Linear regressions were used to analyze the data. Most participants were female (91.2%) and senior high school students (60.0%). Many of the participants had neither heard about HPV (92.3%) nor HPV vaccinations (95.4%). Significant predictors of adolescents’ likelihood of getting the first dose of HPV vaccination were perceived beliefs and change in a physical environment (p < 0.001), with each variable accounting for 6.1%and 8.8% of the variance respectively. Significant predictors of adolescents’ likelihood of completing HPV vaccination recommended series were perceived beliefs, practice for change, and emotional transformation (p < 0.001), with each variable accounting for 7.8%, 8.1%, and 1.1% of the variance respectively. Findings underscore important opportunities for developing educational interventions for adolescents in Ghana to increase the HPV vaccination uptake.HPV vaccines are efficacious in preventing HPV related cancers. However, the vaccination uptake in Ghana is very low. Studies that utilize theoretical frameworks to identify contributory factors to HPV vaccination uptake in Ghana are understudied. We used multi-theory model (MTM) constructs to predict initiation and completion of HPV vaccination series in Ghanaian adolescents. Adolescents ( African governments, non-governmental, and philanthropic organizations have concentrated much of their efforts on preventing malaria and HIV/AIDS in the African continent. However, less attention has been given to Human Papillomavirus (HPV) related cancers, which are silently claiming the lives of Africans in great numbers. HPV is the most common sexually transmitted diseases in the world ,2. HPV i®, Gardasil®, and Gardasil 9® are efficacious in preventing HPV related cancers [® is a bivalent vaccine that targets HPV 16 and 18, Gardasil® is a quadrivalent vaccine that additionally targets HPV 6 and 11 which cause genital warts. Gardasil 9® is a nonavalent vaccine that has been licensed in the United States of America (USA), Europe, and other high-income countries and targets additional oncogenic HPV serotypes: 31, 33, 45, 52, and 58. Studies show that the HPV vaccines produce a higher immune response in 9–14 year-olds than they do in 15–26 year-olds, and as such to increase efficacy the vaccination is recommended in the early years of adolescence [® and Gardasil®, are sold in Ghana. Since 2013, there have been two HPV vaccination demonstration projects completed in Ghana, one by Global Alliance for Vaccines and Immunizations (GAVI) [® Access Program (GAP) [Fortunately, prophylactic HPV vaccines such as Cervarix cancers ,11,12,13lescence ,18. Howelescence . Since tlescence ,21. Low-lescence . In Ghanlescence  and currs (GAVI) ,24 and tam (GAP) . A complam (GAP) .Barriers to HPV vaccination in sub-Saharan Africa include structural factors such as inadequate infrastructure, limited trained health workers, vaccine cost, stigma, and lack of knowledge ,27,28. FThe multi-theory-model (MTM)  is a behThe continuation of a given behavior also involves three key constructs, emotional transformation, practice, and social environment. The emotional transformation construct is changing individuals’ emotions toward a given behavior change . The construct of practice for change highlights the individuals’ deep consideration of a given behavior change and engaging in iterative processes to overcome barriers and remaining focused on sustaining a behavior change . The construct of changes in the social environment stipulates that creating social support from the environment  helps in sustaining a given health behavior . Unlike The inclusion of MTM in HPV vaccination research constitutes a new approach well supported in health promotion and public health research. The use of MTM has consistently shown to have high predictive power across multiple health-related behaviors . Since iA target sample size was calculated in the G*Power software, specifying an effect size of 0.10, an alpha of 0.05, power of 0.95, and 7 predictors. Based on the criteria, a total sample size of 226 was determined adequate for the study. To account for potential missing data the sample size was increased by 25% and therefore our recruitment target was 283 participants.A convenience sample of adolescents was recruited from four schools in the Ashanti Region in Ghana to complete a 44-item survey based on MTM constructs. To be eligible, adolescents had to be 12 to 18 years and be able to read or understand English. The study protocol went through a series of approval processes before recruiting adolescents for the study. First, study approval was also obtained from the Ghana health services, Ghana education service, and school administrators. After obtaining these approvals, we worked with the school administrators to identify interested students and obtained parental permission before the child could assent to the study. All assenting adolescents were given a hard copy of the survey to complete and return to the researchers.We used a modified version of multi-theory model (MTM) questionnaire . The relThe predictor variables were the constructs of MTM and the perceived beliefs subscale. The MTM constructs consisted of three subscales that predict the initiation of the HPV vaccination: participatory dialogue, behavioral confidence, and physical environment, and three subscales to predict the completion of the HPV vaccination recommended series: social environment, practice for change, and emotional transformation.The participatory dialogue consists of the perceived benefits of HPV vaccination (5 items) and perceived barriers of HPV vaccination (5 items). The perceived benefits of HPV vaccination items included protection against cervical, head and neck, mouth and throat cancers and genital warts, improve the immune system, and feel better about overall health. The perceived barriers of the HPV vaccination items included side effects, the cost of the vaccine, inconvenience, pain, and possible effects on the reproductive system. To calculate the participatory dialogue score, the disadvantages scores were subtracted from the advantages scores. This scoring approach is consistent with the recommendation by theory’s author , The parThe changes in social environment subscale (3 items) includes certainty of getting support from a family member, friends, and health professionals for all the recommended series of HPV vaccination. The practice for change subscale (3 items) included certainty of being able to keep a record to monitor getting the recommended series, overcome barriers to getting recommended doses, and change schedule to be able to get the recommended doses. The emotional transformation subscale (3 items) included certainty related to being able to direct emotions, motivate oneself, and overcome self-doubt to getting the recommended doses. The composite scores were computed for the subscales and the possible score range was 1–15 where higher scores indicated the likelihood of completing the recommended doses of the vaccine.We also included a perceived belief variable as an additional predictor. The three perceived belief items assessed participants’ perceived belief about whether HPV can cause cancer, HPV vaccination can prevent HPV related cancer, and HPV vaccination was effective when adolescents are vaccinated in their pre-teen years. The possible score was 1–15 where higher scores indicated higher beliefs.The two outcome variables for the study were the likelihood of adolescents getting the first dose of HPV vaccination within the next month and the likelihood of adolescents completing the recommended series of HPV vaccination with the next 12 months. The response options were somewhat likely, moderately likely, likely, very likely, and completely likely. The possible score range was 1–5 where 1 indicated less likelihood and 5 indicated a high likelihood of initiation or completion.Descriptive statistics were calculated on the demographic data to describe the sample. Univariate analysis was used to compare the mean difference of participants’ likelihood of getting the first dose and completing the recommended series of HPV vaccination. Hierarchical multiple regression analyses were conducted to predict HPV vaccination behaviors. All data were analyzed using the IBM Statistical Package for Social Sciences (SPSS) version 25.0.The protocol was reviewed and approved by the Institutional Review Boards (IRBs) of Baylor University (IRB# 1355428) and Kwame Nkrumah University of Science and Technology in Ghana (IRB# CHRPE/AP /126/19).The sample characteristics for adolescents are given in Participant scores for change in a social environment , change in the physical environment , emotional transformation  and practice for change  were relatively high. However, participants’ participatory dialogue  of HPV vaccination  was very low .r) ranging from 0.65–0.92 . Female participants were more likely than males to report a willingness to get the first dose of the vaccine . Participants between ages 12 and 15 years were more likely than those between ages 16 and 18 years to report a willingness to get the recommended doses of the vaccination .Univariate analyses for initiation showed that participants’ sex and age were strongly associated with the likelihood of getting the first dose of HPV vaccination  = 8.488, p < 0.001). After controlling for the sample characteristics in the final model (see Model 3), the perceived beliefs and change in physical environment were the significant predictors of the adolescents likelihood of getting their first dose of HPV vaccination (p < 0.001), with each variable accounting for 6.1% and 8.8% of the variance respectively.In hierarchical multiple regression analyses for initiation see , the resp < 0.001). Participants between ages 12 and 15 years were more likely than those between ages 16 and 18 years to report a willingness to get the recommended series of the vaccination . Females were more likely than males to report a willingness to complete the recommended doses of the vaccines .Univariate analyses for completion showed that age, sex and education were strongly associated with the likelihood of getting the recommended doses of HPV vaccination  = 15.544, p < 0.001). After controlling for the sample characteristics in the final model (Model 3), perceived beliefs, practice for change subscale, and emotional transformation subscale were the significant predictors of the likelihood of completing the recommended series of HPV vaccination (p < 0.001), with each variable accounting for 7.8%, 8.1%, and 1.1% of the variance respectively.In hierarchical multiple regression analyses see , the resIn the current cross-sectional study, we investigated predictors of Ghanaian adolescents’ intention to accept HPV vaccination. Key predictors for initiating vaccination included age, gender, perceived beliefs, and change in the physical environment. The predictors for completing the recommended doses of HPV vaccination included practice for change and emotional transformation.Most of the participants in our study reported that they had not heard about HPV nor HPV vaccination. We also found age, gender, and perceived beliefs about the HPV to be significant predictors for initiating vaccination uptake. These finding are consistent with other studies conducted in low middle-income countries. Researchers report a general lack of awareness about HPV and the availability and need for the HPV vaccine ,41,42,43New findings of interest from our study are (1) the effects of the physical environment on HPV vaccination uptake, (2) how practicing for change predicts completion of the HPV vaccination series, and (3) the effects of emotional transformation on willingness to complete the vaccination uptake. Specifically, we found that participants’ ability to make arrangements for payment, having a place to receive the vaccination, and transportation for HPV vaccination were significantly associated with intentions to obtain the vaccine. Other studies identified physical environment and transportation as a barrier to HPV vaccination ,53; howeWe also found that practicing for change was a significant predictor of completing the vaccination series. Adolescents practicing for change, such as keeping a journal, increases the likelihood of completing the vaccination series. Similar ideas, such as health care providers sending calendar reminders, have been used to increase health behaviors ,55,56.Finally, we found that emotional transformation  significantly influences the adolescent’s willingness to complete the recommended doses of the vaccination. Lloyd et al.’s  study amThe current study has important implications for health care providers and interventions aimed at promoting the initiation and completion of HPV vaccination among adolescents in Ghana. We observed low knowledge and awareness about HPV despite the fact that there are vaccinations available to prevent the effect of HPV diseases. Therefore, it is critical to design and implement intervention studies to educate Ghanaian adolescents. In other studies, effective interventions have focused on education regarding the etiology and provided epidemiological evidence of HPV, its related diseases, available vaccines, and their effectiveness to prevent those diseases ,58,59,60Our findings underscore the gender differences in the likelihood of accepting HPV vaccination. Girls were more likely to report a willingness to initiate and complete the vaccination series. While it is