SDHC, COX7A1, COX16, COX17, ELOVL6, GOT2, and SLC16A2 were correlated with improved patient survival only in the HPV-positive group. This work suggests that specific transcriptional alterations in metabolic genes may serve as predictive biomarkers of patient outcome and identifies potential targets for novel therapeutic intervention in HPV-positive head and neck cancers.Human papillomavirus (HPV) causes an increasing number of head and neck squamous cell carcinomas (HNSCCs). Altered metabolism contributes to patient prognosis, but the impact of HPV status on HNSCC metabolism remains relatively uncharacterized. We hypothesize that metabolism-related gene expression differences unique to HPV-positive HNSCC influences patient survival. The Cancer Genome Atlas RNA-seq data from primary HNSCC patient samples were categorized as 73 HPV-positive, 442 HPV-negative, and 43 normal-adjacent control tissues. We analyzed 229 metabolic genes and identified numerous differentially expressed genes between HPV-positive and negative HNSCC patients. HPV-positive carcinomas exhibited lower expression levels of genes involved in glycolysis and higher levels of genes involved in the tricarboxylic acid cycle, oxidative phosphorylation, and β-oxidation than the HPV-negative carcinomas. Importantly, reduced expression of the metabolism-related genes  As of 2018, head and neck squamous cell carcinomas (HNSCC), namely cancers of the oral cavity, oropharynx, nasopharynx, larynx, and hypopharynx, had the 8th highest combined incidence rate and the 5th highest 5-year prevalence as interpreted from GLOBOCAN data . InfectiHPV-positive (HPV+) HNSCCs are distinct from their HPV-negative (HPV-) counterparts from a molecular perspective, with characteristic genetic, epigenetic, and protein expression profiles ,8,9. In Altered metabolism is a cancer hallmark that was recognized decades ago with the discovery of the Warburg effect, also known as aerobic glycolysis . AerobicSDHC, COX7A1, COX16, COX17, ELOVL6, GOT2, and SLC16A2—correlated with markedly improved patient survival in HPV+, but not HPV- HNSCC. The products of these genes could potentially be exploited as targets for therapeutic intervention. Furthermore, the low expression of these seven genes appears useful in predicting improved overall survival in HPV+ HNSCC and could serve as biomarkers of patient outcome.In this study, we used RNA-seq data from over 500 HNSCC primary tumour samples from the TCGA to comprehensively compare the expression of genes across key cellular metabolic pathways between HPV+, HPV-, and normal-adjacent control tissues. Expression of a number of metabolic genes were significantly altered in HPV+ versus HPV- HNSCC. Specifically, genes involved in glycolysis were expressed at lower levels in HPV+ compared to HPV- HNSCC. In contrast, genes involved in the TCA cycle, oxidative phosphorylation, and β-oxidation exhibited higher expression in HPV+ samples compared to their HPV- counterparts. Importantly, we identified that low expression of multiple metabolic genes—In order to identify differences in metabolic gene expression between HPV+ and HPV- HNSCC, we analyzed the TCGA Illumina HiSeq RNA expression dataset from the HNSC cohort for expression of 229 metabolic genes in central metabolic pathways . This clThis analysis illustrates that glycolytic genes in HPV- HNSCC tissue are more upregulated in comparison to normal tissue than in HPV+ HNSCC when compared to normal tissue. This is particularly evident between HPV+ HNSCC and HPV- HNSCC tissues, since most glycolytic genes are downregulated in HPV+ HNSCC when compared to HPV- HNSCC. However, genes involved in the TCA cycle, oxidative phosphorylation, and β-oxidation were downregulated in both HPV+ and HPV- HNSCC in comparison to normal control tissue. This means that, despite differences in metabolic gene expression between HPV+ and HPV- HNSCC, the metabolism of both types of tumours could resemble the Warburg effect, with lower cellular respiration rates compared to normal control tissue. When compared to one another, HPV+ HNSCC had generally higher expression of these genes than HPV- HNSCC. Other metabolic pathways appeared to have a more similar split between upregulated and downregulated genes in all comparisons, suggesting that the presence of HPV may have minimal impact on transcriptionally mediated changes in these pathways. Overall, it appears that HPV+ HNSCC may be more reliant on cellular respiration than HPV- HNSCC.SDHC, part of the mitochondrial respiratory complex II; COX7A1, COX16, and COX17, all part of the mitochondrial respiratory complex IV; ELOVL6, involved in fatty acid elongation; GOT2, involved in amino acid metabolism; and SLC16A2 , which encodes a thyroid hormone transporter. We performed a pathway enrichment analysis of our seven significant genes utilizing a PANTHER overrepresentation test which indicated that 5 of these 7 genes were significantly associated with the mitochondria . These genes were SDHC, COX7A1, COX16, COX17, and GOT2. Detailed studies of the relative expression of each of these genes in HPV+, HPV- and normal control tissue, as well as their association with overall patient survival are presented below.Tumour-associated metabolic alterations have functional consequences that impact disease progression, response to therapy, and patient survival . We dichSDHC, which encodes a component of the mitochondrial cellular respiration complex II, correlated with increased HPV+ HNSCC patient survival    B. Howeve = 0.45) C.COX7A1 and COX17, which encode structural components of complex IV, and COX16, whose product is involved in complex IV assembly. Consistent with the possibility that HPV+ HNSCCs are more reliant on cellular respiration than HPV- HNSCCs, three genes encoding components of the mitochondrial cellular respiration complex IV were also correlated with HPV+ HNSCC patient survival . These gCOX7A1 expression was significantly lower in both HPV+ and HPV- HNSCC samples compared to normal control tissues    B, but no = 0.59) C. COX16 expression was lower in HPV+, but not in HPV- HNSCC    E, but no = 0.59) F. COX7A1 and COX16, COX17 expression was higher in HPV+ than HPV- HNSCC   (In contrast to V- HNSCC G. Expres tissues G. Howeve= 0.012) H, but no = 0.59) I.ELOVL6 expression in HPV+ HNSCC was not significantly different from HPV- HNSCC. However, both HPV+ and HPV- HNSCC had significantly lower overall levels of ELOVL6 expression than normal control tissues   ( tissues A. HPV+ H= 0.084) B. Reduce = 0.83) C.GOT2 expression was significantly lower in HPV+ than HPV- HNSCC or normal control tissues and significantly lower in HPV- HNSCC than normal control tissues .  tissues D. The hiSLC16A1 (MCT1) was recently reported to be associated with poor survival outcomes in HNSCC [SLC16A1 was significantly higher than normal head and neck tissues for both HPV+ and HPV- HNSCC, and expression of SLC16A1 was higher in HPV- HNSCC than HPV+ HNSCC, the impact of differential expression of SLC16A1 on overall survival in either HPV+ or HPV- HNSCC was not significant (p > 0.05). Of the various family members, only expression of SLC16A2 (MCT8), whose main function is thyroid hormone transport, appeared to be associated with altered overall survival   (Expression of  tissues G. HPV+ H= 0.047) H, but no = 0.48) I. To determine the extent that each of the HPV+ HNSCC survival-associated genes could influence patient outcomes, we generated a hazard ratio (HR) for each gene and a variety of clinical variables by univariate analysis . Each HRAs the contribution of these genes to overall survival might not be independent of one another, we also analyzed the relationship between survival and gene expression for all survival-associated metabolic genes and clinical variables concurrently by multivariate analysis . The hazCOX16 and COX17 in their tumours was compared to survival of patients with high expression of both genes, survival was significantly greater in the COX16 and COX17 double low expression group (p = 0.0015)  than samples expressing high levels of both genes  A. In patth genes B. In pat SLC16A2 C, survivSLC16A2 and COX16 or SLC16A2 and COX17. This provides further evidence that the improved survival associated with low expression for each of these genes may occur independently of one another. Additionally, the multivariate analysis indicates that COX16 and COX17 independently contribute to survival despite the correlation between these two genes  is a vitamin E analogue, which has selective growth inhibitory properties for some human cancer cells [SDHC expression and HPV+ HNSCC survival outcomes we observed.As shown in our results, limiting er cells . α-TOS cer cells . α-TOS ier cells  in in vier cells . Given tCOX7A1 and COX16 gene products have not yet been identified. However, as both are part of mitochondrial respiratory complex IV, it is possible that the complex IV inhibitors ADDA 5 [Specific small molecule inhibitors for both the s ADDA 5  and tetrs ADDA 5  could prs ADDA 5 . E6 has s ADDA 5 , which ms ADDA 5 . Whethers ADDA 5 , perhapsTwo inhibitors of ELOVL6 were able to reduce the fatty acid composition of hepatocytes and the liver in a murine model of obesity ,37, but GOT2 was associated with statistically significant survival in both groups (p < 0.05). However, only expression of GOT2 in our HPV+ HNSCC group met the FDR cut-off of q = 0.1. This means that while GOT2 may be important for survival outcomes in both HPV+ and HPV- HNSCC, it is likely that it has a more substantial contribution to patient survival in HPV+ HNSCC. In breast cancer, sensitivity to a nucleotide synthesis inhibitor, methotrexate, has been linked to high GOT2 expression [GOT2, may be sensitive to methotrexate, but this remains to be explored.In our study, we found that low expression of pression . This ispression . It is pSLC16A2 encodes a plasma membrane T3/T4 transporter. Once inside the cell, T3/T4 can bind nuclear and mitochondrial-localized thyroid hormone receptors, which are key regulators of mitochondrial biogenesis [SLC16A2 at high levels. We extracted data from our previous study of 27 HNSCC cell lines (6 HPV+ and 21 HPV- HNSCC cell lines) examining the effects of a variety of agents on cell growth and proliferation, including the TKI inhibitors mentioned above [SLC16A2. A flavonoid, silychristin, also inhibits SLC16A2 [ogenesis . As HPV+ogenesis . These Togenesis . As suched above . Of the  SLC16A2 , but has SLC16A2 . High le SLC16A2 , and thy SLC16A2  and T ce SLC16A2 .COX16, COX17, and SLC16A2 were independently associated with overall survival. This suggested that the collective changes in the expression of these three genes could be a powerful predictor of clinical outcome. This was supported by our observation that the overall survival for the group of patients exhibiting simultaneously low expression of any two of these genes was far better than those simultaneously expressing high levels. Thus, expression of these genes may have prognostic utility. Furthermore, COX16, COX17, and SLC16A2 may represent attractive therapeutic targets for HPV+ HNSCC that warrant further exploration, especially considering that treatment with specific inhibitors may phenocopy the effects of low expression of these metabolic genes. While our univariate analysis of all seven genes confirmed that their expression was significantly correlated with survival, our multivariate analysis indicated that It is important to be cognizant of the limitations to this kind of study. One limitation to this study was the lack of protein data in the TCGA to corroborate our HPV+ HNSCC mRNA expression data. This is an important consideration, as levels of protein expression do not necessarily mirror mRNA expression . As withhttps://gdac.broadinstitute.org/). Patient survival data for the TCGA HNSC cohort, as reported by the Pan-Cancer Atlas [https://www.cell.com/cms/10.1016/j.cell.2018.02.052/attachment/f4eb6b31-8957-4817-a41f-e46fd2a1d9c3/mmc1.xlsx.Level 3 RNA-seq by Expectation Maximization normalized Illumina HiSeq RNA expression data (build 2016012800) for the TCGA HNSC cohort, was downloaded from the Broad Genome Data Analysis Centers Firehose server  reflecting drug activity were extracted from a previously conducted high throughput drug screen using 27 HNSCC cell lines . The aveSDHC, COX7A1, COX16, or COX17 was associated with better survival outcomes. Low expression of ELOVL6, involved in fatty acid elongation; GOT2, involved in amino acid metabolism; and SLC16A2, involved in thyroid hormone transport, were also all associated with better survival outcomes in HPV+ HNSCC patients. However, of these genes, only COX16, COX17 and SLC16A2 were independently correlated with survival outcomes according to our multivariate analysis. Importantly, COX16, COX17, and SLC16A2 were associated with near 100% survival in all patients with low expression of any two of these genes. The products of these genes may represent useful new therapeutic targets for HPV+ HNSCC, as inhibition of their functions could phenocopy the metabolism of those tumours with low levels of metabolic gene expression, leading to improved survival in HPV+ HNSCC patients.In summary, our analysis of HNSCC TCGA data stratified by HPV status indicated that the metabolic profile of HPV+ and HPV- HNSCC are strikingly different. HPV- HNSCCs may utilize glycolysis to a greater extent than HPV+ HNSCCs, while HPV+ HNSCCs may be more reliant on the TCA cycle, cellular respiration, and β-oxidation than HPV- HNSCCs. Despite this difference, both types of HNSCCs likely exhibit far less cellular respiration than normal head and neck tissues, consistent with a cancer-associated Warburg phenotype . Importa


