Clitoria ternatea plant is commonly grown as an ornamental plant and possesses great medicinal value. Its flower is edible and also known as blue pea or butterfly pea flower. The unique feature of anthocyanins present in blue pea flowers is the high abundance of polyacylated anthocyanins known as ternatins. Ternatins are polyacylated derivatives of delphinidin 3,3′,5′-triglucoside. This review covers the biosynthesis, extraction, stability, antioxidant activity, and applications of anthocyanins from Clitoria ternatea flower. Hot water extraction of dried or fresh petals of blue pea flower could be employed successfully to extract anthocyanins from blue pea flower for food application. Blue pea flower anthocyanins showed good thermal and storage stability, but less photostability. Blue pea flower anthocyanins also showed an intense blue colour in acidic pH between pH 3.2 to pH 5.2. Blue pea flower anthocyanin extracts demonstrate significant in vitro and cellular antioxidant activities. Blue pea flower anthocyanins could be used as a blue food colourant in acidic and neutral foods. The incorporation of blue pea flower anthocyanins in food increased the functional properties of food such as antioxidant and antimicrobial properties. Blue pea flower anthocyanins have also been used in intelligent packaging. A comparison of blue pea flower anthocyanins with two other natural blue colouring agents used in the food industry, spirulina or phycocyanin and genipin-derived pigments is also covered. Anthocyanins from blue pea flowers are promising natural blue food colouring agent. Both sps Ellis) . Genipins Ellis) . TherefoClitoria ternatea flower is one anthocyanin source containing stable blue colour polyacylated anthocyanins  to form delphinidin 3-O-β-glucoside. O-β-glucoside, only after malonylation of delphinidin 3-O-β-glucoside. Accordingly, delphinidin 3-O-β-glucoside is malonylated in the presence of anthocyanidin 3-O-glucoside 6″-O-malonyltransferase (A6″MaT). Then, two glucose molecules are added to delphinidin 3-O--β-glucoside, first to 3′ position followed by 5′ position (O-glucosyltransferase (UA3′5′GT) in two subsequent steps -β-glucoside-3′,5′-di-O-β-glucoside and can be called as ternatin C5. Ternatin C5 is the simplest ternatin. Other 14 ternatins are synthesised by adding acyl and glucosyl groups to ternatin C5 in the presence of acyltransferases (ATs) and glucosyltransferases (GTs). Acyltransferases (AT) also plays a major role resulting in the blue colour and the stability of ternatins, because polyacylation of ternatins with p-coumaroyl groups results in a shift of colour of anthocyanins to bluish region due to intramolecular co-pigmentation among acyl moieties and between acyl moieties and anthocyanin chromophore  of a blue pea flower anthocyanin extract . SelectiConsidering substrate: solvent ratio, 1:20 (g/mL) was reported by O-glucoside equivalent/g) obtained by ultrasound-assisted water extraction was higher than that  obtained by conventional ethanolic extraction  with increasing temperature. This study also showed that the addition of catechin as a co-pigment to the anthocyanin extract of blue pea flower at pH 3.5 increased the half-life of anthocyanins from blue pea flower to 8.39 at 90°C . CatechiAnthocyanins from blue pea flowers are less stable to photo stress compared to thermal stress . When anThe good thermal stability up to 70°C, the storage stability at 25°C and the intense blue colour demonstrated at pH 3.6 to 5.4, makes anthocyanins from blue pea flowers, suitable to be used as a blue colour food colourant. Comparing the thermal stability of phycocyanin, genipin-derived pigments and anthocyanins of blue pea flower , phycocyin vivo and in vitro antioxidant activity (50 > 900 μg/mL) and showed a protective effect in human erythrocytes and inhibited the oxidation of pBR322 plasmid DNA  of the blue pea flower anthocyanin extract (0.47 mg/mL) was significantly higher compared to that of ascorbic acid (0.002 mg/mL)  and ABTS assays (IC50 – 6.51 μg/mL) were significantly lower compared to blue pea flower anthocyanin extract. This indicates that ascorbic acid and Trolox possess higher antioxidant activity compared with the blue pea flower anthocyanin extract.In one study, it was found that blue pea flower anthocyanin extract demonstrated significant antioxidant activity against DPPH and peroxyl radicals. Using DPPH assay, the IC2 mg/mL) . Similar2 mg/mL)  and the 2O2-induced cytotoxicity and UV-induced mtDNA damage in human keratinocytes. The cellular antioxidant activity of blue pea flower extract is clearly demonstrated in these studies. Overall, the results of in vitro antioxidant assays should not be interpreted directly as the antioxidant property inside the body of a living being (ng being . Therefop < 0.05) in the sucrose treatment with blue pea flower anthocyanins when compared to the sucrose treatment with water. Plasma insulin level increased in sucrose only treatment, but plasma