important to educate females about HPV vaccination, it is equally important to develop intervention studies to address other HPV related cancers. Researchers have found that adolescent boys’ willingness to accept vaccination may be enhanced by knowledge of penile, anal, mouth, and throat cancers and the effectiveness of the vaccination in preventing those types of cancers. That said, this result in gender differences should be interpreted with caution because of the small sample size for the adolescent boys.Our study findings also highlight the importance of the physical environment. Our data suggest that changing transportation, payment for vaccination bills, and a place for vaccination may influence adolescents’ vaccine intentions. Though these physical environments are modifiable, for most people in low-and middle-income countries, access to transportation to vaccination facilities, and paying for vaccination bills are challenges that would need government support or policy change to allow health insurance coverage to include HPV vaccination .Additionally, our findings underline the need for clinicians and intervention studies to encourage adolescents to practice the habit of keeping records or journals to remind themselves of the next vaccination scheduled visit, practice the strategies to overcome HPV vaccination barriers, and adjust their regular schedules to accommodate vaccination schedule to enhance the chances of adolescents completing of the recommended series. We also observed that emotional transformation is a significant predictor of adolescents’ likelihood of completing the HPV vaccination series. Future intervention is needed to address the adolescents’ emotional transformation by helping them channel their feelings and emotions, teaching self-motivation, and strategies to overcome self-doubt to enhance their probability of completing the HPV vaccination series.The current findings should be considered in light of the study’s limitations and strengths. The limitations include the nature of the study design as a cross-sectional study because it can introduce response, recall, and selection biases. Our outcomes are based on self-reported intention to initiate and complete the HPV vaccine series. We were unable to verify vaccination status through vaccine records. The use of a convenience sample limits the generalizability of the study findings to only study participants. The small sample size of 25 adolescent boys is a limitation in the study. A future study should include a larger sample size of boys to better understand the association of sex and HPV vaccination uptake. Finally, the lack of data from parents who are key in decision making for vaccination is a major limitation. However, knowing factors that influence adolescents’ likelihood of accepting the HPV vaccination can help guide a future intervention to facilitate adolescents-parent communication about HPV vaccination . Also, pIn conclusion, the findings underscore the lack of awareness of HPV related cancers among adolescents in Ghana. The likelihood of the HPV vaccination initiation key predictors included perceived beliefs and a change in the physical environment. Perceived beliefs, practice for change, and emotional transformation were key predictors of HPV series completion. Future interventions should address these modifiable factors to increase HPV vaccination uptake among adolescents in Ghana.


Background: The number of cases with cervical cancer is rapidly increasing in Sub-Saharan Africa driven by inadequate rates of human papilloma virus (HPV) vaccination and screening programs and accompanied by poor health delivery systems. There are other factors to contend with such as lack of awareness, social myths, reluctance to vaccine acceptance and stigma with sexually transmitted diseases. Here, we formulate strategies to implement intervention programs against HPV infections and other risk factors for cervical cancer in these countries. Methods: We searched PubMed, Web of Science, and African Journals Online for this review. The current status of anti-HPV vaccination and precancerous screening programs in Mali and Senegal has been assessed by onsite visits. Collaborators from Mali and Senegal collected data and information concerning HPV vaccination and screening programs in these countries. Findings: We found that anti-HPV vaccination and cervical cancer screening have been conducted sporadically mainly in urban areas of Mali and Senegal. No known population-based programs are in progress in either of the two countries. We highlighted the advantages and drawbacks of currently available screening tests and proposed that screening by visual inspection with acetic acid (VIA) accompanied by self-sampling is the most cost-effective, culturally acceptable and most feasible strategy to implement in primary care settings. In addition, HPV DNA testing would be affordable, if local laboratory facilities could be established. We found that many of the factors that increase HPV acquisition and promote the oncogenic effect of the virus are largely widespread in both Senegal and Mali. These include infections with HIV and other sexually transmitted infections (STIs), immunosuppression, polygamous marriages, high parity, early sexual activities, early pregnancies, and multiple sexual partners. Interpretation: Neither vaccines nor screening tests are within the reach of the population in Mali and Senegal because of the high cost. The effective intervention measure would be to integrate anti-HPV vaccines into the Extended Program for Immunization (EPI), which has saved 3 million young lives per year in Africa with the support of GAVI, to implement cost control mechanisms for HPV vaccinations via price negotiations with manufacturing companies, as has recently been done by Rwanda. The collective efforts by local governments, researchers, private sector, and donors may lead to the introduction of affordable screening tests. A robust awareness campaign coupled with sustained and regular engagement of local communities about the prevention and risk factors is extremely important. The projected solutions may be well applicable to other Sub-Saharan countries that face similar challenges containing cervical cancer. The incidence of cervical cancer has been greatly reduced in high-income countries by the use of human papilloma virus (HPV) vaccines and cervical cancer screenings. However, its incidence is on the rise and is second only to breast cancer in low-income countries, especially in Sub-Sahara ,2. CerviThis paper reviews the current status of anti-HPV vaccination and precancerous screening programs in two Sub-Saharan countries, namely Mali and Senegal. We advocate for cost control mechanisms, increased awareness, and the implementation of extended vaccination and screening programs. We formulate strategies to implement intervention programs against HPV infections and other risk factors for cervical cancer in these countries. Despite the current challenges, the proposed strategies present feasible solutions to eliminate cervical cancer in the coming decades from this part of the world.pRB, which are known to control a board range of cellular processes [HPV is a highly infectious virus transmitted through oral, anal, or genital sexual contact, as well as through nonpenetrative sex involving skin-to-skin contact . It is erocesses .HPV is not the only cause of cervical cancer . The proThree vaccines, namely Cervarix , Gardasil, and Gardsil9 , were developed to prevent HPV-16- and -18-related cancers. Cervarix and the first-generation HPV vaccine, Gardasil, can prevent about 70–84% of cervical cancers . A next-The best time to get the vaccine is before young girls or boys become sexually active. This is because the vaccine works best before there is any chance of infection with HPV.Over the last decade, schoolgirls across the USA and other developed countries have routinely received HPV vaccines, when they are 11–13 years old . StudiesThe Strategic Advisory Group of Experts (SAGE) of the World Health Organization (WHO) recommended a two-dose schedule of an HPV vaccine for girls below 15 years of age in the year 2014. An ongoing study from India reported that the immunogenicity in 15–18-year-old recipients with two doses of a quadrivalent HPV vaccine was noninferior to that in the 15–18-year-old recipients with three doses . The comThe literature suggested the effect of a vaccine lasts from six to 10 years ; howeverScreenings can detect cancer at an early stage, ensuring the treatment and follow-up of those cancers with confirmed HPV infection . VariousInvasive cancers are rare before the age of 30 years; screening women, who are too young, could cause adverse reactions through the detection and unnecessary treatment of spontaneously regressing low-grade lesions. The WHO recommends that countries ensure women from 30 to 49 years are screened at least once in their lifetime .In Mali, cervical cancer is the most common form of cancer with the majority of infections occurring in the 15–24 years age range . The incCervical cancer happens to be the most commonly occurring form of cancer in Senegal as well; the incidence rate is comparable to that of Mali, which is 37.8 per 100,000 women . ReportsVarious risk factors, some of which promote HPV acquisition and others that propagate the oncogenesis of cervical cancer, are rampant in both Mali and Senegal . For exaChlamydia trachomatis and Neisseria gonorrhea have all been linked to an increased risk of cervical cancer [Malnutrition in conjunction with infections endemic to Sub-Sahara  has left the population with a compromised immune system. Reproductive tract infections are also prevalent in the region. Recent studies have implicated other STIs besides HPV with cervical cancer; Herpes simplex type 2, l cancer . InfectiSeventy percent of the world’s cases of HIV are diagnosed in Sub-Saharan Africa . The preThere is a significant shortage of trained healthcare personnel, which affects access to health services in Sub-Saharan countries. In 2006, the WHO estimated that Africa had a needs-based shortage of 818,000 healthcare professionals  based on a country needing 2.28 healthcare professionals per 1000 population . Adding Anti-HPV immunization has been conducted sporadically in Mali and Senegal (mainly in urban areas); however, the short-term or long-term impact on the incidence of cervical cancer is not yet known. Ongoing immunization programs against common infectious diseases have been implemented in Mali through the support of GAVI. This program has provided Gardasil vaccine since September 2012 for girls aged 9 to 13 years. A pilot study for the introduction of HPV vaccines has been conducted in two districts, one in an urban area of Bamako and another in a rural area of the Fana district. The results of these studies are not yet available.Similarly, the Senegalese government has also launched an HPV vaccination campaign with the support of GAVI, which started in November 2017. However, HPV vaccines are not yet widely available for the implementation of population-based vaccination programs. Few data are available regarding the prevalence and distribution of HPV types in Senegal. These epidemiological studies have potential implications for vaccination programs.Overall, cervical cancer screenings are seldom performed in Mali. One study revealed that 82.5% of participants (n = 166) tested positive for either of the high-risk HPV types 16 or 18 . The samLimited access to health care services has left Mali with low rates of coverage for many life-saving and preventative and curative interventions. Mali lacks economic resources and healthcare infrastructure to implement routine cervical cancer screens; these services are currently only offered at a limited number of district health centers, where physicians or midwives could provide screening by VIA upon request . TreatmeNo known population-based screening program is in progress in Senegal. Random screenings are occasionally performed in urban areas and less frequently in rural areas. It is estimated that 4.43 million (out of a population of 15.85 million) females over the age of 15 are at risk of contracting cervical tumors . About 2Studies revealed an incidence rate of precancerous lesions that ranged from 5.7% to 28% in Senegal, which is comparable to a 10.1% incidence rate in similar low-income countries . A cohorLike Mali, the rate of routine cervical cancer screening throughout Senegal is very low, especially among older women and those living in rural areas . Only abp < 0.01) than seronegative sex workers [A higher proportion of sex workers in urban areas of Dakar, the capital of Senegal, is affected by HPV infection; these rates are estimated to be nearly 10 times higher than that reported among women in the general population . It was  workers . ClearlyCervical cancer is not yet recognized as an important public health problem in Sub-Saharan countries. Promoting awareness regarding causes and potential remedies should be considered as a critical component of any program tackling cervical cancer . StudiesAn important lesson learned from HIV–AIDS responses is that civil society and communities have to be at the center of humanitarian responses. Community meetings and considerations of local customs could be helpful prior to undertaking vaccination and cancer screening; this process would demonstrate support from community leaders and provide a safe space for community