Long control region (LCR) of human papillomavirus (HPV) has shown multiple functions on regulating viral transcription. The variations of LCR related to different lineages/sub-lineages have been found to affect viral persistence and cervical cancer progression differently. In this study, we focused on gene polymorphism of HPV16/18/58 LCR to assess the effect variations caused on transcription factor binding sites (TFBS) and provided more data for further study of LCR in Southwest China.LCR of HPV16/18/58 were amplified and sequenced to do polymorphic and phylogenetic anlysis. Sequences of each type were aligned with the reference sequence by MEGA 6.0 to identify SNPs. Neighbor-joining phylogenetic trees were constructed using MEGA 6.0. Transcription factor binding sites were predicted by JASPAR database.The prevalence of these three HPVs ranked as HPV16 (12.8%) > HPV58 (12.6%) > HPV18 (3.5%) in Chengdu, Southwest China. 59 SNPs were identified in HPV16-LCR, 18 of them were novel mutations. 30 SNP were found in HPV18-LCR, 8 of them were novel. 55 SNPs were detected in HPV58-LCR, 18 of them were novel. Also, an insertion (CTTGTCAGTTTC) was detected in HPV58-LCR between position 7279 and 7280. As shown in the neighbor-joining phylogenetic trees, most isolates of HPV16/18/58 were clustered into lineage A. In addition, one isolate of HPV16 was classified into lineage C and 3 isolates of HPV58 were classified as lineage B. JASPAR results suggested that TFBS were potentially influenced by 7/6 mutations on LCR of HPV16/18. The insertion and 5 mutations were shown effects in LCR of HPV58.This study provides more data for understanding the relation among LCR mutations, lineages and carcinogenesis. It also helps performing further study to demonstrate biological function of LCR and find potential marker for diagnosis and therapy. According to the data worldwide in 2012, cervical cancer is the fourth most common cancer in women, both for new cases and deaths. The data show large difference between developed and developing countries. It is the second most common cancer in less developed regions and covered 87% cervical cancer worldwide, and ranks 11th in developed regions [https://www.hpvcenter.se/human_reference_clones/). Most oncogenic or high-risk HPV types are members of several species of Alpha-papillomavirus genus [Human papillomavirus (HPV) is a prevalent, globally distributed group of circular double-stranded DNA virus which can infect cutaneous and mucosal epithelia throughout the human body . Persistus genus , 5 whichus genus . Alpha-pus genus . HPV16/1us genus . Compellus genus . The relus genus , 11. HPVus genus . Furtherus genus .The whole genome of HPV contains three regions, an early region , a late region  and a regulatory region called long control region or upstream regulatory region (LCR or URR) . The LCRThe LCR has been shown to be the most variable region of HPV genome, mainly because it does not encode any gene and therefore able to accumulate and tolerate more mutations , 17. TheIn this study, we collected the positive samples of HPV16, 18, 58. Analysis of polymorphism, phylogenetic and functional prediction were performed on LCR which were rarely reported in Chengdu, Southwest China. The data helped us to determine the prevalence of lineages/sub-lineages and novel mutations/isolates of each type. It is useful for epidemiological survey and biological function research of LCR.This study was approved by education and research committee and Ethics Committee of Sichuan University, China . All works were followed the guideline of Ethics Committee of Sichuan University. Informed Consent Right was confirmed by patients enrolling. The privacy of patients was assured to protect carefully.All 8244 gynecological outpatients’ cervical swab samples were collected between September 2017 and June 2019 in Chengdu SongZiNiao Sterility Hospital, Sichuan Reproductive Health Research Center Affiliated Hospital, Chengdu Western Hospital Maternity Unit, and Angel Women’s and Children’s Hospital. The samples were collected from women aged 20 to 59 who have normal cytology, low-grade squamous intraepithelial lesion or cervical intraepithelial neoplasia. Each sample was stored at − 20 °C in cell preservation fluid. Specimen collection to DNA extraction was in a week.DNA was extracted using nucleic acid extraction kit  in accordance with the manufacturer’s instructions. Via capillary electrophoresis method by multiple PCR, DNA extraction was amplified using HPV nucleic acid assay and genotyping kit . Genotyping was accomplished by Applied Biosystems 3500Dx genetic analyzer. The kit can distinguish 25 types  of HPV. HPV 16/18/58 positive samples were selected for the following study.LCR sequences of HPV16/18/58 were amplified by primers which were designed based on the reference sequences from GenBank (Table https://blast.ncbi.nlm.nih.gov/Blast.cgi) to detect the novel sites or isolates.To identify the single nucleotide polymorphisms (SNP) in LCR, HPV prototype reference sequences were used as the standard to compare with the valid LCR sequences of each type by MEGA 6.0 . The phyhttp://jaspar.genereg.net/) were used to predicted transcription factor binding sites (TFBS) in LCR of HPV16/18/58 [The online database JASPAR  of them were HPV-positive. Among them, 302(12.8%), 823.5%) and 299(12.6%) were tested as HPV16, HPV18 and HPV58, respectively . A7175C, T7177C, T7201C and C7270T were detected in 32 isolates. No insertion or deletion mutation was found. After Blast on NCBI, we found that 18 mutations and 9 variants were never reported by anyone else.  was detected between the nucleotide sites 7279 and 7280. The most variable site was 7714 (25/67). All four kinds of nucleotide were found on this position, including 8 of A7714C, 15 of A7714G and 2 of A7714T. The second most prevalence mutation was C52T (19/67). Two groups of mutations had a tendency to show together, which were C7265G (12/67), A7714G (15/67) and C7266T (16/67); G7194C (8/57), A7304G (8/57), the insertion (8/57) and A7714C (8/67). 18 novel sites were found in HPV58 which composed 12 novel sequences. . The A branch contained 10 patterns (17 isolates) of sub-lineage A1, 4 patterns (7 isolates) of sub-lineage A3 and 18 patterns 32 isolates) of sub-lineage A4. None of patterns belonged to sub-lineage A2  were divided into sub-lineage A1, 4 patterns (4 isolates) were classified as sub-lineage A2, 1 pattern was identified as sub-lineage A3 (3 isolates), A4 (1 isolates), respectively. Two patterns (pattern No.34 and No.33) were not clearly identified, while No.33 was more like sub-lineage A5, No.34 was more close to sub-lineage A3 in the tree  of sub-lineage A1, 10 patterns (16 samples) of sub-lineage A2 and 8 patterns (9 samples) of sub-lineage A3. Three patterns were classified as lineage B, 2 for Sub-B1 and 1 for sub-B2 Fig. .Fig. 4NJASPAR database was used to investigate the potential binding sites for the transcription factors in the HPV-LCR. Also, it was applied to assess wherther the mutations affected the transcription factor binding sites.For HPV16, the results showed 7 mutations potentially affected the binding sites of transcription factors. A potential binding site of PHOX2A located at nucleotide position 7730 which was detected as the second most variable site of HPV16-LCR. More transcription factors like HOXC11, CEBPB, FOS, ETS1 and YY1 were detected on position 7219, 7310, 7366, 7394/7395 and 7595, respectively  and invasion cervical cancer. HPV variants may have co-diversified with human populations and thus obtain an intrinsic geographical difference on prevalence and infections . The knoThese three types of HR-HPV showed different prevalence and mutation rates in our study. More HPV16 and HPV58 were detected than HPV18 in this study. Only one HPV16 isolate was completely same with HPV16 REF sequence, while 16 (24.2%) isolates were same with REF sequence of HPV58. It may attributed to the high prevalence and active interaction in viral genome integration and cancer development of HPV16, so the LCR were able to occur and reserve more variations. HPV18 is less popular than HPV16/58 in Southwest China, and we didn’t find as many mutations as those two types in 18. T7592C was a general mutation for HPV18 in this area. It was found in all isolates of HPV18 and also the only variation of 35.3% (12 isolates) HPV18 LCR sequence. HPV58 showed different prevalence through geographical regions. The unusual high prevalence had been reported in East Asia, Africa and some other areas . A few sThe distribution of the sub-lineages were basically same with the data had been reported previously. In this study, A lineage accounted for a large proportion of all three types. It has been reported that lineage A related to pathogenicity more than other lineages.For HPV16, a worldwide phylogenetic analysis indicated that the EUR lineages (A1 ~ A3) were epidemic in many regions. A4 was largely found in Eastern Asia . 56% of All HPV18 isolated were identified as lineage A. The global data of lineage A showed that sub-lineage A1 predominated in Eastern Asia and Pacific, while sub-lineage A3/A4 were prevalent in many region around the world. The isolates of A5 were detected principally in Africa . Our datFor HPV58, globally, the sub-lineage A2 was the most widespread variant, whereas sub-lineages A1 and A3 were rarely found out of Asia. A1 were the most prevalence sub-lineage of HPV58 in our study, A2/A3 were also be found. 95.4% of the HPV58 isolates were clustered in lineage A which showed much more relation with CIN2/CIN3+ rather than lineage BCD . We alsoThe HPV LCR which contain the binding sites for both viral and cellular factors, has shown regulatory functions on replication of HPV, transcriptional activity of the E6/E7 and the other interaction through the virus life cycle , 13, 28.In conclusion, this study investigated the gene polymorphisms, phylogeny, and relevant functional prediction of high-risk HPV-LCR from Southwest China. Although our study showed some limitations on sample capacity and source, it provided more data for understanding the intrinsic geographical relatedness of HPV-16/18/58 variants, the complicated relation among HPV-16/18/58 LCR mutations, transcription factors and carcinogenesis. It also helps performing further study to demonstrate the biological function of HPV-16/18/58 LCR variants and the effect of multiple infection of high-risk HPV on tumor progression. The TFBS we found is still need deeper exploration for the potential of them to be marker in diagnosis and therapy.


