Cervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein.Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 μg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP.The recombinant HPV 52 L1 was expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.The recombinant L1 HPV 52 protein is successfully expressed in  Cervical cancer has emerged as a leading cause of cancer-related mortality in women, ranking fourth globally with a distribution of cases and death of 6.5% and 7.7%, with cancer cases often occurring in women aged between 15 and 44 years . In IndoHuman papillomavirus (HPV) belongs to the Papovaviridae family; it is relatively small, 55 nm in diameter, and a non-enveloped virus and has an icosahedral capsid consisting of 72 capsomers . HPV infOne method to prevent cancers caused by HPV is through the early vaccination of girls aged 9–14 as recommended by the WHO . A virusE. coli [HPV L1 protein has been expressed in several bacteria, such as E. coli , insect,E. coli –20. The Pichia pastoris have become a viable option for biopharmaceutical recombinant proteins [P. pastoris expression system was selected for heterologous protein expression due to its ability to utilize methanol as a carbon source and induce foreign protein expression through methanol use [P. pastoris possesses promoter derived from the AOX1 gene, which is well-suited for regulating the expression of foreign genes. Furthermore, this yeast exhibits physiological properties that enable it to produce a high cell density compared to other fermentative yeasts [Yeast protein expression system is widely employed in industrial-scale production, exemplified by the approved vaccines Gardasil® and Gardasil-9® . In receproteins . This phanol use . This sye yeasts .P. pastoris expression system, but only in strain BG10 [P. pastoris strain GS115 (Mut+), which has a faster growth rate and a high metabolic rate, resulting in higher productivity. In Indonesia, cervical cancer cases caused by HPV 52 are the focus of national vaccination program. Consequently, the development of HPV vaccine targeting type 52 is a logical step in combating against this global pathogen. In addition, P. pastoris is a cost-effective and volume-efficient alternative to superior eukaryotic expression systems for VLP production. Currently, available vaccines are still not accessible to the majority of the economically disadvantaged population in Indonesia, as the commercial HPV vaccine costs at least US $492.32 for one person (three doses). In order to reduce the incidence of cervical cancer in developing countries, particularly Indonesia, there is a need for a strategy to generate a less expensive HPV vaccine that the government can provide to everyone.Previously, the L1 HPV 52 protein was produced in the ain BG10 , and it P. pastoris. This research can provide alternatives and broader perspectives on tackling the HPV 52 pathogen by producing VLP-based vaccines urgently needed in developing countries.Herein, we describe the design and construct of a recombinant L1 HPV 52 protein, as well as its production and purification by fast performance liquid chromatography (FPLC), and the subsequent characterization of the L1 HPV 52 structural protein in P. pastoris GS115 strain and pD902-HPV 52 as a vector expression  were used in this study. The Escherichia coli BL21 DE3 strain  was used as the host for plasmid propagation.The methylotrophic yeast P. pastoris pre-induction growth medium, and induction medium, respectively.Low-salt LB medium is as follows: 1% tryptone, 0.5% NaCl, and 0.5% yeast extract at pH 7.5; YPD: 1% yeast extract, 2% peptone, and 2% dextrose; YPDS: 1% yeast extract, 2% peptone, 2% dextrose, and 1-M sorbitol; BMGY: 1% yeast extract, 2% peptone, 100-mM potassium phosphate buffer pH 6, 1.34% yeast nitrogen base (YNB) without amino acid, 1% glycerol, and 0.2% biotin; and BMMY: 1% yeast extract, 2% peptone, 1.34% YNB, 100-mM potassium phosphate buffer pH 6.0, 0.2% biotin, 0.5% methanol as Pichia pastoris and commercially synthesized by ATUM Company DNA 2.0 Gene Design & Synthesis . It was then inserted into pD902 between EcoRI and NotI sites and was copied by Escherichia coli, which persisted at 25 μg/ml Zeocin on a low-salt LB medium. The gene was amplified by a polymerase chain reaction  using the specific primers shown in Table The HPV 52 L1 gene was designed according to our previous study . The genSacI enzyme  to transform P. pastoris. The linear plasmid pD902-L1HPV 52 was purified with the QIAquick Gel Extraction Kit . The transformation was performed using the Groningen method [P. pastoris from the Gene Pulser Xcell electroporation device  at 7.5 kV/cm, 50 μF, and 129 Ω. Transformants were cultured on YPDS agar plates containing 100 μg/mL Zeocin and incubated for 3–5 days at 28 °C. Transformant colonies were screened on YPD agar plates containing a Zeocin concentration gradient (100–1000 μg/mL). Genes from colonies that survived the highest Zeocin concentration were extracted, and PCR amplification was performed, followed by gene sequencing analysis. Amplification conditions were performed the same as described above.Recombinant plasmids were extracted and linearized using the n method , with slP. pastoris cells were grown in test tubes containing 5-mL YPD and 100 μg/mL Zeocin and incubated at 30 °C with shaking at 250 rpm until OD600 reached ~2–6. Then, 1 mL of culture was inoculated into 25 mL of BMGY medium and incubated under the same conditions for 24 h with 1:10 aeration to the total volume. After incubation, cells were centrifuged at 3000 × g at 4 °C for 5 min, resuspended in BMMY medium, and incubated at 30 °C with shaking at 250 rpm. To induce expression of recombinant proteins from P. pastoris, 0.5% (v/v) methanol was added every 24 h, followed by a culture sampling to analyze optical density.Recombinant Cells were harvested after 72 h of growth, centrifuged at 3000 × g for 5 min, and lysed according to previously reported methods , with slP. pastoris was analyzed by SDS-PAGE and Western blotting. Samples were mixed with Laemmli sample buffer and denatured for 10 min at 95 °C. Proteins were fractionated on 10% SDS-PAGE and stained with Coomassie brilliant blue [The characterization of HPV 52 L1 expression levels in ant blue . AfterwaP. pastoris HPV 52 clones were inoculated into 2 mL of YPD containing 100 μg/mL Zeocin and grown at 30 °C and 250 rpm for 18–24 h to get ~2 × 107 cells/mL. Two subcultures of 10 μL were made, with one of the media containing a selection marker while the other did not. From the grown culture, serial dilutions were made and spread on nonselective and selective solid medium, incubated at 30 °C for 2–3 days [Recombinant 2–3 days . New subThe total protein concentration was quantified using the Pierce BCA assay , following the manufacturer’s protocols with slight modifications. An anti-L1 HPV 52 antibody  was used as the positive control. The results were calculated based on the standard curve, with three replicates of each sample analyzed.The HPV 52 L1 protein was examined by ELISA using specific antibodies . Briefly, the ELISA plate was coated with the anti-L1 HPV 52 antibody (1:5000) overnight at 4 °C. The coated plate was washed three times with washing buffer  and blocked with blocking buffer  for 1 h at room temperature. Unabsorbed proteins were removed by washing buffer. Samples were then added to the plate for 2-h incubation at room temperature. After being washed three times with wash buffer, the anti-HPV 52 L1 (1:1000) as a primary antibody was added and incubated for another 2 h at room temperature. The unbound primary antibodies were removed by washing. The anti-mouse IgG-HRP conjugate  was added to the well, incubated for 2 h at room temperature, and washed with wash buffer. The proteins were stained with the substrate ABTS , and the optical density was detected using a microplate reader  at 602 nm.HPV 52 L1 VLP formation was analyzed using TEM. The precipitated L1 protein was dialyzed with PBS prior to TEM preparation. Samples were then absorbed on carbon-coated grids and negatively stained with 2% phosphotungstic acid . Samples+, 20-mM NaCl, pH 6.2) to 100% elution buffer . The total volume of the gradient was 20 column volumes. The fractions were collected and analyzed for HPV 52 L1 protein using SDS-PAGE and Western blotting.Cation exchange chromatography, as part of fast performance liquid chromatography (FPLC), was used as the first step of purification. The sample was filtered with 0.45 μm and then purified with HiTrap Capto SP ImpRes column 5 × 5 mL  on ÄKTA Pure . After the sample application was complete, the column was washed with five column volumes of binding buffer. The column was eluted with a linear gradient from 100% binding buffer  on ÄKTA pure  at 5 mL/min, which was equilibrated with ten column volumes with running buffer. The running buffer for this column was 50-mM MOPS NaP. pastoris, as indicated by the presence of these colonies. Using the universal primer AOX (Invitrogen) and a specific primer for the HPV 52 L1 gene, the transformant colonies were confirmed via PCR and sequencing. The L1 gene was detected at 1500 bp with both the AOX primer and the HPV 52 L1-specific primer in PCR products performed on an agarose gel. The result of sequencing also revealed sequences corresponding to the target gene. Following a multi-copy colony selection with graded Zeocin concentrations, three colonies, clones 6, 7, and 15, survived and grew well up to 2000 μg/mL Zeocin. Three clones were then utilized to optimize the expression of L1 recombinant proteins.On YPDS solid media containing Zeocin, transformed colonies were observed after 5 days of incubation. The absence of colonies on the solid medium containing Zeocin as compared to the solid medium lacking Zeocin as the positive control indicates that cell viability was in good condition. Colonies on solid media containing Zeocin indicated that pD902 carrying the L1 gene was effectively introduced into P. pastoris host lacking an insert (serving as a negative control) and a P. pastoris carrying recombinant HPV 52 L1 were expressed and stimulated with 0.5% methanol for 96 h at 30 °C. Western blotting was used to characterize the collected samples. L1 protein was observed to have a size of approximately 55 kDa in the recombinant clone. On Western blotting, the 55-kDa protein reacted with an anti-rabbit HPV 52 L1 polyclonal antibody (Fig. Simultaneously, a P. pastoris