members to ask questions and gain a greater understanding about the purposes of these procedures . The devThe Extended Programs for Immunization (EPI) were introduced into many low-resource countries, and the EPI is believed to save 3 million young lives per year in Africa. The integration of anti-HPV vaccines into the EPI is likely to increase this number by preventing thousands of deaths from cervical cancer yearly . An effeIt is important to strengthen local cervical cancer screening capacity and raise national-level policy stakeholder awareness in Sub-Saharan countries. The introduction of combinational VIA and HPV DNA testing for cervical cancer screening needs to be considered. Both testing permits self-sampling with VIA being easy to perform, and once lab facilities are created for HPV testing, several samples can be analyzed in a single day. VIA may be used as an approach for a population-level epidemiological prevalence study in areas, where other screening modalities are rarely available or are too expensive . OrganizThe literature suggested that self-sampling is just as effective as clinician-obtained samples and has a high rate of acceptance among women ,51. A stDue to the lack of routine screenings, most women in these resource-constrained countries are present with such an advanced disease, and the only suitable treatment options are palliation. In most Sub-Saharan countries, palliative treatments are extremely limited. For instance, oral morphine is only available in 11 African countries . Those, The authors of this article (while onsite visits) found that the cost of HPV vaccination and screening tests such as Pap tests (if available in private clinics) is exceedingly high in Mali and Senegal and thus is beyond the reach of the general population. The yearly GDPs per capita in Senegal and Mali are $1546.50 and $901, respectively. Gardasil has been approved by Malian Food and Drug Administration since 2008 but is still not available in the public health sector. Both Cerverix and Gardasil are only available in a few private pharmacies in these countries and are not affordable even to middle-class Malian or Senegalese individuals. The retail prices for Gardasil and Cervarix are as high as US$150–$190 per dose . Merck aClearly, cost poses the most significant barrier to low-income countries for developing broadly available HPV immunization and cancer screening services; negotiating vaccine prices with vaccine manufactures could be one way to address this barrier. For example, Merck has a patient assistance program in the USA for individuals, who do not have health insurance; in Rwanda, this vaccine manufacturer has already agreed to supply vaccines at a lower price, demonstrating that such negotiations are indeed feasible.In the last decade, there has been significant growth in the capabilities of suppliers and vaccine manufacturers located in developing countries for various other vaccines; however, this trend did not include HPV vaccines . OpportuSub-Saharan countries are unduly burdened by cervical cancer. National HPV vaccination and screening programs could significantly reduce the incidence of cervical cancer. Until these programs are effectively introduced, measures to raise awareness about the prevention and risk factors implicating local communities are extremely important.Presently, the exorbitant cost of vaccines and screening tests is beyond the reach of the majority of Sub-Saharan citizens, and in fact, cervical cancer is considered as a disease of poverty. It can be hoped that through the collective efforts between governments, communities, donors, the private sector, innovators, researchers, and pharmaceutical companies, productive synergies could save thousands of lives from a cancer that is realistically eradicable.


According to their oncogenic properties, Human Papillomaviruses (HPVs) are classified into two types: Low-Risk (LR-HPVs) and High-Risk Human Papillomaviruses (HR-HPVs). The immune system naturally controls the majority of HPV infections; however, when the HR-HPV infection is persistent, the risk of developing cervical cancer increases. Previous studies indicate that multiple-infection or coinfection with HR-HPV occurs frequently and can potentiate the development of cervical lesions. This study aimed to establish the HPV coinfection rate in squamous intraepithelial lesions from Mexican patients. For HPV detection, we performed PCR on 55 cervical lesions diagnosed by colposcopy. We detected the presence of HPV infection in 87.27% (48/55) of the lesions; interestingly, HPV coinfection was observed in 70.83% (34/48) of these samples. We also evaluated HPV infection in adjacent areas without morphological changes from 25 samples. The results showed that 80% (20/25) of these were HPV-positive and, curiously, all presented HPV-16 infection. In conclusion, our results revealed a high prevalence of HPV coinfection in cervical lesions in Mexican patients, and these results contribute to future research focused on the role that HPV coinfection plays in the development of cervical cancer. Cervical cancer (CC) is the second type of cancer among women in developing countries and is the primary neoplasm associated with Human Papillomavirus (HPV) infection . Late deThere are more than 200 HPV types, of which 12-15 are classified as HR-HPV , 7. CervIn this study, we analyzed multiple HPV types of infection in biopsies from Mexican patients diagnosed with cervical lesions and CC. Our data revealed the presence of multiple HPV-type infections in the majority of samples with lesion (70.83%), but only 30% in adjacent tissue without morphological changes.Biopsies were obtained from Mexican women seen at Hospital IMSS “La Raza” (Mexico City) and “Hospital Juárez de México” (Mexico City) and who were pathologically diagnosed with Atypical Squamous Cells of Undetermined Significance (ASCUS), Low-grade Squamous Intraepithelial Lesion (LSIL), High-grade Squamous Intraepithelial Lesion (HSIL), or Cervical Cancer (CC). The present study conducted according to the Declaration of Helsinki for Medical Protocol and Ethics, and the Institutional Committee of Research and Ethics approved the study (registration no. HMJ 2231/13-B). Expert colposcopists performed all examinations, and samples were classified according to the International Federation of Gynecology and Obstetrics (FIGO) . Fifty-fBiopsies were placed in Tissue-Tek medium  for their preservation and stored at −80°C prior to DNA extraction.gapdh gene was performed to test sample sufficiency employing the template 100 ng of DNA and targeting a 140-bp sequence with the following primers: forward: 5′-AGGTGACACTATAGAATAACCGTCAAGGC-3′; and reverse: 5′-GTACGHACTCACTATAGGGATGGTGGTGA-3′. To detect HPV, we performed PCR assays using the PGMY09/PGMY11 primers  targeting a 450-bp sequence of the L1 gene [Samples were mechanically disrupted and subsequently homogenized in lysis buffer for DNA extraction according to the established protocol by the DNA/RNA All Prep Kit . Amplification of the  L1 gene . Accordit-test independence among variables, analysis of variance (ANOVA) was utilized to compare groups. Odds ratios (OR), calculated with the Medcalc® and StatSoft 8.0® software, were determined to estimate the association of genotypes with cervical carcinoma. P values of < 0.05 were considered statistically significant.A woman (biopsy) infected with more than one HPV genotype was considered to have a multiple HPV-type infection or coinfection. To determine Student's This study included biopsies from 55 patients with a mean age of 42.5 years. Samples were grouped according to their pathological diagnosis. Thus, we analyzed eight biopsies diagnosed with ASCUS (15%), 25 biopsies diagnosed with LSIL (45%), 11 biopsies diagnosed with HSIL (20%), and 11 biopsies diagnosed with CC (20%). Moreover, from 25 of these biopsies , we obtained a sample of adjacent tissue without morphological changes.Overall, 48 of 55 biopsies with cervical lesions were HPV positive; therefore, HPV prevalence was 87.27%; 95.83% (46/48) of HPV-positive biopsies contained HR-HPV  correspoInterestingly, we found that 70.83% (34/48) of HPV-positive samples presented more than one HPV genotype (coinfection), while the remained only had a single infection . Of the In that, the most frequent HR-HPVs in CC at worldwide are HPV-16 and HPV-18 ; we evalThe OR statistical analysis revealed that coinfection with more than one HPV type has a probability of 97.6% of developing LSIL . This esn = 8) contained a single infection; while 40% (4/10) of biopsies without morphological changes from patients diagnosed with HSIL presented coinfection, and one of these showed four different HR-HPV genotypes. Finally, the two biopsies without morphological changes from patients diagnosed with LSIL and who were HPV-infected revealed coinfection , 89% (8/9) from patients with CC, and 33% (2/6) from patients with LSIL were HPV-positive. Although the percentages of HPV-positive samples were similar between cervical lesions and adjacent regions without morphological changes, the proportion of HPV coinfection was different. Samples without morphological changes but that were HPV-positive exhibited only HR-HPV genotypes with eight different HPV. All of these samples contained HPV-16 ; 70% . Moreover, the percentage of negativity (12.73%) in our samples could be related to the PCR type employed (simple PCR with primers MY09/MY11). In addition, we cannot rule out that negative cases in our study may be applicable to viral load, which could be below the severity of the technique. Other studies have concluded that utilizing a nested PCR using the MY09/MY11 and GP5+/GP6+ primers can significantly increase the sensitivity from 10 to 100 times, compared with the simple PCR employing only the MY09/MY11 primers [Here, we analyzed the presence of HPV single infection and coinfection in Mexican patients with different cervical lesions. Our data showed an 87.2% HPV detection rate in these samples, while previous studies reported rates as high as 98% , and thegativity .73% in o primers .P = 0.235). The data suggest that, although HPV infection is involved in the development of cervical lesions, it is not sufficient to predict the injury type that could be produced. In agreement with previous reports, these results suggest that other factors might be implicated in the development of each lesion type, including the viral genotype and the presence of more than one viral type [Nevertheless, we found an HPV incidence greater than or equal to 80%, regardless of the injury type diagnosed. However, we did not detect a correlation between the presence of infection and lesion type  than HSIL  or cervical cancer . This result supports the hypothesis that the subsequent accumulation of different infections can be a marker of persistent infection and, eventually, a possible risk factor for the progression of the lesion.Studies of HPV coinfection in Mexico are scarce to date; Aguilar-Lemarroy et al.  reportedAnother key factor in the development of cervical lesions is the presence of simple or mixed coinfections . We obseIt is noteworthy that 36% of the observed coinfection contains HPV-16; similarly, López-Rivera et al.  reportedP ≤ 0.05) in mind, we were able to predict that all these regions have a 91% chance of developing cervical cancer. It is noteworthy that these results are not comparable with those found in the literature, because majority of studies only genotypify the HPV found in intraepithelial injuries, but not in the areas adjacent to the cervical lesion. In this regard, it is important to note that these results highlighted the importance of studies about viral genotyping, even if patients do not exhibit any injury by colposcopy, as it would aid in timely diagnosis and appropriate treatment for the prevention of the development of intraepithelial lesions and cervical cancer. In recent years, several epidemiological studies have mentioned the presence of single infections and coinfections in control groups. For instance, Aguilar-Lemarroy [Regarding results about the adjacent regions without morphological changes, the prevalence of HPV infection (80%) was similar to that found in lesion biopsies. However, coinfection was low (30%) in the adjacent areas without morphological changes of patients with cervical lesions; although, all of these HPV-positive areas could develop some injury over time, considering that the prevalence of HPV-16 in these regions was 100%. Bearing these data and the OR results (HPV-16/CC, OR = 10.3, Lemarroy  reportedIn summary, our data demonstrate the incidence of multiple HPV-type infections in the different grades of the cervical lesion. These data may help to develop better clinical strategies against cervical lesions caused by HPV coinfections and in the search for a more efficient treatment.A total of 87.27% of Mexican patients with cervical lesions studied here demonstrated HPV infection, and the most frequent genotypes were HPV-16, HPV-31, HPV-51, and HPV-18. The prevalence of HPV coinfection in the cervical lesions was 70.83%, and coinfection with certain types correlates with the type of cervical lesion, determining that these factors can be used as a predictor of the degree of the injury; in particular, HPV-16 increases the chance to develop cervical cancer. Moreover, HPV detection in healthy areas could be a predictor for the development of injury, given that prevalence of infection in these regions was 80%.


