazil) and covered by the FHS was conducted. Data were collected from September 30 to November 5, 2019. The questionnaire “Knowledge and Acceptability of HPV and Its Vaccine” was used. Data were analyzed through binary and multiple regression analyses.p=0.008), having sons , inadequate knowledge about doses of the HPV , and knowledge of anyone who received the HPV vaccine .A total of 247 participants were included in this study, and 85 of which (34.4%) declared that they did not vaccinate their children. Hesitation to vaccinate was related to few years of schooling (odds ratio [OR]: 0.79, Results suggested the low efficiency of FHS in increasing the HPV vaccination coverage in the assisted communities. A strategy involving a dialog with assisted families and continuous health education to health professionals should be implemented to combat fake news and increase HPV vaccination coverage. However, of the 194 member countries of the WHO, only 107 implement HPV vaccine programs and only 5 achieve the goal of 90% vaccination coverage.8In Brazil, the National Immunization Program adopted the quadrivalent HPV vaccine only in 2014. Initially, only girls aged 11–13 years are included in the vaccination program. Currently, the HPV vaccine is available for girls aged 9–14 years, boys aged 11–14 years, and immunocompromised individuals. The vaccine is delivered predominantly through the Primary Health System at a two-dose schedule for free.8 In Western Amazon, 53.9% of females are not vaccinated.9 The midwest and north regions of Brazil have 14.57% and 9.24% vaccination coverage, respectively.10The Primary Health System, which is integrated in the community and responsible for public vaccination, is an important tool for the massive vaccination of a population, but has failed to expand the HPV vaccination coverage. Brazilian studies obtained alarming data. Only 58.4% of females have completed the two-dose schedule, and 71.1% received only one dose in seven Brazilian cities, including Belém.12 In a specific territory, VH can vary among subpopulations due to influencing socioeconomic aspects. For example, VH can be high among people with low socioeconomic conditions and education, restricted access to information technologies, limited knowledge about effectiveness, risk and safety of the vaccine, disease that the vaccine covers, and risk for the disease.13Parents or guardians play important roles in vaccine hesitancy (VH) because their permission is needed for the vaccination of children. VH is defined as delaying vaccine acceptance or complete refusal even on conditions that information and vaccination services are available.14 low knowledge about the HPV and the vaccine,15 absence of knowledge about other parents with children vaccinated against HPV and the antivaccine movement, and the belief that the HPV vaccine stimulates a precocious sexual life.17 The primary health care system, which is in close contact with communities, has an important role on decreasing the VH.Worldwide, regarding HPV VH, many factors have been associated with low income and education,18 Therefore, the FHS is expected to increase HPV vaccination coverage considerably in FHS territories. However, to date, no study has evaluated how FHS influences the parent's HPV vaccine acceptance. Therefore, this study aims to analyze the parents' HPV vaccine acceptance and its associated factors in a peripheral community of Belém covered by FHS.In Brazil, the Family Health Strategy (FHS) is the main entry point of people in the public health care system and is in close contact with the assisted population in the community where it is integrated. The FHS has three main attributes for health promotion: family orientation, community orientation, and cultural competence. Each FHS team is composed by a medical doctor, a nurse, a nursing technician, and community health agents (CHAs).19 In 2020, Pará is the eighth Brazilian state to report a large number of CC cases (780). CC, the prevalent cancer type in women in the state, has incidence rates of 18.41 and 12.41  in Belém.20Belém, the largest city in the Brazilian Amazon region, is the capital of Pará and has an estimated population of 1,506,420 and area of 1,059,466 square kilometers. Approximately 38.5% of the population lives in subnormal agglomerate characterized by an irregular urban pattern, lack of essential public services, and housing located in areas considered unsuitable for occupation.21 The population of girls and boys within the age range of HPV vaccination in Montese is composed by 5556 children.19This observational, cross-sectional, and community-based study was carried out in the Montese neighborhood of Belém. This area is a subnormal agglomerate, has an estimated population of 16,000, and has the largest number of FHS teams: FHS-I, FHS-II, FHS-III, and FHS-IV. Each FHS team assists 4000 inhabitants.In our study, we selected a microarea covered by the FHS-IV team because it has the highest number of CHAs (08). In this microarea, each team covered 756 houses. Data were collected from September 30 to November 5, 2019. Inclusion criteria were as follows: (1) parents or guardians, (2) aged 18 years or older, (3) living in a neighborhood, and (4) having children in the HPV vaccination age range. Parents or guardians unaware of their children's vaccination status were excluded.22 and 19% missing data.The StatCalc (population survey) and Epi Info version 7.2.2.16  were used to calculate the sample size on the basis of the 756 houses and an acceptable margin of error of 5%, 95% confidence interval (CI), design effect of 1.0, 20% expected frequency of unvaccinated persons against HPV,After sample size calculation, we accessed the record of all families assisted by the FHS-IV team. All houses of families (379) with children in the age range of the HPV vaccine were visited. Finally, the number of such children in each house was entered into a digital spreadsheet, and the simple random probability sampling was carried out using a computer-generated list in the Biostat 5.3. When no one was at home on the day of the visit, the next house on the list was selected. A sample loss was the 34.82% (132 families/houses).Data collection was conducted by trained researchers who understood the purpose of the study, form of data collection, and the need for confidentiality. These researchers were accompanied by CHA. Data were collected in the morning because of low rate of violence, and the average duration of data collection was 30 min.During the domiciliary visit, the interview was limited to only one of the parents or guardians. When a house had more than one child, participants were asked to answer questions about the vaccination status of the oldest child (referred to as the “indicated” or “selected”).The formulary used in this study was composed of two parts. The first part was used to characterize the sample of the study, whereas the second part was used to evaluate the parents' knowledge regarding the HPV vaccine and barriers and their acceptability of the vaccine. The sociodemographic formulary contained the following sociodemographic questions about children  and parents or guardians .23 This version contained 31 questions divided into six domains: (1) knowledge about HPV (7 questions), (2) knowledge about the HPV vaccine (11 questions), (3) vaccination barriers for HPV (2 questions), (4) acceptability of the HPV vaccine (3 questions), (5) personal background (3 questions), and (6) health professionals (4 questions). All questions had three options for answers: “no,” “yes,” and “I do not know.” In the adapted version of the formulary, we used only domains 1, 2, 3, and 4. Domains 5 and 6 were excluded because they were directed to health workers.To evaluate the HPV knowledge and the vaccine acceptance of the children's parents, we used the adapted version of the formulary “Knowledge and Acceptability of HPV and Its Vaccine.”In addition, we revised question 16 of domain 2 from “Are 3 doses required for complete vaccination?” into “Are 2 doses required for complete vaccination?” due to the update on the number of vaccine doses. All modifications were consented previously by the author of the formulary.Following the modifications, a pretest of the formulary was done in the study area with a random sample of 25 parents or guardians who were excluded from the study. After this pretest, no adjustment in the form or the content was necessary. During the formulary application, the interviewer cleared possible parents' doubts concerning the questions.All participants were asked about their children's vaccination status. Responses were categorized into two groups: “vaccinated” (at least one dose) and “not vaccinated” (no dose). If the participant did not know the vaccination status, he was excluded. Vaccination status was the binary dependent variable. The response event selected was “not vaccinated.” Independent variables were sociodemographic factors , knowledge about HPV, knowledge about the HPV vaccine, barriers to HPV vaccines, and acceptability of the HPV vaccine. No recompense was offered to volunteers who participated in the study.Data were entered into the Epi Info, and analyses were performed using the Minitab 20  and BioEstat 5.3  software. Categorical variables were expressed as percentages, and numerical variables were presented as means and standard deviations. Parameter estimation was used to describe the proportion of children that were not vaccinated against HPV. Multiple logistic regression