was evaluated based on the analysis of inheritance, gDNA changed over 100 generations, and L1 expression in six groups in sequence. The stability of recombinant HPV 52 L1 clones in P. pastoris was determined by observing how they were transmitted, how the gDNA altered over 100 generations, and how the L1 gene was expressed in six distinct groups in sequence. The recombinant HPV 52 clone exhibited stable inheritance in nonselective marker media for the first 60 generations of 100 generations, after which the inheritance efficiency decreases marginally. On an agarose gel, PCR results of gDNA from 100 generations revealed a precise band at 1500 bp, indicating that pD902 carrying HPV 52 L1 had been irrevocably integrated into the genome of P. pastoris (Fig. The stability of recombinant HPV 52 L1 clones in HPV 52 L1 protein was detected in fractions 3–7 (Fig. The yeast lysate was clarified and loaded into a cation exchange chromatography column. The chromatography curve for the separation of L1 HPV and 52 VLP from monomers and pentamers in elution fractions is depicted in Fig. 3–7 Fig. A, and th3–7 Fig. B. The amP. pastoris was purified by ammonium sulfate precipitation, and 50-nm VLPs were observed by transmission electron microscopy. This study demonstrates that recombinant HPV 52 L1 can self-assemble into VLPs of the same size as natural virions.The HPV 52 L1 protein expressed in HPV infection is a pathological condition that is closely linked to development of cervical cancer, which can be fatal. Vaccination against HPV infection is the most effective cervical cancer prevention , 29. As P. pastoris using an integrative vector so that L1 protein could be produced. Colony transformants with high and stable expression levels are ideal and beneficial for large-scale protein production. Multi-copy transformants produce higher yields than transformants with a small number of copies [In this study, we investigated the expression of HPV 52 L1 in f copies . In addif copies , 31. Incf copies , 33 thatPichia pastoris is known to have high stability due to chromosome integration that avoids segregation instability [tability . Based otability  discovertability .The previous study examined the correlation between copy number and stability, revealing low copy transformants exhibit high stability when methanol induction is disregarded. In contrast, the stability of high copies is contingent upon the presence of methanol, as previous research has indicated that a significant decrease in stability occurs upon methanol induction as a result of metabolic burden . This stPurification facilitates the complex assembly reaction of the L1 protein into VLPs. VLPs of purified HPV L1 protein have excellent antigenicity and immunogenicity. The purification process has an effect on how antibodies are made to recognize the L1 capsid protein of HPV 52 . The pars far from pH can increase protein aggregation due to the protein’s open structure [Size-exclusion chromatography, which is commonly used in protein aggregation analysis for peptide and protein biotherapeutics , is the tructure . By incrtructure . HPV VLPtructure .P. pastoris cells or their occurrence during ammonium sulfate precipitation remains uncertain. Another study revealed that VLPs originating from P. pastoris-derived HBsAg were assembled during the purification step [P. pastoris-derived VLPs may be a potential problem in strategic approaches. However, the findings of this work demonstrate that the expression of L1 HPV 52 in P. pastoris can result in the production of self-assembled VLPs, which have the potential to be utilized as a prophylactic vaccine.The self-assembly of VLP with a diameter of 50–100 nm is observed when expressed L1 HPV 52 protein expressed, precipitated with ammonium sulfate, is used. This self-assembly occurs without any additional processing steps, as depicted in Fig. ion step . The proP. pastoris using 0.5% methanol induction and demonstrated favorable stability, making it a viable candidate for the generation of HPV vaccine. The L1 HPV 52 protein was obtained within a size range of approximately 55 kDa, rendering it a promising target for vaccine development. This study demonstrated the successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.In conclusion, the recombinant L1 HPV 52 protein is successfully expressed in


This study aimed to assess the value of an HPV E6/E7 mRNA assay and HPV 16 18/45 genotype assay combined with age stratification for triaging women negative for intraepithelial lesions or malignancy (NILM) cytology.From January 2017 to December 2021, a total of 162,309 eligible women underwent cervical cancer screening at the Affiliated Hospital of Jining Medical University, China. Excluding those with negative HPV E6/E7 mRNA, abnormal and unsatisfactory cytology, and those who failed to undergo colposcopy, 6,845 women were ultimately included in our study. We analysed the triage guidance for different subtypes of HPV in the presence of NILM cytology.2 = 653.214, P < 0.001). The percentages of LSIL + and HSIL + in the HPV16 + group  and HPV16+/18/45 + group  were much higher than those in the HPV18 + group  (P < 0.001). However, there was no significant difference in the percentage of histopathological results between the HPV16 + group and HPV16+/18/45 + groups (P > 0.05). The above results were consistent after stratification according to age.Among 162,309 women, 19,834 (12.2%) were positive for HPV E6/E7 mRNA. Of the 6,845 women included in the study, 1,941 (28.4%), 561 (8.2%), 55 (0.8%) and 4,288 (62.6%) tested positive for HPV 16, HPV 18/45, HPV16/18/45 or other HR-HPV genotypes, respectively. The proportions of LSIL+  and HSIL+ (including HSIL and ICC) pathological results in the HPV 16/18/45 + group were 57% and 34.1%, respectively, higher than 36.3% and 11% in the other HR-HPV + group (χThe rate of histopathological abnormalities was still high for the other HR-HPV subtypes with NILM cytology, although the rate of histopathological abnormalities was much higher for the HPV 16/18/45 positive subtypes. Therefore, colposcopy should be performed in women with HPV E6/E7 mRNA positivity and NILM cytology, regardless of age and HPV genotype. Cervical cancer is a serious cancer that threatens women’s health. Cervical cancer incidence and mortality have increased yearly in China over the last 20 years, and the impact on young women has also increased –3. The mTraditional Pap screenings have performed poorly in underdeveloped countries, with a sensitivity ranging from 30 to 40% . DespiteThe E6/E7 oncogenes are well known to play an important role in the development of cervical cancer. Because E6/E7 overexpression occurs after HPV integration into the genome, direct detection of HR-HPV E6/E7 in cervical samples may be more specific in detecting high-grade cervical lesions than HR-HPV-DNA testing . CompareIn this study, we analysed the pathological diagnosis of cervical biopsy in patients positive for different genotypes of HPV E6/E7 mRNA in the presence of normal liquid-based cytology. We preliminarily discussed the need for referral colposcopy and cervical biopsy in patients positive for different genotypes of HPV E6/E7 mRNA to provide feasible suggestions and a basis for clinicians for follow-up management.From January 2017 to December 2021, a total of 162,309 eligible women aged 18–75 years underwent cervical cancer screening in the physical examination centre and gynaecological clinic of the Affiliated Hospital of Jining Medical University, China. Women were excluded from the study according to the following criteria: (1) previously confirmed cervical cancer or other malignancies; (2) pregnancy; or (3) unsatisfactory cytology. A total of 19,834 HPV E6/E7 mRNA-positive women were detected among the 162,309 women. A total of 4939 women were excluded because of abnormal liquid-based cytology (LBC) and unsatisfactory cytology. Because colposcopy and cervical biopsies were not conducted in 8050 women, they were excluded from the study. Therefore, valid data for all tests were available for 6845 women, as shown in Fig. LBC technology was used for cytological detection. Thin-layer slides were created using the Thin Prep 2000 Processor  according to the manufacturer’s instructions. Cytologic data were simultaneously and independently diagnosed by two expert cytopathologists without knowledge of the other examination results. The results of LBC were evaluated according to the Bethesda system 2014.HR-HPV E6/E7 mRNA was detected using the Aptima® HPV assay , an FDA-certified HPV E6/E7 mRNA assay that detects 14 h-HPV types , according to the manufacturer’s instructions. All HR-HPV-positive samples were further genotyped by the Aptima® HPV 16 18/45 Genotype (GT) assay (AHPV-GT) . AHPV-GT can detect HPV16 and a subset of HPV18 and HPV45 cases .A senior colposcopist (5 years of experience or more) performed the colposcopy utilising an electronic colposcopy system , and the results were reviewed by another senior colposcopist (5 years of experience or more). The cervical transformation zone  and high-grade lesion (HGL)) was evaluated using standard colposcopic procedures according to the International Federation of Cervical Pathology and Colposcopy (IFCPC) 2011 criteria. A biopsy was taken at the most severe lesion in the four-quadrant transformation zone of the cervix for those with suspicious or abnormal colposcopy; if no abnormality was seen, a random biopsy was performed in the four-quadrant transformation zone of the cervix. If the transformation zone was not completely visible, an endocervical curettage (ECC) was performed.Two experienced pathologists independently diagnosed histological slides without knowing the results of the other assays. If the two pathologists gave different diagnoses, then a discussion was held by all pathologists in our hospital until a consensus was reached. The diagnosis result of histological slides meets the standard of the current World Health Organisation classification. The pathological classification included the following: (1) normal or chronic inflammatory changes were classified into the normal group; (2) CIN1- and p16-negative CIN2 were classified into the LSIL group; (3) p16-positive CIN2, CIN3 and cervical carcinoma in situ were classified into the HSIL group; and (4) invasive squamous cell carcinoma and adenocarcinoma were classified into the ICC group .SPSS Statistics 21.0  was used for statistical analysis. A