Background. Human papillomavirus (HPV) infection has been recognized as an important risk factor in cancer. The purpose of this systematic review and meta-analysis was to determine the prevalence and effect size of association between salivary HPV DNA and the risk of developing oral and oropharyngeal cancer. Methods. A systematic literature search of PubMed, EMBASE, Web of Science, LILACS, Scopus and the Cochrane Library was performed, without language restrictions or specified start date. Pooled data were analyzed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). Quality assessment was performed using the Newcastle–Ottawa Scale (NOS). Results. A total of 1672 studies were screened and 14 met inclusion criteria for the meta-analysis. The overall prevalence of salivary HPV DNA for oral and oropharyngeal carcinoma was 43.2%, and the prevalence of salivary HPV16 genotype was 27.5%. Pooled results showed a significant association between salivary HPV and oral and oropharyngeal cancer , oral cancer  and oropharyngeal cancer . Significant associations were also found between salivary HPV16 and oral and oropharyngeal cancer , oral cancer  and oropharyngeal cancer . Conclusions. Our meta-analysis demonstrated the association between salivary HPV infection and the incidence of oral and oropharyngeal cancer indicating its value as a predictive indicator. Human papillomavirus (HPV) infection has been recognized as an important risk factor in a subset of head and neck squamous cell carcinomas, independently of traditional risk factors such as tobacco or alcohol use ,2. GlobaTP53 and Rb (retinoblastoma tumor suppressor gene) [HPVs are small, non-enveloped, close-circular, double-stranded DNA viruses of approximately 8000 base-pairs which present a specific tissue tropism infecting epithelial cells of the skin and mucosae of the anogenital and upper aero-digestive tract . More thor gene) . A subseor gene) . In addior gene) ,11,12 asor gene) . Currentor gene) , howeveror gene) . Nowadays, a variety of molecular biological methods have been developed for the detection and genotyping of HPV at DNA, mRNA, and protein levels by polymerase chain reaction (PCR), real-time PCR, in situ hybridization, immunohistochemistry and serum antibody assays . In addiThis study was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines  and the The systematic literature search was performed in PubMed, EMBASE, Web of Science, LILACS, Scopus and the Cochrane Library through 9 January 2020, without language restrictions or specified start date. The following combinations of keywords and medical subject headings were used: (human papilloma virus OR HPV) AND  AND . All studies were screened based on the title and abstract, and eligible manuscripts were retrieved for full-text review. Additionally, we manually searched the reference lists in each original and review article in order to avoid missing potential studies. The literature search was performed independently by two researchers (ORG and MMSC), and any disagreements were resolved by consensus. The studies selected through the search strategy and other references were managed using RefWorks software, and duplicated items were removed using the associated tools.We included the studies that met the following criteria: (1) case-control studies of patients with oral and/or oropharyngeal cancer and healthy controls, (2) HPV DNA prevalence determined in salivary samples , and (3) sufficient data to calculate odds ratios (ORs) with 95% confidence intervals (CIs). The exclusion criteria were as follows: (1) in vitro or animal study, (2) reviews, letters, personal opinions, book chapters, case reports, and conference abstracts, and (3) duplicate articles or suspicion of data overlap.Two researchers (ORG and MMSC) independently assessed each eligible manuscript, extracted data using a pre-established form, and collated the data into a Microsoft Excel spreadsheet . Any disagreement among reviewers was resolved by consensus. The following information was extracted from each study: author, publication year, country, type of sample, method of collection, tumor location, sample size, HPV detection method, number of cases and HPV-positive cases, number of controls and HPV-positive controls, HPV-positive genotypes, overall HPV DNA prevalence (number of subjects testing positive for any HPV type) and type-specific HPV DNA prevalence . If the required data were incomplete, attempts were made to contact the authors to obtain the missing information.The Newcastle-Ottawa Scale (NOS)  was usedhttps://www.r-project.org). Firstly, to evaluate the statistical model applied to the meta analytic database, heterogeneity was assessed using the Cochran’s Q statistic test-based Chi-squared test and I2 statistics. Heterogeneity was considered significant when I2 > 50% and/or presence of a p < 0.10 for the Cochran’s Q test. The prevalence of HPV DNA and HPV genotypes in oral and/or oropharyngeal cancer was calculated using fixed or random effects depending on the heterogeneity. The relationship between saliva HPV DNA infection and oral and/oropharyngeal cancer risk was evaluated by pooled odds ratio (OR) and 95% confidence intervals (CIs) comparing cases to controls. If significant heterogeneity was detected, the DerSimonian and Laird random-effects model was applied to calculate the pooled OR with 95% CIs; otherwise, the Mantel–Haenszel fixed-effects model was used. Then, subgroup analyses were performed to explore the potential sources of heterogeneity among studies according to the anatomic tumor location and HPV genotypes. Additionally, publication bias was checked with Begg’s and Egger’s tests and by visual inspection in funnel plots demonstrating the relationship between the individual log ORs and their standard errors [p-values of <0.05 were considered to indicate statistical significance. Statistical analysis was conducted using the meta package of free R software ; controls under risk conditions (2); suspicious of data overlap (3); insufficient data (3); and reviews, letters, and meta-analysis (4). Finally, 14 articles met all the inclusion criteria and were included in the final analysis. A detailed flowchart showing the selection process is shown in n = 7, 50%, respectively) were analyzed for HPV positivity and genotyping. The methods most used for saliva HPV-DNA determination were conventional PCR, nested PCR and quantitative PCR. However, other analytical strategies such as next generation sequencing or immunoassays were also employed for salivary HPV genotyping  while the infection rate in the healthy control group was of 8.9% (n = 2304). Salivary HPV16 was the most common type of HPV DNA positive cases (n = 1116), representing 27.5%   . p = 0.159; Begg’s test, p = 0.298) distribution of the studies, indicating no evidence of publication bias (A random-effects model was used because heterogeneity was identified among the 14 studies (I2 = 82%). Visual inspection of the funnel plot revealed a symmetrical . However, salivary HPV18 did not show any significant increased risk for oral and oropharyngeal cancer with a pooled OR of 1.80 (95% CI = 0.66−4.90). In addition, a significant association was found for salivary HR-HPV with OR of 5.94 , whereas salivary LR-HPV did not show any significant increased risk with OR of 1.45 (95% CI = 0.70−2.98). The respective funnel plots are represented in For the type-specific analysis , salivarp < 0.01)  . p < 0.01) whereas salivary HPV18 showed no significant association with a pooled OR of 1.92 (95% CI = 0.63−5.91). In addition, a significant association was found for salivary HR-HPV with a pooled OR of 26.69  whereas no significant association was found for salivary LR-HPV with a pooled OR of 2.08 (95% CI = 0.75−5.81). Their respective funnel plots are shown in According to type-specific analysis , salivarp < 0.01) (p = 0.02), whereas no significant association was observed for salivary HPV18 with a pooled OR of 1.51 (95% CI = 0.45−5.15). In addition, a significant association was observed for salivary HR-HPV with a pooled OR of 4.44 . However, salivary LR-HPV did not show any significantly increased risk for oral cancer with OR of 1.79 (95% CI = 0.67−4.74). Their respective funnel plots are shown in Our subgroup meta-analysis consisted of 12 studies, including 658 cases and 2210 controls. In the pooled analysis, salivary HPV DNA infection and oral cancer showed a significant association with a pooled OR of 2.58  . Accordi < 0.01) , salivarIn the present study the overall pooled prevalence of salivary HPV-related to oral and oropharyngeal cancer was 43.2%. Similarly, a meta-analysis based on 11 case-control studies evaluating the HPV infection in oral and oropharyngeal cancer found an HPV DNA prevalence of 39.27% . In termOverall, in our study, salivary HPV16 was the most commonly detected oncogenic type, accounting for around 28% of cases. As we expected, salivary HPV16 showed a higher prevalence in oropharyngeal cancer 39.6%) than oral cancer (18.6%), in accordance with previous studies ,39,40. I.6% than To the best of our knowledge, this is the first meta-analysis evaluating the association between salivary HPV and oral and/or oropharyngeal cancer. The pooled OR showed that oral and oropharyngeal cancer patients had an almost five-fold higher risk of HPV infection than controls. A previous meta-analysis evaluating the presence of HPV in oral and oropharyngeal cancer detected by different methods  reported a significant association with an OR of 2.82 . OverallAll the studies included in the present meta-analysis addressed HPV status in oral exfoliated cells collected from saliva with or without oral rinses. In this sense, the first association between oral HPV and oral cancer was reported by Smith et al. . These aOur study has several strengths. It is the first meta-analysis highlighting the association between salivary HPV infection and oral and/or oropharyngeal cancer. Moreover, we examined both the overall and the specific prevalence of salivary HPV DNA in oral and/or oropharyngeal cancer. In addition, we performed a comprehensive literature review without language restrictions and the results of our study were in concordance with the scientific evidence. However, the present study is not exempt from limitations. Firstly, the studies included in our meta-analysis were heterogeneous, which could be explained by different factors such as ethnicity, sample size, geographic region, anatomic tumor location, method of HPV detection and different HPV genotypes. Although we performed a subgroup analysis by anatomic tumor location and HPV genotypes, we were unable to elucidate the potential sources contributing to this heterogeneity. Secondly, data such as age, smoking, drinking, sexual habits or diet were not provided by the studies in our sample, hampering the assessment of these confounding variables. Thirdly, some studies included in our analysis could be biased due to the fact that cases and controls were not matched for demographic variables such as age, sex and lifestyle habits. In addition, although almost all these studies analyzed HPV16 and HPV18, we observed high variability regarding HPV genotypes and HPV detection methods, which could substantially affect the results of our analysis. To the best of our knowledge, this is the first meta-analysis addressing the association between salivary HPV infection and oral and oropharyngeal carcinoma. The findings of this meta-analysis provide additional evidence that salivary HPV is associated with oral and oropharyngeal cancer, suggesting that salivary HPV infection is a risk factor for oral and oropharyngeal cancer. However, to validate our findings, future research should focus on prospective cohort studies that explore the occurrence of salivary HPV infection in oral and oropharyngeal cancer. In addition, it is necessary to analyze confounding variables that might be associated with an increased risk of HPV infection in oral and oropharyngeal cancer.