was used in examining the factors associated with vaccination status.p-values <0.20 were entered into the multiple logistic regression. Multicollinearity was verified using the variance inflation factor (VIF), and correlations were tolerable at VIF <5. Crude and adjusted odds ratios (ORs) and their respective 95% CIs were used in evaluating the effects. p-Values <0.05 were considered statistically significant. We did not use any hierarchical model, and all tests used were two-sided.Binary regression analysis was used in assessing the association of the independent variables with the dependent variable. All variables with This study was approved by the Research Ethics Committee of the Federal University of Pará under protocol no. 3968426 and followed the principles of the Declaration of the Helsinki. All participants provided written informed consent to participate in the study. None of the participants received rewards for participating in the study.In this study, we visited 379 houses and the sample of this study was composed by 247 parents or legal guardians  having in average of 2.8 children per household in the age range of the HPV vaccine. Although, the sample calculus resulted that we had to interview a number of family covering at least 360 children in specific age range for HPV vaccine, the number of houses visited covered about 692 children.The children's parents or guardians had a mean age of 41.4±12.1 years (range: 18–79 years). Most of the participants were females , had a monthly family income of less than two minimum wages , were mothers , and had high school or university education . Only half of the participants were unmarried . The average number of children in the house was 2.8, and 53.8% of the children (133/247) were girls with a mean age of 12 years. Approximately 98.4% attended school 243/247; .p=0.00). The probability of having an unvaccinated status decreased with increasing number of years of schooling of children . Participants with male children were twice as likely to not vaccinate their children compared with participants with female children  were then explored through multiple logistic regression analysis . Male children were three times likely to be unvaccinated than female children . Participants with inadequate knowledge about the doses of the HPV vaccine were twice more likely to have unvaccinated children than those with adequate knowledge . Participants who answered “no” or “not sure” to the question on whether they know anyone who already had the HPV vaccine were seven times likely to refuse the vaccination of their children .The likelihood of being unvaccinated decreased with increasing years of schooling of children  are covered by FHS services.19The HPV vaccination coverage in the Montese (65.6%) was higher than that in Belém (58.4%), which was possibly because of the influence of the FHS in this area. However, the HPV vaccination coverage in Montese remains far from the target goal of 90% proposed in the global strategy for eliminating CC. Considering that FHS is in close contact with people living in the assisted communities, we expect a high HPV vaccination coverage. However, our results showed that the HPV vaccination in this neighborhood was quite close to that in Brazil. A previous study showed HPV vaccination values of 75.6%, 79.7%, and 65.9% in children aged 13, 14, and 15 years, respectively.25 In Germany, this percentage is even smaller (17.4% of 4747 vaccinated girls).26 However, through being in close contact with the families, the FHS team has the preponderant role of demystifying myths, earning the trust of the assisted population, and promoting an increase in the vaccination coverage in the community. In Georgia, United States of America, a study showed that the caregivers' inability of earning the trust of the children's parents is the reason to HPV VH.27About 34.6% of our sample did not accept the HPV vaccine. This problem is not restricted to Brazil. In Uganda, of the 6093 girls who participated in a study, only 22% are vaccinated against HPV.29 Another study involving Latin American mothers from Colombia, Guatemala, El Salvador, Dominican Republic, United States, and Puerto also showed a low acceptability of the HPV vaccine for boys.30Parents or guardians with male children had a high rate of nonacceptance of the HPV vaccine (44.7%) because of their insufficient knowledge about the effect of HPV on men's health. In Sweden and United States, the low level of vaccination in boys is associated with their parents' absence of knowledge about the importance of vaccination to boys and their belief that their sons are not at risk of acquiring the HPV.6 These facts show the necessity to reinforce to the society the benefits of vaccination for boys to prevent the several types of cancer in men promoted by HPV.The societal historical construction restricts HPV as a problem limited to women, and this belief needs to be demystified. Progress has been made toward the HPV vaccine among boys but remains lacking. Globally, 107 countries introduced the HPV vaccine, but only 33 countries, including Brazil, reported a gender-neutral HPV vaccination program in their vaccination calendars. In these countries, the HPV vaccination coverage for males often rapidly converges with that for females.31 In addition, given their knowledge about HPV and vaccine benefits, students are likely to accept the vaccine and convince their parents. A previous study on Chinese university students showed that a high educational attainment results in low HPV vaccine hesitation.32The likelihood of not being vaccinated decreases with increasing years of schooling. This finding suggests the success of the school health program launched in 2007 by the Brazilian Ministry of Health. The program's primary goal is health promotion in students, including reproductive health. This program is associated with the FHS, and one of the functions of professional workers is to keep vaccination cards updated.33 In Italy, the parents' HPV VH is directly associated with lack of knowledge about HPV, vaccine, and usefulness of the vaccine for HPV-related cancer prevention.34In this study, parents or guardians who did not have their children vaccinated were likely to have no knowledge on the number of doses needed. This fact could be related to the lack of knowledge about the HPV vaccine. In a district of Utah, low HPV vaccination coverage is directly associated with parents lacking knowledge about the benefits of HPV vaccine.35In the multiple regression analysis, participants who answered “no” or “not sure” to questions about knowing anyone who already got the HPV vaccine were likely to have vaccinated children. Given that the HPV vaccine programs have been launched worldwide, HPV antivaccine campaigns are spreading fake news and acting as barriers to the acceptance of the vaccine. In the United States, the followers of an antivaccine group in Facebook has increased to ∼298% since 2019 with more than 31,000,000 followers.36 Knowledge of people who are vaccinated and have not shown side effects increases the acceptance of the vaccine. A previous study in the Brazilian Amazon region showed that adolescents who know someone vaccinated are twice as likely to be vaccinated.9In 2017, a study on 1263 parents in the United States showed 19% refusal to vaccinate their children after reading stories about the vaccine's harmful effects.37 suggesting the need for health authorities to provide health updating and standardize the process of professionals.Being in close contact with the community, the FHS needs to strategize a dialog with families to reduce myths and promote health in communities. However, although health professionals have a preponderant role on the HPV HV elimination, a study in Italy showed that pediatricians working in Primary Healthcare System do not have expected conduct regarding the HPV vaccine prescriptions. For example, some of pediatricians revealed not prescribing the vaccine for boys for considering it unsafe or unnecessary to them,HPV Vaccination NOW campaign on social media has been successful in correcting misinformation about the HPV vaccine by promoting peer-to-peer dialog.35 Most of the neighborhood of Belém has pages on Facebook and Instagram. In Germany, a social media campaign was launched in 2017 to increase the HPV vaccine acceptance. From 2017 to 2018, the positive comments on the social media page increased from 50% to 75%, and the page reached more than 8,020,000 followers in 2019.38FHS should also promote the reduction of HPV HV through social media. For example, the Although our results showed that the HPV vaccination coverage in an FHS-covered community is higher than that in Belém, parents still refuse the vaccine. Nonacceptance is directly associated with limited knowledge about the HPV and its vaccine. Being integrated into communities and in close and constant contact with families, the FSH needs to hold dialogs with families to combat antivaccine campaigns. The use of social media should be considered as a strategy. Furthermore, continuous health education should be ensured to enable CHAs to deal with this subject with families.Community educational interventions and health workers' continuing education highlighting the importance and efficiency of the HPV vaccine should be carried out to increase HPV vaccine acceptance. Brazilian Municipal, State, and Federal health authorities should provide resources and infrastructure to FHS for improved performance.