P value < 0.05 (two-sided) was considered statistically significant. Pearson’s chi-square or Fisher’s exact probability test was used to compare the differences in percentages between different groups. The 95% confidence intervals (CIs) of proportions were calculated based on the following equation: As shown in Fig. The median age of the 6845 women recruited was 41.42 ± 10.69 years. We calculated the age distribution of all enrolled cases. As shown in Table As shown in Table To determine the impact of HPV E6/E7 mRNA at different ages, we stratified the analysis by age ≤ 40 and > 40 years and found that the proportion of LSIL+  was higher in the HPV16/18/45 + group than in the other HR-HPV + group, regardless of age ≤ 40 or > 40 years . We also compared the pathological findings in the HPV16+, HPV18/45 + and HPV16+/18/45 + groups in both age groups. In either age group, the proportion of LSIL + cells in the HPV16 + and HPV16+/18/45 + groups was higher than that in the HPV18 + group , while there was no significant difference between the HPV16 + group and HPV16+/18/45 + group. See Tables As E6/E7 mRNA is only expressed in actively infected cells, total transcript levels increase during CIN occurrence and progression. The E6/E7 mRNA-based HPV test was found to be more specific in detecting high-grade lesions than the DNA-based HPV test , 20. ThePrevious large population-based studies in China reported that the HPV infection rate was 9.9-27.5% . In thisThe use of the AHPV-GT test, which can detect HPV E6/E7 mRNA in HR-HPV genotypes 16 and 18/45, to triage women with NILM cytology has been reported in very few studies. In this study, the immediate risk of HISL+ (including HSIL and ICC) in women with HPV16/18/45-positive NILM cytology was significantly higher than that in other HR-HPV-positive women . However, the immediate risk of HSIL + remains high in other HR-HPV-positive women, and this result does not change with age stratification. The 2019 ASCCP guideline recommends colposcopy when immediate CIN3 + risk is 4–24%. , 29. TheMeanwhile, we grouped the three HPV16/18/45 subtypes again and found that the detection rates of HSIL and SCC were significantly higher in the HPV16 subtype-positive group than in the HPV18/45 subtype-positive group , whereas the detection rates of HSIL and SCC did not increase when HPV16 was combined with HPV18/45 infection compared with single HPV16 infection . Similarly, the results did not change with age stratification. We should therefore be more alert for patients with positive HPV16 subtypes.This study has several limitations. First, because our data were derived from a clinic-based population rather than a general population, biases may exist. Second, this study did not exclude patients with a previous diagnosis of CIN, and there may be a bias, such as a higher proportion of histological LISL+ (42.7%) in HPV E6/E7 mRNA-positive patients. Third, the analysis of this study was limited to baseline data and did not include follow-up surveillance data; therefore, our study does not represent a conventional screening program and it may be more suitable for countries that have no active screening program. What’s more, follow-up studies of women with positive HPV E6/E7 mRNA and NILM cytology are needed to further verify the conclusions of this study.In summary, this study found that the rate of histopathological abnormalities remained high for the other HR-HPV subtypes with NILM cytology, but the rate of histopathological abnormalities was much higher for HPV 16/18/45 subtype positivity. Therefore, in clinical work-up, colposcopy should be performed in women with HPV E6/E7 mRNA-positive NILM cytology, regardless of age and HPV genotype. There should be more vigilance for HPV 16 subtype infection.


HPV-31, -33, and -58, along with HPV-45 and -52, account for almost 11% of HPV-associated cancers. Our previous studies showed that after HPV-16 and -51, HPV-58 was common and HPV-31 was as frequent as HPV-18 among Iranian women with normal cytology. Hence, in this study, we aimed to investigate the intra-type variations in L1 genes of HPV-58, -31, and -33 to find the predominant lineages circulating in women with normal cytology.Complete coding sequencing of the L1 gene was amplified and nucleotide and amino acid sequences were compared to those of the references. The selective pressure on L1 protein and whether the variations of the L1 genes embed in L1 loops, or N-glycosylated sites were also investigated.B1, A, and A1 (sub)lineages were common in the HPV-58, -33, and -31 samples, respectively. Ninety nucleotide mutations were observed. Twenty nine nucleotide changes corresponded to nonsynonymous substitutions in which seventeen mutations were located in L1 loops. Only one codon position in HPV-58 sequences was found as the positive selection. No difference was observed in N-glycosylation sites between reference and understudied amino acid sequences.In the current study, we reported, for the first time, the (sub) lineages, amino acid, and genetic diversity in the L1 gene of circulating HPV-58, -33, and -31, in women with normal cytology, in Iran. Such studies can not only have epidemiological values, but also aid to set vaccination programs.The online version contains supplementary material available at 10.1186/s13027-023-00499-7. Human papillomaviruses (HPVs) are small, non-enveloped, double stranded DNA viruses with a genome consisting of approximately 8000 bp, which contains of early region  and late region  . HR HPVsConsidering the fact that HPV types are able to diversify their genetic content from their whole genome, they can also be separated into lineages and sub-lineages, diverging from 1 to 10% and 0.5 to 1%, respectively , 7. WithThe current study was approved by the ethics committee of Iran University of medical sciences, Tehran, Iran with ID IR.IUMS.REC 1396.32308. According to Helsinki declaration, all patients were informed by a written consent prior to sampling and their information was attentively protected.In this cross-sectional study, 32, 21, and 11 Thin Prep pap samples with normal cytology were included that were positive for the single infection of HPV-58, -31, and -33, respectively. Indeed, the samples were collected from women who were referred to hospitals affiliated to Iran University of medical sciences in Tehran province for routine screening tests from October 2019 to September 2021.HPV DNA was isolated by using QIAamp DNA Mini kit . The positive samples were approved by High + Low PapillomaStrip kit  for HPV genotyping, according to the manufacturer’s instructions. The L1 gene in samples were amplified, using 50 µl of total PCR reaction, including 33.9 µl DNase-free water, 5 µl 10 × reaction buffer, 4 µl dNTP mix (10 mM for each), 0.8 µl of each forward and reverse specific primers (10 pmol/µl), 0.5 µl mi-Pfu DNA polymerase , and 5 µl DNA template. Also, β-globin primers were used as control and to determine the quality of DNA. The primers and thermocycler programs that used in this study are shown in Table Multiple sequence alignment (MSA) of sequenced samples was done and phylogenetic trees were drawn by using maximum likelihood method, Kimura 2-parameter method in the bootstrap test, 1000 replicates, by Mega 10 software . The seqhttps://www.ebi.ac.uk/Tools/st/emboss_transeq). Complete amino acid sequences of HPV-31, -33, and -58 isolates were compared with the references UniProt accession numbers P17388, P06416, and P26535, respectively, to determine the changes. Consequently, NetNGlyc 1.0 server was applied to predict N-glycosylated sites through artificial neural networks and threshold 0.5 defined by the conserved Asparagine-Xaa-Serine/Threonine sequons [The understudied L1 gene nucleotides were translated to amino acid sequences through an online tool of the European Bioinformatics Institute  , in which the rate of synonymous  and nonsynonymous (beta) substitution is used to estimate the negative and positive selection at each codon based on the neutral model  and hylogeny , 13. Thehylogeny  in compaThe average age of infected females with HPV-58, -31, and -33 were 33.6, 29.8, and 33, respectively. Two samples from HPV-58 and one sample from HPV-33 were excluded due to the undesirable sequencing, thus we continued the study with 30 and 10 samples for HPV-58 and HPV-33, respectively.The phylogenetic trees were constructed by maximum likelihood method with Mega 10 software  of each G430A, G1258A, C1263A, and A1264G, as well as G840A 83.3% (n = 25) as major substitutions  of nonsynonymous mutations of our samples occurred out of the loops and among those mutations occurring in loops  they were mostly located in DE and FG loops (70.6%). The FG and DE loops of L1 proteins contribute to the epitopes for cross-neutralizing antibodies. Deletion at positions 281 and 282, which are located in the late region of the FG loop, leads to the loss of cross-neutralizing antibodies, which are produced through vaccines containing HPV-16 virus-like particles (VLPs)  of nonsy. More stThis study had some limitations which should be addressed; (1) According to the report of Ministry of Health of Iran, cervical cancer did not rank in ten common cancers among Iranian women. As it was highly unlike to collect adequate positive cancerous samples for HPV-31, -33, and -58, we designed our study on samples from women with normal cytology to estimate the predominant circulating genotypes of interest in the population. However, our sample size of positive specimens for the three HPV genotypes was small. (2) Our study contained only samples with normal cytology. In contrast, some specific-lineage variations in coding sequence regions or the long control region (LCR) of HPV-16 are associated with severe lesions . Thus, iIn the current study, phylogenetic analysis of HPV-58, -33, and -31 showed B1, A, and A1 as the frequent (sub)lineages circulating among women with normal cytology. We also reported, for the first time, the genetic diversity in the L1 loops of these HPV genotypes and the effect of amino acid changes on selective pressure and N-glycosylation sites. Additional studies with larger sample size, containing cancerous samples, in different geographical regions in Iran are required to provide a reliable insight into the distribution of these HR HPV genotypes.Additional file 1. Single nucleotide mutations and neutral variations in the understudied HPV genotypes 31, 33, and 58 isolates.