Callinectes sapidus, the 3D-nanoarchitecture is color-specific, while the blue and red-orange pigments interplay in different nano-sized channels and pores. Thinnest pores of about 20  nm are found in blue shell. Additionally, the blue pigment co-existence in specific Bouligand structure is proved for the green crab Carcinus aestuarii, although the crab does not appear blue. The pigments interplay, simultaneously detected by Raman spectroscopy in color-specific native cuticles, overturns our understanding in crustaceans coloration and may trigger the selective use of particular colored natural nanoarchitectures for broaden area of applications.3D-engineered nano-architectures with various functionalities are still difficult to obtain and translate for real-world applications. However, such nanomaterials are naturally abundant and yet wasted, but could trigger huge interest for blue bioeconomy, provided that our understanding of their ultrastructure-function is achieved. To date, the Bouligand pattern in crustaceans shell structure is believed to be unique. Here we demonstrated that in blue crab  Nikola Tesla would have been surprised to find out that thinking “in terms of energy, frequency, and vibration”, the secrets of the blue crab colors could be elucidated.5, seen by seafood producers both as a threat to benthic shellfish cultures6 and a potential new commodity in the invaded areas6, loved by gourmands as a delicacy, the Atlantic blue crab, Callinectes sapidus, poses an increasing interest, as its wasted shells could be potentially turned in valuable by-product, not only because of chitin, proteins7 and biogenic calcite content, but also as natural, porous, pigmented biomaterial, yet poorly understood as highly ordered nano-architecture posing inspiration for biomimetics8.Accounted by ecologists among 100 worst invasive alien species in eastern Mediterranean coastC. sapidus cuticle simultaneously features bright blue, red and white anatomical exoskeleton counterparts , an orange carotenoid is non-covalently bonded in crustacyanins21. UV/Vis absorption band shifted from 480  nm in ATX monomers12 to 632, 585–595 and 610  nm in isolated α-, β- and γ-crustacyanin12 respectively, suggests the opportunity to exploit vibrational resonance Raman (RR) spectroscopy to selectively detect free or non-covalently bound ATX in custacyanin in intact crab shell by-product.Interestingly, 15, and more recent computational and experimental approaches26. For instance, Gamiz-Hernandez et al.19 showed that β-crustacyanin (not α-) is the responsible pigment for the blue color. Based on theoretical calculations19, it was found that β-crustacyanin (comprising two stacked ATX molecules), induced a bathochromic shift of ATX arising from the polarization effects and steric constrains of the ATX-protein binding site. On the other hand, based on the available models of crustacyanins, van Wijk et al.17 proposed a protonation model involving the keto-groups of the ATX terminals near a water molecule. Controversial conclusions underlying the comparative results of the cuticle ultrastructure in lobster H. americanus raised additional questions regarding its crystalline nature28. The “universal” nature of the 3D- hierarchical calcite arrays which follows a twisting plywood path (Bouligand-type pattern) in crustacean exoskeleton has been suggested, based on studies on several species of crabs32, but lacking any connection with the pigments populating the morphological pattern. Katsikini33 detected ATX only in C. sapidus blue crab cuticle, due to the use of a single laser line (488  nm), which fulfilled the RR conditions for the respective carotenoid only, but suggested that Br and Sr are involved in two different mechanisms contributing to the blue color.It is also surprising that the current knowledge on the blue coloration in crustaceans remains controversial, considering the numerous earlier studiesCurrently, comprehensive understanding of chemical structure-morphology relationship in blue crab shell is absent, although of high interest for transforming such aquatic porous by-product into added-value material within the blue bioeconomy goal.C. sapidus is not exclusively determined by the presence blue pigment, astaxanthin bounded in crustacyanin. Moreover the blue pigment is present in other crab cuticle colors, not necessarily blue.Here, relying on combinatory multi-laser RR micro-spectroscopy and imaging, assisted by electron microscopy, X-ray diffraction and computational chemistry, we show that two interplayed pigments and their distribution in the 3D-nanoarchitecture of the plywood path is color-specific and the blue shell of 2 species, Carcinus aestuarii green crab. The ultimate goal is to properly address such wasted biogenic material within the blue bioeconomy concept.Comparatively, we employed a co-inhabitant, native33  and, yet limited, RR scattering data of one blue shell33. Exoskeleton photonic crystal would be sustained, provided that the ultrastructure periodicity influences the propagation of certain light wavelengths and its dielectric properties are tuned by its morphological biochemistry.We hypothesize an additional photonic color component to the overall blue cuticle appearance, sustained by its optical propertiesC. sapidus, and the green C. aestuarii crab. The type, number of samples and experimental purpose of all the samples used in the present study are summarized in the Supplementary Table The complete experimental approach to extract information on pigments identity and their native distribution in crabs exoskeleton ultrastructure is summarized in the Fig. ν1(C=C) + ν2 (C-C) linear combination showed distinct position for ATX and ncb-ATX, the latter being 22  cm−1 downshifted. Comparative display of the carotenoid ν1(C=C) Raman band positions in spectra acquired from the four shell types with the three laser lines are showed in the Supplementary Fig. Raman spectra collected from each crab shell color are strongly dependent on the laser excitation wavelength and highlights the free or non-covalently bond astaxanthin (ncb-ATX) occurrence in the colored shells when proper laser excitation is applied  comprised two Raman band components, one strong band assigned to the ν1(C=C) skeletal stretching mode of ATX above 1500  cm−1 and a weaker band (or shoulder) in the 1491–1493  cm−1 range, attributable to the same ν1(C=C) mode in ncb-ATX in carotenoprotein. By contrary, full resonance Raman excitation of carotenoprotein with red laser (632.8  nm) line , which are neither resonant to ATX nor its protein complex, always featured a weak shoulder of free ATX band around 1514  cmThe peaks position was reproducible among animals  involved in strong dipole-dipole interactions, which simulated non-covalent binding to the protein carrier, exhibited v1 mode red shifted to 1494  cm−1  for the RR spectrum of non-covalently bond ATX (ncb-ATX) and compared it to its free counterpart, to correctly assign the experimentally observed Raman data. Computed Raman modes of isolated ATX featured theoretical v1, v2, v3 and v4 modes of ATX or ncb-ATX in the high wavenumber range (2100–3100  cm−1) confirmed the existence of two pigments in the blue shell: they corresponded to ATX modes . The difference in position of C=C modes above 1500  cm−1 can be attributed to stretching modes of conjugated C=C bonds of free carotenoids. The C=C mode arising from the ncb-ATX complex consistently appeared 1492  cm−1  of the two main modes contributing to the overall v1 Raman band, assigned to the ncb-ATX and ATX. The near-infrared 785  nm laser line was used for this purpose, as it provides normal Raman scattering only, thus avoiding resonance Raman contribution of either pigment. As the intensity of normal Raman scattering is directly proportional to the analyte concentration, for similar excitation conditions and collecting optics, the intensity ratio (R = I1492/I1514), gives an estimation of relationship between the content of ncb-ATX and free ATX chromophores in each native shell color. The calculated values of the R ratios defined above are shown in the Table To further clarify the different colors of shells which contain the same pigments, we calculated the relative Raman intensity ratio R = I149/I1514 ofNote the highest value of R = 8.487 ± 0.283, for the blue color, almost 3 times higher than in the green shell, and more than 11 times higher than in the red shell, meaning that blue shells contain the most ncb-AXT chromophores relative to AXT. Due to the natural inhomogeneity of shell surface color, the considerable standard error values are not surprising. Carotenoid signal was most frequently absent from white shells spectra with non-resonant 785  nm excitation  collected from blue shell cross section at 20, 80, 140, 200, 240, 300, 360 and 420 μm distance from margin showed in the Fig. v1 band was tracked, as showed in the Fig. The ATX and ncb-ATX exhibited well-defined Raman signal collected from the fracture surface and the intensity evolution of carotenoid v1 Raman band  or ethanol to probe pigments extraction, Raman analysis of same shell fragments showed that intact ncb-ATX in crustacyanin complex is prerequisite for maintenance of shell native color. Each treatment resulted in complete dissociation of the complex 35 in calcite, ranging from 1085 to 1088  cm−1, the ν4in plane bending mode36 around 712  cm−1 and well-defined calcite lattice bands at 155 and 281  cm−1 in all cuticle color types  stretching mode indicated the highest calcite crystallinity in red dactyls, followed by green dactyls, white, and blue palm shells. Significant broadening of the v1 mode to the lower wavenumbers, paired with broadening of the v4 and lattice modes indicates the co-existence of amorphous37 CaCO3 in blue