We aim to describe the relationship between the immunohistochemical expression patterns of HPV E4 markers and the presence of HPV major capsid protein (L1) in cervical tissues obtained by biopsy of patients with abnormal liquid-based cytology (LBC) results, HR HPV infections, or clinically suspicious cervix. A novel HPV-encoded marker, SILgrade-E4 (XR-E4-1), and an HPV (clone K1H8) antibody were used to demonstrate the expression in terminally differentiated epithelial cells with a productive HPV infection in the material. A semiquantitative analysis was performed based on light microscope images. The level of E4 protein decreased with the disease severity. Patients with LSIL-CIN 1 and HSIL-CIN 2 diagnoses had significantly lower levels of HPV major capsid protein (L1) than those without confirmed cervical lesions. Our analysis confirms a higher incidence of L1 in patients with molecularly diagnosed HPV infections and excluded lesions of LSIL-CIN 1 and HSIL-CIN 2. Further studies on the novel biomarkers might help assess the chances of the remission of lesions such as LSIL-CIN 1 and HSIL-CIN 2. Higher levels of E4 protein and L1 may confirm a greater probability of the remission of lesions and incidental infections. In the cytological verification or HPV-dependent screening model, testing for E4 protein and L1 expression may indicate a group with a lower risk of progression of histopathologically diagnosed lesions. The human papillomaviruses (HPVs) are a heterogeneous family of viruses with over a hundred known subtypes . ProfessHPV is characterised by tropism to the multilayered epithelium of the skin and mucous membranes. As a result of microdamage to the skin or epidermis, the cells of the basal layer are infected . The repα, β, γ, μ, and ν. A group of HPVs that are genitally transmitted comprises supergroup A (known as α-papillomaviruses) [The clinical outcome of infection varies depending on the type of HPV. Human papillomaviruses are responsible for forming warts, condylomas, precancerous skin and mucous membrane conditions, intraepithelial neoplasia, squamous cell carcinoma, and adenocarcinoma of the cervix. The HPV family of viruses is heterogeneous and can be divided into viruses) , and we The microscopic evaluation of tissue preparations obtained from a cervical biopsy or loop electrosurgical excision procedure (LEEP) by experienced pathologists is crucial for diagnosing precancerous lesions. There are clearly defined differences and guidelines for distinguishing between LSIL-CIN 1, HSIL-CIN 2, and HSIL-CIN 3 lesions; it is a subjective assessment. Researchers prove that, especially in the case of HSIL-CIN 2 diagnosis, it is easy to make mistakes—the aspects of underestimation and overtreatment are essential.The use of p16 IHC staining enables classifying CIN 2 lesions as the LSIL or HSIL type. It could limit the number of surgical procedures, such as LEEP conisation or cold knife conisation . Due to The E4 protein’s function is thought to induce cell cycle arrest and disrupt the keratin filaments . It may The present study aims to characterise the relationship between the immunohistochemical expression patterns of HPV E4 markers and the presence of HPV major capsid protein (L1) in cervical tissue obtained by biopsy. Additionally, we would like to assess their relationship with HPV genotypes in LBC from women referred for colposcopy due to abnormalities in previous punch biopsies. Moreover, we examine the relationship between E4 protein and the status of HPV infection. Another research question is whether the genotyped subtypes are related to the presence of HPV major capsid protein (L1) in the tissue. All the examination and follow-up groups are under regular oncogynaecological care and underwent proper treatment.We provide a prospective, ongoing 24-month, non-randomised pilot study to assess the level of HPV E4 protein and the presence of HPV major capsid protein (L1) in patients reporting to the District Hospital in Poznań and Specialised Individual Practise due to abnormal LBC results, the presence of highly oncogenic HPV genotypes, or clinically suspicious cervix in the years 2019–2021. The Poznan University of Medical Sciences Bioethical Committee approved the study protocol (540/22). We obtained written consent for the study from all the patients. We included patients who met the following criteria: (i) aged over 18; (ii) non-pregnant subjects, at least six weeks after the puerperium for patients who gave birth; (iii) patients not treated with immunosuppressive drugs; (iv) expressing informed and written consent to participate in the study; (v) agreeing to the proposed surgical diagnostics in the case of indications and possible surgical treatment. The exclusion criteria were (i) the refusal of the possible treatment of SIL and (ii) a lack of technical possibility of performing the test. A total of 85 women met the above criteria. All the subjects from the study group underwent a verification diagnostic analysis of abnormal LBC results. Either punch biopsy or, in the case of histopathologically confirmed HSIL (CIN 2 and CIN 3), LEEP-conisation and curettage of the cervical canal was performed.® . Then, the probes were passed to an independent, standardised laboratory. Cervical swabs were analysed according the Bethesda system. PCR was performed, followed by a DNA enzyme immunoassay and genotyping with a reverse hybridisation line probe assay for HPV detection. The lab technicians performed sequence analysis to characterise HPV-positive samples with unknown HPV genotypes. The molecular test detected the DNA of 41 HPV genotypes to identify the following genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 68a, 68b, 69, 70, 71, 72, 73, 81, 82, 83, 84, 87, CP6108, and 90 in vitro. A positive result in molecular tests confirms the presence of DNA of at least one of the mentioned above oncogenic types of HPV in the collected specimens. We collected liquid-based cytology (LBC) and molecular assessment samples with an endocervical cyto-brush preserved in PreservCytThe LINEAR ARRAY HPV Genotyping Test uses biotinylated primers defined to nucleotide sequences within the L1 region of the HPV genome. The HPV primer pool in the master mix reagent is used to amplify the DNA of 41 genotypes of the virus. Within the L1 regions, bound by the primers, sequences connect with the probes. An additional pair of primers targets the human beta-globin gene to ensure the adequacy of the cellular material, extraction, and amplification of the genetic material. AmpliTaqR Gold DNA Polymerase is used for “hot start” amplification of the HPV target DNA and the control beta-globin gene. After PCR amplification, the HPV amplicon and beta-globin amplicon are denatured with a denaturation solution to create single-stranded DNA. Sequential volumes of the amplicon are transferred to holes containing a hybridisation buffer and a single LINEAR ARRAY HPV genotyping strip (HPV and beta-globin-specific probes are present on the strip). Only an amplicon containing highly matched sequences (only 1–3 mismatches) with the probe can hybridise to it. After hybridisation, streptavidin–horseradish peroxidase conjugate is applied to the LINEAR ARRAY HPV genotyping strip, followed by hydrogen peroxide and TMB. The reaction produces a blue-coloured complex that shows up at the probe position where hybridisation occurred. Then, the technicians perform a visual analysis by comparing the blue line pattern to the LINEAR ARRAY HPV genotyping test reference guide result template.The Polish Society of Colposcopy and Cervical Pathophysiology recommended the classification of the International Federation of Cervical Pathology and Colposcopy. Each time, we performed a punch biopsy from clinically suspect sites and curettage of the endocervix ,16. ® Manual Tissue Microarrayer. Specimens were retrieved from selected regions of donor tissue and precisely arrayed in a new recipient paraffin block. The tissue cores were 5.0 mm in diameter and ranged in length from 1.0 to 6.0 mm, depending on the depth of tissue samples available in the donor block. Cores were inserted into 14 × 14 × 5 mm recipient blocks. The initial sections were stained for haematoxylin and eosin to verify the histopathological findings. To test the expression of HPV and E4 markers, 30 slides were stained using immunohistochemistry methods. They contained tissue samples from 85 patients. On each slide were samples from six patients assessed by two independent pathologists. A pathology specialist identified and marked areas of tissue containing lesions for the construction of the slides. Patients’ blocks were assembled using a UNITMA Quick-RayThe presence of HPV in cells was detected using a human papillomavirus (HPV) Ab-3 (Clone K1H8) antibody . E4 expression was detected using a SILgrade-E4 (XR-E4-1) . The expression levels of both proteins were evaluated based on visualisation by immunohistochemical staining, described below.Serial 4-micrometre tissue sections were cut from the donor blocks containing cores of lesions and applied to adhesion slides (Epredia™ SuperFrost Plus™). A human papillomavirus (clone K1H8) antibody  was used to visualise cells infected with HPV types 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58. The HPV major capsid protein (L1) in the tissues were detected using an antibody to the nonconfirmational internal linear epitope of the major capsid protein of HPV 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58. The specification from Dianova is enclosed. A novel HPV-encoded marker, SILgrade-E4 (XR-E4-1) , was used to demonstrate expression in terminally differentiated epithelial cells with productive HPV infection in the tissue material. Slides were stained on a fully automated immunohistochemistry slide