Regardless of the disease burden of human papillomavirus (HPV), the vaccine has not been included in the Indonesia National Immunization Program. Since 2017 there was a demonstration program of the HPV vaccination in Yogyakarta Province. This vaccine was given free to female primary school students in the 5th and 6th grades (11–13 years old). This study aimed to assess whether a structured-educational intervention focus on HPV increases the parental awareness, knowledge, and perceptions toward HPV and the vaccine acceptability.We conducted a pre-post structured-educational intervention study from July to August 2017 before the implementation of the HPV vaccination demonstration program, in Kulon Progo District, Yogyakarta Province, Indonesia. Parents of female primary school students grades 5th and 6th were selected using a school-based proportional random sampling. A pediatric resident provided a structured-educational intervention, which consists of the burden and risk of HPV disease, as well as the benefit and safety of the vaccine. Parents were required to complete validated self-administered questionnaires before and after the structured-educational intervention.p < 0.001). HPV vaccine’s acceptability increased from 74.3 to 87.4% (p < 0.001). There was a significant correlation between increasing HPV vaccine acceptability with the improvement of awareness, knowledge, and perception toward HPV infection, cervical cancer and HPV vaccination . After the structured-educational intervention, better knowledge and positive perceptions of HPV vaccination were predictive of HPV vaccine’s acceptability with OR 1.90 (95%CI:1.40–2.57) and OR 1.31, respectively.A total of 506 parents participated. Before receiving the structured-educational intervention, parents’ awareness of HPV infection and the vaccines were low. Only 49.2% of parents had heard HPV infection, and 48.8% had heard about the vaccine. After the structured-educational intervention, there were significant improvements in parent’s awareness, knowledge, and perceptions of HPV infection, cervical cancer, and HPV vaccination (all .A structured-educational intervention may improve parental awareness, knowledge, and perceptions toward HPV and the acceptability of the vaccine. Further study, a randomized control trial with longer follow-up are needed to evaluate the long-term and actual effectiveness of improving parents’ knowledge, perceptions and HPV vaccine acceptability Globally, cervical cancer ranks the third most common cancer in women, with a total estimated 569,847 diagnosed cases of invasive cervical carcinoma with 311,365 deaths from the disease annually . CervicaAs one proactive way to increase HPV vaccine coverage, the Global Alliance for Vaccines and Immunization (GAVI) supports low-middle income countries in Southeast Asia, including Indonesia, to conduct the HPV Demonstration Program .  The HBecause children in this age need parental consent for the vaccination, the acceptance of the HPV vaccine is highly dependent on the knowledge, perceptions, and approval of their parents , 8, 9]..9]. A syThe WHO recommended countries which are introducing the HPV vaccine into their NIP should invest in a comprehensive communication plan to first build community awareness and acceptance for the vaccine and the program. This plan includes clear program and communication objectives, understanding community knowledge, attitudes and practices, and defined target audiences. Communication efforts should reach all key target audiences, especially parents . ParentsTo our knowledge, no study in Indonesia has assessed the impact of a structured-educational intervention on parental knowledge, perceptions, and acceptability of HPV vaccination. This study aimed to evaluate whether a structured-educational intervention about HPV risks and the HPV vaccine increases the parental awareness, knowledge, and perceptions regarding HPV and the HPV vaccination acceptability.We conducted a one group pre-test post-test design study in KulonProgo District, one of districts in Yogyakarta Province, Indonesia, where the demonstration program was implemented. The study was conducted from July to August 2017, before the implementation of the HPV vaccination demonstration program. This research was part of a larger study assessing knowledge, perceptions, and acceptability of parents and teachers regarding HPV related to the disease and the vaccine. Parents of female primary school students grades 5th (11–12 years old) and 6th (12–13 years old) were selected using a school-based proportional random sampling. A study information statement was sent to the parents, and they were invited to participate in the study. We excluded parents who cannot read and write.After collecting socio-demographic and baseline data on knowledge, perceptions, and acceptability on HPV infection and the vaccine, a structured-educational intervention was provided to groups of parents in the classroom. Each group consisted of 40–50 parents. The researcher developed the intervention based on literature review and using the framework of the health belief model (HBM), which emphasize that a person’s perception, such as the perception of disease severity, susceptibility, barriers and benefits, influence the intention and actual health-seeking behavior . The intData were collected using validated self-administered questionnaires, which consisted of awareness (4 questions), knowledge and perceptions about HPV infection (14 and 6 questions), knowledge and perceptions about cervical cancer (3 and 2 questions), and knowledge and perceptions about HPV vaccination (5 and 2 questions). For the assessment of knowledge scores, if the answer was correct then it was given a value of 1 and if the answer was incorrect then a value of 0. The total score of awareness ranged from 0 to 4, the score of knowledge on HPV infection ranged from 0 to 14, the score of knowledge on cervical cancer ranged from 0 to 3, and the score of knowledge on HPV vaccine ranged from 0 to 5. Questions about perceptions used a 5-point Likert scale, ranging from strongly disagree  to strongly agree . The total score of perception on HPV infection ranged from 6 to 30, the score of perception on cervical cancer ranged from 2 to 10, and the score of perception on HPV vaccine ranged from 2 to 10. Acceptability was measured by a dichotomous item asking whether they are willing their daughters to get the HPV vaccine, free of charge (yes or no answer options). The full item questioner was presented in supplementation File , seven parents were excluded due to inability to read and write, resulting in a total data set of 506 participants. The majority of the respondents are mothers (92.8%), with a median age of 40 years, and the majority are Moslem (98.2%). Forty-three participants (8.8%) experienced a history of any type of cancer in their family, and 4.4% mentioned that their daughter had already received the HPV vaccination  72.7%) and cervical cancer (73.7). However, only 46.2% of them had ever heard about HPV infection, and 46.2% of them mentioned that HPV caused cervical cancer. The majority (76.5%) of respondents already knew that the vaccination could prevent infection, but only 44.1% of parents had ever heard of the HPV vaccination , and every woman was at risk of having cervical cancer (72.6%). However, the perception of the risk of having HPV infection was low, with only 40.3% strongly agree or agree that they are at risk of having HPV infection. The perceptions of the benefit and safety of the HPV vaccine were high; 78.6% of respondents strongly agree/agree that they believe HPV vaccination is useful for preventing cervical cancer, and 69.7% of them strongly agree/agree that they believe HPV vaccine is safe  and OR 1.31(95%CI:1.05–1.63), respectively , scientifically safe (97.0) and effective in preventing the disease (97.7%). The three most important considerations were free of charge, the vaccine is halal, and the vaccine is effective.Considerations for accepting HPV vaccinations were free of charge (according to 95.5% of respondents), the vaccine was included in NIP (according to 97.1% of respondents), the vaccine is considered safe by Moslem standards or p < 0.001(Table p < 0.001). There was a significant correlation between increasing vaccine acceptability with improvement of awareness , knowledge of HPV infection , cervical cancer  and HPV vaccination  and perceptions of the HPV infection , cervical cancer  and HPV vaccination .After the structured-educational intervention, there were significant improvements in parents’ awareness, knowledge, and perceptions of HPV infection, cervical cancer, and the vaccine, all 001Table . ParentaThis research was a school-based study to assess whether a structured-educational intervention can improve parental awareness, knowledge, and perceptions about HPV infection, cervical cancer, and HPV vaccination as well as acceptability of HPV vaccination. Before the intervention, most of the parents had poor baseline knowledge and some negative perceptions of HPV. After the intervention, there was a significant improvement in parents’ HPV-related knowledge and perceptions as well as their intention to permit the HPV vaccine for their children.At the baseline, we found that most of our participants had heard about STIs and cervical cancer, but less than half of them had heard about HPV infection and the vaccine. A previous qualitative study assessing parental knowledge and perceptions of HPV and cervical cancer prevention in rural Central Java, Indonesia 2015) found that most respondents have limited knowledge of HPV and the vaccine as well as the relationship between HPV and cervical cancer  found th. Since tThere were several factors related to the acceptability of HPV vaccination. From the regression analysis, we found that better knowledge and positive perceptions of the HPV vaccination were significantly associated with higher acceptability. In line with our study, previous reports have shown that parents’ knowledge about HPV was positively correlated with HPV vaccination acceptability , 12. Perhalal (permissible under Islamic Shariah Law) were important considerations for accepting the vaccine. Including HPV vaccine in the NIP and providing health insurance could eliminate the cost barriers. Because the majority of our study population is Muslim, the halal issue is an important consideration for accepting the vaccine. Previously, the non-halal status was reported as a barrier to the rotavirus vaccine acceptability in Yogyakarta, Indonesia. Engagement of religious leaders is needed to facilitate the bridge between immunization programs and the community, particularly during the introduction of a new vaccine [We found that besides the vaccine’s benefits and safety, free of charge, the vaccine being included in NIP, and the vaccine considered  vaccine .Earlier studies found that parents need HPV-related information to decide whether to accept or refuse the HPV vaccination , 13, 30.Previous studies conducted in more developed countries showed the effectiveness of digital media/ website to educate parents. A randomized cluster trial, parents of 11–17 years old girls watched a digital video outlining the risks and benefits of the vaccine was conducted in Indiana, United States. It showed that adolescents whose parents watched the video had a 3-times higher odds of receiving a dose of the HPV vaccine .. Anothehow to facilitate an interactive discussion”.In developing countries such as Indonesia, which internet access is not always available, providing a more “conventional” education would be more feasible and acceptable. Our study found that a structured-education intervention using a standard PPT presentation followed by an interactive discussion focused on HPV disease and the vaccine improved parents’ knowledge, perception, and acceptability. This result emphasized the effectiveness of “conventional” educational interventions to target consumers/ parents in improving the acceptability of vaccines proven by previous studies. A hospital-based, brief, HPV-focused intervention, using a PPT presentation, improved the awareness and acceptability of HPV vaccine among rural Chinese woman’s . Another. Another systematic review conducted by Radisic et al. (2017) found that uptake of HPV vaccination may be facilitated by encouraging health care provider endorsement [The structured-educational intervention conducted in our study, in the form of a presentation and interactive discussion by professional health educators, was shown to significantly improve the parents’ knowledge and perceptions about HPV. Giving educational campaigns could increase parental knowledge and perceptions about HPV, concurrently with physician recommendations and nationally funded vaccination programs , 28. A sorsement .This research has some limitations. This one group pre-test -post-test design study without a control group did not allow us to confirm any causal relations between the intervention and outcomes. A pre- and post-test without follow-up could not determine the long-term effectiveness of the educational intervention on HPV vaccination acceptability. Further study, a randomized control trial with longer follow-up are needed to evaluate the long-term and actual effectiveness of improving parents’ knowledge and perceptions about the HPV vaccination as well as the vaccine acceptability. The strength of this study is that it assessed the effectiveness of an education intervention before the implementation of the HPV vaccination demonstration program, which included a representative sample of parents in Kulon Progo, a rural District. The results of this study can provide important evidence to the policy-makers about the importance of health education.A structured-educational intervention may improve parental awareness, knowledge, and perceptions toward HPV as well as the acceptability of the vaccine. There was a significant correlation between improving the parental knowledge and perceptions of HPV vaccine with increasing acceptability of the HPV vaccine. A structured-educational intervention designed for parents may have important implications for improving vaccine acceptability. Further study, a randomized control trial with longer follow-up are needed to evaluate the long-term and actual effectiveness of improving parents’ knowledge, perceptions and HPV vaccine acceptability.Additional file 1: Table 6 The full item questioner