To identify the optimal cost-effective strategy for cervical cancer screening program in Thailand by comparing the different algorithms which based on the use of primary human papilloma virus (HPV) assay. We use a Microsoft Excel–based spreadsheet to calculate the accumulated cases of preinvasive and invasive cervical cancer and the budget impact of each screening program. The model was developed to determine the cost-effectiveness of 3 screening strategies: pooled HPV test with reflex liquid-based cytology triage, HPV genotyping with reflex p16/ki67 dual stain cytology, and pooled HPV test with dual stain. The main outcomes were the total cost, incremental quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICERs). Strategy entailing primary HPV genotyping and reflex dual stain cytology is the least costly strategy  and provides the similar QALY gained compared to pooled high-risk HPV testing with reflex dual stain (Average QALY 24.03). Pooled HPV test with reflex dual staining is more costly compared to strategy without reflex dual staining. The ICER was US$353.40 per QALY gained. One-way sensitivity analysis showed that the model is sensitive to the cost of dual stain and the cost of cancer treatment. Decreasing the incidence of cervical cancer case and increasing the QALYs can be successful by using dual stain cytology as the triage test for pooled HPV test or HPV genotyping. The result of our analysis favors the use of HPV genotyping with the reflex dual stain as it offers the most QALY at the lowest cost. Human papilloma virus (HPV) testing has been used in conjunction with cytology and used for triage of cervical cytology showing atypical squamous cells of undetermined significance (ASCUS). To improve the efficacy and increase the coverage of screening, several new strategies have been studied.611 Our previous study compared the cost and benefit of 4 different cervical cancer screening strategies involving primary HPV 16/18 genotyping, high-risk HPV testing, liquid-based cytology, and conventional cytology.12 Model predictions indicated that the most cost-effectiveness strategy is primary high-risk HPV testing by reducing cost and also increase the detection of cervical intraepithelial neoplasia 2 and 3. However, the primary HPV testing still has some problem because of its high sensitivity and low specificity. This may cause overtreatment. The concept of dual stain has been introduced to decreased overtreatment case. One systematic literature review showed that across all age groups in a screening population, dual staining was significantly more sensitive than and equally specific as cytology.13 Specificity gains resulted in fewer false positives and an increase in the number of correct referrals to colposcopy. Dual staining with p16/Ki-67 cytology is an attractive biomarker approach for triage in cervical cancer screening.13Cervical cancer is the fourth most common cancer in women of about 570 000 of new cases in 2018.14 Based on the available results of cost-effectiveness analysis study in Thailand, the objective of this study is to evaluate the cost and effectiveness of HPV-based primary screening strategies with different triage including Papanicolaou (Pap) cytology and p16/Ki67 dual stain cytology in Thai population 30 to 65 years old in order to implement the most appropriate strategy to our country.Our recent study assessed the clinical and cost-effectiveness of HPV primary screening triage with p16/Ki-67 dual stain cytology compared to cytology. The preliminary result suggests that screening by use of HPV genotyping test and dual stain cytology as the triage test for other high-risk HPV positive women as a primary screening test in Thai population 30 to 65 years old is expected to be more cost-effective than cytology.We developed a computer simulation model (A Microsoft Excel–based spreadsheet) to calculate the number of accumulated cases of cervical intraepithelial neoplasia, invasive cervical cancer, and budget impact of each screening program. The model of natural history was constructed . Individ16 Based on the total number of Thai population (in 2015) and excluding the women with HIV infection, history of hysterectomy and pregnant, 7 953 963 were eligible for screening.17The study population for our simulation was a closed cohort representing the Thai population 30 to 65 years of age which is an approximately 26.6% of the total population. The screening coverage was 50% of the target population.14 According to the clinical guideline of the National cancer institute of Thailand, all strategies were considered a 5-year interval. The decision tree models of screening and management algorithms are displayed in We examined 3 different algorithms of primary HPV assay based on the evidence of cost-effectiveness data from our previous studies.High-risk HPV testing with reflex liquid-based cytology (LBC) triage: Pooled high-risk HPV testing is a primary tool, followed by LBC for women with the high-risk HPV positive result. A cytology of ASCUS or worse leads to immediate colposcopy. A repeat HPV testing at 12 months will be performed for HR-HPV-positive women with normal cytology. If the result of high-risk HPV is negative, the women will return to routine screening in 5 years .Human papilloma virus with 16/18 genotyping and reflex dual stain: Screening with HPV genotyping then refers to colposcopy if the result is positive for HPV 16 or 18. The dual staining is performed in cases of other 12 high-risk HPV positive and those with positive result undergo colposcopy. For those who negative dual staining, HPV testing will be done at 12 months interval. Women with a negative result of HPV genotyping return to screening in 5 years .High-risk HPV testing and reflex dual stain: using high-risk HPV testing alone every 5 years followed by dual stain if the result is positive for high-risk HPV. The women with positive results of both high-risk HPV testing and dual stain will refer to perform colposcopy. Repeat the HPV test in 12 months for a woman with HR-HPV-positive and -negative dual staining .18Colposcopy was considered to be a gold standard for diagnosis of a precancerous lesion with 100% sensitivity and specificity. Disease progression and regression were assumed to be constant over time and not to be age-specific. The model assumes that the loss to follow-up rate in all strategies were the same and thus will not affect the comparative result. Based on expert’s opinion, we assumed that all cervical intraepithelial neoplasia 3 cases and 50% of cervical intraepithelial neoplasia 2 received treatment. Women with cervical intraepithelial neoplasia 1 or posttreatment cervical intraepithelial neoplasia 2+ returned to follow-up every 6 months. Cervical intraepithelial neoplasia 1 patients with 2 times of negative results and cervical intraepithelial neoplasia 2+ patients with 4 times of negative results would return to routine screening.19; they would stay in the current status or progress to more severe states in the model. We considered both deaths from cervical cancer and all-cause mortality.Four percent of cervical intraepithelial neoplasia 2 to 3 and 20% of invasive cervical cancer were assumed to be recurred after treatment20 We conducted the analysis from provider’s perspective. The cost component used for the cost evaluation are depicted in 21 Only direct medical cost was included. Indirect costs such as loss of productivity and transportation costs were assumed to have the same among patients. The cancer treatment cost is the median cost across all stages. All clinical and cost inputs were discounted at an annual rate of 3.5%.22 The results of cost-effectiveness analysis were presented by incremental cost-effectiveness ratios (ICERs) per quality-adjusted life years (QALY) which is the additional cost divided by additional QALY, compared to the next most costly strategy. We used Thailand’s gross domestic product (GDP) per capita to evaluate the most cost-effectiveness strategy which is suggested by the World Health Organization (WHO). An ICER of less than 3 times the per capita GDP would be considered cost effective.23Based on case values used in the model were mainly based on the data from the published literature referenced in A one-way sensitivity of cost parameter of the screening tools in each strategy was performed in order to evaluate the uncertainty in which parameters might affect the ratio. The costs were varied 10% below and 3 times above the base case estimation.24 Comparing strategies 3 and 1, pooled high-risk HPV test with reflex LBC was less costly than with dual stain cytology, whereas pooled high-risk HPV test with reflex dual stain provided a more effective strategy at an ICER of US$353.40 per QALY gained. The cost-effectiveness frontier between total cost and QALY gained of each strategy is presented in Supplemental Material 2. Strategy 1 or pooled high-risk HPV testing with reflex LBC was not on the frontier and fell to the right side of the solid line, meaning that it was not an efficient use of resources. Comparing strategy 3 and strategy 2, pooled high-risk HPV test with reflex dual stain cytology was clearly dominated by HPV genotyping with reflex dual staining (same benefit but higher cost).The screening performances are shown in The model sensitivity was measured by the absolute percentage change in ICER induced by decreasing 10% below and increasing 3 times perturbation in the parameters . More significant changes were found in scenarios related to changes in the costs of dual stain cytology test and cancer treatment. However, HPV genotyping test with reflex dual stain still was the optimal strategy.25 It is still not clear what the best strategy for triage of screen-positive women is. To find an optimal integrated screening and triage strategy for Thai women, we conducted the cost-effectiveness analysis study and reported the primary high-risk HPV testing with reflex LBC was preferred over primary liquid base cytology as it was more effective and less costly.12 We then applied the HPV model by using p16/Ki67 dual stain cytology as triage for high-risk HPV-positive women (non16/18 HPV positive) and compared with primary cytology. The result showed the good performance of p16/ki67 dual stain cytology and reported that HPV genotyping with reflex dual stain was more costly but more effective than cytology method with an ICER was 1395 per QALY.14Evidence suggests that primary HPV testing can be more effective than cytology and additional triage tests are needed to identify women with progressing disease. The triage strategies include HPV genotyping for HPV 16/18, cytology, p16/Ki67 dual stain cytology, host methylation, and viral methylation testing.16 Uijterwaal et al reported dual stain cytology had a sensitivity of more than 70% for cervical intraepithelial neoplasia 2/3 and the cumulative 5-year cervical intraepithelial neoplasia 3 risk was significantly reduced in dual stain negative women.11 It is therefore suitable for triaging to colposcopy. From our study, the use of dual staining in either strategy 2 or 3 increased the number of precancerous cases detected which resulted in decreased cancer cases detected. One prospective study in HPV-positive women revealed that the dual stain cytology had similar sensitivity and higher specificity compared with cytology for detection cervical intraepithelial neoplasia 2/3 cases.9 Until now, there are no data that how long dual stain negative women remain at low risk of precancerous. Women with positive HPV and negative dual stain in our model