stainer, BenchMark ULTRA . The staining protocol parameters were based on HIER using CC1 , protease 3 (760-2020) for 4 min, 32 min of incubation with the primary Ab, and OptiView (760-700) with amplification (760-099) as a detection system. The antigen was localised using chromogen DAB-3.3 applied in all the preparations. The slides were stained with hematoxylin II (790-2208) for 8 min and bluing reagent (760-2037) as a post counterstain for 4 min. The slides were passed through a series of alcohols and, finally, xylene before the coverslips were mounted. Before staining our study group, we prepared slides with the HPV-infected cervix as a positive control and the tonsil as a negative control.2 in the MS Exel file. The analyses included photos of the examined tissue fragments from patients who showed symptoms of HPV infection. DAB  staining was quantified by phase analysis. The brown-coloured staining was indicative of the expression of the analysed protein. Automatic classification and measurements were performed and used for further statistical analysis [The slides were photographically documented using a 3DHISTECH pannoramic MIDI scanner . The magnification was set at 400×. The staining was evaluated semiquantitatively using an Olympus BX 43 light microscope and the cellSens Dimension software from Olympus. Automatic detection of objects with a colour intensity greater by limit value enabled the identification of areas with a positive immunohistochemical reaction occurrence. The software detected the coloured area in the required intensity and recorded it as a numerical value in mmanalysis . p value as 0.05. Nominal variables are presented as n and %; quantitative ones, as the median (Me) with quartiles 1 and 3; and the median difference (MD) is specified with 95% confidence intervals. The normality of the variables’ distributions was analysed with Shapiro–Wilk tests. All the tests that were used in the analysis were nonparametric; to compare the level of E4 protein and the level of HPV antibodies, the Mann–Whitney U test or Kruskal–Wallis test was conducted (depending on the number of groups). Kendall’s tau-b was used as a correlation coefficient to analyse the dependency between two quantitative variables. Boxplots were constructed with the Python programming language using the Plotly Graphing Library.We conducted the analysis in the statistical program SPSS and set the 2, and the average level of HPV major capsid protein (L1) reached 5008.66 µm2. Regarding the LBC results, most of the patients were diagnosed with LSIL (43.5%). A similar percentage of patients had the following LBC results: ASC-US, ASC-H, and HSIL. Only three patients had no pathology found in the LBC. In one patient, the cervical swab turned out to be non-diagnostic. Therefore, we did not use these data in the analysis. A significant majority of the women were HPV-positive (88.2%); the most frequent genotypes were HPV 16 , HPV 31, and HPV 6. Other HPV genotypes were present in less than 10% of the study group. As shown in p = 0.017). Women with the HPV 6 genotype had lower E4 protein levels than women without it . No other significant differences in the level of E4 protein between the selected groups were observed , other HPV genotypes, and comorbidities), as presented in Subjects with the HPV 18 genotype had significantly higher levels of E4 protein than subjects without this genotype of HPV . Moreover, it should be noted that the level of E4 protein decreased with increasing intraepithelial lesions; it reached 226, 120, and 45 for CIN 1, CIN 2, and CIN 3, respectively. Patients from the CIN 3 group had significantly lower levels of E4 protein than patients with CIN 1 and 2 analysed as one group ; p = 0.002 for mail analysis; p < 0.050 for both post hoc analyses). After excluding the “norm” group from the analysis, patients with CIN 3 diagnoses also significantly differed from the CIN 1 and CIN 2 groups—the level of L1 was significantly higher in this group, both before and after removing extreme outliers from the CIN 1 group . Patients with CIN 3 diagnoses also had significantly higher levels of L1 when the CIN 1 and CIN 2 groups were analysed as one group ; p = 0.006). Moreover, the patients with normal biopsy results had significantly higher levels of a dependent variable than patients from the CIN 2 group , as shown in When analysing all four groups with outliers, patients with CIN 1 and CIN 2 diagnoses had significantly lower levels of HPV major capsid protein (L1) than patients whose biopsy results were normal (p = 0.952), as shown in No correlation between the HPV major capsid protein (L1) and E4 protein level was detected  in cervical tissue obtained by biopsy. Additionally, we assessed their relationship with specific HPV genotypes found in patients. Our study confirmed that subjects with the HPV 18 genotype had significantly higher E4 protein levels than subjects with a different genotype of HPV. By contrast, women with HPV 6 had lower E4 protein levels. Interestingly, for the HPV 16 genotype, the As expected, we observed that the level of E4 protein decreased with an increase in intraepithelial lesions; it reached 226, 120, and 45 for CIN 1, CIN 2, and CIN 3, respectively. These results are in line with Leehman’s work, which confirmed a decreasing amount of E4-positive staining from 41% in LSIL to 3% in HSIL lesions (CIN 3). In addition, Leehman’s work touches on using another immunohistochemical staining, e.g., p16. The spread of p16 is closely related to the epithelial development cycle, and its expression is related to the production and transformation of HPV. Additionally, it has to be emphasized that the intensity of p16 expression and the spread to higher layers of the epithelium is correlated with the disease severity . The research by Vink et al. also confirms low E4 protein levels in HSIL/CIN3 cervical lesions. E4 protein expression was present in 9.8% of CIN 3 .The HPV-encoded marker panE4 might be considered a novel marker for the initiation of the viral productive phase and, hence, the completion of the papillomavirus life cycle . It is eMore publications support the idea that extensive E4 protein expression decreases with an increasing CIN grade. According to Zummeren et al., E4 protein expression is most frequent in classically graded CIN 1 and absent in carcinomas . High-grThe second aspect of our work was to assess the presence of HPV major capsid protein (L1) in cervical tissues, which, to date, is not explored in the literature. Patients with CIN 1 and CIN 2 diagnoses had significantly lower levels of L1 than patients with normal histopathological results. Our analysis confirms a higher incidence of HPV in patients with molecularly diagnosed HPV infections with excluded cervical lesions of LSIL-CIN 1, HSIL-CIN 2, and CIN 3. For this reason, the above results exclude the use of the test detecting the HPV major capsid protein (L1) in cervical tissues for a more accurate detection of groups at high risk of developing lesions such as HSIL and cervical cancer. On the other hand, such a test may help in detecting incidental HPV infections. As Hilfrich et al. proves, the L1 capsid protein is detectable only at that stage of the life cycle. Therefore, detecting L1 capsid protein synthesized in the cells of the superficial layer of the epithelium might be easy to obtain while taking a cervical swab . HoweverWe believe that it would be valuable for science and beneficial for patients in the future to develop diagnostic tests detecting L1 and E4 protein in cervical smears, which would significantly reduce the invasiveness of the test. However, it should be borne in mind that the cervical smear is superficial and we could obtain few L1 or E4-containing cells, while in tissue sections, the assessment of layers and individual cells is much easier due to the amount of material.A small group of studies indicate the possibility of using an immunohistochemical test detecting HPV major capsid protein (L1) and the E4 protein in a population of HPV-positive patients to identify incidental HPV infections with excluded cervical intraepithelial lesions. In the future, they may also facilitate the diagnostic and therapeutic process for intraepithelial lesions in the cervix and the vagina, vulva, anus, and nasopharynx. Interestingly, in tissues with confirmed pathology, observed highest levels of L1 in CIN 3 lesions might be associated with the development of THIN HSIL. THIN HSIL lesions characterized by faster development within the metaplastic immature epithelium or glandular epithelium with rapid proliferation of reserve cells may be characterized by high expression of L1. Alternatively, coincidental infection with a new oncogenic type of HPV virus during the development of a CIN 3 lesion and the patient’s current immune status could impact the level of replication, and thus expression of L1.In the cytological verification or HPV-dependent screening model, testing for E4 or L1 expression may indicate a group with lower risk of progression of histopathologically diagnosed lesions of CIN 1 and 2 type. Additionally, it could extend the observation time of CIN 1 and CIN 2 without the need for surgical intervention .A significantly higher diagnostic value characterizes E4 protein expression analysis compared to the L1 expression analysis. The study showed only the fact that the level of the expression of the E4 protein decreases with the disease severity. Finally, it may be a marker stratifying women with histopathological diagnosed cervical intraepithelial neoplasia into a progressive or non-progressive group, but further analysis will be needed to confirm this thesis. The final decision, regarding a surgical diagnosis, is always made by experienced physicians using an adequate colposcopic protocol.