In Bolivia the incidence and mortality rates of uterine cervix cancer are the highest in America. The main factor contributing to this situation is the difficulty of establishing and maintaining quality prevention programs based on cytology. We aimed to evaluate the effectiveness of HR-HPV testing on self-collected samples to detect cervical intra-epithelial neoplasia and identify the best combination of screening tests.A total of 469 women, divided in two groups, were included in this study. The first group included 362 women that underwent three consecutively primary screening tests: self-collected sampling for HR-HPV detection, conventional cervical cytology and visual inspection under acetic acid (VIA). The second group included 107 women referred with a positive HR-HPV test that underwent conventional cervical cytology and VIA. The presence of high grade intraepithelial lesion (CIN 2+) or invasive cancer was verified by colposcopy and biopsy.In the screening group the sensitivity to detect high grade intraepithelial lesion (CIN 2+) or invasive cancer were 100, 76, 44% for the VIA, HR-HPV test and cytology, respectively. In the referred group, the sensitivity to detect high grade intraepithelial lesion (CIN 2+) or invasive cancer by VIA and cytology were 100 and 81%, respectively.VIA and HR-HPV self-sampling were the best combination to detect CIN2+ lesions. Cytology analysis gave the poorest performance. Worldwide, cervical cancer is the fourth leading cause of death among women . In 2018The situation in Bolivia is alarming regarding the incidence and mortality rates of uterine cervix cancer, with the highest incidence in America  and a mortality rate of 19 per 100,000 women (rates standardized by age) , 12.Screening, offered by the first level of care, responsible for prevention, is available free of charge, for sexually active women until 64 years old . NeverthTo improve this situation, our group has recently evaluated the acceptability of introducing an HR-HPV test on self-collected samples with cheap and simple devices , 15. IndThe purpose of this study is to determine the clinical effectiveness of our HR-HPV DNA detection strategy on self-collected samples to detect high grade intraepithelial lesions and cervical cancers, comparing it to Pap smear analysis and VIA, in order to identify the best combination to screen Bolivian women or to sort women at risk.Between January 2016 and April 2018, a cross-sectional study was carried out in Cochabamba (Bolivia) within the framework of the inter-institutional cooperation agreement between the “Universidad Mayor de San Simon” and the Public health service of Cochabamba (SEDES). Through these two institutions, a cervical pathology clinic was equipped at Hospital Cochabamba, where the present study was carried out. The staff consisted of a nurse assistant and a gynecologist trained in colposcopy and VIA during the two previous years.The Bio-ethical committee of the “Universidad Mayor de San Simon” approved the study protocol . A total of 469 women were included in this study, divided in two groups.The first group phase 1) included 362 women that underwent three screening tests: HR-HPV DNA detection, Pap smear, and VIA, performed consecutively at the consultation. The women in this group assisted spontaneously or were referred from general medicine clinics to the cervical pathology clinic of Cochabamba hospital due to their gynecological symptoms. This phase 1 study was named “the screening group”. The second group (phase 2) included 107 women referred with a positive HR -HPV DNA detection test, these women were identified in cervical cancer prevention campaigns conducted in different geographical areas of the city of Cochabamba . They underwent additional Pap smear and VIA tests, and this study group was named “the referred group”. All women (phase 1 and 2) were examined by colposcopy after the three (screening group) or two (referred group) screening tests. Biopsy was performed when necessary, according to the colposcopy observation results.  to perform vaginal self-sampling in order to analyze their samples in a HR-HPV DNA detection assay . The kitSamples for conventional cytology analysis (Pap smear) were taken from the cervical transformation zone by the gynecologist. The smear was spread on a glass slide and stained, according to the Papanicolaou method . The anaThe Visual Inspection under Acetic Acid (VIA) was made with white wine vinegar with an acetic acid concentration of 5%, one minute after the application. The solution was changed every 2 weeks. The cervix was observed under a white LED light and the test was considered positive when the presence of aceto-white lesions was found in the squamo-columnar junction or near to an opening in the center of the ectocervix known as Os. Otherwise it was considered negative . VIA resColposcopy was a separate independent examination, performed after the three screening tests, on the same day (VIA and colposcopy were done by the same examiner). The decision to take a biopsy was based on the results of the colposcopy, using the classification of the IFCPC  . A biopsBiopsies were processed in the reference laboratory of Pathology of the Bolivian-Japanese gastroenterological hospital. Histological classification (CIN) of epithelial lesions of the cervix were used according to the description made by Richart . SamplesA CIN 2 severity grade was determined as the “cut off” in the biopsy result in this study, considering that people whose lesion were exceeding this cut off had a positive gold standard result while those having a lower severity grade results were considered as negative in this gold standard assay..Statistical analyses were performed independently on available results using cross tables to determine the sensitivity, specificity and predictive values of each test. Number of true positives, true negative, false positives and false negative cases were calculated considering the biopsy analysis result (obtained in Bolivia) of CIN2 as a cut off to detect CIN2+ and cervical cancer (gold standard for positive cases) and considering negative colposcopy result or normal or CIN1 biopsy analysis result as negative cases. The confidence limits for each proportion were calculated with OpenEpi software n = 362) or the referred group (n = 107) are presented in Table Characteristics of the women, aged between 25 and 64 years, participating either to the screening group  in our study population. This phase 1 study, was performed on 362 women, named “the screening group”, that underwent, consecutively, HR-HPV DNA detection by self-sampling, Pap smear and VIA, before colposcopy. A biopsy was performed when necessary, according to the colposcopy observation results. In this screened group, 72 biopsies were taken, out of which16 had high-grade intraepithelial lesions (CIN2+) and 8 had invasive cancer. The rest of the biopsies had a result lower than CIN2. The sensitivity, specificity and predictive values of the three screening tests to detect CIN2+ lesions or worse, based on the biopsy results (gold standard) were determined through the analysis of the results of 362 patients. The sensitivity and specificity were respectively: 44 and 93% for the PAP, 100 and 86% for the VIA and 76 and 78% for the HR-HPV test. The positive and negative predictive values were, respectively, 30 and 96% for PAP, 35 and 100% for VIA, 18 and 98% for HR-HPV test  and 4 to invasive cancers, the rest of the biopsies had a result lower than CIN2. The sensitivity, specificity and positive values of the two screening tests to detect high-grade intraepithelial lesions and cervical cancer (CIN2+ or worse) were determined through the analysis of the results of 107 patients. The sensitivity and specificity were respectively: 81 and 88% for the PAP, 100 and 85% for the VIA and the positive and negative predictive values were 64 and 95% for the PAP, 53 and 100% for the VIA, respectively  gave a concordance of 84% for the CIN2 + lesions and an agreement of 70% for the lesions CIN 1 lesions  but also mainly in the cytology analyses . As VIA was performed immediately, on the place, all the results were available for this test . It is worth noting that the HR-HPV test was robust, giving results with no inter-observer variation . The maiThe cytology in the screened group showed a low sensitivity 21–67%) and a specificity of 92%, to detect CIN2 + lesion or worse, giving the lowest performance amongst the evaluated tests. Similar results were obtained in an extensive study conducted in European and North American countries, reporting a low sensitivity (53%) and a high specificity (96%) of the PAP staining to detect CIN2 + lesions [7% and a The screening of patients with VIA allowed to detect high-grade intraepithelial lesions (CIN2 + or worse) with 100% sensitivity and 86% specificity. Other studies reported similar results with 79% sensitivity and 89% specificity in Africa and India , 83% senVIA generally gave a large number of false positive, leading to low positive predictive value (34%)  .This inWe also assessed the performance of the VIA and the Pap smear test as triage method for HR-HPV positive women (referred women). Although the number of samples with CIN2 + lesions was small, because losses of samples in cytology (around 50%), the results suggested that both tests give better positive predicted value in triage compared to their use in screening.Bolivia is one of the poorest countries in South America and its public health system had difficulties in the monitoring and treatment of pre-malignant lesions of the cervix detected by cytology. This is partly due to the fact that 38% of the cytology results were lost , as obseA limiting factor in our study was that a confirmatory biopsy was not performed on women with a normal colposcopy result. The final result on the condition of their cervix was based only on colposcopy impression (that could have led to the higher specificity of the VIA). This limitation was due to ethical and technical reasons, as our intervention followed the official guidelines from the Bolivian Ministry of Health . This coOur results suggest that the VIA and our low cost HR-HPV DNA PCR detection assay have better sensitivity to detect cervical cancer and its true precursor lesions, compared to standard Bolivian PAP smear test. Among all evaluated test the cytology analysis showed the lowest performance to detect CIN 2+ lesion or worse, even at the lowest cytological cut-off (ASCUS).Since our self-sampled HR-HPV detection test (HPV screening) could overcome sociocultural barriers and to increase screening coverage , an alsoFinally, to improve the screening coverage, it is also essential to better sensitize the population through the education and facilitate access to screening results. Furthermore, it is essential to establish monitoring of the flow of exams, to identify the cause of delaying results and to develop new strategies that could insure delivery of the results in time but also that could improve the patient adherence to their gynecological health follow-up.Additional file 1. Bolivian anatomo-pathology analyses, by rereading 29 biopsies out of 101 biopsies chosen at random in a Belgian laboratory.