were retested with HPV testing, while women with positive HPV and negative cytology were returned to routine screening. If the women with positive HPV and negative dual staining were returned to routine safety, the total cost of strategies 2 and 3 would decrease and these 2 strategies would be more cost-effective than cytology triaging.From this cost-effectiveness analysis, it can be concluded that HPV primary plus genotyping with reflex dual stain cytology every 5 years for Thai women at age of 30 to 65 was the most effective strategy according to a cost-effectiveness threshold based on per capita GDP. Comparing to pooled high-risk HPV test with reflex LBC, the higher cost of diagnostics test was compensated by higher screening performance and lower cost of treatment. Wright et al assessed the performance of dual stain cytology compared with cytology for triaging HPV-positive women undergoing primary HPV screening. The result showed sensitivity, positive predictive value, and negative predictive value were higher for dual staining than cytology, in a similar way to our result.Comparing between primary pooled high-risk HPV primary testing with dual stain cytology (strategy 3) and primary HPV genotyping with dual stain cytology (strategy 2), the clinical outcomes were comparable but strategy 3 was more costly. Both strategies included dual stain cytology which was one of the most expensive diagnostic tests. A larger number of women in strategy 3 were sent for dual stain cytology while HPV 16/18-positive women in strategy 2 were referred directly to colposcopy. This may cause strategy 3 to be the most expensive strategy. One-way sensitivity analysis also confirmed this reason, if the cost of dual staining increased 3 times, the ICER between strategy 3 versus 1 and 3 versus 2 would increase to 114% and 23.97%, respectively. The cost of cancer treatment was another factor that affected the ICER result, the ICER between strategies 3 versus 2 increased almost twice when cancer treatment cost increased 3 times. In the first screening round, the total cost in strategy 2 may be slightly higher than strategy 3 because more cancer cases were detected and treated. However, the number of women would be reduced in the next screening round and the detection of cancer cases would be lower. This results in decreasing the cost of cancer treatment in long term for strategy 2.To the best of our knowledge, this is the first economic study to evaluate an optimal triage strategy for HPV-positive women in Thailand. Our analysis included both direct costs of screening and treatment, and we presented the screening performance, a number of cases detected, QALY and screening budget of each strategy. However, there were some limitations of our study. First, several clinical input parameters of the model were derived from Western literature and the percentage of cervical intraepithelial neoplasia cases received treatment were based on the expert’s opinion. Second, since there were no Thai data available, we did not use the age-specific progression rates for HPV acquisition, clearance, progression from HPV infection to cervical intraepithelial neoplasia 2/3 and cancer. Third, our analysis calculated the cost from Thai Government Hospital which limited applicability to health-care settings in other countries. We tried to minimize this limitation by using the asymmetrical distribution of the cost to right-hand tail in sensitivity analysis because the costs we used were lower compared to the average cost of overall hospital in Thailand. To clarify the most suitable strategy for implementation of cervical cancer screening in Thailand, further studies are necessary. The clinical parameters should be based on the updated data from several regions of Thailand. Indirect cost such as cost of day loss should be incorporated to estimate the total cost of illness based on social perspective. Finally, increasing HPV vaccination coverage will lead to lower prevalence of HPV16/18 infection or higher prevalence of other high-risk HPV infection, which will further reduce the efficacy of strategy 2.In summary, the findings from this study emphasizes the importance of dual stain cytology as a triage test for high risk HPV-positive women. The strategy of primary HPV genotyping test with dual stain cytology every 5 years interval was the most cost-effective screening method and should be considered for implementation in practice and for future guidelines.Click here for additional data file.Supplementary for Cost-Effectiveness of Primary HPV Screening Strategies and Triage With Cytology or Dual Stain for Cervical Cancer by Tanitra Tantitamit, Nipon Khemapech, Piyalamporn Havanond and Wichai Termrungruanglert in Cancer Control


Infection with human papillomaviruses (HPVs) can cause benign and malignant tumours in the anogenital tract and the oropharynx both in men and women. The aim of the presented study was to investigate cervical, anal, and oral HPV-detection rates among women referred to colposcopy for abnormal Cervical Cancer (CaCx) screening results and assess the concordance of HPV-types among these anatomical sites.Women referred to colposcopy at a single centre due to abnormal cytology, conducted for CaCx screening, were subjected to cervical Liquid-based Cytology (LBC) smear testing, anal and oral sampling. Routine colposcopy consisted in multiple biopsies and/or Endocervical Curettage (ECC). HPV-detection was performed by PCR genotyping in all three anatomical sites. In high-risk (hr) HPV-DNA positive samples either from anal canal or oral cavity, anal LBC cytology and anoscopy were performed, or oral cavity examination respectively. Descriptive statistics was used for the analysis of HPV-detection rates and phi-coefficient for the determination of HPV-positivity concordance between the anatomical sites.p < 0.001).Out of 118 referred women, hr. HPV-DNA was detected in 65 (55.1%), 64 (54.2%) and 3 (2.5%) at cervix, anal canal and oral cavity respectively while low-risk HPV-DNA was detected in 14 (11.9%) and 11 (9.3%) at cervix and anal canal respectively. The phi-coefficient for cervix/anal canal was 0.392 for HPV16, 0.658 for HPV31, 0.758 for HPV33, − 0.12 for HPV45, 0.415 for HPV52 and 0.473 for HPV58. All values were statistically significant . Persistent infection with high-risk human papillomaviruses (HPVs) is necessary, but not sufficient, for the development of Cervical Cancer (CaCx) –3. HPV hcondylomata acuminata), dysplastic lesions , or a combination of both [Anal HPV-related cancer is strongly associated with the immunosuppression secondary to HIV-infection and associated with a history of anal-receptive intercourse; however, autoinoculation of vulvar warts to the perianal skin can occur. Clinically, anal HPV-infection can be either asymptomatic or present as benign warts  (58.8%), for any HPV-type, whereas it is 30.7% for women . MoreoveLess is known concerning oral HPV-infection, its prevalence and its implications including the involvement of HPV in oropharyngeal carcinogenesis , 15. HPVProphylactic HPV-vaccines have been implemented in the national vaccination programs of many countries for the primary prevention of CaCx and other HPV-associated diseases . BivalenWomen who already have a cervical HPV-infection and especially women who have a precancerous cervical intraepithelial lesion (CIN) are considered to be susceptible to HPV-infections in other anatomical sites . To inveThe presented analysis was conducted on samples collected from 118 women referred to the Colposcopy/Cervical Pathology Clinic of the 2nd Department of Obstetrics/Gynaecology , Greece) for either an abnormal Pap-test screening result or for follow-up testing after surgical treatment for high-grade CIN between January 2015 and November 2016. Exclusion criteria had been pregnancy, prior total hysterectomy due to various reasons, and prior chemo-radiation treatment. All women with anal hr-HPV positivity where referred to the Anal/Colorectal Clinic of the 1st Department of General Surgery, Medical Faculty AUTh, for anoscopy. Women with oral hr-HPV positivity were referred for oral examination to the Stomatology Clinic, School of Dentistry, Faculty of Health Sciences AUTh.Each woman was subjected to cervical/anal/oral sampling. All samples were processed at the Molecular Cytopathology Laboratory of the 2nd Department of Obstetrics/Gynaecology. Ectocervical and endocervical sampling was done using a plastic Ayre’s Spatula and a Cytobrush®  respectively, and anal sampling was done using the Anex® Brush . After collection each device was directly immersed in a vial containing PreservCyt® collection fluid , according to manufacturer’s instructions. Oral samples were collected by an oral rinse with 15 mL Listerine® for 30 s , 22 in sCytology testing was performed on cervical/anal samples using Liquid-Based Cytology (LBC) , prior to molecular testing. Cytology results were classified according to Bethesda 2014 classification . ASCUS oDNA extraction/purification was performed using CLART-HPV2 Extraction/Purification kit . CLART-HPV2 detect infections and coinfections of 35 most clinically hr- (hr) and low-risk (lr) HPV-types (6/11/16/18/26/31/33/35/39/40/42/43/44/45/51/52/53/54/56/58/59/61/62/66/68/70/71/72/73/81/82/83/84/85/89). Protocol was carried out according to manufacturer’s instructions. One ml of PreservCyt solution was used for HPV-DNA extraction with purifying columns and 5 μl of that was used for PCR-Amplification. Prior to visualization, PCR-products were denaturated at 95 °C for 10 min. Visualization was performed using 5 μl denaturated product on CLART-microarray. Analysis and interpretation of results was automatically performed by GENOMICA’s reader (CAR®).Colposcopic evaluation was performed according to 2011 colposcopic terminology of International Federation for Cervical Pathology and Colposcopy . RoutineHigh-resolution anoscopy (HRA) was performed at the Anal/Colorectal Outpatient Clinic of the 1st Department of General Surgery, Papageorgiou Hospital, AUTh, Thessaloniki, Greece. An anoscope was inserted and a colposcope was used to examine the squamocolumnar junction, the anal canal including the transformation zone and the perianal skin in a systematic manner. After examination with acetic acid (3% or 5%), and subsequent application of Lugol’s iodine solution, a decision was made regarding the anoscopic impression, which could be either within normal limits, or indicative of a LSIL, HSIL or cancer. In case of an abnormal anoscopic impression a biopsy was taken . If HRA Oral visualization was conducted using an adequate light source in a systematic way. All mucosal surfaces were examined and lesions were recorded on a diagram. The lips were inspected first, followed consecutively by the labial mucosa, buccal mucosa, floor of the mouth, ventrum of the tongue, dorsal surface of the tongue, hard and soft palates, gingivae and teeth .p-value. The level of significance was set to 0.05.The main purpose of the analysis was to assess the concordance of different HPV-types among different anatomical sites in women. Phi-coefficient was used to determine HPV positivity concordance between cervical/anal, cervical/oral, anal/oral and was calculated for each HPV-type or HPV-types group concerning these anatomical site pairs. The HPV-type groups were hr-HPV, lr-HPV and the nonavalent HPV vaccine types group (HPVs 6/11/16/18/31/33/45/52/58). Phi-coefficient can range from −1 (strong negative correlation) to +1 (strong positive correlation). Values near zero indicate no correlation. For each phi-value calculated we report the respective Smoking habits were assessed by Smoking Intensity Index (SII), a new variable created as below: Number (cigarette/day) × 365 × Number (years) ÷ 1000 .All analyses were performed using statistical software SPSS Statistics 22.0.The age of the recruited women ranged from 21 to 61 years (mean age 38.6 years). Table As expected, due to the characteristics of the referral population in this study, cervical HPV-prevalence was high. In particular, hr-HPV prevalence was 55.1% Table . Among hAnal HPV-prevalence presented similar trend to the cervical one with minor differences. The overall anal hr-HPV prevalence was 54.2%. The most common hr-HPV type was HPV53 (16.9%), followed by HPV31 (15.3%), HPV51 (10.2%), HPV16 (9.3%), HPV66 (6.8%), HPV70 (5.9%), HPVs 33/58 (each 5.1%). Remaining hr-HPVs presented prevalence below 4.2%. Lr-HPV-types were detected in 9.3% of the cases; the most common lr-HPV was HPV6 (8.5%), followed by HPVs 42/61/81 (4.2% each), HPV40 (3.4%). The prevalence of the remaining lr-HPVs was less than 