The Director-General of the World Health Organization has called for global action towards elimination of cervical cancer as a public health problem. Cervical cancer is caused by human papillomavirus (HPV), an infectious agent with no non-human reservoir. One way to achieve this is through very high levels of vaccine coverage that could enable global eradication of vaccine-type HPV. Using the case study of India, we show that HPV eradication can meet all the Dahlem and Strüngmann criteria for feasibility of eradication. It can be achieved with 90% gender-neutral HPV vaccine coverage together with 95% coverage in high-risk groups such as female sex workers. Such a strategy would likely be cost-effective compared to no vaccination. Although it would be more costly in the short-term than achieving cervical cancer elimination alone, it would save costs in the long-term by removing or at least sharply reducing the need for preventive measures. •Eradication of vaccine-type HPV is a way to permanently maintain cervical cancer elimination.•Eradication is technically feasible, likely cost-effective and equitable.•Eradication requires high gender-neutral vaccine coverage, particularly in high-risk groups such as sex workers. ProphylThe widespread availability of both vaccines and screening methods has the potential to bring about large reductions in cervical cancer incidence globally. Countries which achieved high coverage in population-based HPV vaccination programmes reported 85% reductions in HPV 16/18 among 15–19 year old females 5–8 years after programme initiation, with large reductions also reported in males and older females due to herd effects . In pracCervical cancer is caused by an infectious agent with no non-human reservoir . Since HThis article discusses the feasibility, public health and economic case for eradication of vaccine-type HPV. First, we define eradication and examine the technical feasibility of HPV eradication according to widely accepted criteria. Then we use epidemiological and economic modelling to explore key criteria around the public health strategies needed to achieve eradication, and the cost-effectiveness of these strategies, using India as a case study.2According to the WHO's generic framework for control, elimination and eradication of neglected tropical diseases, a pathogen is eradicated when its prevalence is reduced to zero, as a result of deliberate efforts, with no more risk of reintroduction . By thatThe current WHO elimination initiative aims to bring cervical cancer incidence to below 4 cases per 100,000 women in every country . This wi3The world has seen a number of infectious disease eradication efforts, beginning with efforts to eliminate yellow fever from the Western Hemisphere in the early 1900s . In 19974Since vaccinating every individual in the world is infeasible, eradication efforts have relied on sustained high vaccine coverage providing herd protection for the vast majority of the population, combined with surveillance and special vaccination programmes to close immunity gaps and contain outbreaks . None ofEpidemiological modelling suggests that achieving 90% female-only vaccination coverage will dramatically reduce cervical cancer incidence in every country . HoweverEven achieving this threshold will not eradicate vaccine-type HPV, particularly not HPV 16 which is responsible for more cancers than any other HPV type. A meta-analysis of predictions from 16 transmission dynamic models of HPV vaccination in high-income countries demonstrated that 90% female-only coverage is insufficient to eliminate HPV 16 transmission, although 90% coverage in both males and females  may be sufficient . Since HThe meta-analysis did not look at model predictions for low- and middle-income countries. Hence we examined different scenarios about HPV vaccination in India from HPV-ADVISE , an indiThese levels of coverage are challenging but not impossible to reach. Demonstration projects in low- and middle-income countries often reach or exceed 90% coverage . Out-of-5HPV vaccination of females at 80% coverage prior to sexual debut has been shown to be highly cost-effective globally . These cEradicating vaccine-type HPV would likely have much higher costs for two reasons. Firstly, the targeted population would double since males would need to be vaccinated also. Secondly, the cost per person vaccinated would increase sharply in the move from 80% to 90% coverage since the remaining unvaccinated population will be harder to reach. Studies have not looked at the cost of scale-up to this level of coverage for HPV, but for measles an assessment has been made of costs of scale-up in hard-to-reach populations to reach 90% population coverage based on interviews with health officials . This esThese higher costs may be justified because eradicating vaccine-type HPV would have larger long-term benefits than eliminating cervical cancer as a public health problem without eradicating the underlying infectious agent. In principle, eradication should allow large cost savings from the cessation of cervical cancer preventive activities (vaccination and screening), since there will no longer be a virus to protect against - the so-called “eradication dividend” . This isTo illustrate, we calculated total costs and cost-effectiveness in order to reach elimination of HPV 16 infection in India, assuming that eradication will then be achieved globally in the year it is reached in India, after which most vaccination activities can be ceased. We used epidemiological outcomes from HPV-ADVISE  and econ6The WHO Director-General's call for action on elimination of cervical cancer as a public health issue is an important and necessary stimulus for more widespread introduction of the tools we already have for preventing cervical cancer, particularly in the regions where cervical cancer burden is highest. However, this article argues that in the long run it is insufficiently ambitious. Eradication of vaccine-type HPV itself is feasible, cost-effective (compared to no vaccination) and will eventually bring greater and more equitable benefits. An eradication objective is not incompatible with elimination as a public health problem - indeed, elimination is a necessary step on the road to eradication. However, elimination is largely a national public good. Eradication is a global public good so there is a much stronger case for international cooperation and resource pooling to achieve it, as was seen in the smallpox and polio eradication initiatives.The distinction between elimination as a public health problem and eradication may seem academic, but it will become increasingly important as we approach the disease end game. Indeed, the term “elimination as a public health problem” may eventually become a counter-productive term if it gives the impression that cervical cancer is a solved problem even though the underlying pathogen continues to circulate. Leprosy offers an important lesson; interest in leprosy research has diminished following declaration of its elimination as a public health problem even though cases continue to occur . Even atGiven the higher costs and number of doses needed to achieve eradication, countries may wish to aim for elimination targets first  but then switch to a more equitable eradication strategy as soon as local resources and global vaccine supplies permit.Eradication of vaccine-type HPV with current vaccines will not reduce the incidence of cervical cancer to zero since 10% of cervical cancers are due to types not in any current vaccine. Hence cervical screening may still be necessary, even though the frequency of screening can likely be sharply reduced . HoweverThe benefits of eradication will take many decades before they are realised. Unlike other infections targeted for eradication in the past , HPV can persist in its host for decades. Treatment of cervical lesions clears the infection in some but not all individuals . However10.13039/100000865Bill & Melinda Gates Foundation (OPP1157270). MJ was funded by the 10.13039/501100000272National Institute for Health Research (NIHR200929) Health Protection Research Unit in Immunisation at LSHTM in partnership with Public Health England. The views expressed are those of the author(s) and not necessarily those of the Bill & Melinda Gates Foundation, the National Health Service, the NIHR, the Department of Health and Social Services, or Public Health England.MJ and KP were funded by the None declared.


Drosophila ‘HPV 18 E6’ model that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; we further provided evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. Here, we report a functional genetic screen of the Drosophila kinome that identified IKKHuman papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, we developed a  Among all high-risk HPVs, HPV 16 and HPV 18 are the most prominent types, causing more than 70% of all invasive cervical cancers. According to World Health Organization (WHO), every year 570,000 women are diagnosed with cervical cancer, with a current mortality rate of more than 50%3.Human papillomaviruses (HPV) are small, double-stranded circular DNA viruses that are obligate epitheliotrophs: they specifically infect either squamous or mucocutaneous epithelia. The latter HPV types can be categorized into two classes: low-risk HPVs and high-risk HPVs. The former induce large and obvious benign lesions—condylomata—of the infected tissues. The latter are carcinogenic, causing most cervical cancers, as well as being strongly implicated in oropharyngeal, anal, vulvar, vaginal and penile cancers4. Furthermore, chronic HPV infection can require several decades to provoke transformation6. Existing vaccines are only prophylactic against new HPV infections and are not effective against preexisting HPV infections, nor can they inhibit cancer progression and malignancy7. As a result of these hurdles, effective treatment for HPV-induced cancer in general remains a major unmet clinical need. Current treatments for invasive HPV-induced cancers are primarily radiation and chemotherapy, which show limited effectiveness and the survival rates of, for example, advanced-stage cervical cancer patients is low9.The availability of effective vaccines against the most prevalent high-risk HPVs is expected to eventually reduce HPV-dependent tumors. However, the number of new HPV-induced cancer cases is not predicted to decline appreciably for the next few decades. Economic and cultural barriers hinder widespread immunization in the middle- and low-income countries that account for the majority of cervical cancers11. E6 and E7 oncoproteins cause cellular transformation through elimination of key tumor suppressors P53, and Rb respectively14. High-risk HPV E6s contain a PDZ binding motif (PBM) at the extreme C terminus that is absent in low-risk E6s15. Interaction of the PBM with the PDZ domains of key host cellular PDZ domain proteins, including Magi, Dlg and Scribble, targets these proteins for ubiquitination and subsequent proteasome-mediated destruction19. These PDZ domain-containing proteins are important components of tight junctions and cell polarity-controlling complexes22. This action of E6 requires the assistance of the host E3 ubiquitin ligase, UBE3A and is necessary for cellular transformation24. Transgenic mice expressing high-risk E6 and E7 in the skin develop cancers at high frequency; those expressing E7 and an E6 deficient in the PBM do not develop cancers. The failure to induce cellular transformation is independent of P53, as PBM-deleted E6 retains the ability to inactivate P5325. However, beyond the cellular targets of HPV oncogenes, we have a limited understanding of how persistent expression of E6 