Blue is a favored color of many humans. While blue skies and oceans are a common visual experience, this color is less frequently observed in flowers. We first review how blue has been important in human culture, and thus how our perception of blue has likely influenced the way of scientifically evaluating signals produced in nature, including approaches as disparate as Goethe’s Farbenlehre, Linneaus’ plant taxonomy, and current studies of plant-pollinator networks. We discuss the fact that most animals, however, have different vision to humans; for example, bee pollinators have trichromatic vision based on UV-, Blue-, and Green-sensitive photoreceptors with innate preferences for predominantly short-wavelength reflecting colors, including what we perceive as blue. The subsequent evolution of blue flowers may be driven by increased competition for pollinators, both because of a harsher environment  or from high diversity and density of flowering plants (as in nutrient-rich meadows). The adaptive value of blue flowers should also be reinforced by nutrient richness or other factors, abiotic and biotic, that may reduce extra costs of blue-pigments synthesis. We thus provide new perspectives emphasizing that, while humans view blue as a less frequently evolved color in nature, to understand signaling, it is essential to employ models of biologically relevant observers. By doing so, we conclude that short wavelength reflecting blue flowers are indeed frequent in nature when considering the color vision and preferences of bees. Why make so much of fragmentary blueIn here and there a bird, or butterfly,Or flower, or wearing-stone, or open eye,When heaven presents in sheets the solid hue?Since earth is earth, perhaps, not heaven (as yet)–Though some savants make earth include the sky;And blue so far above us comes so high,It only gives our wish for blue a whet. , calcium oxide (CaO), and copper oxide (CuO) was used to make Egyptian blue (irtyu), a long-lasting entrancing pigment representing the color of the sky and heavens that was used for the decoration of statues that can still be observed . Our visual system compares the responses of these photoreceptors by means of an opponent system , which cWhile much of human history is surrounded by blue skies and waters, this fascination may nevertheless have also emerged from its perceived rarity in the biological world  and remaSurprisingly, it is not only humans for which short wavelength blue coloration has a special allure: honeybees , bumbleb+, Mg 2+, 2 Ca2+) can be key elements for the production of blue flowers , a group of flavonoids . Althoug flowers . It take flowers , it rema flowers . This is flowers , and/or  flowers .Ipomea gesnerioide, which shows that the bird-pollinated flowers are most frequently evolved from bee-pollinated plants. Thus, floral color can evolve through quantitative variation in the production of several types of pigments , cyanidin , and delphinidin  . These ppigments , leadingpigments . Recent pigments , suggest3+, combined with a low soil pH can induce a color change from purple to blue in some plant species   suggestsAdditional factors including nutrient availability  Bedford and habiDiversity is often driven by land-use intensity . IncreasDracocephalum ryschiana, Campanula barbata, Campanula rotundifolia, Jasione montana, Polygala serpyllifolia, Polygala amarella, Gentiana pneumonante, Gentiana nivalis , Hepatica nobilis [Anemone hepatica], and Viola hiarta (as well as the more common violets). These meadows are threatened by climate warming, fertilization, and increased mowing frequency  (C. rotundifolia (J. montana (H. nobilis (or A. hepatica)   and C. rndifolia , are sho montana . Also, t montana , 2011. Oepatica) .Viola, Euphrasia, Polygala, and some Veronica species to be particularly weak competitors with very low tolerance to nitrogen increase. Thus, many species with blue flowers show some evidence of being susceptible to land-use changes, especially since they are expected to be restricted to low nitrogen and low phosphorus but high micronutrient  environments. It is also worth asking whether the presence of blue flower may act as biomarkers of healthy land/ecosystem that have intense land use pattern with uses of different fertilizers.Lysimachia arvensis perform better in terms of seedling mass or reproductive age in dry environments compared to a red morph  shows a preference for blue  which we perceive as a range of colors from violet through red. Three types of wavelength selective photoreceptors are responsible for our normal color vision. The blue (or S for Short) photoreceptor with a peak sensitivity at 421 nm, green (or M for Medium) photoreceptor at 530 nm, and red (or L for Long) photoreceptors at 559 nm . Notablefor blue , other lbilities . Taken tbilities , this shDelphinium are preferred by bees compared to relatively rarer white flower morphs, and the blue color appears to allow bees to see the flower better and thus results in the collection of more nectar rewards per unit time (Antirrhinum majus appear blue to bumblebees, and are both preferred and processed faster than genetically modified mutant flowers of bee-white appearance (The main adaptive advantage promoting the evolution of specific flower colors is to capture the attention of preferred pollinators , while anit time . Indeed,nit time . In a sipearance . Neverthpearance . In Germpearance  than altpearance , and so According to To explore the importance of blue flower color and considering bee pollinator color vision, it is interesting to compare reflectance spectra of plants from different latitudes and geographic regions. Datasets from Australia ,b, NepalIn the bee color space proposed by Color is a perception resulting from the particular way in which a brain processes visual information; therefore, it is important to use a model enabling for the interpretation of opponent processing to fully understand how color signaling is perceived . Of the Flowers in higher elevations may also need to have more efficient color signaling to maximize the chance of attracting pollinators. Bees are efficient pollinators but do, in general, tend to be more frequent at lower elevations, while Diptera and Lepidoptera are more frequently observed at higher elevations . Arnold Previous authors have observed and reported, based on human color vision, that with increasing altitude there appeared to be more blue flowers , and by Our human color vision  enables All authors contributed equally based on their expertise in each section of this review manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: Anal cancer is a rare disease. However, its incidence is increasing in some population groups. Infection caused by Human Papillomavirus (HPV) is strongly associated with the risk of anal cancer, whose variability depends on samples, histology, and HPV detection methods. The aim of the study was to assess prevalence and distribution of HPV genotypes in patients diagnosed with anal carcinoma. Methods: An observational, retrospective study was carried out in a tertiary care hospital in North Sardinia, Italy. Specimens of anal cancer diagnosed from 2002–2018 were selected. Demographic, epidemiological, and clinical variables were collected to assess their relationship with the occurrence of anal cancer. Results: The overall HPV positivity was 70.0% (21/30), with HPV-16 being the predominant genotype (~85%). The highest prevalence of anal cancer was in patients aged ≥55 years. HPV positivity was higher in women  and in moderately differentiated samples (G2) . p16INK4a and E6-transcript positivity were found in 57% and 24% of the HPV positive samples, respectively. The OS  showed a not statistically significant difference in prognosis between HPV positive sand negatives . Conclusions: HPV-DNA and p16INK4a positivity confirmed the role of HPV in anal carcinoma. Our findings could support the implementation and scale-up of HPV vaccination in males and females to decrease the incidence of HPV-associated cancers. Further studies are needed to better clarify the prognostic role of HPV/p16 status. Anal cancer, which accounts for 4% of all lower gastrointestinal tract tumors, caused 48,541 new cases and 19,129 deaths in 2018 worldwide [Anal intercourse, multiple sexual partners , and earIn light of relevant similarities between anal and cervical cancers, numerous countries have adopted HPV-vaccinations for both sexes. It has been estimated that >95% of HPV-associated anal cancers are potentially preventable by nonavalent-HPV vaccine . A potenA higher incidence of anal cancer was described in men who have sex with men (MSM), people who are HIV-positive and immunocompromised (non-HIV) patients, and women with a previous HPV-related disease  . HoweverSince the relationship between anal dysplasia and the HPV infection was proved, new classification criteria were adopted based on nomenclature used in cervical cancer  I, II, II). High/low grade squamous intraepithelial lesion (HSIL/LSIL), a precursor of anal cancer, are usually HPV-positive with a prevalence of HPV-infection, mainly HPV-16 genotype, that increases in cases of severe lesions ,9,10. AnTo the best of our knowledge, no previous studies have investigated the relationship between HPV infection and anal cancer in the general population in Italy. On this basis, an observational, retrospective single-center study was conducted in an Italian university hospital to describe the prevalence of HPV infection, its genotypes and positivity to biomarkers of HPV infections E6 transcript and p16 proteins in samples of anal cancer diagnosed between 2002–2018.We conducted an observational, retrospective study on patients with a diagnosis of anal cancers at the tertiary-care university hospital in Sassari, Italy. Anal cancers are classified as anal if their epicenter is ≤2 cm from the dentate line. An ad hoc electronic form was prepared to collect demographic, clinical, and epidemiological variables in order to assess their relationship with anal cancer in patients with and without HPV infection.Histological sections of anal cancer samples were performed from the archived formalin-fixed paraffin-embedded (FFPE) specimens. In total, 10 consecutive sections of 3 μm were cut from 30 FFPE blocks. Pathologists evaluated hematoxylin and eosin stained sections to confirm diagnosis and tumor staging, which follows the recommendations of the classification system of the American Joint Committee on Cancer (AJCCC) . Anal caImmunohistochemistry was performed for p16 on formalin-fixed paraffin-embedded tissue sections using the kit CINtec p16 Histology , according to manufacturer’s instructions . CINtec Cases were positive if a sequence of proliferating cells showed a strong positive signal. Although strongly positive, few isolated cells cannot confirm the positivity of a case .TM II HPV28 Detection  [Prior to nucleic acid purification, samples were initially deparaffinized. Paraffin was removed when adding 320 µL of deparaffinization solution  . NucleicSamples positive for HPV-16 were tested for E6-mRNA by reverse transcriptase polymerase chain reaction (RT-PCR), using a set of primers designed by Sotlar et al.  for the 2, 10 mMTris-HCl, 50 mMKCl, and 0.2 mM dNTPs. The amplification was based on an initial denaturation step at 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 s, and 60 °C for 1 min. Nuclease-free water and previously tested HPV16-positive liquid-based cytology samples were added in each experiment as negative and positive controls, respectively.RNA was extracted using an AllPrep DNA/RNA FFPE Kit,  . A cDNA INK4a, and E6 proteins expression. Qualitative variables were summarized with absolute and relative (percentages) frequencies, whereas quantitative variables were summarized with means , based on their parametric distribution. The Chi-squared and Fisher exact tests were computed, when appropriate, to assess differences for qualitative variables. A student t-test was