2.5% for each. Out of the 64 women recruited for the current analysis positive for hr-HPV types and negative for intraepithelial lesion or malignancy (NILM) (42.2%) or LSIL (57.8%) anal cytology, only 15 women underwent anoscopy  with no pathological or suspicious lesions detected.Oral HPV was detected much less frequently than cervical and anal. The overall hr-HPV and lr-HPV prevalence was 2.5 and 0.0% respectively. The only hr-HPV types detected were HPV16 (1.7%) and HPV53 (0.8%). No lesions were detected in the oral cavity of the women referred to oral examination.A hr-HPV and lr-HPV co-infection was documented in 7.6, 19.5 and 0.0% concerning cervical/anal/oral samples respectively Table . The prep < 0.001). For cervical/oral and anal/oral phi-coefficient values were −0.071 and 0.04 with p-values 0.443 and 0.662 respectively, indicating no correlation for the detection of these HPV-types in cervix or anus compared to oral cavity. HPV16-detection yielded similar results. Phi-coefficient values for this type were 0.392 (p < 0.001), 0.088 (p = 0.341) and −0.042 (p = 0.646) for cervix compared to anus or oral cavity and for anus compared to oral cavity respectively.Most of HPV-types detected in cervical samples were detected also in anal samples, and for the majority of them there has been a statistically significant correlation. The phi-coefficient, regarding the cervical/anal anatomical site pair, for hr-HPVs was 0.402 (p < 0.001), for cervical/oral − 0.023 (p = 0.8), and for anal/oral −0.041 (p = 0.658). All remaining hr-HPV types yielded results only for the cervical/anal anatomical site pair. The strongest correlations, with a phi-coefficient above 0.7 were noted for HPV33 , HPV35 , HPV51 , and HPV56 . Regarding the nonavalent-vaccine HPVs, correlation was significant for the cervical/anal samples , and cervical/oral samples  . Specifically, the strongest correlation was reported for HPV11 , no correlation was reported for HPVs 62  and 84 . The rest lr-HPVs presented weak (0.3 < phi < 0.7) correlations  cannot provide the analysis with the power necessary for robust conclusions concerning the interpretation of these associations.The higher anal HPV-prevalence in individuals with genital HPV-infection has been attributed to self-inoculation. This phenomenon has been shown in men in a sub-analysis of the HIM study reported by Pamnani et al. , and it Genital HPV-infection prevalence in women decreases with increasing age , and theIn the recent years, there has been significant research on HPV-infection in anatomical sites other than the anogenital area. In particular, there has been an increasing interest in the detection of HPV in the oral cavity and the possible relation of HPV-infection to oropharyngeal cancer. This concerns both sexes but mostly men since this type of cancer is quite more frequent in men than women . HoweverAs a conclusion, our results suggest that most HPV-types, high and low-risk, detected in the cervix of women with prevalent cervical dysplasia, correlate with the ones detected in the anal canal of these women. This particularly applies for the HPV-types related to the nonavalent HPV vaccine (HPVs 6/11/16/18/31/33/45/52/58), implying that this vaccine could be used as the “first line of protection”, apart from cervical cancer, for anal cancer as well.


The Human Papillomavirus (HPV) Prevention and Control Board is an independent multidisciplinary board of international experts that disseminates relevant information on HPV to a broad array of stakeholders and provides guidance on strategic, technical and policy issues in the implementation of HPV control programs. In response to drastic drop of vaccine coverage following the adverse event crisis in Carmen del Bolivar, Colombia, the HPV Prevention and Control Board in collaboration with the Colombian National Cancer Institute and Colombian League Against Cancer convened a meeting in Bogota, Columbia (November 2018). The goal of the meeting was to bring together national and international group of experts to report the disease burden, epidemiology and surveillance of HPV and HPV-related cancers, to discuss the successes and especially the challenges of HPV vaccination and screening in Colombia, as well as the lessons learnt from neighbouring countries. The meeting provided a platform to confer various stakeholder’s perspectives, including the role of the Colombian healthcare system and to catalyse various parts of the public health community in Colombia into effective action. The conclusion of the meeting included following suggestions to strengthen HPV prevention and control: 1) Re-introducing school-based vaccine programs, 2) Integrating primary and secondary prevention programs, 3) Developing an innovative crisis communication plan targeting healthcare workers, teachers and general population, 4) Building trust through efficient and timely communication, 5) Building strong relationship with media to ensure a stable vaccination campaign support, and 6) Promoting empathy among healthcare professionals towards patients to build trust and communicate effectively. Shortcomings of the program seem to be lack of clear stewardship with no collaborative plan between territories and insurers, lack of organized quality assurance, and unjustified risk of screening adolescents as allowed in the new regulation, for whom detection of transient HPV infections may lead to unnecessary treatment.In Colombia, HPV vaccines were authorized before national policy recommendations; within the context of the decentralized decision-making process, HPV vaccination activities were driven by pressure from local political actors . ParentsIntroduction of the HPV vaccine into national immunization program was prepared in close cooperation with Colombian scientific societies. In 2008, a cost-effectiveness study was performed , which lIn 2014, and thereafter, the target population was again girls in 4th degree of primary school older than 9 years of age. In June 2014, reports of adverse symptoms  were received from girls residing in Carmen de Bolivar who received the 2nd dose of the HPV vaccine. After a few days, similar symptoms were reported from more than 600 girls. This news was in the headlines for 2 weeks. Despite studies showing that there was no correlation with vaccination, a big drop in coverage ensued, to 60% in 2015 and to 6% in 2016. In addition, the National Academy of Medicine recommended that a screening for autoimmune diseases be performed before the administration of the HPV vaccine. Due to these events, school-based vaccine programs were discontinued.To increase vaccine confidence, a media campaign was initiated, involving high-level medical figures, which resulted in a slight increase (17.6%) in vaccine coverage in 2018.In April 2018, the Ministry of Health decided to change the HPV vaccination scheme to two doses (0 and 6 months), for all immunocompetent girls between 9 and 18 years of age. For optimal impact of the vaccination program, the following has been considered necessary: 1) acceptance of the vaccine by the adolescents/teenagers and their parents; 2) appropriate knowledge and understanding of the vaccine by healthcare workers; 3) correct management of information about vaccine safety; and 4) immediate response to rumors about the vaccine [22–23].In 2008 the Colombian National Institute of Health and the Colombian Institute of Surveillance of Drugs and Food (INVIMA) started the Eventos Supuestamente Atribuibles a la Vacunación o Inmunización (ESAVI) program to perform the surveillance for potential vaccine adverse events in Colombia. This surveillance system is one of the strongest in America, it is nominal or individual and it is based on weekly reports from nearly 5200 health institutions, representing 1117 municipalities . In addiFor the HPV vaccine, the Carmen de Bolivar incident resulted in 240 adverse events (AEs) per 100,000 doses in 2014 and thereafter the reported rate of adverse events decreased gradually, to a level similar to other vaccines, 7–8 events per 100,000 doses. From 2012 to 2018, only one severe AE was reported: a case of complicated urticaria, in which case the subject was hospitalized. All other cases were considered as mild and/or not related to the HPV vaccine.Surveillance is performed worldwide in all countries that have introduced HPV vaccination.As vaccinations are common, even a small increased risk of multiple sclerosis (MS) or other acquired central nervous system demyelinating syndromes (CNS ADS) could have a significant effect on public health. However, no long-term association of the HPV vaccine with MS or any other CNS ADS was found, arguing against a causal association , 24. SimFindings from a qualitative review of 14 safety studies on CNS ADS, MS, and optic neuritis strongly supported the absence of an association between HPV vaccines and central demyelination . Owing tSimilarly, for postural orthostatic tachycardia syndrome (POTS) the Vaccine Adverse Event Reporting System (VAERS) database for reports of POTS following HPV vaccination 2006 to 2015. Among 40,735 VAERS reports, 29 POTS reports that fully met diagnostic criteria were identified: approximately one POTS case per 6.5 million HPV vaccine doses. No unusual or unexpected reporting patterns was detected that would suggest a safety problem . These cIn conclusion, the issues of safety as well as the need for the HPV vaccine must be addressed with healthcare workers, adolescents and parents. HPV vaccines were developed to prevent cancer and save lives. To this effect, it may help to train (future) healthcare workers (HCWs) to provide a strong recommendation of the vaccine.To evaluate the effectiveness of the HPV vaccination program, type-specific HPV prevalence was compared between 1451 vaccinated and 951 non-vaccinated women aged 18–25 years old, from a single Colombian city, 5 years after vaccination . SignifiBrazil is a large country with great geographical differences between regions. The target population for HPV vaccination comprises 17 million boys and girls, aged between 9 and 14 years. Cervical cancer is the 3rd most frequent cancer among women, with an incidence of 15.4 cases /100,000, and 5000 deaths per year.From 2014, HPV vaccination was introduced in a scaled-up fashion, first targeting 11–13-year-old girls, in a 3-dose schedule, followed in 2015 by vaccination of males and females living with Acquired immunodeficiency syndrome (AIDS). In 2016, the target age for girls was expanded to 9–14-year-olds and beginning in 2017, boys were targeted between the ages of 11–14 years, as well as males and females, aged between 15 and 26 years, with weakened immune systems. Since the start of the vaccination program, 37.5 million doses of vaccine have been given.n = 1774) and women (n = 5812), aged 16 to 25 years. 