OCA2-HERC2 region is strongly associated with human pigmentation, especially eye colour. The HERC2 SNP rs12913832 is currently the best-known predictor for blue and brown eye colour. However, in a previous study we found that 43 of 166 Norwegians with the brown eye colour genotype rs12913832:AA or AG, did not have the expected brown eye colour. In this study, we carried out massively parallel sequencing of a ~500 kbp HERC2-OCA2 region in 94 rs12913832:AA and AG Norwegians (43 blue-eyed and 51 brown-eyed) to search for novel blue eye colour variants. The new candidate variants were subsequently typed in a Norwegian biobank population  for population specific association analysis. We identified five new variants, rs74409036:A, rs78544415:T, rs72714116:T, rs191109490:C and rs551217952:C, to be the most promising candidates for explaining blue eye colour in individuals with the rs12913832:AA and AG genotype. Additionally, we confirmed the association of the missense variants rs74653330:T and rs121918166:T with blue eye colour, and observed lighter skin colour in rs74653330:T individuals. In total, 37 (86%) of the 43 blue-eyed rs12913832:AA and AG Norwegians could potentially be explained by these seven variants, and we suggest including them in future prediction models.The  OCA2) and its neighbouring gene the HECT domain and RCC1-like domain 2 (HERC2) are of special interest because of their strong genetic influence on human pigmentation, especially eye colour variation [OCA2 expression is regulated by the intronic SNP rs12913832, which is situated in a conserved enhancer region in HERC2 [OCA2 promotor and the enhancer. This enhances OCA2 expression and thereby, melanin production. In contrast, the derived G-allele reduces chromatin looping and thereby, OCA2 expression and melanin production [OCA2, both in regulatory and coding regions, have shown associations with eye colour, albeit with minor additive effects [OCA2 SNP rs1800407 was included in the IrisPlex prediction tool and improved the prediction accuracy of eye colour in a European population [OCA2 could affect eye colour formation in individuals with the rs12913832:AA and AG genotypes. Therefore, in this study, we sequenced the OCA2-HERC2 region in a Norwegian biobank population in order to find new candidate variants that may explain blue eye colour in individuals with the rs12913832:AA and AG genotype.Many pigmentation genes have been extensively studied to explain human eye colour variation . Oculocuin HERC2 . The ancoduction . The stroduction ,6,7. Accoduction . Other p effects ,12. The pulation , whereaspulation . In a prpulation . HoweverOCA2-HERC2 region was sequenced in a selected study cohort of 94 individuals, previously genotyped as HERC2 rs12913832 AA or AG [n = 446). All samples were collected with informed consent and subsequently anonymised. The project was approved by the Faculty of Health Sciences, UiT-The Arctic University of Norway (reference number 2021/2034).A Norwegian biobank population of 540 volunteers (presumably unrelated) residing in northern Norway from 2015 to 2017, was used to search for candidate variants that may explain blue eye colour . The OCAAA or AG . For theAA or AG ,13 (Tabln = 43), and brown and intermediate-brown were grouped into the brown category (n = 51). A quantitative eye colour score (Pixel Index of the Eye (PIE)—score) was calculated for each individual’s eye photograph using the custom-made Digital Analysis of Iris Tool software (DIAT) v.1 [p < 0.001). For further details, see Salvo et al. [High resolution photographs were taken of the participants’ eyes, and categorisation of eye colours was performed in the recent study from Salvo et al. . In shorIAT) v.1 . All theo et al. .L* a* b* . The L* value measures skin lightness, ranging from 0 to 100, where 0 is the darkest and 100 is the lightest. For a consensus measurement, at least three measurements were made, avoiding pigmentation spots and hair.Quantitative measurements of skin colour were performed using a NCS  Colour Scan 2.0 on the inside of the upper arm on 507 individuals in the Norwegian biobank population. The output metrics consisted of The software SureDesign  was used to design capture-probes for a ~500 kbp region on chromosome 15  for SureSelectXT HS2 Target Enrichment System . The design included 15,627 probes targeting 427,954 bp. The library preparation was carried out according to the SureSelextXT HS2 DNA System protocol, version D0. All samples were sequenced on an Illumina MiSeq  according to the manufacturer’s instructions with paired-end sequencing (2 × 150 bp) using the MiSeq Reagent Kit V2 (300 cycles).The sequencing output was automatically converted to FASTQ files with the MiSeq Reporter Software. Agilent’s molecular barcode (MBC) sequences were extracted using the Agilent Genomics NextGen Toolkit (AGeNT), and the FASTQ files were trimmed using AdapterRemoval version 2.1.3 ,18 with fisher.test and Kruskal.test commands in R: A Language and Environment for Statistical Computing version 4.2.1  [www.real-statistics.com, accessed on 16 January 2022). Hardy–Weinberg equilibrium (HWE) and pairwise r2 values for linkage disequilibrium (LD) testing were calculated using Haploview 4.2 [The Austria)  were useview 4.2 . For preview 4.2 . Haplotyview 4.2 ,28, usinn = 540) using the SNaPshotTM multiplex system . The SNPs rs543971307 and rs191109490 were in complete LD. Qiagen Multiplex PCR kit  was used to amplify DNA in one multiplex reaction in a final volume of 10 µL. The following PCR conditions were used: 95 °C for 15 min, 35 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s, and final extension at 72 °C for 10 min. A total of 5 µL amplified DNA was treated with 2 µL ExoSAP-ITTM PCR product clean-up reagent  at 37 °C for 60 min and 75 °C for 15 min. Single base extension (SBE) was performed with 1 µL purified PCR product, 1 µL MilliQ-water, 2 µL SNaPshot™ Multiplex Ready Reaction Mix  and 1 µL SBE primer mix (® Size Standard (200:1). The results were analysed with GeneMapper® ID-X v.1.5 , with a minimum peak height threshold of 50 RFU. In total, 425 individuals were successfully typed for all variants and were, together with the 94 sequenced individuals, used for population-specific analysis .Eight candidate variants for blue eye colour, rs551217952, rs543971307, rs191109490, rs78544415, rs74409036, rs62008729, rs72714116 and rs12913832, were typed in the remaining 446 individuals in the Norwegian biobank population . Across the target region, the median read depth ranged from 116 to 498 reads. In the captured 427,954 bp, a total of 2571 variants were identified after excluding 16 variant positions with less than 10 reads in more than 5% of the samples. Of the 2571 variants, 146 showed significant deviations from HWE , and many variants were in strong or complete LD (pairwise r2 ≥ 0.8). A total of 419 independent variants were identified using the Tagger function in Haploview. Among the rs12913832 AA individuals, 24 variants were statistically significantly associated with both the PIE-score and eye colour categories .Approximately 500 kbp of the OCA2 variants were observed in the study cohort . The SNPs rs74653330:T and rs121918166:T were observed in 24 individuals (56%) from the study group, whereas they were only observed in five individuals (9.8%) from the control group. These variants were statistically significantly associated with both quantitative and categorical eye colour . Thus, rs74653330:T and rs121918166:T could potentially explain blue eye colour (Six missense e colour . These tn = 65), we selected the six OCA2-HERC2 candidate variants rs74409036, rs78544415, rs72714116, rs191109490, rs551217952 and rs62008729 as the most promising candidates to explain blue eye colour , and the variant (minor) alleles were primarily observed in the study group and not in the control group . Notably, rs191109490 was in complete LD with rs543971307 (r2 = 1), which was also found to be in the cCRE in HERC2, close to rs12913832.The six candidate variants were all intronic, either in or HERC2 . Only onn = 519), the six candidate variants had low frequencies (0.002–0.082) and were mainly observed in blue eyed individuals . All haplotype pairs, except AA, AG, AA3 and AG1, had a median PIE-score higher than −0.3, indicating blue eye colour  of 507 individuals were measured and used as a metric for skin colour. The L* value in the population varied from 46.92 to 77.58, with a median of 68.51. No overall significant associations were observed between the haplotypes and L* values. However, a trend of lighter skin colour was observed in individuals with rs74653330:T on a background of rs12913832:AA and AG genotypes. In total, 21 (91%) of 23 individuals with rs74653330:T (Haplotypes A3 and G3) had L* values higher or equal to the median skin colour in the population (L* = 68.51), indicating light skin colour. Furthermore, individuals with A3G had significantly lighter skin colour than the rest of the population .Because SNPs in OCA2-HERC2 to search for candidate blue eye colour variants in individuals carrying the rs12913832:AA or AG genotype. In the Norwegian biobank population, 43 individuals (study group) did not have the expected brown eye colour based on rs12913832 [OCA2-HERC2 in blue-eyed Europeans [In the present study, we sequenced a ~500 kbp region of 12913832 . BecauseOCA2, and one of them overlapped with a deletion that was previously identified in 35% (15/43) of the study samples, which could potentially explain the low read depth [Assessment of the sequencing quality showed that the captured region was successfully sequenced with good quality (median read depth between samples ranged from 116 to 498 reads across the target regions). Only 16 variant positions had less than 10 reads in more than 5% of the samples and were excluded. Four of these variants were