computed to detect statistical differences for quantitative variables with normal distribution. The Kaplan–Meier survival analysis was performed to assess the mortality in study subgroups. A two-tailed p-value less than 0.05 was considered statistically significant. All statistical analyses were performed with the statistical software STATA version 15 .The following variables were collected: gender, age at diagnosis, year of diagnosis, comorbidities, histopathological classification  and adenocarcinomas), HPV-DNA positivity, HPV genotype, p16A total of 30 patients with anal cancer, diagnosed between 2002 and 2018, were retrospectively recruited; their mean (SD) age was 64.2 (12.3) years and 16 (53.3%) were males. Characteristics of the study sample are detailed in the Anal cancer was mainly diagnosed in persons aged ≥55 years ; 63.3% and 36.7% were SCC and adenocarcinoma, respectively; 43.3% of the samples were moderately differentiated (G2), followed by those well differentiated , and those poorly differentiated . The most prevalent comorbidities were colic adenoma and bladder carcinoma.HPV was found in 21 (70.0%) anal samples, with the HPV-16 genotype accounting for >85% (18/21) of the positive samples. Only one case showed multiple infections caused by the genotypes HPV-16, -35, and -6. One sample was positive for mono-infection caused by HPV-35 and two cases were caused by HPV-6.p-value = 0.69), respectively. No statistically significant HPV differences were found according to age and year of diagnosis.Women showed a higher HPV positivity  and HPV-16 genotype prevalence  than men. Adenocarcinomas and SCCs showed an HPV-DNA positivity of 30.4% and 66.7% .All  poorly differentiated anal samples were HPV-positive, even if the result was not statistically significant . All cases positive for p16INK4a were HPV-positive. The association between p16INK4a and HPV-DNA positivity was found in 12 (57.1%) anal cancers. SCC and adenocarcinomas were positive for p16INK4a in 36.8% (7/19) and 45.5% (5/11) of the cases, respectively. All HPV-negative samples  were p16INK4a negative.Positivity of p-16E6 transcripts were found in 5 (27.8%) HPV-16 positive samples. Although samples of other comorbidities were negative for high- or low-risk HPV genotypes, anal HPV-positivity was found in 7 (77.8%) patients with comorbidities.n = 9) were male and more than half  had a diagnosis of SCC histological subtype. Among the 14 patients who died during the follow-up, 10  were HPV-positive. The overall survival (OS) did not significantly change when HPV-DNA positivity, p16 status, gender, and histological subtypes stratified patients. No statistically significant differences were observed in OS and OS at five-years between HPV-positive and -negative groups    patients were alive at the end of the follow-up; 56.2% ( > 0.05) . However = 0.03) .The HPV prevalence in 30 anal cancer samples, diagnosed between 2002 and 2018, in an Italian university hospital was 70.0%, with most of them associated with the HPV-16 genotype (85.7%).Although global data estimated a higher prevalence of anal cancer in women , our cohThe overall HPV prevalence observed in our study, is slightly lower than findings of a systematic review and meta-analysis conducted by Lin et al., where prevalence of HPV infection reached 80% in all groups , whereas it ranged from 42% to 76% from normal cytology to high-grade intraepithelial lesions . NeverthRavenda et al. (2014)  found HPThe higher prevalence of the HPV-16 genotype was similar to the high prevalence described in other Italian studies, where it was the most prevalent genotype in anal and cervical cancers ,31, probThe HPV-DNA prevalence was similar in both histological subtypes , differing from other findings where it was proven that adenocarcinoma had a low HPV prevalence. However, the low percentage of adenocarcinoma in other studies might have underestimated the impact of HPV infections ,26.A higher HPV positivity was found in women , which can probably be explained by a persistent cervical HPV infection, which can increase the risk of anal HPV transmission ,33. HoweINK4a in more than half of HPV positive cases and in comparison with its negativity in HPV-negatives, showing its high specificity and strong association with the carcinogenesis mediated by the inhibition of p53 and pRb tumor suppressor proteins. Our results confirmed that p16 INK4a over expression could be adopted as an immuno-histochemical prognostic marker, as reported in head and neck HPV-associated cancers [Another remarkable finding of this study was the positivity of p16 cancers ,35.Probably, the poor sample size did not prove the statistically significant difference in terms of OS between the HPV-positives . HoweverSeveral study limitations, most of them attributed to the retrospective nature of the study, can be highlighted. Firstly, we did not assess a suitable sample size to prove a specific prevalence, even if all consecutive cases of anal cancer were enrolled in the study. Some known and unknown epidemiological variables  were not evaluated; the retrospective data collection hindered a comprehensive collection of key variables. Some statistically significant differences on the lower prevalence of HPV infection in cases of adenocarcinoma  and highTo the best of our knowledge, this study evaluated for the first time the role of HPV in anal cancers including demographic, histological, and molecular variables in our region. Despite the poor sample size, the etiologic role of HPV in some anal cancers can be proved by the HPV-DNA positivity and p16 over expression.Both females and males could have a reduction of their risk after the implementation of an HPV vaccination program. However, observational prospective/retrospective and experimental studies should be carried out in the near future to better clarify the role of HPV in the carcinogenesis of anal cancer  and the efficacy/effectiveness of HPV vaccines, respectively. Moreover, considering the low frequency of the disease in general population, a multicenter design could likely improve the statistical power and could discriminate statistically significant differences we did not find. The added value of point-of-care molecular methods could be showed when evaluating their diagnostic and prognostic accuracy in the routine.


In 2018, there were an estimated 570,000 new cases of cervical cancer globally, with most of them occurring in women who either had no access to cervical screening, or had not participated in screening in regions where programs are available. Where programs are in place, a major barrier for women across many cultures has been the requirement to undergo a speculum examination. With the emergence of HPV-based primary screening, the option of self-collection (where the woman takes the sample from the vagina herself) may overcome this barrier, given that such samples when tested using a PCR-based HPV assay have similar sensitivity for the detection of cervical pre-cancers as practitioner-collected cervical specimens. Other advantages of HPV-based screening using self-collection, beyond the increase in acceptability to women, include scalability, efficiency, and high negative predictive value, allowing for long intervals between negative tests. Self-collection will be a key strategy for the successful scale up of cervical screening programs globally in response to the WHO call for all countries to work towards the elimination of cervical cancer as a public health problem. This review will examine self-collection for HPV-based cervical screening including the collection devices, assays and possible routine laboratory processes considering how they can be utilized in cervical screening programs. Cervical cancer is a major public health problem with approximately 570,000 new cases, and 311,000 deaths globally in 2018, the majority of these in low- and middle-income countries ,5. Participation in a screening program involves a woman knowing the reasons for screening, believing that screening is relevant to her, and that being screened provides benefits that outweigh any potential risks or costs. Being able to access a trusted service provider and the ability to undergo a speculum examination are also contributors to women’s decisions about participating in a screening program . Women wTraditional cervical cytology relied on healthcare practitioners visualizing the cervix and sampling cervical cells from the transformation zone of the cervix . Human pSelf-sampling has been shown to increase participation in never- or under-screened populations . SeveralSelf-sampling facilitated cervical screening can be performed in a variety of settings including health care facilities, homes or workplaces facilitated by community health outreach teams , with HPThis flexibility of self-collection based cervical screening can substantially increase participation in cervical screening in rural, remote or low resource settings , and theIn this review, informed by key papers in the literature and our emerging experience in the HPV-based screening program in Australia, we examine a range of different assays and collection devices and consider other pragmatic issues for introducing self-collection into the laboratory as part of an organized HPV-based cervical screening program. We focus not only on emerging research findings but also on how this evidence can be utilized in the paradigm of clinical testing undertaken at pathology laboratories with the sample volumes that could be expected from an organized screening program.There are a large number of HPV testing technologies available but some of the most common are polymerase chain reaction (PCR)-based assays. Major manufacturers such as Abbott, BD, Cepheid, Qiagen, Roche, and Seegene all produce medium- to high-volume, PCR-based HPV assays for use in cervical screening programs. The meta-analysis by Arbyn et al.  clearly There is a wealth of data, including a recent meta-analysis , that deA recent methodological paper trialled a different protocol for diagnostic testing of self-collected vaginal samples on the Aptima. Borgfeldt and Forslund  used an Another aspect of the Aptima assay is that it lacks a control for cellularity (sample adequacy), and as a result there is no way of knowing how many of the HPV negative specimens actually contained cellular material. In a cervical screening environment where co-testing (HPV and cytology) are always undertaken, there is no need for a cellularity control as a lack of cellular material is easily identified on cytology. However, with self-collection there is an inherent difficulty in knowing the validity of a negative result unless evidence is available to demonstrate that a sample was actually taken by the woman. Even in clinician-collected cervical screening there is a failure of the cellularity control of between 0.1–0.3% ,32,33, aThe current data appears to suggest that the Aptima assay may not be optimised for self-collection using the current manufacturer’s instructions for use. Further studies using the endpoint of histologically confirmed CIN2/3+ would be useful to confirm the current findings of the Arbyn meta-analysis relating to Aptima . There rcareHPV is an adapted form of the Hybrid Capture 2 assay and was developed as an affordable HPV-DNA detection technology ,36. It iTherefore, is careHPV an appropriate assay for self-collection, when meta-analyses have clearly demonstrated that the Hybrid Capture 2 it was developed from is not ? We acknThere is certainly evidence to suggest that self-collection tested on careHPV facilitates screening by being more acceptable than traditional, speculum-assisted, cervical cytology . A numbeThe careHPV assay lacks a control for sample adequacy, or for inhibition (such as by blood). The meta-analysis by Arbyn  determinOf the current devices in use for self-collection, the Evalyn brush  is possibly the most established and evidence-based device. It is currently used in a number of jurisdictions, including