35% of participants had a high-risk infection and 31% had an infection with more than one HPV type. In September 2014, 80 girls presented with symptoms  vaccine: the vaccination scheme was changed from three to two doses; a training program for health care workers about HPV vaccination was initiated; training was organized for directors of education and directors of schools in 24 regions on HPV and HPV vaccine; strategies were developed to involve parents and make it possible to obtain parental consent through the Internet; and a communication campaign was started, involving politicians and the media, and having a TV actress as the face of the campaign. Finally, a contest was held between 24 regions, to see which region was going to be the champion for a new generation of women without cervical cancer.Future points of attention to further improve vaccination coverage are underway including development of an electronic vaccination card; facilitating electronic access for mothers to obtain their children’s vaccination records; changing vaccination informed consent from opt-in to opt-out; and consideration of catch-up, gender-neutral, and 1-dose vaccination strategies. Furthermore, measurement of vaccine impact is being considered.In 2008, Panama began a school-based HPV vaccine program, administering 2-valent HPV (2vHPV) vaccine, given at the age of 10 years according to a 3-dose schedule . Beginning in 2015, the 4-valent HPV vaccine was used instead of 2-valent HPV. If the mother took her daughter for vaccination, she would be offered a Pap test herself. That same year, catch-up vaccination was offered to girls aged 16–17 years, in higher-risk regions, using the remaining 2vHPV doses. In 2016, the 4vHPV was also offered to boys from 10 years of age. In 2017, the vaccine coverage was 69%, which was relatively low, due to lack of 4-valent HPV vaccine stock, not due to AEs and its consequences. The vaccination crisis in Colombia was covered in the news, but there was a quick response in Panama to any anti-vaccine movement, with journalists involved. The Panama experience demonstrated that a political decision is needed to effectively set up HPV vaccination.www.hpv.ie [In Ireland, a school-based HPV vaccination began in 2010 targeting 12–13-year-old girls . In the first 5 years, vaccine coverage was very high, but in 2015 there was a decline in coverage, followed by a massive drop in 2016. Some parents raised major concerns about the vaccine. They were active on social media as well as TV. In response to this, the Health Service Executive (HSE) Ireland developed a website: w.hpv.ie , with inFurthermore, the HPV Alliance was established in August 2017, which now has 37 member institutions, including healthcare workers as well as women’s and children’s groups, all promoting the vaccine. Fact sheets were prepared, for schools, general practitioners and parents. HSE took up social media listening, to be aware of the rumors going on. In response to the rumors, HSE became active on social media, including twitter, using the hashtag #protectourfuture. In all of this, the Health minister was very supportive. And as a positive outcome, vaccine coverage increased from 50 to 65%, which is still lower than before the crisis, but going in the right direction.This region, along the border of Ecuador, has the highest vaccine coverage in Colombia. Where it aimed for a 17% increase, it managed to accomplish an increase to 47%.This success was due to a number of actions: audits of healthcare workers, including education on HPV vaccination, both in public and private IPS; actively going out to rural areas with low access to healthcare to provide vaccinations; organizing peer-to-peer discussions on HPV vaccination in schools; using conventional and non-conventional media, including local radio stations to increase awareness; as well as, allocating a specific nurse for follow-up of women with abnormal Pap smear results, to guarantee treatment when a lesion was found.In 2014, before the crisis, vaccine coverage was 100% in 4198 girls. However, after the 2nd dose, the first cases appeared. A few days later, more than 600 girls started to have the same symptoms . Many hypotheses have been formulated to explain these symptoms, but once vaccination was raised as option, there was little attention for other potential explanations. In fact, the advocate leading the anti-vaccine movement approached the doctor in charge of two girls who were being attended at the hospital, with the request to change the diagnosis to syncope induced by the HPV vaccine.An independent study carried out by the National Institute of Colombia ruled out a direct relation to the vaccine, but this led to much abuse towards spokespeople of the vaccination program, as it was interpreted as a callous denial of the symptoms of the girls.Currently, 4 years later, everything has calmed down. Sixty-five girls of the group with symptoms are still being followed closely, but they have not had further crises. In order to be able to provide the best possible care for these girls, a mobile unit is being used to reach those girls in remote areas, who have limited access to healthcare. Furthermore, meetings with the parents have been organized.In this region, 98% of the population lives in urban areas, and the population has a high level of education. To identify barriers and facilitators for HPV vaccination, a population-based survey was performed on girls born in the years 2003–2005, as well as their parents. Vaccination status was defined by girls’ report, parental report, and the online official vaccine registry. This showed good concordance between girls’ self-report of vaccination and official data. Their school was an important source of information for those girls who were aware of the vaccine. Importantly, 40–60% (unvaccinated) to 70–90% (vaccinated) of the girls were aware that the vaccine prevents cervical cancer and HPV infection, respectively, and these were strong reason for uptake of the vaccine. Nevertheless, 40% of the girls that were vaccinated thought the vaccine may be dangerous and produces AEs.The most important barriers for vaccination were recommendations against vaccination by relatives or friends, as well as media attention related to AEs in Carmen de Bolivar or in other countries. Finally, not being offered vaccination in the school was an important barrier to completion of the vaccine schedule: these girls started but often did not complete the vaccine series.Extensive knowledge of the epidemiology of HPV is available in Colombia, due to participation in scientific studies. Due to these studies, there is appreciation of the burden of cervical cancer in Colombia and a sense that country’s scientists played a key role in the development of the HPV vaccine.Colombia has almost universal insurance coverage and an extremely effective immunization program for all vaccines. A strong surveillance program is available, which is necessary to monitor safety of the vaccination program.There is strong political support and political will to continue and expand the HPV vaccination program, with consensus among different stakeholders on safety and effectiveness of the vaccine. The HPV vaccination program is a well-structured program, which, despite low vaccine coverage, is equitable. The vaccines are affordable at the country-level, and free for those targeted by the program.HPV vaccination in Colombia is hindered by fragmentation in communication and continues to recover from a slow reaction to the Carmen de Bolivar crisis. In general, a communication plan for dealing with crises is lacking and there is inadequate capacity for communication with different groups , leading to a lack of HPV / cervical cancer education of HCWs, the public and media. Moreover, communication, especially with the public, is at the wrong level, being too complex and too scientific.Following the Carmen de Bolivar crisis, Colombia abandoned the school-based system for HPV vaccination, while it has been shown to be the most effective system to achieve high coverage. Finally, HPV vaccination and cervical cancer screening have not been appropriately integrated.The recent reports on safety of the vaccine provide an opportunity to bring the vaccine under the attention of the media and the public in a positive way, aiming to increase vaccine coverage. Furthermore, the adapted E-learning course on vaccine safety (see below) is an excellent opportunity to educate HCWs on HPV and cervical cancer. Given the extensive experience from neighboring countries with the HPV vaccination program, Colombia can learn from these countries, copying successful strategies and avoiding pitfalls.Finally, it would be appropriate to try to show sympathy to those affected by alleged AEs, regardless of whether these reactions are directly linked to vaccination. This is likely to increase the respect and appreciation of the public for the vaccination program and the HCWs involved.The geographical distribution of the Colombian population is a potential threat to the vaccination program: a part of the population has very limited access to healthcare. Furthermore, in some parts of the country, the social situation is (still) unstable. And there is an increasing burden on the healthcare system due to immigration.Moreover, frivolous, predatory litigation towards the HPV vaccination program is likely to harm vaccine coverage, a problem that is aggravated by the strong activism of anti-vaccine groups in Colombia. This anti-vaccine sentiment may spillover to other countries and to other vaccine programs in Colombia and may lead to a higher burden of cervical cancer and other diseases due to reduced vaccine coverage.Finally, there is uncertainty about political support in higher political circles, some opinion leaders are against the HPV vaccine and the sustainability of the HPV vaccination program is not guaranteed.Extensive knowledge, and appreciation, of cervical cancer is available in Colombia, due to participation in scientific studies. Furthermore, there is strong political will to eliminate cervical cancer as well as technical leadership, which can be shared with others in the region.Colombia does not have a population-based program, leading to disparities and difficulties in access to screening. Furthermore, there is a lack of surveillance and no program for follow-up of abnormal Pap smears, which prevents screening from providing any added value. Finally, changes in the screening program cannot be introduced due to regulatory stifling.The new technologies for HPV detection may provide an opportunity to boost cervical cancer screening in Colombia. This is also an opportunity to integrate vaccination and screening. Furthermore, using different approaches for different populations , screening may become more equitable.http://www.e-oncologia.org) [New legislation is coming up, integrating different actions. Finally, an excellent tool has been developed to train the healthcare workforce. E-oncologia (gia.org)  is a virThe module can be calibrated to local needs, such as an adapted course for Colombia after the vaccine crisis, with a focus on the safety of the HPV vaccine. This course, jointly developed by the ICO and the Colombian Cancer Institute, is directed towards doctors, nurses and/or midwives, and in general all health professionals involved in the primary and secondary prevention of cervical cancer. The chapters have been co-authored by Spanish and Colombian experts. So far, close to 5000 participants (of whom > 80% are Colombians) have registered, of whom OVER 70% have completed the course and completed the final exam successfully.In fact, the course could possibly be adapted to be used for non-medical people, such as journalists.The geographical distribution of the Colombian population is a potential threat to the screening program, a part of the population has very limited access to healthcare. Next, in some parts of the country, the social situation is (still) unstable and, owing to the political crisis in Venezuela, there is an increasing burden on healthcare system due to immigration. The cost of screening is very high and failure to reach an agreement to implement new techniques may further hamper the screening program. Finally, there is no systematic roll-out of information to the public.In response to the Carmen de Bolivar crisis, the ‘Mesa de Concertacion’, a roundtable for the elimination of HPV-related cancer in Colombia, was organized. This roundtable brought together many stakeholders: the Colombian League against Cancer; Profamilia; universities; scientific societies ; the association of cities; the National Academy of Medicine; parent associations; the Ministry of Health; pharmaceutical and diagnostics industry; as well as foreign organizations, including the American Cancer Society. Actions following from the roundtable include education to healthcare providers, regaining confidence in the vaccine by giving lectures throughout the country, and reactivation of the school-based system.Many believe that there is a trend towards sensation journalism, exacerbated by social media, where everyone can be a journalist. A bit of background helps to understand the media better: national and regional media often have very different editorial policies of accepting topics for dissemination. Furthermore, the way news is covered depends on more than just the journalist: the inclusion of photos; the impact of the layout; the editor, the choice of title, all aspects that may create a completely different perception of the news.Scientists often distrust journalists, but it is necessary to acknowledge that journalists are actually very powerful and have a large outreach. Therefore, scientists need to reach out the journalists, with factual information, keeping in mind that if journalists receive accessible and clear information, they are more likely to transmit them to the public. A relationship with the media is not a one-time affair, it is a permanent relationship, that needs to be nurtured. Public health actors must consider journalists their communication partners.It is the duty of a journalist to discuss both sides of a story, but sometimes this results in an incorrect balance; it may help to point this out, showing the potential negative consequences (e.g. deaths due to non-vaccination) of publishing a news item. Well-trained media personnel will make fewer mistakes. While there are