and white shells, while broadening of v1 to higher wavenumbers indicates small inclusions of Mg atoms in calcite crystalline lattice36. The area ratio r of the two Raman band components of calcite stretching mode in blue shell showed a value r = area(1085)/area(1075) of 2.82  data obtained with the 1064  nm laser line excitation revealed the prominent symmetric stretching mode of carbonate νet al.40 reported for H. americanus and Cancer pagurus. The highest Mg-calcite pattern was observed in red claw (even higher for its corresponding teeth) followed by the white, green and blue cuticle, as showed in the Fig. 42.The X-ray powder diffraction data Fig.  confirm 32. A series of SEM images collected from fractured cuticles of blue shell is showed in the Fig. C. sapidus and green cuticle of C. aestuarii are given in the Fig. Performing morphology analysis, an ordered, stacked mineral structure was observed, similar to those reported for other crab speciesThe mineral layers are stacked quasi-parallel to the outer surface and their arrangement defines the epicuticles . Furthermore, the spotted pattern of the pigments interplay from Fig. 33. To further check any optical correlation, we measured a series of selected cuticle morphological features  of 477, green 486, red 321 and white 356  nm while the distance between canals was 977  nm in blue, the lowest among the other color cuticles exceeding 1 μm Table . The walThe finding overturns our understanding of the 3D-nanomaterial with Bouligand patterns from blue crab shells, simultaneously exhibiting blue, red and white color.C. sapidus blue, red and white and C. aestuarii green cuticle, respectively. Semi-quantitative EDX elemental analysis  in endocuticle; lowest C, O and Mg level in endocuticle and highest P content in exocuticle. Such feature, could suggest distinct protective role of endocuticle as the thickest load-bearing layer. In line scan of elemental composition, EDX data constantly showed higher content of C associated to corresponding lower content of Ca, Mg and P, particularly in epicuticle, which mostly contains amorphous minerals and waxy lipoproteins45. None of the collected EDX spectra showed any trace of Br or Sr to support the previous consideration33 on the role of these elements in blue crab coloration  supports the shell color distinct feature. Relative mass fractions (wt%) for the 5 main elements  were obtained from 2 to 4 scans  and within certain groups (comparing for certain characters) there were no significant differences. Then we compared series of two groups by ANOVA and by t-test as well, to differentiate the significance between groups. The results were the same  using t test or using ANOVA. Multiple t test was not used here.Statistical analysis revealed the following: channel diameter measurements gave highly significant difference when comparing white-blue (p = 0.0025), white-green (p = 0.0033), blue-red (p = 0.0001) and green-red (p = 0.0001) but no significant difference between white-red (p = 0.0586) and blue-green (p = 0.8017). The distance between channels have extremely significant difference between white-red (p = 0.0001), highly significant between blue-green (p = 0.0127), blue-red (p = 0.002), green-red (p = 0.0014) but no significant difference comparing white-blue (p = 0.15), white-green (0.14). When we analyzed pore diameters we obtained extremely significant difference comparing white-blue (p = 0.0001), white-red (p = 0.0001), blue-green (p = 0.0001), red-green (p = 0.000), but no significant difference between white and green (p = 0.3392). The distance between pores have extremely significant difference between all groups: white-blue (p = 0.0001), white-green (p = 0.0065), white-red (p = 0.0001), blue-green (p = 0.0001), blue-red (0.0001), green-red (p = 0.0001).Comparing exocuticle thickness between pairs, all groups revealed extremely significant difference: white-blue (p = 0.0001), white-green (p = 0.0003), white-red (p = 0.0045), blue-green (p = 0.0003), blue-red (p = 0.002), except when we compared green and red (p = 0.651) exocuticles where there was no significant difference. The epicuticles gave statistically relevant differences when comparing green with white (p = 0.0001), blue (p = 0.0001) and red (p = 0.0001), but for the others: white-blue (p = 0.3028), white-red (p = 0.0814) and blue-red (p = 0.1696), no relevant difference was observed. Endocuticle thickness had extremely significant difference between all groups (p = 0.0001), and good statistical relevance for the white-blue comparison (p = 0.0295).H. americanus and H. gamarus)19, little is known about the occurrence and localization of astaxanthin-crustacyanin complexes in various crustacean species. Nevertheless, a genetic study conducted to prospect occurrence of genes that encode crustacyanin subunits34 established that crustacyanin is a lineage-specific adaptation developed by common ancestors of certain crustacean groups, however, the genome of C. sapidus was not considered.Red ATX and its blue protein complex presence not only in blue, but in red or green crab cuticle is a proof of the complex mechanisms underlying the crustacean colors. Since the previous studies targeted mostly lobsters  could add additional contributions to the overall properties48 and color appearance not only for human vision, but also for crustacean’s vision. Other proteins can contribute with their specific absorption and/or fluorescence emission along the canals confined between cladding nanopillars walls network, while the nanopillars themselves defining the canals walls structure can act as 3D diffraction grating whose operability in certain diffraction order still has to be understood.The highly ordered porous calcite 3D-supergrating, specific to each crab cuticle and gradually populated with balanced pigments appeared more complex than the extensively investigated structural colors in insects. In the latter, the optical properties of their cuticles stem from highly ordered Bouligand pattern comprising line gratings and/or vanes of their wings structure, with helicoid stacking of chitinous fiber layersC. sapidus cuticle may inspire further biomimetics avenues, since there are no clear-cut borders between blue, red and white shells. Since the crab cuticle can manipulate light with its combined chemical structure and morphology, light absorption and propagation in wavelength dependent manner could warrants attractive applications of the porous magnesian calcite supergratings. In the light of present findings, crab shells could potentially trigger the starting point of new, effective, advanced materials that could serve as porous layer for preventing infections spreading49, control molecular solutions loading and slow releasing, bacterial therapies, or develop new, effective, porous biostimulants for soils remediation and agricultural strategies, in line with the most innovative blue bioeconomy approach50.C. sapidus from invaded Adriatic Sea contain both the aggregated and the non-covalently bound astaxanthin in carotenoproteins. Further investigation of red and white C. sapidus and green C. aestuarii claw shells revealed that the two pigments are ubiquitously present in investigated shells, even in seemingly unpigmented white shells, albeit to much lesser extent. Thus, non-covalently bound astaxanthin in crustacyanins is not the only factor responsible for dark blueish and purplish color, but rather participates in formation of apparent color alongside free carotenoids and the 3D nanostructured regular array of Mg-calcite on organic scaffold. The pigments were found to co-localize over the cuticle surface and in its cross-section, with highest distribution in exocuticle layer. C. sapidus and C. aestuarii also may have a structural color component, derived from the 3D-nanostructured regular array of Mg-calcite on organic scaffold.Non-destructive multi-laser micro-Raman spectroscopy and imaging revealed that native claw shells in the Atlantic blue crab C. sapidus) and Mediterranean green crabs (C. aestuarii) were obtained from fishermen who caught them by gillnets and traps in Parila Lagoon 6. Crabs were brought to laboratory and frozen at −20 °C until needed. Before analysis, the crabs were thawed and their claws were dissected and cleaned from soft tissue that was inside using scissors, tweezers and tap water. Shells were then thoroughly washed in cold running tap water. 10 mature C. sapidus individuals (carapace width about 12  cm), comprising 5 males and 5 females  level of theory56. The solvent effects have been considered by using the implicit SMD model56.Geometry optimizations and Density Functional Theory (DFT) frequency calculations of the isolated astaxanthin molecule and astaxanthin near water molecules were performed with the Gaussian 09 software package−1 for visible laser line and 1  cm−1 for NIR lines. WiRETM 3.4 software  was used for data acquisition. The spectra were acquired either in single-point scan, or in StreamLine imaging mode (described later), on a mobile XYZ HSES (high-speed encoded) stage.Micro-Raman analysis was conducted on a Renishaw InVia confocal Raman microscope . Four laser lines were used on this device: 532  nm excitation was provided by a Cobolt diode pumped solid state (DPSS), air cooled laser, He-Ne- laser provided the 632.8  nm excitation, and near-infrared excitation was provided by two diode lasers emitting at 785 and 830  nm. Spectral resolution was 0.5  cm−1 and 300 scans were co-added.Bruker Equinox 55 FT-IR spectrometer with integrated FRA 106/S IR Raman module was used for to record FT-Raman spectra. A Nd:YAG laser emitting at 1064  nm with 350  mW laser power was used for excitation, and signal was detected by a liquid nitrogen cooled Ge detector. Spectral resolution was 4  cm−1) and 10  s and 20  mW for extended range scans (100–3200  cm−1), for 532, 632.8, 785 and 830  nm lines. The inhomogeneity of claw surface, both in relief and pigment content, made it challenging to record optimal signal-to-noise ratio. Spectra acquired with 532 and 632.8  nm excitation lines featured incremental fluorescence background typical for carotenoid pigments while spectra acquired with 785, 830 and 1064  nm laser line excitation featured low or negligible spectral background.Due to specific sample surface characteristics (as discussed in Results section) various signal intensity was recorded from different parts of shells. Nevertheless, to keep signal acquisition parameters uniform, we used single scans with integration time and laser power to 1 s and 100  mW, respectively, for short-range .Spectra of liquid sample, i.e. the astaxanthin-enriched ethanol extract, were acquired on a DeltaNu compact, dispersive Raman spectrometer . The 532  nm excitation line was used, and spectra were recorded with 8  cmTo support the presented data, Raman spectra were collected from 10 blue crabs  and 