as the device used in the self-collection pathway of The Netherlands national cervical screening program. The Evalyn brush is marketed as a customized device designed to be used for self-collection for HPV-based cervical screening. There are a wide range of studies examining the use of Evalyn brush in detecting 14 oncogenic HPV genotypes on multiple clinically validated PCR-based HPV assays, including the Roche cobas 4800 ,14,15, AIn the Arbyn meta-analysis  there weAn interesting study published by a Norwegian group  examinedAs highlighted above, the one major drawback of the Evalyn brush is its cost. Rovers Medical Devices have sought to address this issue by introducing a more affordable version of the Evalyn collection device called the Viba-brush. The Viba-brush utilises the same collection head but mounts it more simply on a straight handle and is reported to have a price equivalent to other low-cost devices. There are two paired sample studies ,44 whichIn the Dijkstra study  undertakIn the study undertaken by Geraets et al.  biopsiesThere are only a few studies in the past ten years which examine the use of the Qvintip device for HPV testing. Four of these studies ,47,48,49There is a direct comparison study by Jentschke and colleagues  which pr®-cartridge, and a clinician-collected specimen. The flocked swab had equal or greater sensitivity than both the FTA®-cartridge and the clinician-collected specimen. The flocked swab also matched the clinician–collected sample for detection of cytological high-grade squamous intraepithelial (HSIL) lesions and cervical cancers and was more sensitive than the FTA®-cartridge method. Both of these studies used the clinically-validated Seegene Anyplex HPV assay. Another option for self-collection being increasingly used in cervical screening programs is the flocked swab, generally the FLOQswab made by Copan . There are a few studies that examined self-sampling using flocked swabs, but it is difficult to assess these studies together as the specific type of FLOQswab used is not always described. A small study  examininThe Copan flocked swab (product number #552) has also be used in the national cervical screening programs in both Malaysia and Australia after successful pilot implementation studies ,20. VALidation of HPV GENotyping Tests) [It is emerging best practice for the use of HPV assays in screening to require the assay to have evidence of being clinically validated, with the two major mechanisms for assessing clinical sensitivity and specificity being the Meijer Criteria  or VALGEg Tests)  uses a sBoth The Netherlands and Australia have self-collection pathways as part of a HPV-based national cervical screening program, with The Netherlands using the Evalyn brush and laboratories in Australia who are accredited currently all utilizing the Copan FLOQSwab. Currently only Roche cobas HPV assays (cobas or cobas 4800) are being used in these national programs for self-collected specimens. In Australia the availability of self-collection was delayed by lack of any suitable clinically validated HPV assays having manufacturer validated claims for the use of self-collected specimens (on label use), which required laboratories to independently validate their self-collection protocols against paired clinician-collected specimens  instructions for use covering self-collected specimens.One of the less commonly addressed issues with self-collection is the actual processing of the sample within an accredited pathology laboratory, as opposed to a research laboratory. Many of the research studies investigating self-collection involve the self-collected specimen being transported to the laboratory as a dry sample where it is then resuspended in a liquid media ,13,16. TAnother important consideration in the resuspension of dry samples is the availability of media. Many countries do not have stable supplies of liquid-based cytology media and even transporting these media can be difficult, as they are alcohol-based and flammable. This means that they must be shipped as a dangerous good (in volumes > 300 mL), increasing costs which may be prohibitive for low- and middle-income countries. Some manufacturers are looking to validate non-alcohol-based media to reduce these issues, although this may in turn result in reduced stability which could restrict use to point of care testing. In addition to a lack of validated HPV assays for self-collection and a consensus elution protocol, another issue for future scale up of its use is that self-collection is currently dependent on a number of manual steps. If self-collection is going to become widely available, and laboratories are going to process hundreds of samples a day, there will be a need for pre-analytic automation—both for workflow and for the reduction in inter-operator error. Current automated pre-analytic instruments, such as the Roche p480, do not have the ability to process self-collected devices. Another possible mechanism would be to elute dry swabs directly into a solution already used for the processing of specimens, such as the BD diluent or the Roche cobas PCR media. There are currently two studies ,56 whichThe updated meta-analysis on the validity of using self-collection methods for HPV testing in 2018 by Arbyn et al.  was of cThis review also aimed to summarize the current evidence on the utility of other technologies in national screening programs. careHPV presents as a viable test because of the very low cost, although the exact cost per test appears to be in question . The datThe Hologic Aptima HPV test uses RNA as its template, which may potentially give better specificity which could be beneficial for managing limited follow-up resources such as colposcopy more effectively. There are only a relatively small number of studies using Aptima and, because of the different mechanism of detection of HPV results compared to other assays, results cannot be pooled with PCR-based tests. All studies comparing self-collection on Aptima indicate that it is a less sensitive test for CIN2+ which suggests that, if there is higher specificity, it is to the detriment of detecting disease.The evidence supporting non-PCR-based HPV tests for use with self-collected samples is not as favourable or as comprehensive as that supporting PCR-based testing. Further evidence on the application and performance of non-PCR-based technologies for self-sampling are likely to emerge and any new information will be critical for any re-assessment of use of these technologies for self-collection.®-cartridge costing more than US$5 whereas a Copan flocked swab is under US$1 [In contrast to the importance of correct HPV assay selection for the testing of self-collected specimens, a wide range of vastly different sampling devices appear to produce good results. The Arbyn meta-analysis  covered der US$1 . EnvironAnother emerging methodology for self-collection is the use of urine for HPV-based cervical screening. This particular method was not covered in this review as there is a paucity of data on the predictive value of a urine HPV positive result as it relates to clinical outcomes such as histologically—confirmed high-grade cervical disease. There is currently a diagnostic test accuracy study being undertaken called Validation of Human Papillomavirus Assays and Collection Devices for Self-samples and Urine Samples (VALHUDES) , which sThere is a need for further research into the acceptance and feasibility of using self-collection in diverse under- or never-screened populations, but this falls outside the scope of the current review which is more focused on the technical aspects of self-collection.Self-collection is an attractive mechanism to increase participation in cervical screening worldwide. The current evidence strongly supports the need for PCR-based HPV assays with internal controls, specifically for both sample adequacy and detection of inhibition.Whilst there does not appear to be major differences in the sensitivity of different collection devices, their cost, acceptability, and scalability as part of population level screening programs need to be considered.This area of clinical testing for HPV as part of organized cervical screening programs will likely be a fast evolving one, with the continued development of new HPV assays, pre-analytic devices, and hopefully, manufacturer-validated claims for the use of self-collection.This review sought to examine how the current evidence base could be used to support self-collection as a method of increasing cervical screening. There were three key findings relating to what type of test should be used, what type of collection devices should be used and what factors need to be considered moving forwards:


This study aimed to: (1) Estimate HPV prevalence and genotype distribution among female sex workers (FSWs) in Mali and Benin as well as the prevalence of multiple HPV type infections in this group, and (2) Identify potential risk factors associated with high-risk (HR) HPV infections.We analyzed baseline data of 665 FSWs aged ≥ 18 years recruited during a prospective cohort of cervical cancer screening in Cotonou (Benin) and Bamako  from 2017 to 2018. The Linear Array HPV genotyping test was used to identify HPV genotypes. Descriptive statistics and multivariate log-binomial regression were used. Adjusted prevalence ratios (APR) with 95% confidence intervals (95%CI) were estimated to identify risk factors associated with HR-HPV infections.HPV data were available for 659 FSWs . The mean age was 35.0 years (± 10.7) in Benin and 26.8 years (± 7.6) in Mali. The overall HPV prevalence rates were 95.5% in Benin and 81.4% in Mali. About 87.7% and 63.4% of FSWs harbored ≥ 2 HPV types in Benin and Mali, respectively. The top three prevalent HR-HPV among FSWs in Benin were: HPV58 (37.5%), HPV16 (36.6%) and HPV52 (28.8%). Corresponding patterns in Mali were HPV16 (15.7%), HPV51 (14.3%) and HPV52 (12.9%). In Benin, the main factors associated with HR-HPV were vaginal douching  and gonococcal infection , while in Mali they were sex work duration ≤ 1 year  and HIV infection .Our study found a very high prevalence of HPV infection as well as high frequency of multiple HPV type infections in FSWs in two countries in West Africa. These findings suggest the necessity to emphasize cervical cancer prevention in this high-risk group. Cervical cancer is the second most frequent cancer among women and the leading cause of cancer-related death in many countries in Sub-Saharan Africa (SSA) . It is wHuman papillomavirus (HPV) infection is the most common sexually transmitted infection (STI) worldwide . Accordith and 3rd, respectively, in terms of age-standardized incidence and 12th and 3rd rank according to mortality of cervical cancer [Benin and Mali are two West African countries where cervical cancer is a major public health issue . Recent l cancer . Furtherl cancer  and 12% l cancer , but litIn regions of the world where the HIV epidemic is driven by heterosexual transmission, FSWs and their clients constitute a core group for the spread of HIV/STI to the general population . In addiWe analyzed baseline data from a prospective cohort study on cervical cancer screening, HPV and HIV infections. From January 2017 to March 2018, the study was conducted in collaboration with three Non-Governmental Organizations (NGOs) in Bamako  namely SOUTOURA, DANAYA SO and ARCAD-SIDA. These NGOs are the most active in the field of STI/HIV prevention in Mali, with a recognized leadership and trusted by FSWs. The details of NGOs from Mali are shown in a previous publication . In BeniFor uniform procedures in both countries, we have carefully planned our study. Before starting the activities, we organized a training session targeting physicians at the “Dispensaire des IST (DIST)” in Cotonou and at the ARCAD-SIDA STI clinic in Bamako. These sessions covered theorical and practical aspects of cervical cancer screening, review of questionnaire and practical issues related to data