and E7 can lead to dysplasia and cancer.HPV 16 and HPV 18 direct cellular transformation through the persistent expression of two viral early genes, E6 and E7Drosophila has proven a strongly useful platform for modeling human diseases including cancer, owing in part to high conservation of genes and signaling pathways, and the availability of a broad array of genetic tools31. One particular advantage of Drosophila disease models is their use in functional genetic screens designed to discover novel targets and pathways that mediate human disease34. In this study we used a recently developed Drosophila model of HPV18 E6 + UBE3A35 in a screen to identify kinases active in aspects of E6/UBE3A-induced transformation. We report that reduced activity of ‘inhibitor of nuclear factor kappa-B kinase subunit beta’ (IKKβ)—which regulates the innate immune response—strongly suppressed E6 + UBE3A-mediated cellular abnormalities, as well as rescuing degradation of E6 targets. We provide evidence that the IKK36, UAS-HPV18-E635, GMR-Gal4, Kinome set, IKK1/TM6B,Tb, UAS-IKKDrosophila stock center.The following fly stocks were used: UAS-hUBE3ATo examine the effect of IMD 0354 on the growth of HaCat (HPV−ve), HeLa (HPV 18 +ve), CaSki and SiHa (both HPV 16 +ve), the cells were plated out and allowed to adhere overnight. Next day their culture medium was replaced and different concentrations of IMD 0354   were added to the plates. Cells were counted 48 h later using a haemocytometer. The assay was repeated 3 times. One-way ANOVA test using the Prism program was used for statistical analysis.HaCat, HeLa and CaSki cells were plated out at equal density, and allowed to attach overnight. The next day they were treated with 500 nM IMD354  for 3 h, then harvested by typsinisation and centrifugation . The cell pellet was resuspended in citrate buffer with 0.1% NP-40, RNase and Propidium iodide, and incubated for 30 min in the dark at room temperature. FACS analyses were then performed  and the cell cycle profiles were analysed . The assays were performed four times. Analysis: the % of events recorded in the subG1 , G1, S and G2 peaks for each run were compared between treated and untreated samples for each cell type and the fold change with treatment was calculated.38. Following incubation with primary antibody, the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako) were used, followed by enhanced chemiluminescence (ECL) detection according to the manufacturer's instructions . Original blots provided in HPV18-positive HeLa cells were seeded on 6 cm dishes and allowed to attach overnight. Fresh medium was then added, containing either 0, 100 nM or 500 nM IMD 0345 (in DMSO). After 5 h the cells were harvested in 2× SDS-PAGE gel loading buffer, run on SDS-PAGE, and analyzed by Western Blot. The membrane was probed with antibody specific for phosphorylated E6, as described previously39, rabbit anti-Baz 1:1000, rat anti-DE-cadherin 1:50 . The appropriate secondary antibodies were conjugated Alexa488, Alexa594, and Alexa 647 (Invitrogen).For immunolabeling pupal eyes, 40–42 h after puparium formation, were dissected in PBS and fixed in 4% formaldehyde. Fixed tissues were washed three times in PBS solution containing 0.1% Triton-X-100 and blocked in 5% normal goat serum for 1 h before incubation with primary antibodies. The primary antibodies used in this study were rabbit anti-Magi 1:20040.The fly eye is a compound eye consisting of 750 unit eyes. These ‘ommatidia’ are clusters of sensory neurons arranged in a precisely repeated hexagonal pattern formed by the precise arrangement of supporting pigment cells. Formation of this highly organized pattern requires precisely regulated cell proliferation, cell differentiation and programmed cell death; disruption of any of these processes leads to a disorganized, rough eye phenotype that is readily scored under a light microscopeGMR-Gal4, UAS-E6, and UAS-hUBE3A (referred to as GMR>E6/hUBE3A) were crossed to flies heterozygous for a mutant kinase. Comparing GMR>E6/hUBE3A; kinase/−+ to GMR> E6/hUBE3A flies, we screened 195 kinases and identified IKKβ as the strongest suppressor of the E6 + hUBE3A-mediated rough eye phenotype  in the developing fly eye leads to a disorganized, rough eye phenotype. To identify loci that modify the E6 + hUBE3A-induced eye defects, we used mutations in the kinome to perform a dominant genetic modifier screen. Flies with stable integration of the transgenes 43. To determine whether IKKβ also has a functional link to E6 + UBE3A in human cells, we assessed the effect of the synthetic IKKβ inhibitor IMD 0354 -5-chloro-2-hydroxybenzamide)44 on the growth of HaCat (HPV−ve), HeLa (HPV 18 +ve), CasKi (HPV 16 +ve), and SiHa (HPV 16 +ve) cells. IMD 0354 is a non–adenosine triphosphate–binding (ATP-binding) competitive selective IKKβ inhibitor that prevents ATP attachment to IKKβ38. Different concentrations of the inhibitor were tested ranging from 250, 500, 750, 1000, to 1250 nM. IMD 0354 reduced growth of all four cell types; HeLa cells were most strongly affected, with a significant effect starting at the lowest concentration of 250 nM. While IMD 0354 had a significant effect on the HPV 18 and 16 positive cells, it had a minor effect on the growth of HaCat cells that lack HPV  led to a strong phenotypic rescue: pigment and cone cell defects were reduced and the overall organization of the ommatidial array improved  revealed that, in comparison with pupal eyes expressing E6 and UBE3A, in which both the junctional and polarity complexes were perturbed in ommatidia  and compared with untreated cells for E6 phosphorylation. Western blot analysis was performed using an antibody to detect phosphorylated E6. We found that inhibition of IKKβ resulted in extensive phosphorylation of E6, which was absent in untreated cells  promotes cellular transformation and malignancy in fly epithelia50. Therefore, we asked whether reducing IKKβ activity can suppress the cellular transformation caused by the cooperation of oncogenic Ras and E6. Co-expression of oncogenic Ras, Ras64BV14 with E6, and hUB3A in eye imaginal discs (GMR>E6/hUBE3A/Ras64BV14) at 25 °C resulted in overgrowth and pupal lethality , the lethality was rescued and flies developed to adulthood. These adult flies, however, still exhibited significant abnormalities in eye morphology, suggesting incomplete rescue 55, suggesting that persistent infection may reflect a defect in the host innate immune system. Conversely, activation of the innate immune system, and consequent inflammation, contributes to HPV-induced tumor progression56. Several studies have shown an increase in the level of inflammatory cytokines in individuals with reduced HPV clearance59. Additionally, upregulation of several TLRs and components of the NF-κB signaling pathway have been implicated in HPV-related cancers. It has been suggested that the malignant phenotype of cervical cancer cells requires the function and activation of IKKβ/NF-κB signaling61. IKKβ phosphorylates the inhibitor of NF-κB, resulting in its ubiquitination and proteasomal degradation. This action of IKKβ frees the NF-κB, which in turn enters the nucleus and activates the transcription of pro-inflammatory, pro-cell proliferation and anti-apoptotic genes63. Increased expression of IKKβ and its association with an aggressive phenotype has been reported in several types of cancers including head-and-neck, ovarian and liver cancers66. It is notable that IKKβ's role in cancer is not only limited to its function in regulation of the NF-κB pathway; IKKβ can also phosphorylate p53, resulting in its ubiquitination and subsequent degradation. Inactivation or loss of p53 has been identified in more than 50% of cancers, including HPV-induced cancers: high-risk HPV E6s target p53 for ubiquitination and proteasomal degradation67. IKKβ-mediated loss of p53 can be suppressed by inhibition of IKKβ in cancer cells69. The inhibitory function of IKKβ on p53 was further found in HPV38E6E7 human keratinocytes. IKKβ phosphorylates and stabilizes a dominant-negative inhibitor of p53, ∆Np7370. Another phosphorylation target of IKKβ is fork-head transcription factor FOXO3a. IKKβ has been found to stimulate cell cycle progression and proliferation of breast cancer cells, through phosphorylation of FOXO3a, leading to its exclusion from nucleus and subsequent degradation in the proteasome. This function of IKKβ could be further overridden by the overexpression of FOXO3a71. All these findings suggest that IKKβ may contribute to HPV-induced cellular abnormalities in several ways, some through the innate immune pathway, and some independent of it.In this study we identify IKKβ as a mediator of the HPV 18 E6 and hUBE3A-induced cellular defects in both fly and human cancer models. This is consistent with previous studies demonstrating that HPV 16 E6 interacts with components of the innate immune pathway, including IKKβ, and it activates the NF-κB transcription factor76. One such inhibitor is IMD 0354, which selectively inhibits IKKβ by preventing ATP attachment to IKKβ. This will in turn block phosphorylation of IκBα, thus preventing nuclear translocation and activation of NF-κB38. Although IMD 0354 was initially designed to inhibit inflammation77, it has recently proved to have strong anticancer properties79 in several cancers. IMD 0354 inhibits breast cancer cell proliferation, progression of mast cells, and it induces apoptosis in prostate, lung and colon cancers82. In our study we found that IMD 0354 inhibited the growth of HPV 16+ and 18+ cervical cancer cell lines. This result is consistent with previously reported function of IMD 0354 in cancer cells and hence suggests a therapeutic potential for IKKβ inhibitors to treat HPV-induced cancers. Although both HPV16+ and HPV18+ cervical cancer cell lines responded to IMD 0354, however, IMD 0354-mediated inhibition of IKKβ exhibited greater effectiveness on HeLa cells (HPV 18+) compared with SiHa and CaSki cells (HPV 16+). It has previously been demonstrated that these cell lines differ in a number of molecular pathways, and in their response to treatments such as therapeutic agents that induce cell death85. HeLa cells exhibit greater apoptotic cell death in response to cisplatin than SiHa and CaSki cells do, a difference that is due to the higher levels of p53 and p21 in HeLa cells84. Additionally, these cell lines show distinct proteomic profiles for pathways connected to p53 activation, mitochondrial function and oxidative stress86. These findings suggest that the stronger effect of IMD 0354 in HeLa relative to SiHa and CaSki cells could in part be due to their difference in molecular pathways affected by IKKβ. Additionally, IMD 0354 caused an extensive phosphorylation of E6, which was absent in untreated cells. This finding is intriguing, as phosphorylation of E6 on the PDZ binding motif results in disruption of its interaction with PDZ domain-containing proteins47. This is consistent with our results, as degradation of Magi was inhibited when IKKβ was reduced. It would be of interest to understand the mechanism by which inhibition of IKKβ results in E6 hyperphosphorylation.IKKβ inhibitors have served as potent anti-tumor agents inhibiting cell proliferation and invasiveness, as well as inducing cell death. Their activity has been demonstrated in several cancer types including breast, colon, ovarian, oral, prostate, liver, melanoma, and leukemiaIn summary this study suggests an important role for IKKβ in E6-induced cellular abnormalities and that targeting IKKβ could be a potential therapeutic option for cervical cancer and, perhaps, for other HPV-related cancers for which there is currently no effective treatment.Supplementary Information.