in ad depth .https://gnomad.broadinstitute.org/, accessed on 8 December 2022). The SNP rs74653330:T is also moderately frequent in East Asia, where it is associated with lighter skin colour [In addition, to confirm the association of the missense variants rs74653330 and rs121918166 with eye colour, we identified five new candidate variants  that may potentially explain blue eye colour in individuals with the rs12913832:AA and AG genotype. Independently, they were all associated with quantitative eye colour (PIE-score) in the Norwegian biobank population, and 37 (86%) of the 43 blue-eyed individuals in the study group had at least one of the seven variants (including rs74653330:T and rs121918166:T). To our knowledge, this is the first time that the variants rs74409036, rs78544415, rs72714116, rs191109490 and rs551217952 are reported to be associated with human eye colour variation. These variants have very low frequencies and are mainly observed in Europeans, and some of them also in Americans , these had higher PIE-scores (perceived as blue) than individuals with the AA and AG haplotype pairs. Notably, one individual carried the rs12913832:AA genotype and was incorrectly predicted to have brown eyes with a high probability  by the IrisPlex tool . These individuals had intermediate-blue eye colour that might be perceived as green or hazel. As discussed in many studies, eye colour is on a continuous scale from dark brown to light blue, and perception of colour varies from person to person e.g., ,30,31). ,31. 30,3p-values . In addition to the one individual with rs191109490:C , two had the rs74653330:T variant, potentially explaining blue eye colour in all these rs12913832:AA individuals.Individuals estimated with the rs74409036:A rs12913832:G haplotype (AG1) had large variations in PIE-scores, and therefore it may not be easy to predict the eye colour of AG1 individuals. However, when rs74409036:A was observed together with other variants in the haplotype pairs A1G, A3G1 and A4G1, the individuals’ PIE-scores were significantly higher (perceived as blue) than AG1 individuals. Individuals with A3G1 and A4G1 haplotypes also carried the missense mutations rs74653330:T and rs121918166:T. These two variants are associated with blue eye colour in Scandinavians [In the present study, we also observed lighter skin colour in individuals with the rs74653330:T rs12913832:A haplotype . This is in line with previous findings that this variant is associated with skin pigmentation variation in East Asians and Scandinavians . AnderseHERC2-OCA2 region and eye colour in the Norwegian biobank population. Their independent effect on human eye colour variation may be smaller than the HERC2 SNP rs12913832. However, including them in prediction models improves the precision for prediction of blue-eyed individuals with rs12913832:AA and AG genotypes. It would be interesting to assess the associations in similar populations, e.g., other Scandinavian or Northern European populations. While the HERC2-OCA2 region explains most of the blue and brown eye colour variability in Europeans, SNPs in other pigmentation genes, such as TYR, TYRP1, SLC24A4, SLC45A2, ASIP and IRF4, are also found to be associated with eye colour [HERC2, rs1800407 in OCA2, rs12896399 in SLC24A4, rs16891982 in SLC45A2, rs1393350 in TYR and rs12203592 in IRF4) and PIE-score in the Norwegian biobank population, and only observed rs16891982 in SLC45A2 to be significantly associated with blue eye colour in rs12913832:AA and AG individuals [SLC45A2 may also be a target of interest to search for new blue eye colour variants. A recent GWAS identified 50 novel loci associated with eye colour, including pigmentation genes and genes involved in iris morphology and structure [Here, we demonstrate high correlation between variants in the e colour ,37,38,39e colour . We previviduals . The proividuals . Thus, Structure . This de


IntroductionThe human papillomavirus (HPV) causes the most sexually transmitted infections (STIs) worldwide. According to the World Health Organization (WHO), cervical cancer is the fourth most common type of cancer among women worldwide and the eighth leading cause of female cancer death in Saudi Arabia, especially in women between the ages of 15 and 44. The HPV vaccine is known to prevent HPV disease and death. Because parents are often the decision-makers regarding receiving HPV vaccination during adolescence, assessing parents’ knowledge about HPV and its relationship with their intention to vaccinate their daughters is highly necessary.Materials and methodsAn exploratory descriptive cross-sectional study design was used. A total of 773 parents, both mothers and fathers, of female students attending sixth grade from all areas of Jeddah city in Saudi Arabia were recruited. A self-administered validated questionnaire was used to collect the necessary data.ResultsThe current study findings showed that 356 (46.1%) parents had poor knowledge about HPV, 119 (15.4%) had a fair level of knowledge, and 298 (38.5%) had a good level of knowledge. While 344 (44.5%) had intention to vaccinate, 337 (43.6%) were not sure and 92 (11.9%) were not intending to vaccinate. A significant association was identified between knowledge level and respondents’ variables such as being the father or mother, nationality, age, educational level, sector of employment, and monthly income. Employment status was significantly associated with the intention to vaccinate against HPV.ConclusionConcerns about the effectiveness and safety of the HPV vaccine as well as lack of knowledge about HPV and the vaccine influenced parents’ negative intention to obtain the vaccine. Health promotion initiatives for the HPV vaccine should be culturally responsive and emphasize the risks and benefits of the vaccine for women. Knowledge and attitudes about HPV can be improved through concise, visually designed, and comprehensive educational intervention programs targeting parents and their children at schools. Despite the proven safety and efficacy of HPV vaccines, more comprehensive strategies may be needed in the future to increase coverage rates of HPV vaccination nationwide. The human papillomavirus (HPV) causes the most sexually transmitted infections (STIs) worldwide . It has The most common high-risk HPVs are HPV-16 and HPV-18, which are associated with cervical cancer. HPV-6 and HPV-11 are the most common low-risk HPVs. Such infections can be threatening because many people who are infected do not manifest any symptoms, and the majority of HPV infections clear from the body without treatment .According to the World Health Organization (WHO), cervical cancer is the fourth most common type of cancer among women worldwide, with an estimated 604,000 new cases of cervical cancer and 342,000 deaths reported in 2020. About 90% of new global infections and deaths in 2020 were in low- and middle-income countries .People in Arab countries are suffering serious health problems because of increasing incidences of cervical cancer due to the limited availability of effective vaccination and screening programs for cervical cancer. One of the objectives of the strategy of the WHO to eradicate cervical cancer by the year 2030 is to vaccinate 90% of girls against HPV before turning 16 [Also, in 2020, around 380 new cases of cervical cancer were diagnosed, and about 179 cervical cancer deaths occurred in Saudi Arabia. Cervical cancer is the eighth chief cause of female cancer death in Saudi Arabia and the eighth main cause of cancer death in women aged between 15 and 44 years old [Vaccinations have played an important role in enhancing health and decreasing the global worry about vaccine-preventable diseases. Infection prevention is the utmost cost-effective public health approach for improving the health of the population. In 2016, the WHO identified the HPV vaccine as a public health priority that should be incorporated into national immunization programs . The HPVUnderstanding the obstacles that parents who vaccinate their daughters face is essential if the HPV vaccine is to be used to its full potential to protect public health and prevent many HPV-related diseases and cancers. Information about vaccine safety and efficacy heavily influences parents’ choices. Parents who do not vaccinate their daughters often fear the vaccine’s side effects and doubt its efficacy, creating a barrier to vaccination .Increased parental knowledge and awareness of HPV may increase motivation to have one’s daughters vaccinated against HPV, leading to improved vaccination coverage in Saudi Arabia . KnowledStudy design, setting, and samplingAn exploratory descriptive cross-sectional study design was used to assess the association between parents’ knowledge and their intention to vaccinate their daughters. After obtaining ethical approval from the Faculty of Nursing at King Abdulaziz University, official approval was obtained from the Department of Public Education in Jeddah city. A convenient sample of 773 parents was recruited from public primary schools in all areas including the north, south, east, and center of Jeddah city in Saudi Arabia between December 2022 and March 2023. The four main education offices in the city of Jeddah  distributed the survey link to all parents registered in the system via email. Included parents, both mothers and fathers, had a daughter attending grade 6 at one of the primary government schools in Jeddah who were able to understand Arabic. While non-Arabic-speaking parents and those who did not complete the survey were excluded from the total sample. The self-administered survey was completed by parents after voluntarily consenting to participate, and they were fully aware of their right to confidentiality and withdraw at any time.Data collection toolsA self-administered questionnaire was developed by the primary researcher