collection procedures in the field.For field work issues, we recruited four PEs from each participating NGO in Mali, two PEs as well as two facilitators in Benin. Specific training was provided to these PEs and facilitators to familiarize them with the survey procedures. A cervical cancer screening awareness campaign preceded the recruitment of FSWs in the two cities. These activities were performed by trained PEs and senior staff from the participating NGOs in the bars, brothels, hotels, etc. Women interested to participate in the study were invited to come to the DIST in Cotonou (STI clinic offering adapted services to FSWs) or to the ARCAD-SIDA STI clinic specialized in FSW care in Bamako. The service package offered in these two FSW-friendly STI clinics includes small talk discussion for behavior change, peer education, condom use demonstration, as well as free distribution of condoms and lubricants. In addition, they provide STI care using syndromic management as well as HIV testing and treatment for free of charge.A FSW was defined as any woman who receives money or gifts in exchange for sex. To be included in the study participants had to fulfill the three criteria as follows: (a) being a FSW in the study settings for at least 6 months; (b) being referred by one of the PEs of the participating NGOs; and (c) being aged between 18 and 65 years old. All FSWs who had previously been diagnosed with cervical cancer, who had a hysterectomy and those who were pregnant were excluded from the study.At the clinics, the study procedures were explained to each FSW and informed consent was obtained. A questionnaire was administered face-to-face by qualified interviewers to collect data on socio-demographic characteristics, including age, marital status, educational level, country of origin; behavioral and sex work characteristics like the frequency of alcohol consumption, drug consumption, number of cigarette packs smoked per week, age at first sexual intercourse, total number of sexual partners in the last week of work, number of paying clients in the last week of work, having regular partner, frequency of condom use, duration of sex work, place where practicing sex work, oral and anal sexual intercourse, vaginal douching; reproductive health variables as well as family and medical histories, such as parity, number of abortions, number of vaginal and cesarian deliveries, history of cervical cancer, ovarian cancer, vaginal cancer or breast cancer, family history of these cancers; history of STIs in the last six months.Following the interview, each woman underwent a gynecological examination performed by a well-trained physician. Vaginal and cervical swabs were obtained from each participant for curable STI testing. A cytobrush  was used to collect cells from the endocervix and ectocervix and stored in tubes containing 3 ml of fresh PBS  for HPV DNA testing. Cervical cancer screening was performed using visual inspection methods (VIA/VILI). Finally, a venous blood sample was taken for HIV testing and CD4 count.Trichomonas vaginalis and Candida Albicans. The diagnosis of bacterial vaginosis (BV) was made using Nugent score.In both countries, the same procedures were used for the following tests. Wet mounts of the vaginal swabs were microscopically examined immediately for Neisseria gonorrhoeae and Chlamydia trachomatis detection, we used different tests. In Benin, these infections were detected using the NG/CT Probetec® assay from Becton Dickenson , while in Mali, these infections were detected using the Abbott Real-Time CT/NG assay. These assays were carried out according to the manufacturers’ instructions. These two tests have similar sensitivity and specificity as reported elsewhere [For lsewhere , 27. AllBoth, Mali and Benin have the same national HIV testing algorithms and we thus used the same tests for the detection of HIV antibodies for all the study participants. Alere Determine HIV-1/2 test  was used as first line and positive specimens were then confirmed with a rapid and discriminatory test SD Bioline . Women testing positive for HIV were immediately offered antiretroviral therapy.HPV testing was performed as part of the study. In both countries, endocervical and ectocervical cells were centrifuged at 3000 rpm for 5 minutes at 4°C for cervical cell concentration. Cell pellets were stored at -20°C until DNA extraction. At the end of the study, all cell pellets were sent to Montreal (Canada) in a laboratory specialized in HPV testing. HPV Genotyping was performed using the Linear Array HPV genotyping test . This test identifies 36 genotypes by hybridization on a linear array of PGMY-generated amplicons with 34 type-specific probes for HPV-6, -11, -16, -18, -26, -31, -33, -35, -39, -40, -42, -45, -51, -53, -54, -55, -56, -58, -59, -61, -62, -64, -66, -67, -68, -69, -70, -71, -72, -73, -81, -83, -84, and -89, two probes for two variants of HPV-82, and one probe that cross-reacts with HPV-33, -35, -52, and -58. Amplification and type detection were performed according to the manufacturer’s recommendations . The croThe dependent variable was “HR-HPV” defined as being positive for at least one HR-HPV type.t test for continuous variables and rank test with median score. We also computed descriptive statistics to estimate HPV prevalence rates. An exact binomial 95% confidence interval (95%CI) was calculated for each prevalence rate. To identify factors associated with HR-HPV infection, we carried out, for each country, univariate and multivariate log-binomial regression models with a robust “sandwich” variance estimator to calculate the adjusted prevalence ratios (APRs) with 95% CI. All variables significant at p ≤ 0.2 . We used descriptive statistics to analyze demographic, behavioral and sex work characteristics. The results were presented as percentages, means with standard deviations (± SD) or medians with inter-quartile ranges (IQR). Pearson’s Chi-Square test or Fisher’s exact test were used for categorical variables, the Student’s election ) in univo2017/93/CE/FMPOS), and the “CHU de Québec-Université Laval” (#2017–3313), as well as the National Ethics Committee for Health Research in Benin (# no01 du 25 janvier 2017). The objectives, procedures and potential risks related to the study were explained to each woman and written informed consent was obtained before enrolment. Consenting participants signed or apposed their fingerprint on the consent forms. In Mali, participants received 5000 CFA (about US$8.40), while in Benin, they received 3000 CFA (about US$5) for compensation of transportation and the time spent at the clinic. The amount of compensation was slightly different between the two countries because of the socio-economic context . Condoms and lubricants were distributed for free to each woman.The study was reviewed and approved by the ethics committees of the School of medicine of Bamako, Mali  were more common in Mali than in Benin (p < 0.0001). HIV prevalence was 26.3% in Benin versus 20.4% in Mali and the proportions of positive VIA/VILI tests were 20.2% and 10.5%, respectively.A total of 710 FSWs were approached . Of these 20 FSWs were excluded in Mali  and 25 FSWs in Benin (9 had their menstruations and 16 pregnancies). A total of 665 women were thus included in the study, 312 in Benin and 353 in Mali. FSWs from Benin were older than those from Mali, the mean (± SD) age was 35.0 ± 10.7 versus 26.8 ± 7.6 years respectively  and 81.4% (95%CI: 77.0–85.4) in Benin and Mali, respectively . HR-HPV HPV genotype distribution varied widely between the two countries with HPV58 (37.5%), HPV16 (36.6%) and HPV52 (28.8%) being the top three in Benin, compared to HPV16 (15.7%), HPV51 (14.3%) and HPV52 (12.9%) in Mali. Concerning LR-HPV the top three in Benin were: HPV62 (35.6%), HPV81 (23.6%) and HPV61 (23.0%). This profile in Mali was HPV62 (15.1%), HPV61 (12.9%) and HPV84 (10.6%).The prevalence of HR-HPV multiple type infections (≥ 2 HR-HPV) among FSWs was high in this study . MultiplTo evaluate the potential effectiveness of the HPV vaccines in FSWs, we estimated the overall prevalence rate and multiple infections with HPV types covered by available vaccines . This anThe country-specific risk factors for HR-HPV types are shown in This is the first epidemiological study on HPV infection among FSWs in Benin and Mali. Our findings show a high overall prevalence rate of HPV infection as well as a high rate of HR-HPV types among this group in both countries. Similarly, there was a high rate of multiple HPV type infections among them. We also noted a wide variation of HPV genotype distribution among FSWs between both countries. Furthermore, we identified several factors associated HR-HPV infections that were different between countries.Prevalence rates of HPV infection of 95.5% in Benin and 81.4% in Mali were several-fold higher than those found among women in the general population  , 15. TheFurthermore, compared with other studies among FSWs in West Africa where HR-HPV prevalence rates varied from 32.9% - 72.5% , 24, theA striking feature of HPV infection in FSWs is the wide variation of genotypes across countries. HPV58 and HPV16 were the most frequent in Benin, while in Mali, the predominant genotypes were HPV16 and HPV51. These two prevalent genotypes are quite different from those observed in Madagascar , but shaLike elsewhere , 21, in In Mali the highest prevalence of HR-HPV was observed in home-based FSWs. While the relation between HIV infection and sex work place is well documented , little We studied the epidemiology of HPV infections among FSWs from a convenience sample. To minimize selection bias, we used different recruitment approaches either by the PEs or FSW leaders. In addition, self-reported information on risky behaviors such as number of sexual partners, age at first sexual interaction, etc., may be subject to recall and social desirability bias. Indeed, due to stigma or social desirability, this information collected may be underestimated. However, such misclassification bias would be limited in our study since the data were collected by trusted qualified interviewers. Failure to measure certain variables could cause residual confounding in our analyses on risk factors. To deal with this bias, we adjusted for a large number of potential confounding variables based on the literature. Furthermore, the associations we found between several factors and HR-HPV cannot be viewed as causal because of the cross-sectional nature of the data. In addition, although our results reflect current HPV infection status, the lack of information about HPV antibodies in the serum, which is reported to be a marker of past infections , constitUnderstanding the distribution of HPV genotypes among FSWs is crucial to estimating the burden of HPV infections as well as the future impact of HPV vaccines within this high-risk group. The high prevalence of HR-HPV and other STIs/HIV found among FSWs implies an increased risk of cervical cancer.Currently, the bivalent and quadrivalent vaccines are approved in both countries, but they are not integrated in the routine immunization program. However, these vaccines are available in private pharmacies. The relative low representativeness of HPV types covered by the quadrivalent vaccine like HPV6, HPV11 and HPV18 may question the potential effectiveness of this vaccine in this group. The presence of HPV16, HPV52 and HPV58 in the top three would favor the nonavalent vaccine for cervical cancer prevention. Since evidences support vaccination against HPV in sexually active women up to the age of 44 years , FSW immOur study, the first in Benin and Mali, showed a high prevalence of HR-HPV infections among FSWs. These results make FSW a priority group for cervical cancer prevention programs adapted to their context. Additionally, the identification of the predominant HPV genotypes in this population shed the light on the vaccine potentially needed in this specific group.S1 File(ZIP)Click here for additional data file.