master’s programs in scientific journalism, science is a very broad topic, much broader than just health, so specific education for journalists remains necessary.Finally, media can be an important factor in discrediting the vaccine. If a good relationship with a journalist is established, he/she could point out how other journalists can be reached, to make them allies, avoiding unnecessary damage to vaccination programs.Re-introduce the school-based system for HPV vaccination, as it has been shown to be the most effective system to achieve high coverage.Adoption of Resolution 3280 might be a good time to integrate primary and secondary prevention programs, as both have the same overall goal: reduction of cervical cancer. This should be done with a focus on equity and sustainability of the programs.Develop a communication plan for crisis situations and adequate capacity for communication with different groups . While the e-course is a good tool for training of HCWs, different ways of educating teachers and the general population need to be developed To this respect an alliance is being organized with the local office of the Open University of Catalonia (UOC), a university with a long tradition in e-learning educational programs in Spanish language to support the initiatives of the national cancer instituteEstablish a good and long-lasting relationship with the media, trying to make them a communications partner for the vaccination program.Bad news is swiftly communicated widely; it is time to start spreading the good news, at an accessible, non-technical level, for everyone to understand. Good news encompasses safety of the vaccine, as shown in a plethora of studies worldwide, as well as impact of the vaccine on HPV infections and HPV-associated lesions, shown in countries with high vaccination rates , but also in countries with lower vaccination rates such as the United States.Show empathy, public health officials must show respect and acknowledge the symptoms of those affected by them, also if these are not vaccine related. The plea from families of alleged victims must not be ignored. Medical Doctors (MDs) and nurses who sympathize will eventually find an opportunity to communicate with victims and their families and better understand their problems and concerns.After one and a half days of interesting presentations and lively discussion, it was concluded that there is still a lot of work to do. Therefore, several action points have been formulated that can be pursued to increase vaccination coverage in Colombia:The overall country HPV vaccine coverage rates increased slightly the year following the meeting from 29 to 34% for the first dose and from 9 to 11% for the 2nd dose. This increase was the result of an increase in a few regions such as the departments of Antioquia and in Arauca, where a special project is being implemented. In other departments, , the rates remain stable or have decreased.  and one of the lowest HPV vaccination rates. A new communication strategy was developed from a bio-psycho-social perspective to educate and sensitize girls to be vaccinated, the parents of the girls, the teachers, health care providers and the local mass media on the benefits and safety of the HPV vaccine. Eighteen months after starting the project, the coverage rate for the 1st vaccine dose in Arauca increased from 4.7% in 2017 to 83% in December 2019. Most of the increase in the 1st dose occurred in the last 6 months of 2019, thus coverage of the 2nd dose will be best measured in the first 6 months of 2020. We hope to reproduce this strategy in other regions of the country.


The vaginal microbiota has been reported to be associated with HPV infection and cervical cancer. This study was performed to compare the vaginal microbiota at two timepoints in women performing self-sampling and had a persistent or transient HPV16 infection. The women were tested for 12 high-risk HPV (hrHPV) types but only women with single type (HPV16) were included to reduce confounding variables.In total 96 women were included in this study. Of these, 26 were single positive for HPV16 in the baseline test and HPV negative in the follow-up test and 38 were single positive for HPV16 in both tests and diagnosed with CIN2+ in histology. In addition, 32 women that were negative for all 12 HPV tested were included. The samples of vaginal fluid were analyzed with the Ion 16S™ Metagenomics Kit and Ion 16S™ metagenomics module within the Ion Reporter™ software.Lactobacillus-dominated groups, one with Lactobacillus sp. and the other specifically with Lactobacillus iners. The two remaining clusters were dominated by a mixed non-Lactobacillus microbiota. HPV negative women had lower prevalence (28%) of the non-Lactobacill dominant cluster in the baseline test, as compared to women with HPV16 infection (42%) . Transition between clusters were more frequent in women with persistent HPV16 infection (34%) as compared in women who cleared the HPV16 infection (19%) .K-means clustering resulted in two The vaginal microbiota showed a higher rate of transitioning between bacterial profiles in women with persistent HPV16 infection as compared to women with transient infection. This indicate an instability in the microenvironment in women with persistent HPV infection and development of CIN2+. Persistent infection of human papilloma virus (HPV) is the main cause for development of cervical cancer . A set oLactobacillus crispatus, L. gasseri, L. iners and L. jensenii, respectively, while CST IV is dominated by non-Lactobacillus bacteria [Lactobacillus spp. and low proportions of strictly anaerobic bacteria, while IV-b having higher proportions of Atopobium, as well as Prevotella, Sneathia and Gardnerella [.In addition to HPV, other risk factors for cervical cancer is recognized, such as smoking, high parity and use of oral contraceptives . The assbacteria . CST IV dnerella .Atopobium vaginae and Gardnerella vaginalis to be associated with cervical interepithelial neoplasia (CIN) [Gardnerella abundance with HPV positivity and increasing grade of CIN is associated [Lactobacillus sp. and higher frequency of CST-IV is associated with HPV infection and grade of CIN in several studies [L. gasseri-dominated) is related to fast HPV clearance rate, and CST IV-B with slow HPV clearance rate, in comparison to CST I [L. Crispatus to be associated with lower HPV detection rate, but finds no association between HPV and relative abundance of other Lactobacillus species or with Lactobacillus as a group [Several studies report ia (CIN) –9. Howevsociated . Lower a studies –13. CST to CST I . Also, o a group .The vaginal microbiota in women is known to also depend on covariates such as the menstrual cycle , 16, preThe present study was based on analyses of seven variable domains of the 16S RNA gene in single samples from women with HPV negative tests and paired vaginal samples collected 4–6 months apart from women with a persistent HPV16 infection and women with transient infections. Only women infected by a single hrHPV type (HPV16) were included to reduce confounding variables in the analysis. These women were also tested for HPV18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 with negative results. The aim was to study the association of the vaginal microbiota with the course of HPV16 infection.Women in this study were selected from a previous randomized intervention study conducted between 2013 and 2015, that included non-pregnant women between 30 and 49 years of age from Uppsala County, Sweden . To be eIn brief, the randomized intervention study consisted of a total of 36,390 women out of which 17,997 were randomized to repeated HPV-testing with self-sampling. 7997 women performed the 1st test with 7443 negative and 554 positive tests. The aim of the previous study was to compare the detection rate of CIN2+ in histology in women performing repeated self-sampling of vaginal fluid for HPV testing, with the rate of CIN2+ detection in women following the regular screening program based on Pap smear cytology. Women who were HPV positive (N = 554) in their first test were asked to repeat the self-sampling in 4–6 months. 501 of the 554 women followed the study protocol and performed a 2nd HPV-test which resulted in 355 positive tests. Women that were HPV negative in the first (N = 7443) or second HPV test (N = 146) were referred to the regular screening program. For the present study we selected 26 women who were single infected by HPV16 in the baseline test and subsequently HPV negative in their follow-up test, and 38 women who were single infected by HPV16 in both the baseline and the follow-up tests, and later diagnosed as CIN2+ based on histology during the follow-up period of 18 months from invitation date. Finally, 32 age-matched women who had a single self-sample with a negative HPV test were also included. Women that were infected by HPV16 in their baseline test and HPV negative in their follow-up test were considered to have transient infections, while women with two single HPV16 positive samples together with CIN2+ histology were considered to have a persistent infection. The study was approved by the Regional Ethics Committee in Uppsala (Dnr 2012/099).The vaginal self-sampling procedure has been described earlier . BrieflyHomo sapiens hydroxymethylbilane synthase; GenBank accession no. M95623.1) as a control for the amount of human cellular material. The LOD for human genomic DNA is 10 copies of HMBS per PCR.A clinically validated qPCR‐based assay, HPVIR , was useThe colposcopic evaluation included an identification of squamocolumnar junction and transformation zone (TZ) with application of 5% acetic acid and iodine solution. Biopsies were obtained from all the identified abnormal areas, and a blind biopsy was taken in women with normal colposcopy. All gynecological examinations were performed at the Clinic of Obstetrics and Gynecology, Uppsala University Hospital. Clinical classifications were according to SNOMED  and the highest histological grade found in each patient was used for interpretation of the results.Ion 16S™ Metagenomics Kit User Guide revision C.0. According to the manufacturer’s instructions, 3.0 μl of the FTA-card eluate was used as input to the amplification. After purification with Agencourt® AMPure® XP beads, amplicons were quantified using Bioanalyzer instrument (Agilent) and DNA-concentrations in the products ranged from 1.6 to 77.3 ng/μl. With the exception of one sample where 50 ng was used, 100 ng of the input material was used for the AB Library Builder™ System (Thermo Fisher Scientific), following the guide Ion Xpress™ Plus and Ion Plus Library Preparation for the AB Library Builder™ System revision 5.0. Prepared libraries were amplified for addition 5 cycles as described in Ion 16S™ Metagenomics Kit User Guide revision C.0. Final libraries were quantified using the Fragment analyzer (Agilent) and prepared in the Ion Chef System (Thermo Fisher Scientific) before sequenced on the Ion S5 ™ XL System (Thermo Fisher Scientific) on five Ion 530™ Chips (Thermo Fisher Scientific).16S hypervariable regions were amplified using Ion 16S™ Metagenomics Kit (Thermo Fisher Scientific) following recommendations in the The primary data analysis was carried out with The Ion reporter version 5.6. Briefly, quality analysis was run requiring primer detection in both ends, the minimum alignment coverage of 90%. The analysis was performed with the built in QIIME pipeline with protocol Metagenomics 16S w1.1. Reference library Curated Greengenes v13.5 was used and a minimum alignment of 97% was required for genus identification and 99% for species identification. If no identification at 99% could be made on species level but was achieved at genus level, genus level annotations were used. Difference between the two top hits was allowed to be maximum 0.2% to confirm species identification.Lactobacillus bacteria were grouped by taxonomic level genus if information was available, otherwise they were grouped on taxonomic level family. Bacterial genus or family that represented less than 1% of the total were grouped as “Others” for non-Lactobacillus bacteria, and as “Lactobacillus sp.2” for Lactobacillus bacteria. K-means clustering were performed with four clusters, using the “kmeans”-function and the results visualized as a heatmap using the “pheatmap”-package [p values were adjusted for multiple testing using the Bonferroni correction and q values were considered significant if the q value < 0.05. Binomial testing was used for comparison of cluster transition proportions.Statistical calculations were performed and figures generated