after conducting an extensive review of the related literature. The distributed questionnaire had three parts.Part I: Sociodemographic Characteristics of ParentsThis part of the questionnaire covered parents’ demographic characteristics such as age, gender, nationality, marital status, number of children, educational level, field  and occupation, monthly income, and family history of cervical cancer.Part II: Parents’ Level of KnowledgeThis part of the questionnaire assessed parents’ knowledge about HPV and the HPV vaccine. It measured parents’ level of knowledge using 21-item knowledge questions with “Yes,” “No,” and “Not sure” answer options. The scoring of the answers was as follows: “Yes” had a score of 1, and “No” or “Not sure” had a score of 0. The total scores ranged from 0 to 21. The scores were categorized as follows: 15-21, good knowledge; 8-14, fair knowledge; and 0-7, poor knowledge.Part III: Parents’ Intention to Vaccinate Their DaughtersThis part of the questionnaire identified parents’ intention to vaccinate based on 13 questions with “Yes,” “No,” and “Not sure” answer options. “Yes” had a score of 2, “Not sure” had a score of 1, and “No” had a score of 0. The score range was from 0 to 26. The scores were categorized as follows: 18-26, has the intention to vaccinate; 9-17, not sure; and 0-8, no intention.The content validity of the questionnaire was assessed by three experts in the field of child and women’s health nursing who hold a doctoral degree. A pilot study was conducted with 77 (10%) parents. The Cronbach alpha coefficients of reliability were found to be 0.919 and 0.901 for knowledge and intention scales, respectively. The results confirm a high level of internal consistency of scale items. Therefore, no modifications were applied to the questionnaire.Data analysisThe collected data were analyzed using Statistical Package for the Social Sciences (SPSS) version 25 . Descriptive statistics included frequency, percentage, and graphs used for categorical variables. The chi-squared test and Fisher’s exact test for association between categorical variables were conducted to examine the relation between HPV knowledge and intention to vaccinate based on the basic characteristics of participants. Spearman’s correlation was calculated between the scores for parents’ knowledge about HPV and their intention to vaccinate. Adjusted odds ratios (OR) of intention to vaccinate for HPV and 95% confidence interval (CI) of adjusted OR were calculated using a multinomial logistic regression model. A P-value of less than or equal to 0.05 was considered significant.Demographic characteristicsAs shown in Table Parents’ knowledge about HPV and the HPV vaccineTable Based on the 21-item knowledge questions that measure parents’ level of knowledge about HPV and the HPV vaccine, Table Parents’ intention to vaccinate their daughters against HPVTable Based on the 13 questions in Table HPV information sourcesFigure Correlation between parents’ knowledge about HPV and the HPV vaccine and their intention to vaccinateTable Basic characteristics of parents according to their level of knowledge about HPV and the HPV vaccineTable Basic characteristics of parents according to their intention to vaccinate against HPVTable Multinomial logistic regression model of intention to vaccinate (reference: no intention)Table The overall model is found to be highly significant, as indicated by the P-value of the chi-squared statistic for the difference in the -2-log likelihood between the final model and the reduced model. The pseudo-R-squared value suggests that 31.7% of the variation in the dependent variables is explained by the variance in the explanatory variables included in the model.The results of the “not sure” category reported that knowledge is a significant predictor of being not sure about vaccination. Participants with good knowledge were 2.72 times more likely to be not sure of having vaccination compared to others.Regarding the predictors of intention to vaccinate, the variables knowledge and marital status were found significant. Participants with good knowledge were 1.24 times more likely to have vaccination.HPV is a prevalent STI that has been linked to a variety of cancers. Vaccination is a safe and effective method of preventing HPV . The SauParents’ knowledge about the benefits of the HPV vaccination was generally poor. Nearly half of the Saudi parents had low knowledge of HPV and its vaccine, which is in line with the results of other studies conducted in Saudi Arabia ,17,18. TIn addition to nationality, other correlations were identified between significant knowledge of HPV and respondent variables such as being a father or a mother, age, educational level, work sector, and monthly income. The current study noted that fathers had a slightly higher knowledge and intention to vaccinate their daughters compared to mothers. However, the number of fathers who participated was much lower than the number of mothers. This contradicts the results of another study, in which mothers were 1.4 times more likely than fathers to agree to obtain the HPV vaccine . HoweverMonthly income was associated with acceptance of the HPV vaccine. Fathers with high incomes were more accepting of the vaccine, consistent with other study findings ,27. MeanSurprisingly, in this study, the results of knowledge about HPV of parents who were employees in the non-health sector was higher than those working in the health sectors. However, parents who were health workers were more willing to vaccinate their daughters. This is also reflected in a study conducted to examine knowledge about cervical cancer among health practitioners in King Fahad Medical City, which showed that awareness about cervical cancer among the practitioners was poor. The authors highlight the importance of establishing awareness programs to educate healthcare workers about cervical cancer and HPV . MeanwhiThe internet was found in this study to be the most common source where parents sought information about HPV. Social media messages are considered a reliable source of information in Saudi Arabia ,28. HoweThe current study found that most participants were not confident about the safety of the vaccine. Several other studies found that the main reasons for mothers’ refusal of the HPV vaccine were concerns about its safety and its negative effects on young adolescents . It is tThe study also found another major reason for parents’ reluctance to vaccinate their daughters against the HPV vaccine, which is the lack of recommendations from healthcare providers. It was noted that the majority of the parents in this study did not receive recommendations from their child’s healthcare provider to get the vaccine, which is in line with the findings of the study of Cunningham-Erves et al. (2018) . This raThe predictors of intention to vaccinate as discussed previously can assist policymakers, the Ministry of Health, and vaccination campaign leaders in planning a delivery strategy to maximize reaching the entire segment of the population putting in mind the factors that account for parents to have no intention or hesitancy to vaccinate their daughters, such as lack of knowledge and awareness about the vaccine and its side effects, employment, and marital status.Demographic variables and individual differences explained the percentage of variance in vaccination intention in the parents of this study. These interesting findings should be investigated further to look at other factors that account for non-intention to vaccinate such as cultural beliefs.Healthcare workers at primary healthcare clinics and school health visits should emphasize the need and safety of the vaccine targeting parents and their daughters to help increase HPV vaccination uptake in the country. Future research should include investigations into the actual uptake and completion rates of HPV vaccination and the barriers to obtaining the HPV vaccine in different regions of Saudi Arabia.Healthcare providers must be taught about HPV, its symptoms, and treatment, as well as the ways to prevent it, with a focus on the HPV vaccine. The role of healthcare centers in raising awareness and disseminating accurate and correct information about HPV and its vaccination should be emphasized. Health promotion programs for the HPV vaccine and warnings about the risks of HPV infection in women should be implemented. The benefits of the HPV vaccine should be taught in all government and private schools across the country, keeping cultural sensitivity in mind.Although this study is one of the first studies in Saudi Arabia to assess parental knowledge and intention to vaccinate against HPV using a large number of participants recruited from government schools covering the perspective of both parents, mothers and fathers, the limitation of this study is that the sample consisted solely of parents from only Jeddah city in Saudi Arabia. This may limit the generalizability of the findings to other parts of the country. Future research could address these limitations by exploring larger and more diverse samples from across Saudi Arabia.Concerns about the effectiveness and safety of the vaccination as well as a lack of knowledge about HPV and the HPV vaccine influenced parents’ negative intention to obtain the HPV vaccine. Health promotion initiatives for the HPV vaccine should be culturally responsive and should emphasize women’s risks of experiencing an HPV infection as well as the benefits of getting vaccinated. Knowledge and attitudes about HPV can be improved through a concise, visually designed, and comprehensive educational intervention experience targeting children in schools and their parents. Despite the proven safety and efficacy of HPV vaccines, more comprehensive strategies may be needed in the future to increase coverage rates of HPV vaccination nationwide.