Nicotiana benthamiana, and transmission electron microscopy (TEM) analysis showed the presence of VLPs and/or capsomeres. Immunogenicity studies were conducted in mice utilizing HPV 35, 52, and 58 and showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous HPV pseudovirions in pseudovirion-based neutralization assays (PBNAs). This work demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries.Cervical cancer is ranked fourth among the top cancers in women and is the second most common cancer in low- and middle-income regions, with ~570,000 new cases reported in 2018, which attributed to 84% of worldwide cervical cancer cases. Three commercially available prophylactic Human papillomavirus (HPV) vaccines are effective at preventing HPV infections. However, these vaccines are expensive due to their complex production systems, therefore limiting their use in developing countries. Recently, the use of plants to produce vaccines has emerged as a cost-effective alternative to conventionally used expression systems. Here, L1 proteins of eight high-risk  and two low risk (HPV 6 and 34) HPV types were successfully expressed in  The HPV L1 capsid protein can self-assemble into virus-like particles (VLPs) that are structurally similar to native virions . Interes®, and Gardasil9®. Cervarix™ protects against HPV 16 and 18 infections, whereas Gardasil® targets HPV 6, 11, 16, and 18   ), with cTM ultracentrifuge tubes and centrifuged for 45 min at 174,587× g, 15 °C in a SW32Ti rotor . All sucrose solutions were prepared in 1× HSPBS. After centrifugation, the 30% cushions were collected with a long needle and the sucrose removed by dialysing the samples using cellulose membrane dialysis tubing with 10 KDa cut-off  overnight at 4 °C in 1× HSPBS with agitation.Clarified crude extracts were loaded onto 5 mL 30% (w/v) sucrose cushions overlaid onto 1 mL 50% sucrose cushions in 38 mL Ultra-Clear®) density gradients  prepared in 1x HSPBS and centrifuged for 3 h and 30 min at 174,587× g, 15 °C in a SW32Ti rotor (Beckman). After centrifugation, the bottom of the tubes were punctured, and 1 mL factions were collected.Dialyzed samples were loaded onto discontinuous Optiprep™ . Membranes were blocked in blocking buffer  at room temperature for 30 min with agitation. The membranes were probed with 1:2000 rabbit-raised anti-Gardasil® sera ® diluted in blocking buffer by incubating at 4 °C overnight with agitation. The blots were washed 4 × 15 min in blocking buffer and incubated for 1 h at 37 °C with a 1:5000 dilution of anti-rabbit IgG alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich®) in blocking buffer with shaking. The membranes were washed 4 × 15 min with blocking buffer not containing milk and L1 proteins were detected with 5-bromo-4-chloro-3-indoxyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) phosphatase substrate  for 30 min.Purified L1 fractions were mixed with 1x sample application buffer  bromothymol blue, 0.5 M EDTA, 5% (w/v) SDS, and 25% (v/v) glycerol) and denatured at 95 °C for 5 min . The L1 TEM was used to determine if the expressed L1 proteins assembled into VLPs. A Model 900 SmartSet Cold Stage controller  was used to glow discharge carbon-coated copper grids (mesh size 200) at 25 mA for 30 s. Samples were trapped onto grids by floating the grids, carbon side down, on the sample for 4 min. Thereafter, the grids were washed 4× with deionized water and negatively stained for 1 min with 2% w/v uranyl acetate. Grids were air-dried and viewed with an FEI Tecnai 20 transmission electron microscope.®.Three oncogenic HPV types  were selected to immunize mice and were quantified and prepared for animal studies. These types were selected as they are relevant in an African context and are not included in or are cross-protected by Gardasil®]) protein standard. BSA standards and 25 µL of samples containing VLPs were mixed with 1× sample application buffer (SAB) and denatured at 95 °C for 5 min. Samples were loaded onto 10% SDS-PAGE gels and Coomassie-stained. Quantification of the protein bands were carried out with StudioTM Lite version 5.2 software .Gel densitometry was used to quantify plant-produced HPV vaccine candidates; the concentration of L1 was determined relative to a Bovine serum albumin  according to the manufacturer’s instructions. Samples were also grown overnight on LB agar at 37 °C without antibiotics and observed for bacterial growth before being stored at −80 °C.® (positive control), equimolar amounts of plant-produced HPV 35, 52, and 58 VLPs, or administered a purified empty vector as negative control; the concentration of the vaccines are summarized in ® has been previously shown to elicit a significant immune response in mice at 1/10th of a human dose ) before incubation overnight at 4 °C in 1:200 mice serum diluted in blocking buffer. The following day, the pre-absorbed sera were removed and used in all ELISAs in this study.To reduce the cross-reactivity of the antibodies present in the sera with host cell proteins the final bleeds from mice immunized with plant-produced L1 VLPs and the empty vector negative control were pre-absorbed using The “Lunchbox” Immunoabsorbent Technique , to pote® sera were added to the wells and the plates incubated for 1 h at 37 °C. The plates were washed as described above and 100 µL of 1:5000 dilution of alkaline phosphatase-conjugated anti-mouse IgG secondary antibody and anti-rabbit IgG for Gardasil® (Sigma-Aldrich®) were added to each well and the plates incubated for 1 h at 37 °C. Plates were washed 4× with , after which 200 μL SIGMAFAST™ p-nitrophenyl phosphate (Sigma-Aldrich®) substrate was added to each well. The plates were incubated in the dark for 30 min before reading the absorbance with a Bio-Tek Powerwave XS spectrophotometer at 405 nm.For ELISAs, 96-well plates  were coated with 100 µL of the L1 antigens diluted to 0.8 ng/µl in coating buffer (10 mM Tris pH 8.5) and incubated overnight at 4 °C with agitation. Plates were blocked with 300 μL blocking buffer for 1 h at 37 °C, before they were washed 4× with 1× TST . Diluted mice sera or rabbit-raised anti-Gardasil® sera (positive control). A plant-produced empty vector was used as negative control for plant-made vaccine candidates only. ELISA data were all normalized by subtracting the background readings and titres are stated as the reciprocal of the maximum dilution with higher absorbance readings than the equivalent pre-bleed serum. Moreover, serum obtained from mice vaccinated with the plant-produced VLPs that produced titres with absorbance readings higher than the empty vector at the lowest dilution (1:200) were considered anti-L1 positive.Controls included: wells coated with coating buffer (to obtain background readings), pre-bleeds (negative controls), and rabbit-raised anti-GardasilHPV pseudovirion (PsV) production, purification, and neutralization were all done as described by Buck and co-workers , with a N. benthamiana plants with recombinant Agrobacterium cultures at a final OD600 of 0.5. Plants were harvested at 5 dpi, biomass presented with slight chlorosis in plants infiltrated with the HPV L1 constructs compared with the negative control, pTRA-rbcs1-cTP-only plants. In all cases, no tissue necrosis was observed. L1 expression was confirmed on western blots and a Coomassie-stained gel, in the case of HPV 52 L1, after Optiprep™ density-gradient purification. The L1 proteins of the 10 HPV types were successfully expressed in N. benthamiana and purified. This is evidenced by the detection of L1 bands (~56 kDa) for all HPV types  negative control was analyzed on a western blot and Coomassie-stained gel and, as expected, no L1 bands were observed on the western blot C,D. Two TEM analysis of purified fractions of each HPV type showed fully assembled spherical VLPs measuring 40–60 nm diameter . The humoral immune response was analyzed by testing type-specific anti-L1 antibodies in ELISA using plant-produced VLPs as antigen. Sera from individual mice in each group were pooled and anti-L1 antibody titres determined by indirect ELISAs , which confirmed the presence of antigens in the coated samples and validated the experiments , was observed.L1 PBNAs were carried out to test the ability of sera obtained from mice to neutralize HPV PsVs. Well-characterized neutralizing antibodies , provided by Neil Christensen , were ustibodies ,39. TherP signal A. This sp = 0.1436).Sera from mice immunized with plant-produced VLPs successfully neutralized type-specific PsVs  C. NeutraNegative controls for the PBNAs included PsVs that were not treated with sera and PsVs treated with sera from mice vaccinated with the empty vector, and no neutralization was observed in the empty vector sera. B,D. TreaTo our knowledge, there is no evidence of cross protection of heterologous HPVs by HPV 35, 52, or 58. However, we wanted to investigate if sera obtained from mice vaccinated with plant-produced VLPs were able to cross protect against other HPV types, and therefore, sera were tested in PBNAs with heterologous PsVs that were available in our lab , to determine if they could neutralize these PsVs. Unfortunately, plant-produced serum did not neutralize any of heterologous PsVs tested in this study at a 1:50 dilution of the sera. Highest neutralization titres for all sera are summarized in N. benthamiana and the immunogenicity of three of these, which were important in an African context, was analyzed in mice.HPV 16 L1 and/or L1/L2 chimaeric VLPs have previously been expressed in plants and have been shown to elicit immune responses in animal models ,22,23,24N. benthamiana using the pTRAkc-rbcs1-CTP plant expression vector that targets expression to the chloroplast [Ten HPV types were successfully expressed in oroplast . Expressoroplast ,22,23,29oroplast ,41,42, woroplast . Among ooroplast ,45.Despite potential proteolytic cleavage occurring for some of the L1 proteins expressed in this study, TEM analysis showed the presence of fully assembled VLPs measuring 40–60 nm in diameter as well as small VLPs and/or capsomeres measuring 25–39 nm . HPV L1 HPV VLPs are highly immunogenic because of their repetitive surface-displayed epitopes, ability to activate B-cells, their particulate nature, and their size, which is ideal for the uptake of nanoparticles by dendritic cells ,50. In t® vaccine and the plant-produced trivalent VLP vaccine candidate, whereas no L1 response was detected in the negative control empty vector sera. PBNA was used to investigate the ability of the VLP vaccines to neutralize PsVs. Sera from mice immunized with the trivalent VLP vaccine candidate elicited type-specific neutralizing antibodies, which successfully neutralized HPV 35, 52, and 58 PsVs , while plant-produced trivalent vaccine was used at 6 µg . However, the plant-produced trivalent vaccine candidate induced a humoral immune response comparable to the immune response elicited by the commercially available HPV Gardasil® vaccine. This was evidenced by the type-specific anti-L1 neutralizing titres observed (The immune response elicited by the plant-produced trivalent VLP vaccine candidate cannot be directly compared to that of Gardasilobserved , suggestIn conclusion, we demonstrated the successful production of a trivalent plant-produced VLP vaccine candidate consisting of HPV 35, 52, and 58. The vaccine successfully elicited a type-specific humoral immune response with neutralizing antibodies. This work has successfully demonstrated that plant-produced HPV VLPs are highly and appropriately immunogenic, demonstrating the potential to use plant-based transient expression systems to produce cost-effective HPV VLP-based vaccines, particularly for developing countries.