using R version 3.4.3 . Read nu-package . Shannon-package . Sankey -package . Binomiap value = 0.35). Also, the mean age was not significantly different between HPV negative women , women with transient HPV16 infection  and women with persistent HPV16 infection and CIN2+  (Table The median time between the two self-sampling occasions were 162 days and the mean absolute deviation (mad) 14.8 days for women with transient HPV16 infection and 155.5 days (mad = 30.4) for women with persistent HPV16 infection. The number of days between the two sample collections was not significantly different between women with transient and persistent infection . The number of reads per sample across these seven regions was between 125,202 and 1,148,323, with a mean of 515,814 ± 190,890 reads. Based on the analysis of all seven variable regions, the analysis identified 723 operational taxonomic units (OTUs), corresponding to 433 species, 188 genera and 102 families. When the analysis was based solely on regions V3 and V4, which is a set of 16S variable regions frequently used for analysis, we identified 547 OTUs, representing 324 species, 157 genera and 66 families , and in the second cluster samples were dominated by OTUs specifically classified as Lactobacillus Iners, with a mean proportion of 86.4% . In cluster 3 and 4, samples were dominated by a mixed non-Lactobacillus microbiota. In the third cluster most samples were dominated by Gardnerella or Prevotella and some samples were dominated by e.g. Atopobium, Snethia or Streptococcus, with a mean proportion for the dominant bacteria of 39.4% . Finally, in the fourth cluster ten of eleven samples were dominated by Gardnerella and the last sample by Aerococcus, with a mean proportion of the dominant bacteria of 69.8%  , but the difference did not remain significant when corrected for multiple testing  (Table Lactobacilli dominant clusters (cluster 3 and 4) than those with persistent infection (29%), but the difference was not statistically significant   of the non-Lactobacilli dominant cluster (cluster 3 and 4), as compared to women with an HPV16 infection (42%). This difference was nominally statistically significant  nor the second sample (the second two groups). All nominal p value = 0.036, Fig. We compared the K-means clusters in the baseline and second samples of individual women to study the transition between clusters as a function of clearance and persistence of HPV16 infection. Transition between clusters were significantly more frequent in women with persistent HPV16 infection (34%) as compared to women who cleared the HPV16 infection in the timespan between the two sampling occasions (19%) . This is consistent with the results of several other studies [q value = 0.0519). We did not detect a difference in alpha diversity between women with or without HPV16 infection, nor did we find a difference between women who cleared their HPV16 infection and those with persistent HPV16 infections. This is consistent with one previous study [The results indicate that transient and persistent HPV16 infections, as compared to being HPV negative, is associated with a vaginal microbiota dominated by non- studies –13. Howeus study , but othus study , 34.The vaginal microbiota profile did not differ significantly when comparing women who cleared their infection with those that had a persistent infection and CIN2+. However, there was a significantly higher rate of transitioning between microbiota profiles in women with persistent HPV16 infection, as compared to women who cleared their infection. This indicate an instability in the vaginal microenvironment in women with a persistent HPV infection and CIN2+.The causative relationship between HPV infection and vaginal microbiota is poorly understood, but the inflammatory process in women with CIN2+ could have an effect on the vaginal microenvironment and the composition of the vaginal microbiota. The initial HPV infection in the basal layer has no productive life cycle before the differentiation of the infected cells, it contains no virus induced cell-death and causes limited inflammatory response . The HPVThe strength of our study is that the women included were single-infected with HPV16, which reduces the complexity of HPV infection with different HPV types. We used HPV16 infected women that developed CIN2+ as a proxy for persistent infection. These women are likely to have harbored their HPV16 infections for some time, since they had developed CIN2+. There are some limitations in this study. The limited sample size reduces the statistical power to detect actual associations between the vaginal microbiota and the presence of HPV16. The HPV test include 12 high-risk HPV types, but the samples could possibly have other HPV infections that could affect the microbiota, which would go undetected with the test used in this study. Self-sampling could also potentially detect HPV-infections not exclusively present in the endocervix which would lead to an overestimation of detected HPV-infections. Here, this does not affect the negative or persistent group with CIN2+ histology diagnosis but may have influenced the positive HPV16 test for the first test in the transient group. A recent meta-review  does howWe also lack information on whether the women participating in the study have been vaccinated for HPV or not. However, the age groups participating in the study have not been included in the organized Swedish HPV-vaccination program which was initiated in 2012 for girls born in 1998–2000  and vaccWe used the seven variable regions included in the Ion 16S™ Metagenomics Kit. As noted, there is a difference in the number of OTUs identified at all taxonomic levels when the analysis is based on all seven variable regions as compared to the frequently employed regions V3 and V4. This can be attributed to the variability of the 16S gene, the differences in primer design and sequencing technology makes direct comparisons between studies of the vaginal microbiota difficult. However, the use of seven regions resulted in a higher number of OTUs identified and generally had a good correspondence with the CSTs previously described for the vaginal microbiota.In summary, to our knowledge this is the first report showing a significantly higher rate of transitioning between vaginal microbiota profiles in women with persistent HPV16 infection and CIN2+ as compared to women who cleared their infection.p value = 0.036) of transitions between microbial profiles than women with transient HPV16 infection. This indicate a higher instability in the vaginal microenvironment in women with persistent HPV infection and development of CIN2+.Women with persistent HPV16 infection and CIN2+ have a higher rate  (blue) and the regions V3 and V4 only (yellow) based on the 16S rRNA Ion Torrent amplicon sequencing kit. Figures represents the taxonomic level: A. Species, B. Genus, C. Family.


Positions and Covering methodology to obtain exact solutions for the two-dimensional, non-guillotine restricted, strip packing problem. In this classical NP-hard problem, a given set of rectangular items has to be packed into a strip of fixed weight and infinite height. The objective consists in determining the minimum height of the strip. The Positions and Covering methodology is based on a two-stage procedure. First, it is generated, in a pseudo-polynomial way, a set of valid positions in which an item can be packed into the strip. Then, by using a set-covering formulation, the best configuration of items into the strip is selected. Based on the literature benchmark, experimental results validate the quality of the solutions and method’s effectiveness for small and medium-size instances. To the best of our knowledge, this is the first approach that generates optimal solutions for some literature instances for which the optimal solution was unknown before this study.We use the  Two-Dimensional Strip Packing Problem (2SP) is composed of a given set of n rectangular items, each one with specific width wi and height hi, for i = 1, …, n, and a strip of width W and infinite height. The aim is to place all the items into the strip orthogonally; without overlapping, minimizing the overall strip’s height jack and  Teng in , the genzuela in , and thetfeld in . These adagli instances. For these instances, we add two columns for the P&C: column “%” represents the packing density which is added to make a comparison with the other approaches, and column “Ratanapan and Dagli in dagli instfeld in  . The Sbb, Sda and Sbp algorithms were coded in C++ and tested on a Pentium M 1.7 GHz with 1G of RAM. The column generation and linear programs were solved with CPLEX and Concert Technology.The experimental results for posed by  are show[Hifi in , the B&B[Hifi in  called Se B&B of , the dice B&B of  named Sde one of  with thee one of , and thee one of  called Shifi instances, we are not comparing the dichotomous behavior that seems to be a promising characteristic for the 2SP, but, we are analyzing the exact algorithms that solve the one-dimensional problem. Indeed, the best algorithms in terms of solution value are P&C, Sda, and Sbb, respectively. To the best of our knowledge, the P&C methodology is the only one finding the optimal solution for instances hifi19 and hifi25. Furthermore, the execution times of the last iteration for the P&C are efficient since they are less than 45 minutes in the worst case (instance hifi18). Nevertheless, the execution times of Hifi’s algorithm seem more competitive.For The experimental results for small-medium size instances proposed by Hopper and Turton in  are presIn this section, we present the experimental results for the other two-dimensional cutting instances which have been adapted to the 2SP Tables –10. The burke instances of [In ances of . We compances of  and its ances of   and the optimal height z* of the strip because some anomalies exist in the instances, and a perfect packing does not exist with the original width. The anomalies exist in the first two instances where the width of some items is larger than the width of the bin. Therefore, to solve these instances, we set the width of the bin as long as the width of the larger item (numbers in bold in column 4 show the modification of W). Furthermore, the optimal solutions obtained by our methodology in the last four instances do not correspond with the results presented by Kenmochi [bottom-left (BL) point and the the staircase (S) strategy. The bounding operations are based on dynamic programming (DP) and linear programming (LP). The P&C shows the optimal results for all the instances.posed by . The firKenmochi . ColumnsKenmochi  that impThe experimental results show that the P&C is an efficient methodology to solve small-medium size instances for the 2SP. Larger instances cannot be solved with the P&C methodology since the combinatorial complexity of the problem also relies on the generation of all the possible positions of the items. In this case, we have detected an opportunity area where we propose to apply a decomposition approach, such as a column generation, to obtain optimal solutions for large instances.In this study, we present a new methodology called “Positions and Covering (P&C)” to obtain exact solutions for the two-dimensional strip packing problem. The methodology is based on a two-stage procedure where first, a set of valid positions is generated in a pseudo-polynomial way representing how each item can be allocated inside of the strip. The height of the strip is computed assuming that a perfect packing exists . In the second stage, a set covering formulation  is solved to determine if the height of the strip is optimal. If the mathematical model is feasible, then the procedure ends with the optimal solution. Else, the height of the strip is increased by one, a new set of valid positions is generated, and a new iteration begins.The P&C methodology was tested using the benchmark for the 2SP solving small and medium-size instances. We verify that many of the solutions proposed by other approaches were the optimal solutions. The main contribution of this study is that we have obtained optimal solutions for instances proposed in the literature where the optimum was not known before. Furthermore, we have modified some other instances of the literature, which were infeasible, and we have solved them, obtaining the optimal solutions.An essential characteristic of the P&C is its simple implementation. Nevertheless, a future research line consists of developing a decomposition approach to enhance the P&C methodology to give optimal solutions for larger instances of the 2SP.