Human papillomavirus (HPV) types 16 and 18, classified as high-risk (HR) HPV genotypes, are considered as risk factors for oropharyngeal cancers, but their role in the development of OSCC remains unclear. We tested the hypothesis of viral etiology in a series of 68 well-characterized OSCCs and 14 potentially malignant disorders (PMD) in non-smoking, non-drinking (NSND) patients using broad-range, sensitive molecular methodologies. Deep-sequencing of the transcriptome did not reveal any vertebrate virus sequences other than HPV transcripts, detected in only one case. In contrast, HPV DNA was detected in 41.2% (28/68) and 35.7% (5/14) of OSCC and PMD cases, respectively. Importantly, 90.9% (30/33) of these belonged to the Betapapillomavirus genus, but no viral transcripts were detected. Finally, high-throughput sequencing revealed reads corresponding to transcripts of the Trichomonas vaginalis virus (TVV), which were confirmed by RT-PCR in two OSCCs. Our results strongly suggest that Alphapapillomavirus genotypes classified as HR are not involved in the development of OSCCs in NSND patients and that known oncogenic infectious agents are absent in these specific OSCCs. Any possible direct or indirect role of Betapapillomavirus genus members and TVV in OSCCs remains speculative and requires further investigation.Head and neck squamous cell carcinomas (HNSCC) are the seventh most frequent cancers. Among HNSCCs, oral squamous cell carcinomas (OSCCs) include several anatomical locations of the oral cavity, but exclude the oropharynx. The known risk factors for OSCCs are mainly alcohol consumption and tobacco use for at least 75–80% of cases. In addition to these risk factors,  In 2015, head and neck squamous cell carcinomas (HNSCC), i.e. cancers of the mouth, nose, throat, larynx and sinuses, affected more than 5.5 million people worldwide and caused more than 379,000 deaths . This grHuman papillomavirus (HPV) types 16 and 18, classified as high-risk (HR) HPV genotypes, are also considered as risk factors for oropharyngeal cancer, but their role in the development of oral cavity cancer remains unclear [versus base of the tongue) was available, which may lead to confusion between OPSCCs and OSCCs, and thus an overestimation of the true burden of HPV in OSCCs [et al. (2010) detected HR-HPV DNA via PCR in only 12.5% of OSCC cases in patients who had no history of smoking or alcohol abuse, whereas Li et al. (2015) did not identify any oncogenic virus in a cohort of 20 OSCCs of the tongue in NS patients [The known risk factors for OSCC are mainly alcohol consumption, tobacco use and chewing betel for at least 75–80% of cases . Althoug unclear . The pos unclear ; since t unclear . The fir unclear . Howeverin OSCCs –22. The in OSCCs , 22, 23.in OSCCs . Therefoin OSCCs –27. To dpatients , 29.Alphapapilliomavirus genus and the second one was based on several PCR systems using consensus HPV primers, followed by amplicon sequencing and analysis of the sequences obtained. Secondly, a broad-spectrum transcriptomic approach (i.e. high-throughput sequencing (HTS) of total RNA), was used to explore other etiological causes. This latter methodology was developed to detect and characterize unknown or variant replicating viruses associated with several human diseases, especially cancers.The aim of this study was to test the hypothesis of a viral etiology due to a known viral agent such as HPV or another, unknown or unforeseen, agent associated with an oncogenic process in a series of well-characterized PMDs and OSCCs in NSND patients using very broad and sensitive methodologies. Firstly, HPV was investigated using two different methods. The first one consisted of the use of a DNA microarray (PapilloCheck HPV genotyping kit) that detects 24 genotypes belonging to the http://www2.voozanoo.net/) on the ICAReB platform at the Institut Pasteur. All samples of patients included in the study were collected during their surgical treatment according to current cancer reference standards. Each investigated tissue sample was harvested from the main tumor removed during surgery. To preserve the nucleic acids as much as possible, the tissues were immediately frozen in dry ice in the operating room and then sent to the Institut Pasteur for long-term storage and molecular investigations. All the bio-resources were managed by the ICAReB platform, comprising RNA/DNA extraction, sample transport, registration and coding with regard to ethical compliance. In parallel to the molecular investigations, the entire tumor was analyzed by anatomical pathologists to determine the histological grade, the margins of the tumor and the two main histological criteria of its severity .At the maxillofacial surgery department at the Pitié-Salpêtrière Hospital , NSND patients with PMD or OSCC were selected for our prospective study according to strict criteria between 2012 and 2015. Patients were included in the study if they were over 18 years of age and were considered NSND following commonly accepted criteria. In addition, we will include in this category all patients whose rare, or very rare, occasional use of alcohol or tobacco suggested that the development of their OSCCs were not related to these two risk factors. Consequently, the patients included in our study were (i) strictly NS or with a consumption equal to or less than 5 packs per year or having stopped smoking at least 15 years prior, regardless of the amount smoked and (ii) strictly ND or with a moderate consumption of 10 or 20 g of alcohol per day or less, with the majority of the cases being less than 1–2 drinks per month for women and men, respectively, or having stopped drinking at least 15 years prior, regardless of initial consumption. The only non-inclusion criteria were previous antecedents of oral cancer. The first criteria for inclusion of PMDs was the clinical diagnosis of leukoplakia. All other PMDs, such as erythroplakia, lichen planus, proliferative verrucous leukoplakia (PVL), etc were excluded. All clinical data related to the patient  or to his/her PMD or OSCC  and histological criteria) were compiled in a database managed using the Voozanoo platform  and stored at -20°C until molecular investigations such as HPV genotyping. Firstly, this extracted DNA was investigated using the PapilloCheck HPV genotyping kit  according to the manufacturer’s recommendations. This assay first included PCR amplification with fluorescent primers (CY5 fluorophore) specific to the HPV E1 gene and the cellular housekeeping gene ADAT1. The amplification products were then hybridized to a DNA microarray containing sequences for 18 HR genotypes  and six low-risk (LR) genotypes . Secondly, three consensus PCR assays were performed using three sets of degenerate primers  [Thermo Fisher Scientific), 3.5 mM MgCl2, 0.2 μM of each deoxynucleotide triphosphate (dNTP), 0.5 μM of each consensus primer GP5+/6+ and 1 U of Ampli Taq Gold DNA polymerase (Thermo Fisher Scientific). The second nested PCR assay was also performed using two sets of degenerate primers (CP65-70 and CP66-69). The reaction was performed in 50 μL including 5 μL of template DNA (either template of DNA or amplicon from PCR using the CP67-70 primers), 1X PCR buffer (Thermo Fisher Scientific), 3.5 mM MgCl2, 0.2 μM of each dNTP, 0.5 μM of each consensus primer and 1 U of Ampli Taq Gold DNA polymerase. Thermal cycling for the three PCRs are described in Frozen tumor tissues samples were stored at -80°C until total genomic DNA extraction using the DNeasy Blood and Tissue kit  according to the manufacturer’s instructions. Extracted DNA was quantified using the QubitCP66-69) , 31. TheTotal RNA was extracted from biopsies using the Trizol procedure (Thermo Fischer Scientific), purified on RNeasy column including a DNase treatment (Qiagen), and randomly amplified  for eachTrichomonas vaginalis virus 1, 2, 3 and 4  were retrieved from [T. vaginalis (TV) were retrieved from [Reverse transcription reactions were performed starting from RNA with the SuperScript III First-Strand Synthesis System (Thermo Fischer Scientific). Then, qPCRs were performed in SYBR Green format with 45 cycles of amplification using primers described in ved from . Additioved from  and desiThis study was approved by both the French Health Products Safety Agency  on 20 February 2012 (B120225-40) and by the Ile de France VI Ethics Committee on 2 March 2012 (CPP no. 13–12 –IDRCB 2012-A00116-37). Written informed consent was obtained from all patients included in this study.in situ carcinomas (n = 6), which represented 42.8% (6/14) of cases. In OSCC samples, histological criteria classified lesions according to the level of cell differentiation and keratinization. More than 83.9% (47/56) of OSCCs were keratinized, but data on this criterion could not be obtained for 17.6% (12/68) of cases. In parallel, in 35.3% and 55.9% of cases (24/68 and 38/68), the cells were moderately or well differentiated, respectively. Poorly differentiated cells were found in only 7.3% of cases. Finally, a history of PMD was found in 17.6% (12/68) of the OSCC cases in this study (This study included 14 PMD and 68 OSCC cases. The sex ratio (M:F) was 0.16 and 0.94 with an average age of 70 years (+/- 15 years) and 69 years (+/- 15 years) for PMDs and OSCCs, respectively , with 86is study . They hais study .Betapapillomavirus genus (β-HPVs) (in situ carcinomas (2/5). Overall, 15 different genotypes of β-HPV were identified in PMDs and OSCCs. However, approximately 86.7% (13/15) of these genotypes were found only once or twice, such as the HPV9 genotype, which was detected only in one PMD case (PM01). Finally, the HPV20 and HPV36 genotypes represented 46.2% (12/26) of the β -HPVs identified in both PMDs and OSCCs.At least one HPV type was detected in 35.7% (5/14) and 41.2% (28/68) of PMD and OSCC cases, respectively (See (β-HPVs)  Tables. (β-HPVs) . All the(β-HPVs) . Among t(β-HPVs) . Finally(β-HPVs) . These β(β-HPVs) . MoreoveT. vaginalis virus (TVV), a double-stranded RNA virus, belonging to the Totiviridae family, were identified. The analyses showed that these reads corresponded to the TVV1-2-3 and TVV3 viruses in CE03 and CE02, respectively , but the others came from either the lip, gum or cheek mucosa. Of these, only three β-HPVs had previously been identified using conventional DNA PCR. With the exception of the detection of bacterial transcripts most likely coming from commensal flora (data not shown), these analyses did not reveal any transcripts corresponding to β-HPV or belonging to other viral agents . In the ectively . The preectively . Moreoveectively .In this study, a search of an association with an infectious agent was investigated using various molecular methods in a well-characterized series of PMD and OSCC in NSND patients. The epidemiology of these OSCCs is different from those with known risk factors . First, these alcohol and tobacco related cancers are generally more common in 60-year-old men and are located mainly in the tongue (25%), the floor of the mouth (17%) and the gums (13%) (unpublished data), whereas