To evaluate whether the presence of human papillomavirus (HPV) DNA and p16 might be associated with better prognosis in patients with hypopharyngeal carcinoma (HPC), especially on overall survival (OS) and disease-free survival (DFS).PubMed, the Cochrane Library, the Web of Science and EMBASE were searched from inception to April 2021 to search for HPV DNA- and p16-related prognostic articles on HPC. Meta-analysis was performed on the selected articles according to the inclusion and exclusion criteria. Publication bias was assessed for the included studies with Egger’s test. All studies were analyzed by using Stata 16.0 statistical software.A total of 18 studies were included, including 12 HPV DNA studies and 11 p16 studies. Meta-analysis showed that HPV DNA positivity was a strong prognostic factor for improved OS in patients with HPC, with a pooled hazard ratio (HR) of 0.61 , but there was no statistically significant difference in DFS . Patients with p16-positive tumors had better OS  and DFS  than patients with p16-negative tumors.This study suggests that the presence of HPV DNA leads to better OS in patients with HPC, and the presence of p16 also corresponds to better OS and DFS. Our results provide up-to-date evidence to clinicians and researchers. Larger studies adjusting for prognostic factors are needed in subsequent studies.The online version contains supplementary material available at 10.1186/s12885-022-09769-w. Hypopharyngeal carcinoma (HPC) is a relatively rare type of tumor and comprises less than 5% of all head and neck squamous cell carcinomas (HNSCCs) . HoweverHuman papillomavirus (HPV) is a type of DNA virus that can infect human mucosa and skin, and it is related to the occurrence of many human diseases. Since HPV DNA was first detected in human head tissue , the relInterestingly, HPV is not only a risk factor for some diseases but may also be a good predictor of some cancers. Multiple studies have reported that HPV is a positive prognostic indicator in oropharyngeal squamous cell carcinoma (OPSCC), and the risk of death in HPV-positive OPSCC patients was reduced by approximately half compared to patients with HPV-negative tumors –12. In aHowever, the effect of either HPV or p16 status on prognosis in HPC has not been clearly concluded. To date, many studies have examined the relationship between HPV infection and the prognosis of HPC, with some suggesting that HPV-positive HPC patients have a better prognosis , 18. SomTo our knowledge, it has not been determined whether HPV and p16 status have prognostic significance in HPC as it does in oropharyngeal cancer. This may be due to the low prevalence of HPV- and p16-positive status in hypopharyngeal cancer patients , 23, and This meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We performed a systematic literature search in the PubMed, the Cochrane Library, the Web of Science and EMBASE databases. The search was restricted to publications in English. We searched all of the literature up to April 7, 2021 for the combined medical subject headings (MeSH) “Alphapapillomavirus” and “Hypopharyngeal Neoplasms”. The search terms included MeSH terms and their entry terms.Two researchers independently selected the articles for full review based on the following inclusion criteria: the study population included patients with hypopharyngeal cancer; the article assessed HPV or p16 status in a population with hypopharyngeal cancer; the total number of patients was more than 20; the study measured at least one primary outcome: overall survival (OS), which is generally considered the best efficacy endpoint for oncology clinical trials; or disease-free survival (DFS), which is most commonly used in adjuvant therapy after radical surgery or radiotherapy, is also widely used in prognosis of hypopharyngeal cancer.The researchers performed a detailed review of the full text of the selected articles. Studies were excluded based on the following criteria: studies that were case reports, reviews, or meta-analyses; studies that included other types of head and neck tumors; HPV was not the prognostic factor; the patients had distant metastatic disease; the exposed group cases were less than 5; studies with insufficient survival data for the calculation of the hazard ratio (HR); studies with low quality assessments; and studies without full-text.Two researchers independently assessed the included articles and conducted the data extraction. If there was a disagreement, help was sought from a third person, Professor Bo Li, who is an expert in evidence-based medicine. Data extraction from each article included (1) first authors, region, year of publication, and year of sample collection; (2) the number of HPV DNA-positive and HPV DNA-negative patients and p16-positive and p16-negative patients; (3) the stage of the cancer in the subjects; (4) the HPV assessment method; p16 testing technique; and (5) the survival outcome (OS or DFS), and hazard ratio (HR) with 95% CIs.For studies without HRs or CIs, we preferentially extracted their Kaplan–Meier curves using Engauge Digitizer 11.1 software to extract the survival data from their survival curves. Then, we used the method reported by Jayne F. Tierney  to estimThe Newcastle–Ottawa Scale (NOS) was used to estimate the quality of the included articles. The following information was evaluated in each study: the study population, comparability, and assessment of outcomes. Articles scoring at least 6 out of 9 points were considered high-quality studies. Low scores (score < 6) were excluded from the study.2 statistics. If the Q test two-sided P value was < 0.05 or the I2 was > 50%, which suggested heterogeneity, we used a random-effect model to calculate the pooled HR. If there was no significant heterogeneity, we applied a fixed-effect model. For the pooled HRs, an HR > 1 suggested that HPV was a risk factor for poor survival, an HR < 1 indicated that HPV infection was a prognostic factor that improved survival, and HR = 1 suggested no significant difference in survival. Since the survival outcomes of the collected studies existed in different years, subgroup analysis of survival indicators in different years will be conducted in this study. A funnel plot and Egger’s test were used to assess publication bias, and we used the trim-and-fill method  to correct outcomes and evaluate the impact of bias on the outcomes. Significance level was set at α = 0.05, all tests were two-sided.Meta-analysis was performed using the statistical software Stata version 16.0. We used HRs and their 95% CIs to measure the survival outcomes in the HPV DNA- or p16-positive group compared with the HPV DNA- or p16-negative group. Statistical heterogeneity across studies was evaluated by Cochrane’s Q statistic and IWe searched a total of 1195 relevant articles from four databases. After duplicates were excluded, we reviewed the titles and abstracts of 754 articles. According to the inclusion and exclusion criteria, 653 articles were excluded. Finally, by carefully browsing the full text of the remaining 101 articles, we included 18 articles in total , 25–37. The characteristics of the 12 included articles on HPV DNA status , 25–31 ap = 0.0001), and HPV infection was beneficial to the survival of hypopharyngeal cancer patients. In addition, we performed subgroup analysis based on survival outcomes in different years, and statistically significant differences were found in the subgroup of 3-year OS , 5-year OS  and > 5-year OS .Eleven studies , 25–31 ep = 0.13).Three studies , 21, 30 p = 0.007). In the subgroup analysis by survival outcomes in different years, significant differences were found for 5-year OS in the subgroup . However, no statistically significant differences were found in 3-year OS  or > 5-year OS .Ten studies , 32–37 ep = 0.001), and p16 infection was beneficial to the survival of HPC patients. In the subgroup analysis by survival outcomes, significant differences were found in the subgroup of 5-year DFS  and > 5-year DFS . No statistically significant difference was found in the subgroup of 3-year DFS . Since there was only one study in this group, the relationship between DFS and p16 status remains to be further explored.Five studies , 32, 33 Publication bias was evaluated by funnel plots and Egger’s test, as shown in Table Multiple studies have highlighted the prognostic significance of HPV status in some HNSCCs, while studies on the relationship between HPV and the prognosis of hypopharyngeal carcinoma have failed to reach a unified conclusion after years of research. Currently, in various HPC studies, HPV has been detected in a variety of ways. At the DNA level, PCR and ISH are often used to detect HPV DNA. At the protein level, p16 is used as the main detection indicator, and p16 is usually detected by IHC. In this study, both of these levels were included to comprehensively evaluate the effect of HPV infection in hypopharyngeal carcinoma.At present, many studies have examined the relationship between HPV infection and survival indicators such as OS and DFS. OS is considered to be the best endpoint to measure tumor efficacy and is the preferred survival indicator. In our study, we found that there was a statistically significant difference in OS between the HPV DNA-positive and HPV DNA-negative groups. HPV DNA has a higher predictive value for the short-term survival of patients with HPC. Axel Sahovaler’s research  points oDFS was defined as the length of time after treatment during which there was no disease recurrence or death due to disease progression. At one institution in the southern United States, 13% of patients with laryngeal or HPC were found to be HPV positive. This institution found that HPV infection was associated with improvements in patients’ OS and DFS, but none of these associations was statistically significant . In our p16 has been a controversial prognostic factor for HPC. According to the article of Meshan et al. , althougSimilarly, our study found significant improvement in both OS and DFS in patients with p16-positive HPC. In the subgroup analysis of OS, only the 5-year OS was statistically significant, and the absence of statistical significance in the other two subgroups may be due to differences between studies in patient population characteristics such as age, sex distribution, race and ethnicity. The pooled HRs of all groups were less than 1, suggesting that p16 positivity had a certain beneficial effect on the OS of patients with HPC., 5-year DFS and > 5-year DFS were statistically significant, but only one of the five articles showed statistical significance. Our study suggested that p16 could improve the prognosis of DFS in patients with HPC to a certain extent, but one group of DFS subgroup analysis results was not statistically significant, so more studies are still needed to confirm the relationship between them.Our results support the hypothesis that HPV-positive and HPV-negative hypopharyngeal carcinoma differ in relation to survival outcomes, which has also been found in other HPV-associated cancers, including laryngeal and oropharyngeal squamous cell carcinomas , 47. SevCurrently, there is no literature on the mechanism by which HPV infection affects the prognosis of patients with HPC, but some studies have proposed the mechanism by which HPV affects prognosis in patients with HNSCC, suggesting that HPV-positive cancers may have a lower degree of serious genetic changes than HPV-negative cancers. Because of impaired DNA repair ability and radiation-induced immune responses, HPV-positive cancers are more sensitive to radiation than HPV-negative cancers, which could affect the response to treatment , 51. OneHowever, our study has some limitations that should be considered. In our study, the HRs for some articles were calculated by extracting the data from Kaplan–Meier curves . There mThe presence of HPV DNA leads to better OS in patients with hypopharyngeal cancer, and the presence of p16 plays a certain role in improving OS and DFS. To the best of our knowledge, this is the largest and most comprehensive meta-analysis of HPV DNA and p16 survival outcomes in HPC, confirming the impact of HPV infection on the prognosis of hypopharyngeal cancer and providing up-to-date evidence to clinicians and researchers.Additional file 1.