Evidence regarding the relationship between vitamin A and HPV infection was limited. Therefore, this study is designed to investigate whether vitamin A was independently related to HPV infection in 13412 American women from NHANES for seven cycles.  The present study is a cross-sectional study. A total of 13412 eligible participants who had available HPV tests and vitamin A intake data were registered in the NHANE database from 2003 to 2016. The targeted independent variable and the dependent variable were vitamin A measured at baseline and HPV infection, respectively. We analyzed the association between dietary vitamin A intake and the prevalence of HPV infection. Besides, GAM and smooth curve fittings were used to address the nonlinear relationship between vitamin A and HPV infection to determine the effect of HPV infection.  The result of fully adjusted binary logistic regression showed vitamin A was not associated with the risk of HPV infection after adjusting confounders . A nonlinear relationship was detected between vitamin A and HPV infection, whose inflection point was 10.5 of log2 vitamin A . One unit increase of log2 vitamin A is associated with the 10% reduced risk of HPV infection when dietary vitamin A is < 1448.155mcg. Conversely, when the dietary vitamin A intake is ≧1448.155 mcg, for each additional log2 of vitamin A, the risk of HPV infection increased by 70%.  We found that dietary vitamin A was quite different from the trend of HPV infection in different confidence intervals. The results suggested that an appropriate amount  of dietary vitamin A may be beneficial to prevent HPV infection. However, excessive intake of dietary vitamin A  may increase the risk of HPV infection. Vitamin A, an essential fat-soluble vitamin throughout the human life cycle, is widely found in eggs, milk, liver, fresh vegetables, and fruits. It cannot be synthesized by itself, so it must be taken through diet . The 201Human papillomavirus (HPV) infection has become a major public health challenge for women. In the United States, HPV infection resulted in about 30,000 cases of cancer each year . 70% of Previous studies have confirmed that dietary factors, vitamin A, in particular, is closely related to decreased HPV-associated tumors  –18. Howehttps://www.cdc.gov/nchs/nhanes/).The National Health and Nutrition Examination Survey (NHANES), a cross-sectional study from 1999 to the present, is designed to assess the health and nutritional status of adults and children in the United States. NHANES covers interviews and medical examinations with a focus on various health and nutrition measurements and is the main program of the National Center for Health Statistics (NCHS). More detailed content can be found on the NHANES official website , 40 people's survey results were less than 1% percentile, so we excluded this part of the population. In total, 13412 out of 35092 women were available for final analysis. More details of the selected sample can be seen in the below flowchart .https://wwwn.cdc.gov/Nchs/Nhanes/2003-2004/DR1IFF_C.htm).Vitamin A was obtained and recorded at baseline as a continuous variable. The public data files on dietary were obtained from the Centers for Disease Control and Prevention (CDC). The automated multiple-pass method (AMPM), a dietary interview computer program, was used to collect the participants' two nonconsecutive 24-hour dietary recalls . Trainedhttps://wwwn.cdc.gov/Nchs/Nhanes/2003-2004/L37SWA_C.htm#LBDHPCR).We described HPV infection as the following detailed process of measurement: (1) extract vaginal cells from the participants with vaginal wipes; (2) the processed samples were stored and sent to the Centers for Disease Control and Prevention, Atlanta, GA, for analysis; and (3) the vaginal swab specimen DNA was detected and analyzed by the Roche prototype line blot assay and the Roche Linear Array (LA) HPV Genotyping kit. More information on HPV measurements can be found on the website (2) (obtained at baseline) and (2) categorical variables: number of boyfriends have had in the past one year (times), race, education level, marital status, smoking more than 100 cigarettes in their lifetime, drink at least 12 glasses of alcohol/1 year, and vaginal or anal intercourse times in the past year (times) (obtained at baseline).Based on published studies, the covariates used in our research can be classified as demographic data or variables that can affect vitamin A or HPV infection used in the NHANES database –26. HencP values for the log-likelihood ratio test was determined.In our study, if the continuous variable was normally distributed, it was presented as mean ± standard, otherwise as medium . Furthermore, we expressed categorical variables as a percentage. The distribution of vitamin A in the population was skewed, so we made a conversion of log2. Our statistical analyses consisted of three main steps. First of all, we employed multivariate binary logistic regression by constructing three models: model 1, no covariates adjusted; model 2, merely adjusted sociodemographic data; and model 3, explored the nonlinearity of vitamin A and HPV infection, and a generalized additive model and smooth curve fitting  were employed. In Step 2, we detected the relationship of nonlinearity between vitamin A and HPV infection, and the inflection point employing a recursive algorithm was calculated. In Step 3, we constructed a two-piecewise binary logistic regression on both sides of the inflection point. The best-fit model based on the http://www.R-project.org, The R Foundation) and EmpowerStats . We determined that P values less than 0.05 (two-sided) were statistically significant.The whole data analyses were employed with the statistical software packages R , 95% confidence intervals, and In the fully adjusted model  and the GAM , race, and other covariates by smooth curve and the effects of the generalized additive model. Both binary logistic regression and two-piecewise binary logistic regression were used to fit the relationship and choose the best-fit model based on P for log-likelihood ratio test.In our present study, we aimed at analyzing the nonlinear relationship between vitamin A and HPV infection . We founTwo-piecewise binary logistic regression for fitting the association between vitamin A and HPV infection was selected because it can accurately represent the relationship. By using two-piecewise binary logistic regression and recursive algorithm, the inflection point was calculated as 10.5. The effect value and 95% CI were, respectively, 0.9 and 0.9–1.0 on the left side of an inflection point. Identically, on the right side of the inflection point, the effect size and 95% CI were 1.7 and 1.1–2.8, respectively.The covariate-adjusted in the two models was the same as the GAM presented in In summary, we found that vitamin A (log2 transformer) was not associated with HPV infection after adjusting other covariates  in the binary logistic regression equation. It appeared that there was a possibility of a nonlinear relationship by turning vitamin A into a categorical variable. Hence, we further considered the nonlinear relationship and found that there was a curvilinear relationship between vitamin A and HPV infection. Furthermore, our findings indicated that the tendency of the effect sizes on both the left and right sides of the inflection point was not consistent ; right OR: 1.7, 95% CI ). This above result suggested an approximately U-shaped curve on the independent association between vitamin A and HPV infection.Shannon et al. performed a case-control study including 50 women with in situ cervical cancer and 125 controls, and 134 invasive cervical cancer cases and 384 controls. They believed that dietary vitamin A intake was found to be related to a reduced risk of HPV infection-associated in situ and invasive cervical cancer . LehtineWe consider the clinical value as the following points: (1) To our best knowledge, it is the first time to observe the U-shaped relationship between vitamin A and HPV infection in American women in a large cross-sectional study. (2) The findings of our research should be helpful to develop dietary guidelines and prevent strategies for HPV infection. (3) With reference to the NHANES database, we have now enrolled more than 3,000 women to conduct a community-based cohort study (Ling Nan cohort), which aims at exploring the association between HPV infection and dietary, psychological, and work factors in Chinese women.Our research has the following advantages. Our sample size is relatively large compared with previous similar studies. To ensure the robustness of data analysis and explore the true relationship between vitamin A and HPV infection, we did a series of sensitivity analyses and fully explained the nonlinear relationship. Our study is an observational study, which is susceptible to potential confounding. Therefore, strict statistical adjustments were used to minimize residual confounders. Furthermore, few studies have elucidated the U-shaped relationship of vitamin A and HPV infection in recent years.However, there are some limitations to our study. Firstly, our study subjects are 13412 American women aged 18–59 years. Hence, the study cannot be applied to men or people beyond the age range. Secondly, we cannot get a causal relationship between vitamin A and HPV infection because our research is a cross-sectional study. Thirdly, dietary vitamin A is obtained from NHANES participants' 24-hour dietary recall interviews. Thus, like other studies, our research has inherent and unavoidable weaknesses and may have recall bias, measurement bias, and so on. However, the directionality  caused by recall bias and measurement bias was fair for each participant, and it did not interfere with our results. Last but not least, our study only discussed the relationship between dietary vitamin A and HPV infection, but did not discuss serum levels of vitamin A (retinol or retinoic acid) and HPV infection. Therefore, the comparison between serum vitamin A and dietary vitamin A with HPV infection should be paid attention in the future.