insulin level did not change significantly with blue pea flower anthocyanins treatment. When sucrose was administered with blue pea flower anthocyanin extract the rise of plasma insulin level was significantly suppressed after 60 min of administration. The plasma antioxidant activity measured by FRAP and ABTS assays increased in all treatments, but subjects treated with blue pea flower anthocyanins with or without sucrose demonstrated a significantly higher plasma antioxidant activity. Ingestion of blue pea flower anthocyanin extract with or without sucrose showed reduced levels of plasma malondialdehyde (MDA) during the postprandial period that indicating a low level of lipid peroxidation. The plasma thiol concentration which is an indicator of plasma antioxidant defence mechanism reduced significantly within 30 min after ingestion of sucrose but administration of blue pea flower anthocyanin extract with or without sucrose increased the plasma thiol level indicating the strengthening of plasma antioxidant defence mechanism by blue pea flower anthocyanins  at a concentration of 1.6 × 10–4 mg anthocyanin/g, the yoghurt drink has a purplish-blue colour and the rice had a dark blue colour. In this study, Brilliant Blue, a synthetic colourant was also added to yoghurt drink at a concentration of 3.13 × 10–4 mg/mL and to rice at a concentration of 3.2 × 10–4 mg/g and the colour of yoghurt drink and rice were green-blue and light blue, respectively  films separately and used to monitor the freshness of milk and fresh orange juice. It was observed that incorporation of anthocyanin extracts did not significantly alter the physical properties of the film and the film with Clitoria ternatea anthocyanin extract was more pH-sensitive in terms of colour change compared with the film with the anthocyanin extract of Carissa carandas. The film with Clitoria ternatea anthocyanin extract showed a distinct change in colour for both milk and juice stored at 25°C after 72 h where the pH of milk changed from 6.2 to 4.1 and the pH of juice changed from 4.2 to 3.4 (In addition to freshness monitoring systems of animal products, 2 to 3.4 . TherefoClitoria ternatea or blue pea flower is an edible flower with medicinal and ornamental value. The blue pea flower is a rich source of polyacylated anthocyanins demonstrating higher stability than non-acylated anthocyanins. Blue pea flower anthocyanins demonstrate an intense and stable blue colour in acidic medium which facilitate their application in acidic food systems as a blue food colouring agent. The cellular and in vitro antioxidant activities of blue pea flower anthocyanins show their potential application in functional foods. Several studies have been conducted on investigating the application of blue pea flower anthocyanins as a food colourant in bakery products, yoghurt and functional beverages. Further research should be carried out on the application of blue pea flower anthocyanins in other food systems. Studies have shown antioxidant and antimicrobial activities of blue pea flower anthocyanins in different applications. Further research could be carried out to investigate the bioavailability and other functional properties of blue pea flower anthocyanins. Since there are limited blue food colourants available, blue pea flower anthocyanins will be a good alternative to be used as a natural blue food colouring agent.GCVG: responsible for writing and editing the manuscript. YYL: responsible for reviewing and editing the manuscript. WSC: responsible for the conceptualisation, funding acquisition, and reviewing and editing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The preventive effect of HPV vaccines against anogenital and oropharyngeal cancers has been proven in both clinical trials and real-world data. We reviewed the published evidence about the long-term efficacy and effectiveness of the HPV vaccine in available papers of clinical trials and real-world data. As far as we searched, the longest period of preventive effect for the bivalent, 4-valent, and 9-valent vaccine were 11 years in the Costa Rica trial, 14 years in the FUTURE II, and 8 years in the LTFU extension study of V503-002 and the Scandinavian study, respectively. The sustained clinical effect during the observation period was longest for the 4-valent vaccine. In real-world data, the longest observation period of the vaccine effectiveness was 12 years in an Australian study for the 4-valent vaccine. On the other hand, the longest period of long-term persistence of HPV vaccine-induced seropositivity was 14 years in FUTURE II for the 4-valent vaccine. For the bivalent vaccine, additional long-term follow-up studies may not have been planned due to the launch of the 4-valent and 9-valent vaccines. In some studies of the 9-valent vaccine, the results have not yet been published because of the short observation period. The additional results are expected in the future. In a national immunization program, most girls