3 green crabs. Examples of shell fragments are showed in Supplementary Fig. C. sapidus and green C. aestuarii claw shells using the StreamLineTM image acquisition facility of the Renishaw instrument. Both the 532 and 785  nm excitation lines were used separately, paired with 20x objective (NA 0.35). Firstly, a rectangular area to be analysed, 90 × 90  µm in size, was defined on the live video image. This area resulted in 9000 spectral acquisition points. The Raman image was acquired under a continuous laser beam by automatic movement of the stage to achieve 1 s exposure time per image pixel. The v1 mode of free ATX, centred around 1514  cm−1 and the v1 mode of ncb-ATX, centred around 1492  cm−1 were selected for creating separate images under the “signal to baseline” criterion. The images were then overlaid to produce composite pigment distribution maps.Raman images were produced on surfaces of blue and red −1 range, estimating the initial half width and obtaining the peaks components position, area and center and yielding the coefficient of determination R2 higher than 0.99. Calculation of Raman intensity ratio (R) of ncb-ATX band to ATX was done after background subtraction from spectra acquired from different shell color types with 785  nm excitation and subsequently comparing the intensity of the mode at 1492  cm−1 (ncb-ATX) and the mode at 1514  cm−1 (ATX) as R = I1492/I1514 . Background subtraction was applied when needed, by creating a user-defined, automatic, 10 points baseline function and iteratively modifying it using the subtraction facility of the Origin software. Lorentzian fit multi-peak was applied for the Raman spectra in the 1425 to 1600 cmC. sapidus and green pieces of C. aestuarii claw cuticle were placed into Petri plate containing boiling water. Water was heated on a magnetic hot plate magnetic stirrer to 100 °C, and pieces of shells were kept in boiling water until their color changed to orange. Shells were then taken out of water using tweezers, dried and subsequently analyzed with the Raman microscope. Raman spectra were acquired from treated shells using the 532, 632.8, 785 and 1064  nm laser lines, and spectra were compared to those acquired from native blue and green shells.Blue, red and white pieces of C. sapidus claw were placed in vials containing 4  ml of ethanol. Raman analysis of both shells and the supernatant was conducted 14 days after the beginning of extraction. Shells and carotenoid microcrystals were analyzed with the Renishaw Raman microscope. The astaxanthin-enriched extract was fast analysed in liquid state with the DeltaNu Advantage 532 Raman spectrometer with 532  nm excitation line and 10  s integration time set via NuSpec software.Pieces of blue shells from K = 0.15418  nm, Ni filter and a LynxEye detector. Corundum (NIST SRM1976a) is used as an internal standard. The data were collected on a 5–64° 2θ interval at a 0.02° 2θ step, measuring each step for 0.5  seconds. The identification of mineral phases was performed with the Diffrac.Eva 2.1 software (Bruker AXS) using the PDF2 (2012) database.Shell fragments were finely ground in an agate mortar and their mineral composition was verified by X-ray powder diffraction (XRPD), using a Bruker D8 Advance diffractometer in Bragg-Brentano geometry, with a Cu tube with λ2 bottom layer to avoid atmospheric water absorption. Before analysis, samples were adherently placed on Hitachi stub SEM holders . A Quorum Q150T sputtering sample coater capable of gold sputtering of controlled thickness for high resolution imaging, and evaporating carbon for EDX analysis was employed. Gold coating thickness was 10  nm (density 19.32  g/m3) at a rate of 14  nm/min. An Oxford energy-dispersive x-ray module  was used for elemental analysis of shells.SEM imaging and EDX measurements were accomplished with a SU8230 Hitachi ultra-high resolution cold-field emission scanning electron microscope. The instrument allows for a combination of topographical and compositional information at a feature resolution of up to 1  nm in optimal conditions. Pieces of claw shells were dried in oven at 40 °C and kept in a container with CaClwere made using unpaired t-test on GraphPad software and one way analysis of ANOVA (one way ANOVA) between groups. Standard deviation was calculated using Excel software. For statistical relevance, values of p ≤ 0.05 were considered significant, p ≤ 0.01 very significant, p ≤ 0.001 extremely significant.We first conducted one way ANOVA to four groups  and within certain groups (comparing for certain characters) there were no significant differences. Then we compared series of two groups by ANOVA and by t-test as well, to differentiate the significance between groups. The results were the same  using t test or using ANOVA. Multiple t test was not used here.Supplementary Information.


We investigated the ability of machine-learning classifiers on radiomics from pre-treatment multiparametric magnetic resonance imaging (MRI) to accurately predict human papillomavirus (HPV) status in patients with oropharyngeal squamous cell carcinoma (OPSCC). This retrospective study collected data of 60 patients (48 HPV-positive and 12 HPV-negative) with newly diagnosed histopathologically proved OPSCC, who underwent head and neck MRIs consisting of axial T1WI, T2WI, CE-T1WI, and apparent diffusion coefficient (ADC) maps from diffusion-weighted imaging (DWI). The median age was 59 years (the range being 35 to 85 years), and 83.3% of patients were male. The imaging data were randomised into a training set (32 HPV-positive and 8 HPV-negative OPSCC) and a test set (16 HPV-positive and 4 HPV-negative OPSCC) in each fold. 1618 quantitative features were extracted from manually delineated regions-of-interest of primary tumour and one definite lymph node in each sequence. After feature selection by using the least absolute shrinkage and selection operator (LASSO), three different machine-learning classifiers  were trained and compared in the setting of various combinations between four sequences. The highest diagnostic accuracies were achieved when using all sequences, and the difference was significant only when the combination did not include the ADC map. Using all sequences, logistic regression and the random forest classifier yielded higher accuracy compared with the that of the XG boost classifier, with mean area under curve (AUC) values of 0.77, 0.76, and 0.71, respectively. The machine-learning classifier of non-invasive and quantitative radiomics signature could guide the classification of the HPV status. Because of differences in the oncogenesis, epidemiology, and prognosis; the eighth edition of the American Joint Committee on Cancer (AJCC) tumour-node-metastasis staging system classifies OPSCC into HPV-positive and HPV-negative tumours2. Therefore, the preoperative differentiation between HPV-positive and HPV-negative OPSCC is critical for patient management as well as prognosis3.Human papillomavirus (HPV) status is a dependable and independent prognostic factor in patients with oropharyngeal squamous cell carcinoma (OPSCC). Patients with HPV-positive OPSCC have better survival rates than patients with HPV-negative OPSCC5, perfusion, and diffusion parameters, which are related to the angiogenesis and cellularity of the tumour. Several studies have reported diagnosis of the HPV status in patients with OPSCC using preoperative computed tomography (CT) or magnetic resonance (MR) imaging8. HPV-positive OPSCC tends to exhibit cystic cervical lymph node metastasis8 and primary tumours with well-defined borders and an exophytic appearance7. Recent studies reported that diffusion-weighted imaging (DWI) may help predict HPV status in patients with OPSCC, as HPV-positive OPSCC reveals a low mean apparent diffusion coefficient (ADC) compared with HPV-negative OPSCC11. Furthermore, a histogram analysis based on dynamic contrast-enhanced MR image showed significantly higher Kep kurtosis values and lower Ve min values in patients with p16-positive OPSCC12. Recently, several published studies had addressed the prediction of HPV status employing a CT-based radiomics approach; however, their diagnostic performance was moderate 15. To date, no studies reported on the application of radiomic machine-learning classifiers on multiparametric MR images to predict HPV status in patients with OPSCC. Therefore, we hypothesise that pre-treatment multiparametric MR image combined with DWI could predict HPV status accurately employing radiomic machine-learning classifiers in patients with OPSCC.The distinct oncogenesis of HPV-positive OPSCC results in characteristic histopathologyOf the 70 consecutive patients with OPSCC, 10 were excluded owing to unknown HPV status (n = 4), post-treatment MR images (n = 4), and loss of MR image data (n = 2). Finally, 60 consecutive patients with OPSCC were enrolled in this study , 0.76 ± 0.12 95% CI 0.47 to 0.97), and 0.71 ± 0.12 (95% CI 0.50 to 0.93), respectively, when using selected features from all sequences  and the random forest classifier (0.76 ± 0.12) demonstrate higher values of the mean AUC compared with those exhibited by the XG boost classifiers (0.71 ± 0.12). The average sensitivity and specificity in the logistic regression classifier were 0.71 and 0.72, respectively. This radiomic signature of HPV status can be used to develop non-invasive tools for discriminating OPSCC patients.19. Previous exploratory studies indicate a correlation between the HPV infection status and CT-based radiomic signature in head and neck squamous cell carcinoma (HNSCC)20. These studies reported AUC values that ranged from 0.70 to 0.86. Although most radiomics studies for classifying the HPV status are based on CT, Ravanelli et al. investigated the correlation between MR imaging texture features and HPV status in OPSCC9. The authors developed a simple predictive model based on mean ADC values and smoking status that yielded an AUC of 0.944. In the present study, we developed a tool for classifying the HPV status using radiomic features from multiparametric MR images and machine-learning classifiers with an AUC of 0.77.Increasing evidence suggests that radiomics, a method that non-invasively extracts quantitative information from medical images, can be used to characterize intra-tumoral heterogeneity22. According to de Perrot et al., diffusion phenotypes based on the histogram analysis of ADC values reflect distinct degrees of tumour heterogeneity in HPV-positive and HPV-negative HNSCCs21. It has been shown that the mean and median ADCs are significantly lower, whereas excess kurtosis and skewness are significantly higher in HPV-positive tumours than in HPV-negative tumours. In their study, HPV-positive tumours exhibit leptokurtic right-skewed histograms, which correspond to homogeneous tumours with densely packed cells, a scant stromal component, and scattered