our NSND cohort was dominated by women over 70 years of age. Moreover, these cancers in NSND patients were mainly located in the mobile part of the tongue and gums, which represented more than 70% of the cases, with localization in the floor of the mouth being rare. These data confirm that cancers in NSND and SD are epidemiologically different. Therefore, in the absence of the two main known risk factors , we investigated HR-HPV as an infectious etiological cause in our NSND cohort. However, our results differ from previous studies because we focused strictly on lesions localized in the oral cavity in NSND patients. Our investigations revealed HR-HPV DNA in only 4.4% of OSCCs in our cohort. Consequently, our study shows that the potential impact of HR-HPV in NSND patients as an etiological agent is lower compared with the majority of other studies in which the OSCCs studied also included patients with the two main risk factors  , 19–22. Nonetheless, this study highlighted the presence of a β-HPV in 35.7% and 41.2% of cases, for PMDs and OSCCs, respectively. Many studies mention that β-HPVs are ubiquitously disseminated throughout the human body, participating in commensal flora . InMoreover, this study identified transcripts of TVV in two OSCCs (CE02 and CE03), including one case co-infected with HPV16 (CE03). TV, a flagellated protozoan parasite, is the main non-viral agent responsible for sexually transmitted infections with more than 276 million estimated cases, 90% of which occur in resource-limited countries . In highet al. (2015) on 20 cases of OSCC investigated in NS patients [et al. (2011), which reported a much higher HR-HPV rate (12.5% vs. 4.4%) in OSCCs in NSND patients [et al. study did not specify the amount of alcohol the patients had consumed, except that they did not suffer from chronic alcoholism. Although the role of moderate alcohol consumption remains uncertain in the development of OSCC, it has a direct or indirect effect on the epithelium of the oral cavity. Alcohol consumption can act via its main metabolite, acetaldehyde, or alter the metabolism of retinoids or increase its permeability to other carcinogenic agents [Finally, this study of the potential infectious cause of this well-characterized series of PMDs and OSCCs, carried out using a wide range of complementary molecular techniques, did not reveal the presence of any other already known oncogenic virus, with the rare exception of HR-HPV. These results are similar to those obtained by Li patients . Howeverpatients . Unfortuc agents –74. Morec agents . Therefoc agents . Hence, In conclusion, all the results of this study strongly suggest that HR-HPV is not involved in the development of OSCCs in NSND patients and that known oncogenic infectious agents are absent in these specific OSCCs. Any possible direct or indirect role of β-HPV and TVV in NSND cases of OSCCs remains speculative and requires further investigation.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Although it is well acknowledged that persistent infection with high-risk human papillomavirus types in genital sites plays a crucial role in the development of squamous cell cervical carcinoma, there is no unanimous consensus on the association between non-HPV sexually transmitted infections and abnormal cervical cytology.In the present study, we evaluated cervical cytology status, sexually transmitted infections and bacterial vaginosis status, and collected social-demographic information among recruited participants to explore the association of STIs and bacterial vaginosis with abnormal cervical cytology.Chlamydia trachomatis, Neisseria gonorrhoeae, and bacterial vaginosis was significantly higher in the ≥ASC-US group than the NILM group (P<0.05). Women with Neisseria gonorrhoeae infection  or bacterial vaginosis  exhibited an increased risk of abnormal cervical cytology after adjusted for carcinogenic HPV-positive status.9,090 women’s specimens were successfully tested, with a total of 8,733 (96.1%) women had normal cytology and 357 (3.9%) women exhibited abnormal cytology. The prevalence of HPV, Neisseria gonorrhoeae infection in genital sites and/or bacterial vaginosis may independently increase the risk for cervical cytology abnormalities after adjusted for carcinogenic HPV-positive status. Besides, these results improved our understanding of the etiology of abnormal cervical cytology and may be useful for the management of women with ASC-US cytology.Our results demonstrated that  Based on epidemiological and laboratory evidence, persistent infection with high-risk human papillomavirus (HPV) types in genital sites plays a crucial role in the development of squamous cell carcinoma (SCC) of the cervix , 2. AfteChlamydia trachomatis [Neisseria gonorrhoeae [Trichomonas vaginalis [Mycoplasma [Ureaplasma species [Chlamydia trachomatis infection is considered as a risk factor for abnormal cervical cytology [Chlamydia trachomatis infection and cervical pre-malignancy after controlled for carcinogenic HPV-positive status.Previous studies reported that there are many other risk factors for SCC including long term use of oral contraceptives , high pachomatis , Neisserorrhoeae , herpes orrhoeae , 11, Triaginalis , Mycoplacoplasma , Ureapla species , 15. Howcytology  or cervicytology , but, Sacytology  found noGardnerella vaginalis, Mobiluncus and Aptopobium vaginae, and other fastidious bacteria such as Megasphaera, Sneathia, and Clostridiales spp [Bacterial vaginosis relates to a remarkable shift in the vaginal microbiota to a dysbiotic state, marked by a diverseness of microorganism and increased loads of aerotolerant and strict anaerobes, including ales spp . Previouales spp , but alsales spp . Neverthales spp .Hence, we investigated the association of STIs and bacterial vaginosis with abnormal cervical cytology among community women, which would improve our knowledge about the etiology of SCC and may be useful for disease control and prevention.Chlamydia trachomatis and Neisseria gonorrhoeae tests), a cervical swab (HPV tests), two vaginal swabs , and an exfoliated cervical cells specimen .Our previous study  has descAfter allocating a unique identification number to each participant, two research nurses were assigned to check the integrity of questionnaire information and instruct participants in urine specimen collection, and guide participants to a specialized room for specimen collection. After collecting participant’s specimens, laboratory testing procedures were started immediately. Laboratory staffs were blinded to the clinical findings.Chlamydia trachomatis and Neisseria gonorrhoeae DNA tests were described in our previous study [The method and procedures of us study .A skilled gynecologist collected the cervical swab for HPV tests. Swabs were taken and placed into a tube containing 3 mL cell preservation medium, and were stored at -20°C until testing. Fourteen high-risk HPV genotypes  and two low-risk HPV types  in all cervical swabs were detected by using the PCR-based MALDI-TOF-MS assays , 24 in tCandidiasis and clue cells were confirmed by Gram staining of vaginal secretions. Trichomonas Vaginalis was diagnosed by microscopic examination of wet mounts immediately once the vaginal secretions swab collected. Amine test, pH of vaginal secretions and leukocytes were further confirmed within fifteen minutes. The diagnosis of bacterial vaginosis was based on Amsel’s criteria [Vaginal secretions swab specimens were collected by two skilled gynecologists and were rolled on to a glass slide for Gram staining immediately. Vaginal cleanliness, detection of hyphae and spores of criteria , which wExfoliated cervical cells specimens were collected by two gynecologists with conical cytobrush and were placed into a tube with 4 mL preservation solution and were temporarily stored at 4°C condition until the required test. Cervical cytology samples were diagnosed by using liquid-based cytology technique  according to the manufacturer’s instruction and were doubled-checked by cytotechnologists without information on STIs test results. The 2001 Bethesda system  was empl2), or two-sided Fisher exact test for 2×2 contingency table was used to evaluate the statistical significance of rates of variables between different groups. A univariate analysis was employed to estimate the associations of variables with different cytological findings. Variables with a significance level of p<0.2 were included in the multivariate logistic regression model adjusted by potential confounders. Crude odds ratios (OR), Adjusted odds ratio (AOR) and corresponding 95% CIs were calculated. A p<0.05 was considered significant. All statistical analyses were performed using SPSS 19.0 statistical software  and R-statistics 3.4.3 software.Anonymous data collection and statistical analysis were performed by Microsoft Excel 2016 and R-statistics software (version 3.4.1) respectively. Abnormal cytology group (≥ASC-US group) was defined as women who had a diagnosis of the following cytology findings: ASC-US, ASC-H, LSIL, HSIL or AGC. Variables were evaluated for comparisons between normal cytology (only NILM finding) group and ≥ASC-US group  or ASC-US group (only ASC-US finding) or LSIL+HSIL group (only LSIL finding and only HSIL finding) for analytical calculations. Prevalence of STIs and bacterial vaginosis were presented as positive rate and corresponding 95% confidential intervals (95% CIs). Chi-square test . A written informed consent was obtained from all participants. All experiments and procedures were performed in accordance with relevant guidelines and regulations of the People’s Republic of China. Participants who tested positive for STIs or abnormal cervical cytology were contacted privately by a nurse for further treatment and other interventions.Chlamydia trachomatis DNA and Neisseria gonorrhoeae DNA and failed to re-sample. 