Justice-involved Veterans experience notable risk for psychosocial stressors  and psychiatric multimorbidity, which can result in complex clinical presentations. However, research examining how such factors coalesce to impact risk for suicide remains limited.We conducted a latent class analysis of 180,454 Veterans accessing Veterans Health Administration (VHA) justice-related services from 2005 to 2018.A four-model class membership solution was identified. Among these classes, risk for suicide was highest among Veterans with greater psychiatric burden, with risk most notable among those with high VA service use. Veterans seeking healthcare primarily focused on substance use disorders or with low psychiatric burden and service use had a lower risk for suicide.Psychiatric multimorbidity is salient as it relates to suicide among Veterans accessing VHA justice-related services. Further evaluation of existing VHA services for this population and methods of augmenting and enhancing care for justice-involved Veterans with histories of co-occurring psychiatric conditions may be beneficial in facilitating suicide prevention efforts.The online version contains supplementary material available at 10.1186/s12888-023-04725-9. According to the most recent Bureau of Justice Statistics report, Veterans comprise approximately 8% of individuals currently in prison within the United States . MoreoveWhile rates of criminal justice system involvement among Veterans do not appear to be elevated relative to non-Veteran adults, justice-involved Veterans differ from justice-involved non-Veterans in several ways. In particular, justice-involved Veterans have higher documented rates of violent offenses compared to justice-involved non-Veteran adults , 3–5. FoJustice-involved Veterans also often experience psychiatric conditions and symptoms . Data suggest that justice-involved Veterans have higher rates of several conditions and symptoms than both non-Veterans involved with the justice system and Veterans without a history of criminal justice involvement –13. In aTo meet the needs of justice-involved Veterans, the Veterans Health Administration (VHA) has developed a number of programs specifically tailored for this population . For exaGiven the clinical complexity and presence of multiple drivers of suicide risk among members of this population, multivariate analytic approaches capable of identifying groups at risk is warranted. Specifically, latent class analytic approaches have demonstrated the ability to identify phenotypes of individuals based on a number of factors . As such, our team combined multiple national datasets to identify Veterans accessing VHA justice-related services who did and did not die by suicide. A latent class analytic approach was used to understand how health comorbidity, psychiatric conditions, and VHA service use coalesced to generate latent classes among the VHA-using justice-involved Veteran population. Such classes were then examined as they related to suicide mortality to inform which Veterans were at greatest risk for suicide.The current study was conducted using data collected via a retrospective chart review of Veterans’ VHA electronic health records (EHR). We identified justice-involved Veterans based on any use of VHA justice-related services 2mental health care - low , or high (> 141); and overall use - low (≤ 65), moderate (> 65 and ≤ 185), or high (> 185) use. Due to high levels of homeless service engagement among justice-involved Veterans, VHA homeless service use was also included [For this cohort, we obtained data regarding sociodemographics , service connection, psychiatric diagnoses , documented history of traumatic brain injury (TBI), military sexual trauma , and medical comorbidity . DiagnoVHA EHR data were combined with data from the U.S. Veterans Eligibility Trends and Statistics (USVETS) and VA/DoD Mortality Data Repository (MDR) National Death Index data. Sociodemographic data from USVETS was cross-referenced with VHA EHR data to enhance the validity of the dataset. MDR was used to determine mortality status within our cohort .In total, we identified 197,737 justice-involved Veterans with available data for the current examination. Given the focus on suicide mortality, we removed 17,283 Veterans classified by MDR as having died by a non-suicide-related cause. This resulted in a final cohort for the latent class analysis of 180,454 Veterans, 822 (0.5%) of whom died by suicide from 2005 to 2018. The current study was approved by the Colorado Multiple Institutional Review Board.Class enumeration was performed using Mplus v.8.5 . We usedα = 0.05. Additionally, due to the size of the sample, both 95% and 99% CI are reported.We then examined the indicator of Veterans’ class membership, identified using posterior membership probabilities, as a predictor of death by suicide. Unadjusted logistic regression was used to model suicide (yes/no) as a function of latent class membership to determine if the risk for suicide differs across classes . Subsequently, we fit an adjusted logistic regression model, where suicide was modeled as a function of latent class membership, age , sex, race , ethnicity, rurality, and service connection (yes/no). Statistical significance of all parameter estimates was evaluated at Table p = 0.333), indicating that adding a third class did not improve model fit to a statistically significant degree compared to the two-class solution. This provided statistical evidence in favor of the two-class solution. However, examination of information criteria  indicated that the addition of classes improved model fit, with incremental improvement beginning to diminish upon adding a fourth class  group was characterized by a high probability of PTSD, adjustment disorder, depression, anxiety, alcohol use disorder, and other substance use disorder. Veterans in this class were likely to receive high levels of mental health  and overall VHA  services and were highly likely to receive VHA homeless services. This class was distinguished from the High psychiatric burden/moderate service user group, which was also prevalent (29.79%), and carried a similar psychiatric burden but were most likely to receive moderate levels of mental health services  and VHA  services. Additionally, this class had a moderate probability (0.502) of receiving VHA homeless services. The Low psychiatric burden/low service user (25.53%) group was characterized by a low likelihood of all psychiatric conditions, and members were likely to receive low levels of mental health  and VHA  services. Members of this class were unlikely to receive homeless services. Finally, the Primarily substance use disorder (13.3%) group had a high probability of alcohol use disorder and substance use disorder, but not other psychiatric conditions, and were most likely to receive moderate levels of mental health and VHA services. Additionally, they were highly likely to receive homeless services.The Low psychiatric burden/low service user group was chosen as the reference given prior research noting the association between psychiatric diagnosis and suicide risk (see Supplementary Table A crude logistic regression model was conducted to determine the relationship between latent class membership and suicide (see Table 2(3) = 22.82, p < 0.001, Nagelkerke R2 = 0.002). The groups experiencing the greatest risk for suicide were the High psychiatric burden/moderate service user  and the High psychiatric burden/high service user groups . Interestingly, in the crude model, Veterans in the Primarily substance use-related group had lower  risk for suicide relative to Veterans with low psychiatric burden and service use (p = 0.049).Overall, the crude model was significant (χ2(12) = 128.95, p < 0.001, Nagelkerke R2 = 0.036). Notably, with the inclusion of these factors, risk appeared to increase largely within the high psychiatric burden and high service use group. Specifically, Veterans in the High psychiatric burden/high service user group had 1.88 times greater risk for suicide compared to the Low psychiatric burden/low service user group (p = 0.001). Similarly, Veterans in the High psychiatric burden/moderate service user group had 1.47 times greater risk for suicide relative to the Low psychiatric burden/low service user group (p = 0.046). However, differing from the crude model, there was no significant difference in risk for suicide between the Primarily substance use-related and Low psychiatric burden/low service user groups (p > 0.05).The model was subsequently adjusted for demographic factors  and service connection Table . Again, Building upon prior work noting risk for suicide among justice-involved Veterans , 17, thiSuch risk is consistent with the notion that psychiatric clinical complexity may indeed be a primary factor in driving suicide risk among justice-involved Veterans. Given the psychiatric multimorbidity and stressors that justice-involved Veterans experience, these Veterans likely present to time-limited care settings with myriad psychosocial concerns. As such, it may be difficult for VHA providers working with these Veterans to balance provision of services, including suicide prevention care . For exaDespite this, the relationship between service delivery and suicide risk in this cohort of Veterans was complex. Specifically, no significant differences in risk for suicide were found between moderate and high service groups of Veterans experiencing high psychiatric burden , TBI, aDespite the strength of findings, results should be interpreted within the context of limitations. First, this examination was specific to Veterans accessing VHA justice-related services. Because of this, findings may not generalize to Veterans who do not qualify for comprehensive VHA care or who interact with the criminal justice system and who do not access VHA justice-related services. Moreover, a sizable portion of our sample was moderately to highly engaged in VHA services, including mental health care. As such, many of these Veterans may have been routinely accessing services which focused on addressing psychiatric conditions as well as which assessed the presence of suicide risk factors. In a similar vein, Veterans accessing mental health services would be more likely to have a psychiatric diagnosis conferred on their EHR.We relied on the VHA EHR, rather than conducting diagnostic interviews. As such, we are unable to determine the accuracy of diagnoses. In addition, we examined substance use disorder diagnoses as a unidimensional construct. This approach limits results given differing risk for suicide based on type of substance , as welAn additional consideration is that we were unable to employ contemporary approaches to predicting distal outcomes while accounting for measurement error in latent class membership. Specifically, while we attempted to use the 3-step BCH method to examine the relation between latent class membership and death by suicide , we wereFinally, we used EHR codes to identify use of VHA justice-related and homeless services, rather than using Homelessness Operations and Management and Evaluation System (HOMES) data . Given tJustice-involved Veterans experience several risk factors for suicide . We provided the first latent class analysis of suicide mortality among members of this population. We found that psychiatric burden and moderate to high service use are factors associated with risk. Investigation of current and novel programing for this population may be a potential method of enhancing suicide prevention efforts for this at-risk population of Veterans.Below is the link to the electronic supplementary material.Supplementary Material 1 Supplementary Table 1: Model fit indices for models with ≤ 8-class solutionsSupplementary Material 2 Supplementary Table 2: Crude and Adjusted Models with Other Latent Classes as Reference Group