comedonecrosis. Meanwhile, HPV-negative tumours exhibit symmetric normally distributed ADC histograms, which correspond to heterogeneous tumours with variable cellularity, a high stromal component, keratin pearls, and necrosis. Meyer et al. investigated the correlation of ADC values with prognostically relevant histopathologic parameters, including the expression of Hif1-alpha, VEGF, EGFR, p53, p16, and Her 222. They found that ADC histogram reflects different histopathological features in HNSCC, and associations between ADC histogram parameters and histopathology depend on the p16 status. In this study, features extracted from ADC maps were attributed the highest weight after LASSO regression, and they were mostly included in the top-performing features.Recent studies have addressed whether the ADC-histogram analysis can be used to identify different histopathological features in HNSCC26. In the present study, using four MR sequences yielded the highest classification accuracy. However, the difference between using four sequences and three or less sequences was significant only in cases not including ADC maps. The selected features after LASSO regression from four MR sequences included features from all MR sequences, whereas features from the ADC map comprised a large percent of top-performing features. Considering a small sample size and imbalance of HPV status in this study, further studies might be needed to confirm whether combining multiple MR sequences enables the detection of more detailed differences between HPV-positive and HPV-negative tumours.Recent studies found that the radiomics signature from multiparametric MR images achieved higher prognostic accuracies compared with a single MR sequence30. Only few studies investigated the effect of different feature selections and machine-learning classification methods on radiomic features30. In these studies, the random forest classifier had the highest prognostic performance for diagnosing cancers from benign tumours. Further, Parmar et al. observed that a generalised linear model exhibits a high prognostic performance in HNSCC and non-small-cell lung cancer types, whereas it shows low stability for HNSCC27. The present study compared three machine-learning classifiers including the logistic regression, random forest, and XG boost model. The logistic regression classifier and random forest classifier demonstrated performance superior to that of the XG boost classifier. The most plausible reason is that the final selected features are highly discriminative in their classification of HPV status, which proves to be most suitable for the logistic regression classifier. However, considering that logistic regression models generally perform better for smaller data sets, compared with tree induction models, and are prone to overfitting32, further validation with large samples might be needed.Machine-learning models have rapidly improved in the past few years. Radiomics is an emerging field for machine-learning that allows the conversion of radiologic images into mineable high-dimensional dataOur study has several limitations. First, it is a retrospective study performed on a relatively small sample with a highly imbalanced dataset for machine-learning (n = 60). Repeated cross-validation and feature selection using the LASSO regression were applied to mitigate the risk of overfitting in this situation. Second, it remains to be validated whether our radiomics signature can be applied to different MR systems, imaging protocols, and software platforms. Therefore, multi-centre studies with large samples and a prospective study design are required to evaluate the true predictive value of the radiomics signature. Third, the regions-of-interest (ROIs) in the tumours were manually delineated based on ADC maps, which tend to be affected by movement artefacts such as breathing and swallowing, along with frequent susceptibility artefacts from the air-tissue interface. Furthermore, the stability analysis, i.e., assessing the robustness of the features, was not properly conducted. To achieve optimal feature selection, the slightly better performing feature can be selected from various kinds of similar features via the wavelet transform, which could lead to low reproducibility of wavelet features. Therefore, the stability and reproducibility of selected features must be investigated in further studies.In conclusion, the present study developed radiomic machine-learning classifiers from multiparametric MR images for the determination of the HPV status in patients with OPSCC. Our results show that logistic regression and the random classifier applied subsequent to feature selection from MR images, including T1WI, T2WI, T1-CEWI, and ADC maps, using LASSO regression exhibit the highest classification accuracy; furthermore, features selected from the ADC map were crucial in classifying the HPV status. This method explores the integration of anatomical and multiparametric MRI radiomics into clinical models, which might have a significant impact in the MR-guided radiotherapy for head and neck cancers.33 and strengthening the reporting of observational studies in epidemiology (STROBE)34.This study was approved by the institutional review board of Asan Medical Center . The local ethics committee, institutional review board of Asan Medical Center, waived off the written informed consent due to the retrospective nature of the study. We reported our results according to the standards for reporting of diagnostic accuracy studies (STARD) 2015 guidelinesWe enrolled consecutive patients with newly diagnosed histopathologically proved OPSCC, who were examined by head and neck MR imaging between April 2012 and November 2017. The eligibility criteria were as follows: (a) patients diagnosed by histopathology with a pre-treatment OPSCC, (b) patients with known HPV status, (c) patients who were examined by head and neck MR imaging including DWI, and (d) patients that were > 20 years old. Patients who had received chemotherapy, radiation therapy, or excisional biopsy prior to the MR imaging were excluded.36. P16 immunohistochemistry was performed using CINtec p16 histology  and HPV DNA detection was performed by PCR/DNA chip scanning 37. HPV-positive OPSCC was diagnosed based on the positive results of either p16 or HPV DNA PCR38.All analyses of the HPV status were performed by the pathology division of our institution without prior knowledge of the MR imaging results. P16 immunohistochemistry or HPV DNA detection by polymerase chain reaction (PCR) was used as the reference standard2; section thickness of 4 mm; no gap; field of view of 192 2, and acquisition time of approximately 5 min. The ADC maps were obtained automatically within the manufacturer console. Imaging data were de-identified in accordance with the health insurance portability and accountability act privacy rule.Head and neck MR imaging was conducted using a 3-T scanner with a 64-channel coil  and the MR imaging protocol as follows: To obtain CE-T1WI, an intravenous dose of 0.1 mmol/kg of contrast agent gadoterate meglumine  was injected into the patient. DWI MR imaging was conducted using multi-shot read-out-segmented echo-planar imaging in the axial plane before the injection. The detailed DWI sequence parameters were as follows: repetition time/echo time, 5450/62 ms; b values of 0 and 1000 s/mmhttps://www.mitk.org, German Cancer Research Center, Heidelberg, Germany)39. Both the primary tumour and lymph node volumes belonged to the same patient. T1WI, T2WI, CE-T1WI, and ADC maps were co-registered with SPM software (https://www.fil.ion.ucl.ac.uk/spm/), using affine transformation with normalized mutual information as a cost function, with 12 degrees of freedom and tri-linear interpolation40. The original ROIs were co-registered on the T1WI, T2WI, and CE-T1WI for the tumour and on the T1WI, CE-T1WI, and ADC maps for the lymph node, then manually adjusted to suit each sequence. All MR images were resampled into isometric voxels of size 1 3 as input data. Field inhomogeneity of MR images was corrected using the N4ITK algorithm41. To ensure just comparison of the extracted features across all patients, intensity normalization was conducted for T1WI, T2WI, and CE-T1WI sequences.Figure 42 at the same institution. The range of mean ± 3 standard deviation of the entire intensity range was quantized into 32 density bin levels for the texture features. The features included seven shape and volume features, 17 first-order features, 162 texture features, and 1432 wavelet features  multiplied by eight images, yielding 1432 wavelet features.From the segmented mask, 1618 total radiomic features were extracted using MATLAB R2015a , using a similar approach to previous study of Yun et al.45. To reduce over-fitting or any type of bias in our radiomics model, LASSO-penalized linear regression was applied to the training data. All radiomics features were centred and scaled to a value with a mean of zero and a standard deviation of one (z-score transformation before applying feature selection). With a linear combination of the selected features weighted by their respective coefficients, a model was used to estimate the HPV status. LASSO regression was implemented using Python  with the Scikit-learn package (https://github.com/scikit-learn/scikit-learn)46. Features with larger contributions to the model were selected.The extracted features may be noisy or highly correlated with each other; therefore, feature selection is required to increase the prediction accuracy and minimise computational cost47 using the Scikit-learn package, and XG boost48 using the Xgboost package (https://github.com/dmlc/xgboost). The algorithms were selected based on their high performance and readiness for application. Three different models were computed and compared to determine the best combination for determining the HPV status in the data set. The models were developed separately for each of the T1WI, T2WI, CE-T1WI, and ADC maps, as well as various combinations of these sequences. Classifiers were trained with a stratified threefold cross-validation procedure repeated 20 times, which allows repetition of experiments for each model up to 60 times. All possible combinations of hyperparameters were investigated by the grid search using GridSearchCV library in the Scikit-learn package. (Supplementary Table Three different machine-learning classifiers were applied: logistic regression, random forest49. AUCs were used to determine the diagnostic performance, with optimal thresholds of the imaging parameters determined by maximizing the sum of the sensitivity and 1 − specificity, i.e., the Youden index, values.The Mann–Whitney U test was used to estimate the relationship between selected radiomic signatures and HPV status, and to compare accuracies between various combinations of MR sequences in a pairwise mannerSupplementary Information.