Zea mays L.) represents the main caloric source for much of the world’s population. Pigmented maize varieties are an excellent source of nutraceutical compounds: blue and yellow maize are rich in anthocyanins as well as carotenoids and phenolic acids, respectively. However, blue maize is usually grown in small quantities as a specialty crop because it lacks the qualities and adaptations of commercial white and yellow varieties. Here, a new high-yield variety of blue maize called Vitamaiz was developed from inbred lines of subtropical blue, white, and yellow maize. The aim of this study was to characterize the nutraceutical and physical properties of 30 Vitamaiz hybrids in two subtropical locations. Kernel physical traits, nutrient composition, and nutraceutical components  were evaluated. The biophysical traits of the hybrids were suitable for nixtamalized and flour maize industries. High levels of FPA (228 mg GAE/100 g), BPA (635 mg GAE/100 g), and AOX  were also detected with elevated TAC levels (274 mg C3G/kg dw) and AOX activity . This is the first study to characterize Blue × Yellow maize hybrids that adapt to subtropical environments.Maize ( Maize is a popular cereal crop that feeds people worldwide ; it provAnthocyanins are natural purple, blue, and red pigments that belong to a major subclass of polyphenols and flavonoids, such as cyaniding-, pelargonidin-, and peonidin-based glucosides, which saturate blue maize with its characteristic color . FlavonoYellow and orange maize varieties, on the other hand, have high levels of carotenoids, tetraterpenoids, lutein, zeaxanthin, β-carotene, β-cryptoxanthin, and α-carotene . CarotenMaize pigmentation is determined not only by plant genotype but also by environmental factors such as altitude, temperature, and abiotic stress. Indeed, it is proposed that the popularity of pigmented varieties declined upon import to Europe because white grains adapted better in Europe . FortunaIncreasingly productive pigmented hybrids have emerged by adapting maize to diverse agroecologies ,12. TradVitamaiz is a collection of Mexican maize hybrids developed through conventional breeding. It was created to produce high-yield blue–yellow and blue–white maize hybrids that retain most of the properties of pigmented grains, such as phenolic acids, anthocyanin, and carotenes. The aim of this study was to evaluate the physical traits, nutrient composition, and nutraceutical compounds found in 30 promising Vitamaiz hybrids that were developed and tested in two subtropical regions. Furthermore, these compounds and their antioxidant capacities were correlated with the physical properties of the kernels of Vitamaiz hybrids.p < 0.001) in all physical traits evaluated were found between the genotypes; however, the environment had no significant effects. The heritability was high (>0.7) only for yield, test weight, and TKW. Differences between the types of crosses (Blue × White vs. Blue × Yellow) were significant only for test weight, TKW, color (b), and kernel thickness. Overall, the Vitamaiz hybrids presented biophysical traits suitable for nixtamalized and flour maize industries.The properties of 30 Vitamaiz hybrids were evaluated in two subtropical environments in 2018 . The maip < 0.001) in genotype were observed for all nutrimental traits evaluated; however, the environment had no significant influence. The heritability was low (<0.5) for proteins, oil, and fiber. Significant differences between the types of crosses were also found for all nutrimental traits; Blue × White had higher nutritional content than Blue × Yellow. Generally, Vitamaiz hybrids contained a high nutritional value for animal or human consumption.p < 0.001) in all nutraceutical traits based on genotype and genotype × environment (G × E) were observed. No environmental effects were detected for free phenolic acids, AOX of anthocyanins, and carotenoid content. The heritability was low (<0.2) for all nutraceutical traits except for carotenoid content, which presented a high heritability value of 0.88.The total soluble and cell wall-bound phenolic acid contents ranged from 125–228 to 359–635 mg GAE/100 g dw, respectively. The antioxidant capacity of soluble and cell wall-bound phenolic acids ranged from 1172–2000 to 4346–8280 μmol TE/100 g dw, respectively. Vm492 × VmM451 (Blue × Yellow) and MzDtp413 × VmM451 (Blue × Yellow) had the highest soluble and bound phenolic contents, respectively. Furthermore, Vm451 × Vm311 (Blue × Yellow) and Vm492 × VmM451 (Blue × Yellow) achieved the highest antioxidant activity for soluble and bound phenolic acid, respectively.The total anthocyanin content and antioxidant capacity ranged from 114–274 mg C3G/kg dw to 1737–3111 μmol TE/100 g dw, respectively. Interestingly, Vm245 × Vm311 (Blue × White) had the highest total anthocyanin content, while Vm451 × Vm311 (Blue × Yellow) showed the highest OAX activity.The total carotenoid content of the Vitamaiz hybrids was also measured; it ranged from 0.46 to 8.28 µg of bCE/100 g dw; Vm327 × Vm451 (Blue × Yellow) had the highest total carotenoid content. From these hybrids, the parent CML451 expressed the hybrid combination whose concentration of nutraceutical parameters was highest.The two types of crosses, Blue × White and Blue × Yellow, were further compared, as shown in A correlation analysis was performed to determine whether nutraceuticals were associated with the physical and nutritional properties of the Vitamaiz hybrids . StatistAs a novel source of nutraceutical compounds, the Vitamaiz hybrids are desirable not only for their physical and nutritional properties; their yield is competitive (7–9 Ton/ha) in local agricultural practices relative to commercially available, conventional blue hybrids grown in subtropical environments  and to oThe physical characteristics of kernels affect their performance in processing and the acceptability of generated products . The tesThe total soluble phenolic acid content of the Vitamaiz hybrids was higher than that of blue kernels evaluated by Mora-Rochin et al.  and in tOverall, Vitamaiz hybrids had low anthocyanin content and AOX compared to previous reports. For example, the ranges of anthocyanin content found here were lower than those of blue kernels studied by Mora-Rochin et al.  and LópeThe genetic profile of Vitamaiz could hinder anthocyanin accumulation because of the combinatory capacity observed previously . AlthougTwo types of crosses were compared in this study: Blue × White was superior to Blue × Yellow in terms of nutraceutical content and activity, excluding carotenoids. This genetic advantage has been previously reported for similar crosses with white maize . This isThe Vitamaiz hybrids contained compounds  that were related to the fiber content and other physical characteristics of the kernels; carotenoids were specifically linked to oil as well as fiber. Maize fiber primarily contains the kernel outer seed coat (pericarp)  because Therefore, Vitamaiz hybrids represent an opportunity to expand the use of pigmented maize to other geographical zones, such as subtropical areas. The grains from these hybrids also contained high levels of nutraceuticals, especially phenolic acid compounds that could be used to produce tortillas and other regional pigmented maize products.Zea mays L. sp. mays var. Vitamaiz). These experimental hybrids of Vitamaiz were developed by the National Laboratory PlanTECC CINVESTAV-Irapuato, as previously described [Improved subtropical blue, yellow, and white maize inbred lines were combined to develop a new high-yield generation of subtropical maize hybrids collectively named Vitamaiz  equipped with a 0.5-HP motor . The ground samples were placed in 20 mL vials and stored at −20 °C for nutraceutical and physical analyses. Color parameters L*, a*, and b* of the ground samples were determined using a colorimeter .The physical properties of the grains were measured using standard procedures: test weight according to Official US Grain Standard Procedures ; TKW by Near-infrared spectroscopy (NIR) was used as a noninvasive method for chemical analysis of the different types of genotypes. Proximate composition was performed with a NIR spectrometer equipped with a diode-array light source  and a Perten calibration package SimPlus . Protein, oil, and starch were determined and expressed as dry matter. Bio-based corrections for NIR data were performed according to the manufacturer’s instructions.2 was 0.99. Data are expressed as mg of gallic acid equivalents per 100 g of dry weight (mg of GAE/100 g dw).Ground samples were used for the microextraction of soluble and bound phenolic compounds, as described previously . BrieflyTotal anthocyanins were assayed according to Abdel-Aal et al.  with som2 was 0.99. Data are expressed as µmol of Trolox equivalents/100 g of dry sample weight (µmol TE/100 g dw).Antioxidant capacity was determined using the ORAC assay. Extracts from soluble and bound phenolic acids and anthocyanins were evaluated against a standard of Trolox with fluorescein as a probe, as described by Ou et al. . Brieflyg to recover the supernatant. This step was repeated twice to complete three hexane washes. The collected superior phases were washed with 300 µL of water and centrifuged for 10 min at 1200 rpm. The superior phase was measured at 450 nm . The experimental samples were compared to a beta-carotene standard curve. Data are expressed as µg of beta-carotene equivalents per 100 g of dry weight (µg of bCE/100 g dw).Total carotenoid content was measured as described by Kurilich et al. . Ground p-value of 0.05 or less was considered significant.Results are expressed as mean ± standard error of three replicates. Analysis of variance (ANOVA) was performed to detect differences between the samples, followed by multiple comparisons with Fisher’s least significant difference (LSD) to identify specific differences between the types of hybrids and interactions between environments (sites). Broad heritability values were calculated according to Hallauer et al. . ANOVA, In this study, the biophysical, nutritional, and nutraceutical properties of 30 pigmented hybrids developed for subtropical locations were evaluated. New hybrids from crosses of blue × white and blue × yellow maize, especially the parental line CML451, proved to be excellent sources of combined nutraceuticals because of their greater potential to accumulate compounds such as phenolic acid. The high antioxidant capacity and endosperm hardness of Vitamaiz hybrids render them suitable for the production of nixtamalized flour, masa, and tortillas, as well as traditional, local maize products. Vitamaiz hybrids represent an opportunity to expand the use of pigmented maize to other geographical zones, such as subtropical areas, to increase food availability and sustainability. Nevertheless, future studies should enhance the genetic variation of naturally pigmented maize to improve its nutraceutical profile.