9,090 (98.3%) women’s specimens were successfully tested, a total of 8,733 (96.1%) women had normal cytology (NILM) and 357 (3.9%) women exhibited abnormal cytology: 181 ASC-US (2.0%), 16 ASC-H (0.2%), 115 LSIL (1.3%), 40 HSIL (0.4%), 5 AGC (0.06%). High-risk HPV infection  had the highest prevalence among detected STIs. The prevalence of bacterial vaginosis and Trichomonas vaginalis was 2.27% (95% CIs: 1.96%-2.58%) and 0.35% (95% CIs: 0.23%-0.47%), respectively (Table 1).Out of the 9,249 women who met eligibility criteria and signed an informed consent, 156 (1.69%) women were excluded because of they met exclusion criteria, 9,093 (98.3%) women completed the questionnaire interview and provided all specimens, 3 urine specimens were invalid for Table 2, HPV-52 (1.8%) was the most prevalent genotype in studied population, followed by HPV-16 (1.1%), HPV-58 (1.0%), HPV-18 (0.5%). Among 357 women exhibited abnormal cytology, 192 women positive for high-risk HPV, with a prevalence of 52.1% . HPV-52 (12.9%) was the most prevalent genotype in ≥ASC-US group, followed by HPV-58 (10.4%), HPV-16 (8.4%), and HPV-51 (5.0%). The prevalence of high-risk HPV genotypes in abnormal groups  were significantly higher than NILM group (Table 2). No cases of AGC were positive for HPV.As shown in Table 3, compared to the NILM group, the prevalence of Chlamydia trachomatis infection, Neisseria gonorrhoeae infection, and bacterial vaginosis were significantly higher in ≥ASC-US group and ASC-US group. In the LSIL+HSIL group, the prevalence of Chlamydia trachomatis infection, Neisseria gonorrhoeae infection, and Vulvovaginal candidiasis were significantly higher than the NILM group.As shown in Table 4, after adjusted for potential confounders, compared to the NILM group, high-risk HPV infection , Neisseria gonorrhoeae infection , bacterial vaginosis  and 40–46 years old  significantly increased the risk of ≥ASC-US cytology. Compared to the NILM group, high-risk HPV infection , Neisseria gonorrhoeae infection  and bacterial vaginosis  significantly increased the risk of ASC-US cytology, and only high-risk HPV infection  significantly increased the risk of LSIL+HSIL cytology.As shown in Neisseria gonorrhoeae infection or bacterial vaginosis exhibited an increased risk of ≥ASC-US and ASC-US cytology after adjusted for carcinogenic HPV-positive status, suggesting Neisseria gonorrhoeae infection or bacterial vaginosis may act as an independent risk factor for atypical squamous cells formation, predominantly for ASC-US cytology.In the present study, we investigated the association of STIs and bacterial vaginosis with cervical cytology abnormalities in the general population. Our main findings were women with Neisseria gonorrhoeae infection in genital sites and/or bacterial vaginosis may increase the risk of cervical epithelial cell abnormalities. A previous study concluded that bacterial vaginosis [In the 2001 Bethesda system, atypical squamous cells does not rule out the diagnosis of abnormal cytology and all atypical squamous cells is considered to be suggestive of squamous intraepithelial lesions (SIL), and atypical squamous cells are qualified as “of undetermined significance” (ASC-US) or “cannot exclude HSIL” (ASC-H) . In the aginosis  was signIn the present study, the prevalence of high-risk HPV in women with abnormal cytology was 52.1%, which is consistent with Northwest China (53.3%)  but therChlamydia trachomatis infection could trigger the formation of cervical lesion, because Chlamydia trachomatis is independently associated with persistence of high-risk HPV [Chlamydia trachomatis in ASC-US group or LSIL+HSIL group was significantly higher than NILM group, a multivariate logistic regression analysis revealed that Chlamydia trachomatis infection may not act as an independent risk factor for atypical squamous cells formation after potential confounders controlled, which reinforces the conclusion of Safaeian et al.’s study [Chlamydia trachomatis infection triggers the formation of oxidative reactive species (ROS), induces genetic instability, and inhibits damaged DNA repair pathways in the endocervical epithelial cells [Chlamydia trachomatis infection may aggravate cervical lesions triggered by high-risk HPV infection and play a possible synergistic action with high-risk HPV in cervical lesion progression [At present, it is unclear whether a single risk HPV . Althoug’s study . Theoretal cells , 37, whigression .Neisseria gonorrhoeae infection or bacterial vaginosis in our study provided a clue that evaluation of Neisseria gonorrhoeae DNA and/or bacterial vaginosis may be a supplementary tool to triage women with ASC-US cytology. Therefore, it is necessary to evaluate the risk of SIL in women with co-presence of ASC-US and Neisseria gonorrhoeae infection or bacterial vaginosis in the future study, which may improve our management of women with ASC-US cervical cytology.Furthermore, management of women with ASC-US cytology confused clinicians in the past decades , 38, 39,Trichomonas Vaginalis and Vulvovaginal candidiasis may limit the strengthening of our results. For example, wet mount microscopy of Trichomonas Vaginalis has low sensitivity, which ranges from 44% to 68%, though with 100% specificity [Trichomonas Vaginalis or Vulvovaginal candidiasis and abnormal cytology. Secondly, without identification and characterization of bacterial vaginosis-associated pathogens in the present study limited the analysis of the association between bacterial vaginosis-associated pathogens and abnormal cervical cytology. Consolaro MEL et al.’s [The current study has several limitations. Firstly, the methodology used for the identification of cificity , but micet al.’s  study reNeisseria gonorrhoeae infection in genital sites and/or bacterial vaginosis may independently increase the risk for cervical cytology abnormalities, which suggests that performing non-HPV STIs tests will be helpful to diagnosis and treatment of patients in routine cervical cancer screening. Meanwhile, these findings enhance our understanding of the etiology of abnormal cervical cytology, and may also be useful for the management of women with ASC-US cervical cytology.In conclusion, this study provided an opportunity to determine the prevalence of STIs and bacterial vaginosis in different cervical cytological findings. We found that S1 File(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Accelerated global control of cervical cancer would require primary prevention with human papillomavirus (HPV) vaccination in addition to novel screening program strategies that are simple, inexpensive, and effective. We present the feasibility and outcome of a community-based HPV self-sampled screening program.In Ile Ife, Nigeria, 9406 women aged 30–49 years collected vaginal self-samples, which were tested for HPV in the local study laboratory using Hybrid Capture-2 (HC2) (Qiagen). HPV-positive women were referred to the colposcopy clinic. Gynecologist colposcopic impression dictated immediate management; biopsies were taken when definite acetowhitening was present to produce a histopathologic reference standard of precancer . Retrospective linkage to the medical records identified 442 of 9406 women living with HIV (WLWH).With self-sampling, it was possible to screen more than 100 women per day per clinic. Following an audio-visual presentation and in-person instructions, overall acceptability of self-sampling was very high (81.2% women preferring self-sampling over clinician collection). HPV positivity was found in 17.3% of women. Intensive follow-up contributed to 85.9% attendance at the colposcopy clinic. Of those referred, 8.2% were initially treated with thermal ablation and 5.6% with large loop excision of transformation zone (LLETZ). Full visibility of the squamocolumnar junction, necessary for optimal visual triage and ablation, declined from 68.5% at age 30 to 35.4% at age 49. CIN2+ and CIN3+ , including five cancers, were identified by histology in 5.9 and 3.2% of the HPV-positive women, respectively , leading to additional treatment as indicated. The prevalences of HPV infection and CIN2+ were substantially higher  among WLWH. Colposcopic impression led to over- and under-treatment compared to the histopathology reference standard.A cervical cancer screening program using self-sampled HPV testing, with colposcopic immediate management of women positive for HPV, proved feasible in Nigeria. Based on the collected specimens and images, we are now evaluating the use of a combination of partial HPV typing and automated visual evaluation (AVE) of cervical images to improve the accuracy of the screening program. Nearly 85% of the annual 570,000 cervical cancer cases and almost 90% of the 311,000 related deaths occur in lower-resource countries  due to tA new candidate for triage of HPV-positive women is automated visual evaluation (AVE) of cervical images using a deep-learning algorithm. A proof-of-principle evaluation of AVE of Cervigrams  for the diagnosis of cervical precancer, demonstrated higher accuracy of AVE than expert gynecologist visual assessment or cytology .Although these are promising results, the Cervigram cervical images were based on film and a discontinued expensive custom camera, and the technology is obsolete. Thus, it is essential to advance the transfer of the method to modern image acquisition devices  . There iThis paper describes the field methods, feasibility, and preliminary descriptive results of Project Itoju in Ile-Ife, Nigeria, designed to evaluate ultimately the combined strategy of self-sampled HPV typing combined with AVE triage on three different image capture devices.Women aged 30–49 years  residinThe study was approved by National Cancer Institute (NCI) and OAUTHC ethical Institutional Review Boards.The screening period was from November 2018 to December 2019. The colposcopy period was from December 2018 to March 2020.The screening visit started when a woman attended one of the three screening clinics (no appointment needed). A nurse-administered short screening questionnaire determined eligibility [age 30–49 years and not pregnant (self-reported)] for screening. If a woman was menstruating and was uncomfortable undergoing screening, she was advised to return later. Known pregnant women were excluded due to an “abundance of caution”, mainly to prevent any possibility that an unrelated adverse pregnancy outcome could be due, or even perceived to be due, to the self-sampling for screening. The lower limit of age for screening was set at 30 years since younger women have a high prevalence of HPV but a very low risk of cancer . The upphttps://www.youtube.com/watch?v=JiNqrDntbTc]   while waThe primary HPV test used in the study was the Digene Hybrid Capture-2 (HC2) HPV DNA (deoxyribose nucleic acid) Test , which is a US Food and Drug Administration approved nucleic acid hybridization assay with signal amplification using microplate chemiluminescence targeting 13 high-risk types of HPV DNA in cervical and vaginal specimens, without distinguishing between them .Trained nurses called participants by phone, when their HPV test results were available (within two weeks of collection for most of the study period). HPV-negative women were informed and educated about the test result over the phone, and their questions were answered. HPV-positive women were asked to visit the colposcopy clinic to receive their test results. A minimum of five contact attempts were made to approach and advise a woman to attend the colposcopy clinic before a woman was declared lost to follow-up.At the clinic, a nurse communicated the positive HPV test result and its clinical meaning to the woman, ensuring privacy. The woman was counseled to undergo a colposcopy examination, preferably during that same visit or later.Each woman was registered and interviewed to determine eligibility for the triage methods research study . Anyone with a history of cervical cancer, hysterectomy, or who was pregnant at the time of enrollment (confirmed with rapid pregnancy test) was excluded from the study. A female nurse took informed consent from all the eligible women for participation in a research study (examining triage methods). An anonymized picture of the cervix was shown to the woman at the