INK4a overexpression as a surrogate marker of transforming HPV infection should consider the prevalence rates of HPV-driven OPSCC as this significantly impacts on its positive predictive value. Health policies should actively promote vaccination against HPV in both females and males also for the prevention of OPSCC.The prevalence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection has a wide geographical variability. In addition, several authors have reported steadily increasing prevalence rates over the past two decades. It is important to know the epidemiological landscape of these tumors both to estimate the reliability of diagnostic tests and to guide public health choices aimed at primary prevention. In this retrospective study, we observed a significant increase in the prevalence of HPV-driven OPSCC from 12% during 2000–2006 to 50% during 2019–2022. The use of p16INK4a overexpression is the standard in determining HPV-driven carcinogenesis, but disease prevalence impacts on its positive predictive value. Methods: This is a multicenter retrospective study enrolling 390 consecutive patients aged ≥18 years, diagnosed with pathologically confirmed OPSCC in Northeastern Italy between 2000 and 2022. High-risk HPV-DNA and p16INK4a status were retrieved from medical records or evaluated in formalin-fixed paraffin-embedded specimens. A tumor was defined as HPV-driven when double positive for high-risk HPV-DNA and p16INK4a overexpression. Results: Overall, 125 cases (32%) were HPV-driven, with a significant upward temporal trend from 12% in 2000–2006 to 50% in 2019–2022. The prevalence of HPV-driven cancer of the tonsil and base of the tongue increased up to 59%, whereas it remained below 10% in other subsites. Consequently, the p16INK4a positive predictive value was 89% for the former and 29% for the latter. Conclusions: The prevalence of HPV-driven OPSCC continued to increase, even in the most recent period. When using p16INK4a overexpression as a surrogate marker of transforming HPV infection, each institution should consider the subsite-specific prevalence rates of HPV-driven OPSCC as these significantly impact on its positive predictive value.Background: The prevalence and incidence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection are increasing worldwide, being higher in high-income countries. However, data from Italy are scanty. p16 The prevalence of oropharyngeal squamous cell carcinoma (OPSCC) varies mainly by geographic area, with it being much higher in Northern Europe and Northern America, and lower in Southern Europe and in low-income countries . HoweverINK4a immunohistochemical (IHC) positivity, which is considered a surrogate for HPV-driven cancer. However, an up-regulation of p16INK4a was identified in 8–20% of tumors with no evidence of HPV transforming infection [INK4a as a stand-alone test for the identification of HPV-driven OPSCC [INK4a positive and p16INK4a negative oropharyngeal tumors in order to improve prognostic stratification, as different aspects of the disease, including category T and N, and the presence of extracapsular lymph node extension, have a different prognostic impact in the two groups [HPV-driven OPSCC represents a separate entity characterized by a distinct genetic profile , differenfection ,10, poinen OPSCC . The VIIo groups .Numerous trials are also underway to evaluate the non-inferiority of de-intensified treatments in HPV-driven OPSCCs whose rationale rests on the observation that HPV-driven OPSCC has a much better prognosis and affect younger patients . Thus, tINK4a IHC in identifying a transforming HPV infection in OPSCC [INK4a IHC? This is the positive predictive value (PPV) and strictly depends on the prevalence of the disease being tested. Therefore, it is of paramount importance to know the current epidemiology of HPV-driven OPSCCs to estimate the reliability of p16INK4a as a surrogate marker of HPV-driven carcinogenesis.A very comprehensive systematic review and meta-analysis estimated a sensitivity and specificity of 94% and 83% for p16in OPSCC . Beyond INK4a IHC in determining HPV-driven OPSCC.The aim of the present investigation was to evaluate the trend in the prevalence of HPV-driven OPSCC in the last 20 years in three centers in Northeastern Italy, with a focus on its impact on predictivity of p16INK4a status. Data on 117 patients have been included in previously published report [This is a multicenter retrospective study enrolling patients in three hospitals from two neighboring areas of Northeastern Italy , which share common lifestyle factors, including the prevalence of tobacco smoking and alcohol drinking ,16. EnroDNA was extracted by the phenol–chloroform method or using a commercial Qiagen kit , in accordance with the manufacturer’s instructions. Search and typing of HPV-DNA sequences were performed by PCR using MY09/MY11 primers and restriction fragment length polymorphism analysis of the amplified products, as described by Nobre et al. , or usinINK4a protein was performed on FFPE sections by immunostaining using the primary antibody CINtec for V-kit , or the BD Pharmingen IHC Detection kit, according to the manufacturer’s instructions. The expression results were scored as positive by using a 70% cut-point and considering the nuclear and cytoplasmic stain distribution. The expression of p16INK4a overexpression. Patients with tumors positive for only one biomarker were included in the group of non-HPV-driven OPSCC. The prevalence of HPV-driven OPSCCs was calculated as the proportion of HPV-driven cases on the total number of OPSCC cases in the period; confidence intervals were calculated according to the Clopper–Pearson method. To evaluate the trends, four periods with similar number of cases were defined: 2000–2006, 2007–2012, 2013–2018, and 2019–2022. Analyses were further conducted in strata according to gender, age , and OPSCC subsite . Differences in prevalence across strata were evaluated through exact binomial test; the Kruskal–Wallis test was performed to evaluate differences across strata of continuous variables.A tumor was defined as HPV-driven when double positive for high-risk HPV-DNA and p16To evaluate the independent factors affecting the prevalence of HPV-driven OPSCCs, accounting for potential factor correlation, a multivariable logistic regression model was adopted. The odds ratio (OR) of being diagnosed with and HPV-driven OPSCC, with the corresponding 95% confidence interval (CI), was calculated, adjusting for potential predictors . INK4a overexpression were calculated as a function of prevalence of HPV-driven OPSCC [PPVs and negative predictive values (NPVs) of p16en OPSCC , rangingen OPSCC .A total of 390 consecutive patients with OPSCC—median age: 64 years, interquartile range: 57–71 years; 290 (74.4%) males—met the inclusion criteria and were included in the study .INK4a overexpression. Seventeen cases  were p16INK4a positive but high-risk HPV-DNA negative, while nine cases  were high-risk HPV-DNA positive but p16INK4a negative.Overall, 125 cases  were HPV-driven based on the double positivity for high-risk HPV-DNA and p16p = 0.018), with a male to female prevalence ratio of 0.68. The median age at diagnosis was similar in patients with HPV-driven OPSCC and in those with non-HPV-driven cancers . A statistically significant increase in the proportion of HPV-driven OPSCC was observed over time from 11.8% (95% CI: 5.6–21.3%) during 2000–2006 to 50.0% (95% CI: 39.8–60.2%) during 2019–2022 (p < 0.001).The prevalence of HPV-driven OPSCC was higher among women (42.0%) than among men .HPV16 was detected in 114 cases , HPV33 in 6 patients , and HPV58 and HPV18 in two cases each; one case showed co-infection with HPV35 and HPV59. HPV16 was more frequently observed among younger patients , while other HPV-types were most prevalent in older subjects  and for cancer localized in the posterior pharyngeal wall/soft palate . As also observed by other authors , this phINK4a-positive OPSCCs [INK4a overexpression would be 96%, 85%, and 58%, respectively. Furthermore, considering the low prevalence of HPV-driven SCC in these subsites, it has almost no validity for SCCs originating from soft palate and posterior oropharyngeal wall. We, therefore, believe that each Institution should know the epidemiological landscape of HPV-driven oropharyngeal carcinogenesis in its own geographic area in order to establish a more reliable diagnostic approach toward OPSCCs. Furthermore, the possibility, which in any case needs further confirmation, that OPSCC driven by non-HPV16 high-risk HPV genotypes may not have a prognostic benefit compared to HPV-negative counterpart [INK4a is low, we strongly recommend defining an oropharyngeal cancer as HPV-driven when double positive for high-risk HPV-DNA sequences and p16INK4a. Indeed, other biomarkers highly indicative of a transforming HPV infection, such as E6 and E7 mRNA detection, are not easily usable in clinical practice [Despite the TNM system having purely prognostic purposes and that it does not aim to suggest a particular therapeutic strategy, and that there are currently no specific treatments for HPV-driven OPSCC, it is known from both in vivo and in vitro observations that HPV-driven OPSCCs are more radiosensitive and chemo-sensitive ,6. Furthe OPSCCs . For thenterpart , underlipractice .Finally, in light of this progressive increase in HPV-driven OPSCC, health policies should actively promote vaccination against HPV in both females and males. HPV vaccines have been shown to be effective in reducing the prevalence of oral and oropharyngeal HPV infections, with an average relative prevention percentage of 83% . In 2020INK4a overexpression as a surrogate marker of transforming HPV infection. The strengths of our study are the use of two biomarkers for HPV-driven determination and the long period of patient enrolment.Some potential limitations have to be acknowledged. A first limitation is the relatively small number of patients included in our cohort. Secondly, this is a retrospective study based on clinical data from participating centers and, different from population-based studies, it may suffer from selection bias. However, the participant hospitals are referral centers for OPSCC in their catchment area, and patients’ health mobility is negligible. Furthermore, FFPE specimens were obtained based on availability within pathology archives, and this limited the sample size, particularly in the early study period. Finally, prospectives are necessary to better clarify the accuracy of p16INK4a overexpression as a surrogate marker of transforming HPV infection, each institution should consider their own subsites-specific prevalence rates of HPV-driven OPSCC as these significantly impact on its positive predictive value.The prevalence of HPV-driven tonsils and base of the tongue SCCs continued to increase, even in the most recent time period with a more marked increase being observed in females compare to males. Consequently, it is urgent to establish politics to increase the information of the Italian population on this aspect. When using p16