L1 and L2 genes were used to investigate the genetic variability of antigenic epitopes. HPV-positive samples were genotyped using the HPVCLART2 assay. The L1 and L2 protein sequences were analyzed using a self-optimized prediction method to predict their secondary structure. Co-occurrence probabilities of the 9v HPV types were calculated. Non9v types represented 49% of the HPV infections; 31.2% of the non9v HPV types were among the low-grade squamous intraepithelial lesion samples, and 27.3% among the high-grade squamous intraepithelial lesion samples, and several genotypes were low risk. The co-occurrence of 9v HPV types with the other genotypes was not correlated with the filogenetic distance. HPV81 showed an amino-acid substitution within the BC loop (N75Q) and the FGb loop (T315N). In the L2 protein, all of the mutations were located outside antigenic sites. The weak cross-protection of the 9v types suggests the relevance of a sustainable and effective screening program, which should be implemented by HPV DNA testing that does not include only high-risk types.Non-nonavalent vaccine (9v) Human papillomavirus (HPV) types have been shown to have high prevalence among HIV-positive women. Here, 1444 cervical samples were tested for HPV DNA positivity. Co-infections of the 9v HPV types with other HPV types were evaluated. The HPV81  Cervical cancer is the fourth most frequent cancer in women . StatistIn developed countries, the cervical cancer screening program started 40 years ago, and it has contributed to reduced incidence of and mortality from cervical cancers . HoweverIn 2006, two L1 virus-like particle (VLP) vaccines were licensed: Cervarix, a bivalent vaccine that contains VLPs of the HPV16 and HPV18 HR types, and Gardasil, a quadrivalent vaccine that includes VLPs of the HPV16 and HPV18 HR types, plus the two: HPV6 and HPV11 LR types. These vaccines prevent 70% of cervical cancers, and the tetravalent vaccine (4v) can also prevent epidermodysplasia associated to HPV6 and HPV11 infections . In DeceThese aspects are particularly relevant among HIV-infected women, who have a high rate of HPV infections and harbor multiple HPV infections more persistently, in comparison to HIV-negative women . In a 4-The aim of the present study was to determine the prevalence of HPV types included and not included in 9v in low-grade squamous intraepithelial lesions (LSIL) and HSIL among HIV-positive women. Based on these observations, a further aim was to determine the probability that the 9v genotypes co-exist with the other HPV types that are circulating among HIV-positive women in Italy. Furthermore, the L1 and L2 genes were analyzed for single infections (SIs) and multiple infections (MIs), to investigate the genetic variability of the antigenic epitopes in these two groups .This was a cross-sectional study that included HIV-positive women who had been screened for HPV-related diseases at the gynecology Outpatient Service of the “L. Spallanzani” National Institute for Infectious Diseases . The Institutional Ethical Committee approved the study protocol (n. 42/13). All of the methods were carried out according to the ethical standards of the Institutional and National Research Committee and the Declaration of Helsinki.Cervical cell samples were obtained using a cytobrush, and were preserved in 2 mL phosphate-buffered saline [24], and stored in ice until they were delivered to the Laboratory of Virology, where they were processed. Cervical smears were prepared for cytological examination (Pap-tests) concomitant with sampling for HPV testing. Cytological diagnosis was formulated according to the 2001 Bethesda system , and clag for 5 min. The pellet was resuspended in 1.5 mL phosphate-buffered saline, and 20 μL proteinase K  was added, with the samples then incubated at 56°C for 10 min. An aliquot of 800 μL was used to extract the nucleic acids , according to the manufacturer instructions. The residual samples were immediately frozen and stored at−80°C until used for further investigations.Approximately 1.8 mL cervical swabs samples were centrifuged at 1,500 × All of the samples were tested for amplifiable DNA using β-globin primers , and HPVn = 6) and a subset of HPV81 MIs (n = 4). HPV81 was chosen because it has been frequently observed in association with LSIL and HSIL grade lesions , and 10 μL DNA extracted from cervical samples. The assay conditions were: 15 min at 94°C, and 35 cycles: 94°C 40 s, 58°C for 45 s, and 72°C for 115 s, followed by a final extension at 72°C for 10 min.To study the L1 genetic variability of HPV81, we selected a subset of HPV81 SIs  and sequenced directly using Big Dye Terminator Cycle Sequencing Ready Reaction kits, and analyzed using a sequencing system . The PCR and sequencing were performed in duplicate.The L2 gene was amplified by nested PCR amplification using the L2 outer sense and antisense primers (round I), and the L2 inner sense and antisense primers . The sequences of the L1 and L2 genes were submitted to the NCBI GenBank database, and were assigned accession numbers (MT547553 – MT547561).All of the sequences were aligned with an HPV81 prototype sequence  probability of an event that is an “estimate” that an event will occur based upon how often the event occurred after collecting data from an experiment in a large number of trials . This tyE is the co-occurrence between the reference 9v non-avalent genotypes  and one of the others , and the total number of trials is the number of times the reference genotype occurs in the given dataset (P(E), as the co-occurrence probability of the event E. Finally, we defined the complementary probability C(E) of event E as the probability of not observing A given B, as in Eq. (2):In our analysis, the event  dataset . Thus, wC(E) = 1] when P = 0, which means that the presence of A rules out the possibility to have B.Thus, the complementary probability is maximal .The analysis of the results here allows us to conclude that infection by each of the 9v HPV types is associated with the presence of a specific set of HPV genotypes. In particular, these data suggest that genotype HPV6 might protect from HPV26, 35, 40, 43, 54, 73, and 85 infections; HPV11 from HPV26, 35, 39, 40, 43, 51, 54, 56, 68, 71, 73, 81, 85, and 89; HPV16 from HPV26, 40, 43, 44, 54, and 85; HPV18 from HPV26, 35, 39, 40, 42, 43, 54, 73, 85, and 89; HPV31 from HPV26, 39, 40, 43, 54, 56, 68, 72, 85, and 89; HPV33 from HPV26, 35, 39, 43, 44, 66, 68, 71, 81, 85, and 89; HPV45 from HPV26, 35, 39, 43, 44, 51, 53, 56, 61, 62, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85, and 89; HPV52 from HPV26, 40, 43, 51, 68, 71, 73, 85, and 8, and HPV58 from HPV26, 40, 43, 71, and 85. The evidence that infection by at least one of the 9v HPV types is never associated with the presence of HPV26, 35, 40, 43, 54, and 73 can lead to speculate that the 9v HPV types might completely protect from co-infections by the six HPV genotypes.y-axis) and the genetic distance (x-axis), where the dots represent the 26 high and low risk HPV genotype subtypes. The co-occurrence probability is the same as for R2 represents the linear regression model fitting index used to quantify how well the co-occurrence probability correlates with the genetic distance. In each panel, the fitting model was built considering only the genotypes with a non-null probability  and two HPV81 MI  were successfully identified . Five HPThe L2 gene was successfully amplified and sequenced in two SI  and in five MI  samples. Eleven nucleotide changes were non-synonymous mutations , and thep < 0.001)  . Increas< 0.001) . The per< 0.001) . The 9v < 0.001) . These d< 0.001) . Among t< 0.001) .Recently, Bogaards et al. re-evaluated the effectiveness of the adjuvanted bivalent HPV vaccine (2vHPV) against type-specific HPV positivity. Here, they demonstrated that cross-protection is maintained up to 8 years post-vaccination, and that the level of protection correlates with genomic distance to HPV16 or HPV18. Indeed they showed that 2vHPV can partially cross-protect against HPV types belonging to HPV α-7 (including HPV18) and α-9 (including HPV16), but cross-protection was unlikely for HPV51, 53, and 56, and for HR types belonging to the HPV α-5 or α-10 species; A bias of this analysis might relate to the different prevalence of HPV types in different geographical areas, and thus we might have included fewer cases of an HPV type because it is not very common in Europe . However, we observed that HPV types that belong to different α-species and that have similar prevalence in Italy co-existed with 9v HPV types with similar frequencies.HPV52 (α-9) infection occurred with HPV59 (α-7) and HPV81 (α-3), which had the same prevalence in a population of women . These fL1 antigenic variants were present in SI and MI, and whether they might influence their cross-protection properties. Overall, the L1 protein harbored a greater number of variable amino acids than the L2 protein . For L1, six amino-acid mutations affected two antigenic sites: the BC loop  and the FGb loop .The sequences of the L1 and L2 genes were submitted to the NCBI GenBank database, and were assigned accession numbers (The studies involving human participants were reviewed and approved by Institutional Ethical Committee L Spallanzani code42/13, 2013. The patients/participants provided their written informed consent to participate in this study.ARG, PDP, and PP conceived and designed the study. CS and DL collected the samples and carried out sample HPV typing. FDN processed the cythological samples. CS, VG, CM, DL, and FDN analyzed the data. ARG and VG writing original draft. ARG, PDP, PP, VG, and MC edited the manuscript. All authors read and agreed to the final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.