time of consent in order to reassure her about confidentiality and privacy of image collection and to minimize refusals.Following the interview, a colposcopy examination was performed by one of the study gynecologists . Cervical images for research were collected one minute after applying 5% acetic acid for each device, sequentially with three different devices: 1) a Samsung Galaxy S8  smartphoAt the beginning of the study, a dry swab sample was collected at colposcopy, before collecting images and applying acetic acid, for the two-type OncoE6 test . The test is known to have high positive predictive value; in fact, three of the five positives  were diagnosed with CIN2+. However, the test was dropped after testing 373 samples due to rare positivity and low yield.Finally, a standard colposcopy examination was performed to assess the presence and possible nature of cervical lesions and to take biopsies of acetowhite lesions, up to a maximum of four biopsies. In addition, endocervical curettage (ECC) was performed in cases where the SCJ was not fully visible, even in the absence of acetowhitening. All women with acetowhite lesions were offered immediate treatment without waiting for histopathology results, following the American Society of Colposcopy and Cervical Pathology 2012 Consensus guidelines , leaningHistopathological confirmation of CIN2 or CIN3 was used as the reference standard for the presence of precancer, against which other experimental tests were evaluated and final clinical review decisions were made. Even though from the clinical management purposes, all high-grade (CIN2+) lesions were treated equivalently; for the true yield of the screening effort, we reported the prevalence of CIN2+ and CIN3+ lesions separately to avoid the ambiguity of equivocal CIN2 lesions  . The stuAll cases were reviewed for adequate clinical management by a US gynecologic oncologist (AN). The more complex cases were discussed in a case conference call. Recall was recommended for women needing further management and such cases were re-reviewed once the recall was completed until the case was determined to be adequately treated. We planned to reach all women needing recall for additional management, a minimum of seven times. However, we restricted our attempts, and recalled only those women at the highest immediate risk of invasive cancer because of the spread of the COVID-19 pandemic in March 2020.All data and images were collected with a HIPAA  compliant smartphone application ‘EVA for research’ . The data platform was custom designed for the project (led by CS) using an advanced barcode scanning system to limit human error from manual key-in. The data and images from different sources on study assigned smartphones were held locally until internet connectivity was available, at which point all data automatically transferred and aggregated to cloud servers and portal for analysis and remote quality assurance.The preliminary data were analyzed using SPSS 20   and Epi Additional details on study methods  are provided in the Additional file A total of 9625 women came for screening, of which 9406 (97.7%) eligible women aged 30–49 years were screened; and 219 (2.3%) ineligible women (1.7% due to age restrictions) were not screened . Roughly 9065 participants provided written feedback. Of those, 80.8% women said that they were able to collect the self-sample without any help from a nurse and 81.2% women said that they would prefer self-sampling to provider’s sampling in the future. Asked to score their impressions, 78.5% women were very confident in their ability to collect the self-sampling, 91% found it very easy, 88.5% found it very comfortable , 95.1% found the video to be very helpful in guiding how to collect the sample, and 97.8% said they are very likely to recommend self-sampling to others. The difficult components of self-sampling reported (one or more issues for 2322 respondents) were: the decision on how deep to insert the brush into the vagina for 52% of 2322, how to insert the brush into the vagina for 49.1%, identifying the vaginal opening for 39.6%, rotating the brush inside the vagina for 23.3%, and proper handling to put the brush into the tube after collection for 18.9% women.P < 0.001, independent chi-square)  [95% confidence interval (CI):16.6–18.1%] of 9406 screened women were HC2-positive. The rate of HC2 positivity was 16.2% among the 8964 women with unknown HIV status and 40.5% among the 442 women living with HIV  enrolled for the study at colposcopy, of which seven cases were excluded due to unsatisfactory colposcopy and difficulty in image collection due to various reasons outlined in the Additional file Following the protocol of immediate management by colposcopic impression, without awaiting histopathology diagnosis of the biopsies taken, 114 (8.2%) women were treated with thermal ablation and 78 (5.6%) with LLETZ.Overall, CIN2+ and CIN3+ (including five cancers), were detected in 5.9% (95% CI:4.7–7.3%) and 3.2% (95% CI:2.4–4.3%) of the HPV-positive women attending colposcopy (85% attendance among the 1630), respectively. Thus, overall yield of the screening effort was 0.9% of 9406 women for CIN2+ and 0.5% for CIN3 + .P = 0.0003, chi-square for trend). No meaningful trend was observed in the prevalence of precancer by age , except for a very high 24.1% (seven CIN2+ including three cancers) overall prevalence at self-reported age of 49 years.The prevalences of CIN2+ and CIN3+ among the HPV-positive women with unknown HIV status attending colposcopy were 5.7 and 2.9%, respectively, whereas the overall yields of the screening effort in these groups were 0.8 and 0.4%, respectively Table . The ageP = 0.31 for CIN2+ and P = 0.03 for CIN3+, independent chi-square test in comparison to women with unknown HIV status)   and increase in HIV viral load , however small numbers precluded trend analysis of precancer/cancer.The prevalences of CIN2+ and CIN3+ among the HPV-positive WLWH attending colposcopy were 7.8 and 6.4%, respectively  were diagnosed through biopsy or LLETZ of acetowhite lesions at colposcopy visit, 35 of which (59.3% of 59) had a high-grade colposcopic impression. QA review of colposcopic images revealed potential under-biopsying of more subtle acetowhite lesions. 16 21.3%) of 75 histopathologic precancer cases were identified, in the absence of visible acetowhitening, only on ECC (1.2% of colposcopy examinations and 0.2% of the screening population). This is important in light of the fact that even amongst women as young as age 30, the SCJ was only partially visible in 8.3% and not visible in 23.1% of women , rising to 12.5 and 52.1%  by age 49 years   were eventually diagnosed with CIN2+ on histopathology Table . ViewingOut of 1138 (82%) patients with full clinical quality assurance review completed, 143 (12.6%) cases were recommended for recall; mainly  for repeat ECC; commonly because the tissue was insufficient for diagnosis on the prior ECC  or because the ECC required dilatation/sedation due to stenotic os or tethered cervix . However, the overall yield of precancer from recall for ECC was only 0.2% for the colposcopy population and 0.02% for the general population of women aged 30–49 years.Our results showed the feasibility and acceptability of self-sampled HPV testing and smartphone-based cervical image collection in Nigeria. Nevertheless, the combination of higher HPV prevalence (17.4%) with a much lower risk of precancer (0.9%) suggests the need for triage to improve the accuracy of the screening program. In addition, any visual triage method would require restricting the upper age limit of inclusion to increase program effectiveness.There was a very positive response and clear acceptability for vaginal self-sampling for HPV testing. The difficult aspects of self-sampling reported by some participants included identifying the vaginal opening, determining depth of the insertion of the brush into the vagina, rotating the brush inside the vagina, and putting the brush back into the tube after collection; these need to be explained more clearly in future self-sampling programs. Compliance with follow-up for colposcopy was also very high.The two previous phases of Project Itoju established the epidemiology of HPV infection in rural Nigeria , validatThe prevalence of HPV and precancer (particularly CIN3+) was markedly high among WLWH as also reported by others . This waAccording to the World Health Organization (WHO), Nigeria has only four physicians per 10,000 population . At presIt is important to note that AVE, like other visual assessment methods, requires that the cervical SCJ be visible. In the current study population, we found that the SCJ was not fully visible for almost 64.6% of women by age 49 years. We also corroborated an earlier poorly understood observation that women with multiple vaginal deliveries were more likely to have fully visible SCJs . No visuIt is worth noting that even though the yield of precancer from ECC at colposcopy visit was 3.2% , the additional yield of precancer from the women recalled for insufficient ECC or difficult ECC collection was only 0.02% for the overall screened population. Another study has also noted overall low yield of ECC with an increase in proportionate additional yield of ECC when fewer biopsies are taken . It is wWe had to halt the recall activities under the screening program as the global spread of the COVID-19 pandemic led to pausing of field efforts in Nigeria, including a very few treatment visits still pending, and changed the risk-benefit ratio for cervical cancer screening. When we reopen, the first step will be to complete these treatment visits. Then, moving forward in the COVID-19 era, we are considering the importance of self-sampled HPV testing with a sterile kit at a household level, avoiding any mass gathering and minimizing the need for speculum examination. A small percentage of HPV-positive women with high-risk types could be triaged and treated, spacing community clinic appointments in a COVID conscious manner .The limitations of this study should be noted. The population selected for the study was a volunteer population of women residing in the university town of Ile-Ife, which may have a lower HPV prevalence than the general population in Nigeria. Also, HC2 is not generally used for self-sampling, as it is slightly less sensitive for detection of precancer than PCR-based HPV test methods . A moderIn future work we will assess misclassification by retesting residual screening samples with a more sensitive whole-genome sequencing method ; review A cervical cancer screening program using self-sampled HPV testing, with colposcopic immediate management of women positive for HPV, is feasible in Nigeria but results in both over-and under-treatment. There are newer HPV tests entering the marketplace that cost less than ten dollars per test, take less than an hour to perform, and provide genotyping, suggesting that a self-sampled HPV based screening program would be feasible in this population in the future . Having Additional file 1.