Despite a well-established cervical cancer (CC) screening program in Norway, the incidence of CC in young women is increasing, peaking at 35 years of age. 25 percent of all women diagnosed with CC had normal cytology within 3 years prior to cancer diagnosis, addressing the need to improve the screening programme to further reduce cancer incidences missed by cytology.We wanted to investigate the detection rate of CIN3+ in women 25–39 years with normal cytology by using a 3-type HPV mRNA test as a targeted quality assurance measure. The control group is women with normal cytology.During 2014–2017, samples from 13,021 women 25–39 years of age attending cervical cancer screening were analysed at Nordlandssykehuset, Bodø, Norway, including 1,896 women with normal cytology and HPV mRNA test (intervention group), and 11,125 women with cytology only (control group). The HPV mRNA testing was performed using a 3-type HPV E6/E7 mRNA test . The women were followed-up according to national guidelines throughout December 2021.Of the 13,021 women, 429 women (3.3%) had CIN3+ confirmed by biopsy in the follow-up, including 13 cases of invasive cervical cancer. Of the 1,896 women with normal cytology and HPV mRNA test (intervention group), 49 women (2.6%) had a positive test. The risks of CIN3+ among women with either a positive or negative HPV mRNA test were 28.6% (14/49) and 0.8% (14/1847). None of the women in the intervention group developed cervical cancer during follow-up. Of the 11,125 women with cytology only (control group), 712 women (6.4%) had abnormal cytology (ASC-US+). The risks of CIN3+ among women with abnormal and normal cytology were 17.7% (126/712) and 2.6% .By testing women 25–39 years of age with a normal cytology result using a specific 3-type HPV mRNA test, an increase in screening programme sensitivity can be achieved without an excessive additional workload. Women with normal cytology and a negative HPV mRNA test have a very low risk of cervical cancer. Human papillomavirus (HPV) is the established cause of cervical cancer . TherefoIn Norway, cytological high-grade lesions are detected in 1.0–1.2% of the population in each screening round of the national cervical cancer screening programme . The majThe HPV genome is divided into three major regions . The major transforming activity of high-risk HPV is shown to be caused by the E6 and E7 oncoproteins, which interfere with the regulators of the host cell cycle and control of transcription , 16. TheThe aim of this study was to evaluate the performance of a 3-type HPV mRNA test used as quality assurance of cytology negative women 25–39 years, by the means of test positivity rate and risk of cervical intraepithelial neoplasia (CIN3+) compared to women with normal cytology without HPV-test.The Department of Pathology at Nordlandssykehuset-Bodø receives about 19,000 cervical cytology samples from women in Nordland County attending the national screening programme every year. During 2014–2017, a total of 41,007 women 25–69 years of age attended screening, including 13,914 women 25–39 years. After exclusion of women with previous high-grade lesions (CIN2+) and women with invalid index cytology, the final study population consisted of 13,021 women including 1,896 women with normal cytology and HPV mRNA test (intervention group), and 11,125 women with cytology only (control group), All specimens were received in PreservCyt solution , a methanol-based preservative and processed by ThinPrep 2000 system  prior to cytological examination to screen for abnormal/dysplastic cells and reported according to Norwegian guidelines.The department used the Bethesda system for classification of cervical cytology  and the The HPV mRNA test used was PreTect SEE , a qualitative assay utilising NASBA technology (targeting full length E6/E7 transcripts) with direct genotyping of amplified mRNA using molecular beacons corresponding to the HPV types 16, 18, and 45, including an intrinsic sample control ensuring sample adequacy. Artificial oligonucleotides corresponding to the viral mRNA were used as positive controls. Negative controls consisted of RNase-free water and were included in each run, according to the manufacturer’s instructions.Total nucleic acids were isolated from 1 ml of the leftover LBC material and analysed with PreTect SEE according to the manufacturer’s instructions. A detailed laboratory manual is available at protocols.io .Index cytology from women with a positive HPV mRNA test were re-evaluated by a second cytology technician (re-examination of the existing slide) and revised cytological diagnoses were confirmed by a pathologist. All women were followed-up in line with the Norwegian national guidelines until December 2021. In the study period, the Norwegian cervical cancer screening programme recommended women aged 25–69 to be screened with cervical cytology every three years, though some women choose to have annual screening outside of the official programme. Women with high-grade cytology were recommended colposcopy and biopsy, while women with low-grade cytology were triaged by a 14-type HPV-test. Women with normal cytology and a positive HPV-test were recommended to be re-screened after 12 months. Women with repeated positive HPV-test (HPV pos. x 2) were recommended colposcopy and biopsy. We used histologically confirmed CIN3+ as the study endpoint.P16(INK4a) (Roche mtm laboratories AG) was used to conclude the histology diagnosis upon cellular uncertainties. If there were any discrepancies between the biopsy and treatment histology, the most severe histology was used as endpoint.SPSS V.28 was used to conduct all statistical analyses, which entailed χ2 tests, Mann-Whitney tests and survival analyses (Kaplan-Meier estimator). A p value <0.05 was considered statistically significant.The Regional Committee for Medical and Health Research Ethics (REC Nord) has approved the protocol as a quality assurance study in laboratory work (2013/497/REK Nord). Such studies are exempt from having a written informed consent from the patients.Of the 13,021 women 25–39 years of age with index cytology in 2014–2017, 429 women (3.3%) had CIN3+ confirmed by biopsy in the follow-up through 2021, including 13 cases of invasive cervical cancer .Of the 1,896 women with normal cytology and HPV mRNA test (intervention group), 49 women 2.6%) had a positive test. The cytology diagnoses were revised from normal to abnormal in 53.1% (26/49) of the HPV mRNA positive cases, while 23 cases remained normal after re-examination . The risks of CIN3+ among women with either positive or negative HPV mRNA test were 28.6% (14/49) versus 0.8% (14/1847), p<0.001 , 712 women (6.4%) had abnormal cytology (ASC-US+). The risk of CIN3+ in women with abnormal and normal cytology were 17.7% (126/712) and 2.6% , p<0.001 Figs .ASC-US+ = abnormal cytology Using the Kaplan-Meier estimator, the cumulative incidence ratios (CIR) during seven years of follow-up for the four categories were: “Normal cytology / HPV mRNA negative” 0.8% (95% CI 0.4–1.2), “Normal cytology / Not HPV-tested” 2.7% (95% CI 2.4–3.0), “ASC-US+ / Not HPV-tested” 18.7% (95% CI 15.7–21.6) and “Normal cytology / HPV mRNA positive” 34.8% (95% CI 17.3–48.6) .ASC-US+ = abnormal cytology  Of the fourteen CIN3+ with a negative 3-type HPV mRNA test at baseline, HPV testing (Cobas 4800) during follow-up identified two HPV 16, two HPV 18, five HPV “others” (not HPV 16/18), two HPV positive (not specified) and three women were not HPV-tested before the CIN3+ diagnosis. For seven of the fourteen women, the CIN3+ was detected 4–6 years after index cytology and might be a result of a new HPV-infection after baseline.Of the 13 women (without an HPV mRNA test at baseline) who developed cervical cancer during the follow-up, 7 had normal and 6 had abnormal cytology at baseline. The risk of cervical cancer in women with normal cytology at baseline was 9.6 per 100,000 women per year.All the six women with cervical cancer after an abnormal cytology (ASC-US+) at baseline had squamous cell carcinoma (SCC). Most of them got their cancer diagnosis a short time after baseline. Of the seven women with normal cytology at baseline there were five women with adenocarcinoma (ADC) and two women with SCC. Most of them were detected in the next screening round after three years (36 months), In this study, we observed that by testing women 25–39 years of age who have a normal cervix cytology result with a specific 3-type HPV mRNA test, an increase in screening programme sensitivity can be achieved without a substantial additional workload being imposed.Cervical cancer is a preventable disease. The main cause of invasive cervical cancer is the deregulated and persistent production of HPV E6 and E7 oncoproteins . Based oIt is well known that cervical cytology is inherently limited in sensitivity and reproducibility due to being a subjective analysis method as opposed to molecular diagnostics which are objective , 25, 26.https://www.kreftregisteret.no/). In our study, the 7-year cumulative incidence of cervical cancer was 14.3 per 100,000 women per year before screening and 9.6 per 100,000 women per year in women with normal cytology at baseline. These findings are in line with the reported incidence among normal cytology screening samples in the Netherlands, as described in the observational population based study by Rozemeijer et al. Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: N/AReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is an interesting paper from a study which examines whether using an mRNA HPV test following a cytology negative result in women <40 years screened as part of the Norwegian cervical cancer screening programme could increase screening sensitivity, identifying more women who are at risk of developing CIN3+.However, not all Figures are needed or clearly presented and correctly labelled. The methods do not contain sufficient details about the screening programme and the follow-up of women in the study including the small group who receive a positive HPV result following normal cytology who have their sample re-examined. The strengths and limitations of the study are not discussed.I hope that my queries and suggestions are helpful.TitleI would suggest rewording the title. It doesn’t make sense grammatically or describe the study very clearly.IntroductionLine 44-46. Consider changing to ‘HPV testing is performed for the early detection of cervical disease, both as a reflex test following cytology or as a primary screening method.’Line 47. Specificity for what? i.e. for identifying women with high-grade lesions, CIN, cancer or something else?Line 52. You say ‘by 2022’ but it is already mid-way through. Do you mean the end of 2022? Was it previously cytology based (I assume?)Line 55. I think you mean that it’s not just that the screening programme hasn’t transitioned to primary HPV testing for this group but that cytology will remain the primary screening tool for this younger age group?Line 58. It is normal practice to avoid starting a sentence with a number.Line 59. I believe that terminology is moving away from using ‘smear’.Line 62-64. Consider rephrasing this last sentence as it is crucial here that the reader understands your main point: i.e., ‘younger women are screened using cytology – given the high number of precancerous lesions in this group an mRNA reflex test following a negative cytology result could be used as a quality assurance measure to increase screening sensitivity’.Line 66: there should be no apostrophe in HPVs.Line 70: you use the word ‘proven’. Suggest rewording to ‘shown’ or ‘found’Line 71: consider using a different word than ‘tool’Line 72: ‘Elevated risk of future’. In line 62 you infer they are ‘high risk’ but here you state low-risk population.Line 73: Consider rewording this for clarity. Also, avoid the use of ‘we’ but state as ‘the aim of this study was…’.Methods:I understand from the methods section that your baseline samples are taken as part of routine screening in 2014-2017. However, you do not state how long the interval is between routine screens for women of this age or what happens to this interval if they have a positive cytology result.Line 108: Your main outcome measure is CIN3+. But I do not understand from the Methods how this outcome is measured, when and in whom? Everyone presumably? But as part of their subsequent screen – and if that’s the case, surely not everyone has a subsequent screen? Some must be lost to follow up? In the Results you mention ‘follow-up through 2021’ but you need to explain more in the Methods what the follow-up means.Line 106: I think you are saying that in women with a positive mRNA HPV result their index cytology  is re-examined by a technician. But you do not then present the numbers of how many are deemed as ‘normal’ when re-examined? What then happens to those with an ‘abnormal’ result on re-examination in terms of their screening internal? Colposcopy? What is their CIN3+ outcome? And what happens or would happen to those who are HPV+ve but cytology ‘normal’ on second reading, is the interval to their next screen shorter?ResultsFigure 1: You split by the group that have mRNA testing and the group that don’t. Have you considered splitting first by cytology normal and cytology abnormal, then splitting the cytology normal into those tested for HPV and those not – this makes a more intuitive tree – but ends up with the same four groups represented at the bottom? The diagram needs and title and footnotes to explain abbreviations.Line 136: Correction ‘a short time’Line 138: Correction ‘nest’ to ‘next’Figure 2: I have no idea what this figure means. It doesn’t add anything to the results so consider removing.Figure 3: Without knowing anything about the follow-up of these women, the screening cycles etc. this seems hard to interpret. The labeling seems insufficient, for example these four groups are actually 1) normal cytology + mRNA HPV positive 2) abnormal cytology (no HPV testing) 3) Normal cytology (no HPV testing) 4) normal cytology + mRNA HPV negative.There is no info in the methods about how these data were obtained or what the graph represents so consider removing this or labeling better.Table 1: See my comment about labeling of Figure 3. You do not mention this table in the Results. I do not think that the label PPV is correct. PPV in a clinical setting means what is the probability, given your positive result, that you really are positive. The cytology ‘abnormal’ result would be considered a ‘positive’ result as would the mRNA HPV positive group, but the other groups would be considered as a ‘negative’ result. I think what these values really are is the percentage who go on to develop CIN3+ within 7 years of the baseline result (i.e. 2014-2021).DiscussionLine 173: why is this in quotations?Line 177: consider using ‘intense’ rather than aggressive?You do not discuss the strengths or limitations of the study. For example, the fact that there is no statistical analysis.Using mRNA HPV testing as a reflex test is only one way to change the screening pathway for this age group, it might be appropriate to suggest alternatives (such as AI to improve cytology) or suggest future research e.g. a cost-effectiveness analysis.I’m not clear on what you are suggesting happens to women who are found cytology normal mRNA HPV positive – colposcopy or a shorter screening interval?Line 210: You say 2.6% of cases in our study needed to be rescreened – i.e. 2.6% of samples with normal cytology but positive mRNA HPV have their original sample re-evaluated by a technician. Using the word ‘rescreened’ suggests they provide a new sample, but I don’t think from the methods that this is the case.Line 218: There is no excessive workload in terms of the number of samples that need to be re-examined but if HPV testing were used as a reflex test then every cytology normal sample would need a reflex HPV test and this would increase the workload in the labs presumably as well as the cost of screening.Reviewer #2: General comments;This study showed that three type HPV mRNA test increases the sensitivity in cervical cancer screening program without an excessive workload. This is interesting and important study to prevent cervical cancer incidence in young women. The authors also said that detection of three HPV types  in this assay might be best choice in the screening of women of 25-39 years of age, since some previous studies suggested that 90% of cervical cancer are positive with HPV16, 18 or 45 in this age group.However, this reviewer does not convince with this hypothesis that this three-HPV mRNA test is the best choice for the above mentioned purpose, since this study showed that the sensitivity of CIN3+ in the intervention group was merely 50% (14/28), although the sensitivity was high enough as it was 97.4% (1847/1896). The authors are likely to say no cancer were found in these CIN3+ cases in the intervention group. However, to prove this assay is the best choice, the authors should show most of 13 cervical cancer cases in the control group in this study are positive by this assay. If possible, they also should examine what HPV types are positive in 14 CIN3+ cases showing negative with this HPV test in the intervention group. Such data may be important to support the findings.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoYes: Toshiyuki SasagawaReviewer #2: **********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  31 Aug 2022Se cover letter and "Response to reviewers"Attachment2022_08_Response to Reviewers_v2.docxSubmitted filename: Click here for additional data file. 26 Sep 2022Impact of HPV mRNA types 16, 18, 45 detection on the risk of CIN3+ in young women with normal cervical cytologyPONE-D-22-12278R1Dear Dr. Sørbye,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ivan SabolAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have addressed each of the many points raised in the first review including removing unnecessary figures, including a strengths and limitations section and providing more details about the national screening programme.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Dr Susie HuntingtonReviewer #1: ********** 9 Nov 2022PONE-D-22-12278R1 Impact of HPV mRNA types 16, 18, 45 detection on the risk of CIN3+ in young women with normal cervical cytology Dear Dr. Sørbye:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Ivan Sabol Academic EditorPLOS ONE