Cervical cancer is the fourth most common type of cancer affecting women globally. In Brazil, it is the third most frequent type of cancer in women and HPV is present in approximately 90% of cases. Evidence suggests that variants of HPV 16 can interfere biologically and etiologically during the development of cervical cancer.Cervix tumor fragments were collected, their DNA was extracted, and nested PCR was used to detect HPV. Positive samples were sequenced to determine the viral genotype. To characterize the HPV 16 strains, positive samples PCR was used to amplify the LCR and E6 regions of the HPV 16 virus.P <  0.05). HPV was present in 88 women (73%). The most prevalent types were HPV 16 (53.4%), HPV 18 (13.8%), HPV 35 (6.9%) and HPV 45 (5.7%). Of the 47 HPV 16 positive cases, variant A (49%) was present in 23 samples, followed by variant D in 20 cases (43%), and variants B and C in 2 cases each (4%). The most prevalent histological type of HPV 16 tumors was squamous cell carcinoma, followed by adenocarcinoma. There was a statistically significant association between HPV 16 variants and the tumors’ histological types (P <  0.001).Data from 120 patients with cervical cancer were analyzed. Most women were between 41 and 54 years of age, had schooling until primary school, a family income between 1 and 2 times the minimum wage and were married/in a consensual union. There was no statistically significant association between HPV or socio-demographic variables and risk factors for cervical cancer (Knowledge of HPV 16 variants will provide data on their influence on the pathological and oncogenic aspects of cervical lesions. Cervical cancer is the fourth most common cancer type affecting women worldwide, with more than 265,000 estimated deaths annually and more than 80% of cases occurring in developing countries , 2. AccoIt is estimated that in the state of Maranhão there will be around 1090 new cervical cancer cases for 2018, with240 of them the capital, São Luís, with a gross rate of incidence of 30.55 cases per 100,000 inhabitants [Human Papillomavirus (HPV) is the main etiological factor for cervical cancer and is associated with the development of diseases ranging from benign warts to invasive cancer . AlthougThe reason why only some uterine cervical lesions associated with high-risk genotypes progress to invasive cancer remains unknown. Evidence suggests that variants of the same HPV type can interact biologically and etiologically during cancer development , 7.The HPV16 is the most carcinogenic HPV type followed by HPV18 and most infections are asymptomatic [Despite of the relationship between different HPV type and the development of cancer being well established, evidence suggests that genetic variations between the same viral type may influence infection potential, viral persistence, the development of precursor lesions and the progression to invasive cancer –12. Due Studies on HPV variants have been developed with the objective of understanding their association with pathological and oncogenic aspects of cervical lesions. Factors influencing HPV infection in cervical cancer are not completely clear, but it is believed that HPV 16 variants playa fundamental role in cervical carcinogenesis and are currently recognized as an important marker for research on viral transmission, persistence and carcinogenicity . VariatiRegarding these aspects, the aim of this study was to evaluate the prevalence of HPV 16 variants in cervical carcinoma samples from Northeastern, Brazil. This is the first evaluation of genetic diversity of HPV 16 in the State of Maranhão, Brazil.This is descriptive, prospective, and transversal study.The study was conducted from January 2016 to December 2017, at the High Complexity Care Unit (UNACON) of the Cancer State Hospital of Maranhão and in the High Complexity Care Center in Oncology (CACON) of the Aldenora Bello Cancer Hospital. This work was approved by the Research Ethics Committee of the Federal University of Maranhão (CEP-UFMA), under Consolidated Opinion N° 1.289.419/2015.The study population was composed of 120 women with a diagnosis of cervical cancer, who were treated at the previously mentioned hospitals.Women older than 18 years diagnosed with cervical cancer who agreed to participate in the study by signing an Informed Consent Form (ICF).Women who presented with surgical indications as an initial treatment or who presented with small lesions, in which biopsy could interfere with staging. Women in psychiatric treatment were also excluded.Initially, the patients were referred for outpatient care at the Oncology Gynecology Service of the Cancer State Hospital of Maranhão and the Aldenora Bello Cancer Hospital and were invited to participate in the study by signing an Informed Consent Form (ICF). A questionnaire was supplied to collect socio-demographic, reproduction, and smoking data.Further, women were submitted to gynecological examination. A cervical biopsy was done and cervical tumor fragments were then placed in microtubes containing 1 mL of RNA Later solution (Life Technologies) at 4 °C and were transported in thermal boxes to the Multiuser Laboratory in the Biobank of Tumors and DNA of Maranhão of the Federal University of Maranhão, University Hospital of the Federal University of Maranhão (HUUFMA). After 24 h, the samples were removed from the RNA Later solution and stored in a freezer at − 80 °C until use.The extraction of the genomic DNA from the samples was performed using the QIAamp DNA FFPE Tissue Purification Kit (QIAGEN®) according to manufacturer protocols.The Nested PCR reactions were performed by using primers PGMY09 and PGMY11 for the first round, and primers GP + 5 and GP + 6 for the second round   at the Molecular Biology Laboratory of the State University of Maranhão located in the Center of Higher Studies of Caxias (CESC-UEMA).Sequencing was performed with an ET Dye Terminator Cycle Sequencing Kit , according to the manufacturer’s protocol.The Chromas program was used to obtain electropherograms of the HPV DNA sequences present in the samples. To identify the HPV type the nucleotide sequences were compared to the Genbank Nucleotide Sequence Database using the BLAST program (NCBI).2, 0.25 μM of each dNTP, 100 pmol/L of each primer, 50–100 ng of DNA, and 2.5 U of Platinum Taq Polymerase. The PCR reaction consisted of denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 1 min; annealing temperature for 1 min and 72 °C for 1 min, followed by a final extension step for 15 min.After determination of the HPV 16 strains, the LCR regions and E6 virus gene of the positive samples were amplified with specific primers. The reaction mixture consisted of a final volume of 25 μL, with 1X PCR Buffer, 2.5 mM of MgClwww.megasoftware.net). BLAST online (Available at: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) was then used to identify the HPV types.The PCR product was subsequently purified and sequenced according to the above protocol. The consensus sequences were merged using the Geneious software (Biomatters Ltd.) and all the sequences generated were aligned with HPV 16 specific strains, using the reference sequences proposed by Burk et al. , using tThe phylogenetic tree of the HPV 16 strains was constructed from the 1300 bp sequences from the E6 and LCR regions using the “neighbor joining” method with p-distance (obtained with pairwise deletion) using the program Mega 4.1. The references proposed by Burk et al. (2013) [We have provided GenBank accession numbers for nucleotide sequences: MT568878 to MT568924.The cervical carcinoma samples were classified according to FIGO staging system .2 (Chi-square) test was used to verify the association between HPV and sociodemographic and clinical variables, and P values ≤0.05 were considered statistically significant. The values corresponding to don’t know/did not respond were excluded from the association analysis.Descriptive statistical analysis was performed using the Stata program (version 14.0). The xp < 0.05) . The majority of these women were in the age group 40 to 49 years of age 28.3%), and self-declared with mixed-race (70%), had schooling up to elementary school (42.5%), had a family income between 1 and 2 times the minimum wage (55%) and were married or in a consensual union (51.7%). There was no statistically significant association between the sociodemographic variables and the presence of HPV , a first pregnancy between 16 and 21 34.2%) and 1 to 3 children (30.8%) were most common. Having only one sexual partner during their lifetime (31.7%) was most common followed by more than 3 partners (30%). Most women reported never having used contraceptive methods (58.3%) (Table 4.2% and Most women (70%) reported having undergone examination before their diagnosis with cervical cancer and most (36.7%) said they received preventive exam annually. Most women reported no tobacco use (57.5%) Table .Table 4HPV was present in 88 women (73.3%). The most prevalent type was HPV 16, which accounts for 53.4%(47/88) and combined with HPV 18 accounted for 67.2% of cases. Most of the samples had HPV types considered high risk, while less than 3.4% had HPV types considered to have low oncogenic risk  and 11 (9.1%) cases of adenocarcinomas. Of the 120 women in this study, 45 (37.5%) had stage IIIB tumors, in cases where HPV 16 was present, stage III was predominant with 23 samples, according to FIGO classification.Of the 47 samples identified as HPV 16, 23 samples were variant A (49%), 20 (43%) were variant D, while variants B and C were present in two samples each (4%).The mean age for women with variant A of HPV 16 was 50.6 years, 58.5 years for variant B, 58.5 years for variant C and 50 years for those with variant D , regarding squamous cell carcinoma and HPV 16 variant “A” and “D”  was predominant followed by stage II (8 cases). For those with variant D, clinical stage III (10 cases) was predominant, followed by stage II (4 cases)   reportedThe virus was highly prevalent (88/120–73%) in the tumor samples examined. The prevalence of HPV in invasive cervical cancer samples can range from 70 to 100%, which may be associated with different techniques used to detect the virus –21.HPV 16 is the most prevalent in cervical cancer worldwide, followed by HPV 18. However, the frequency of HPV types may vary according to the geographic region of the population under analysis. Other studies point out that types 16, 18, 31, and 45as the four most prevalent HPV types in South and Central America , 21.Studies have sought to evaluate the role of intratype variants of HPV16 and HPV18 in the persistence of viral infection, the risk of cervical intraepithelial neoplasia development, and the development of invasive cervical cancer , 13. ForIn the present study in São Luís do Maranhão, the most prevalent strain in the population studied was variant A for HPV 16, according to the literature. In a case-control study conducted by Hang et al. (2016) , we atteIn the study conducted by Volpini et al., (2017)  in BraziIn a study conducted in Brazil by Vidal et al. (2016)  on a cohA study by Villa et al. (2000) , also inp < 0,001).The most prevalent histological type in tumors with HPV 16 was squamous cell carcinoma (SCC) in 38 samples. There was a statistically significant association between histological type and HPV variant  was predominant.Studies also indicate a variation in the distribution of HPV types among histological types. HPV 16 has been associated with squamous cell carcinoma and HPV 18 has a higher prevalence in adenocarcinoma than HPV 16. However, few studies have attempted to identify associations between HPV 16 variants and the histologic type of cervical tumors , 25.In the study by Hang et al. (2016) , it was Treatment response also depended on the HPV 16 variant. For example, 12 cases with variant A showed complete remission, compared to only 7 cases with variant D. Likewise, there were 2 deaths with variant A, and 3 who had variant D. In 12 cases it was not possible to evaluate disease status at the end of the first treatment.Tan et al (2019) reported that European prototype E-T350 was the most prevalent (82.76%) followed by Asian (As) variant. In patients with suspected cervical lesions the most prevalent variant was As variant (54.9%) by increasing significance with severity of cervical diseases .In another study conducted by Ortiz-Ortiz et al (2015), the variants more frequently found in women with cervical carcinoma were E-G350, AA-a, AA-c, E-C188/G350 and E-A176/G350. All of them are associated with the development of cervical carcinoma, however, AA-a showed the highest association .Few studies have been conducted to evaluate the effect of HPV 16 variants on the response to oncologic treatment. After analyzing 155 cases of HPV16 positive cervical cancer (132 cases of variants E × 23 cases of NE variants) Zuna et al (2011)  came to In our study we found a greater number of NE variants . Of the 47 cases we found 23 with variant A (E) and 24 with variants B, C, and D. The mortality of patients with the NE variants was 13% (3/24), while it was 9% (2/23) in those with variant A (E). The number of cases of complete remission was also lower with the NE variants (33% × 52%). These findings can be explained by the large number of cases of variant D in our study (20 cases). A study by Burk et al (2013) [We observed the predominance of sublineage A1 (21 cases), followed by sublineage D3 (18 cases). In the study by Hang et al (2016) , the A4 In a study by Alfaro et al., (2016)  of 462 wA study by Nicolas-Parraga et al. (2017) , sought The data also showed an increase in the prevalence of A1–3 in Europe , variant D in South-Central America , A4 in Asia  and variants B and C for Africa (from 28 to 66.7% and from 12.3 to 37.5% for ECC and ADC) .The most prevalent HPV in cervical carcinoma samples in São Luís was HPV 16. This study evidenced for the first time the prevalence of HPV variants ant the most prevalent was variant A. There was a statistically significant association between histological type and HPV variant.Further studies are needed to understand the role of HPV variants in the origin and progression of cervical cancer, as well as the relationship between HPV 16 and its variants with the development of precursor lesions. The data presented here may help the development of future epidemiological studies on HPV 16 variants, as well as in the creation of strategies to combat the types that remain circulating, and which were not included in the currently available vaccines against HPV.