and boys are inoculated with HPV vaccine by the time puberty begins; thus, it is important to monitor the vaccine effect at least until the sexually active period in their 20s and 30s. Human papillomavirus (HPV) is mainly transmitted through sexual activity. In fact, HPV sequences have also been identified in chorionic villi tissues from pregnant females and other districts of the reproductive tract ,2. HPV iMore than 200 types of HPV have been identified, which are classified into high-risk types  that are carcinogenic, and low-risk types  that cause anogenital warts and benign tumors, such as condyloma acuminata. High-risk HPV types are thought to be responsible for 7–8% of human malignancies, including 96% of cervical cancers, 93% of anal cancers, 64% of vaginal cancers, 51% of vulvar cancers, 36% of penile cancers, and 63% of oropharyngeal cancers. Among them, the risk of carcinogenesis from type 16 and 18 infections is particularly high [HPV is estimated to cause nearly 36,000 cases of cancer in men and women every year in the United States. HPV vaccination can prevent 33,000 of these cancers by preventing the infections that cause them ,14,15,16The HPV vaccine has been available since 2006 and targets mainly adolescent girls ., and moThree databases  and references or related articles were used to review the efficacy and effectiveness of HPV vaccination. In the database, related articles were identified using the following search string: (“human papillomavirus vaccination” OR “HPV vaccination” OR “human papillomavirus vaccine” OR “HPV vaccine”) AND . Given the search results, we added the terms “long term”, “follow up”, “extension”, and “antibody” to cover all relevant articles.PubMed and Google Scholar literature searches yielded a total of 1751 records. In addition, we found 35 literatures listed in the package insert and 150 studies in ClinicalTrials.gov of the HPV vaccines. After that, we screened 1936 records at the abstract/title level for eligibility according to exclusion criteria. Subsequently, 108 records were screened in full-text. A total of 36 publications reported relevant data of long-term effects of HPV vaccination. For this review, 32 clinical trials and 4 observational studies were included .®) against HPV 16/18 (high-risk HPV type: HR-HPV), 4-valent vaccine (Gardasil®) against HPV 6/11 (low-risk HPV type: LR-HPV) and HPV 16/18 (HR-HPV), and 9-valent vaccine (Gardasil 9®) against HPV 6/11 (LR-HPV), and HPV 16/18/31/33/45/52 (HR-HPV) [® and Gardasil® could prevent 70% of cervical cancer cases, and Gardasil-9® almost 90% [Currently, three types of HPV vaccines are used worldwide to prevent the development of cervical cancer. The bivalent vaccine (Cervarix(HR-HPV) . Cervarimost 90% . Table 1most 90% ,26,27.Numerous clinical trials have been conducted to date for each vaccine. We considered the difference in efficacy depending on the vaccine used and target age .According to a previous review on the long-term effect on precancers , the obsThe extension HPV023 trial on a bivalent vaccine was an initial double-blind, randomized, placebo-controlled study in women aged 15–25 years with normal pre-vaccination cytology . As a reIn the Costa Rica Vaccine Trial, a randomized, double-blind, controlled trial that assessed the efficacy of the initial bivalent vaccination, women aged 18–25 years were enrolled. As a result, cumulative VE against HPV 16/18-associated CIN2+ (CIN2 or worse) and CIN3+ (CIN3 or worse) over the 11.1-year period was 97.4%  and 94.9% (73.7–99.4), respectively . This ren = 2198) and 5747 women were assigned to the total vaccination cohort . The vaccine showed efficacy in preventing persistent HPV16/18 infection, cytological abnormalities, and CIN1+ [The longest follow-up period so far was 7 years in the VIVIANE study. In the VIVIANE study, a 7-year follow-up of a double-blind, randomized controlled study involving women age > 25 years; women were randomly assigned (1:1) to receive a bivalent vaccine or aluminum hydroxide control, using an internet-based system. In addition, 4407 women were assigned to a cohort following the protocol for efficacy  or hepatitis B virus (HBV) control vaccine (n = 89) at 0, 1, and 6 months, and were followed for 7 months. The antibody titers of HPV 16/18 in men aged 10–18 and 10–14 years at 7 months after vaccination were higher than those in women aged 15–25 and 10–14 years in previous reports [The P011-NCT00309166 trial was the first to investigate the efficacy of the bivalent vaccine in boys . In this reports .The observation period was 3.6 years in the FUTURE I , 5 yearsIn the V501-018-11 trial on men and women aged 9–15 years, during the 10-year follow-up period no genital warts, cervical and/or genital pre-cancers or cancers were observed .n = 2121) during the entire study period. VE of 100%  was demonstrated at 12 years or more, with a trend toward sustained protection up to 14 years after vaccination.Recently, a final report of the FUTURE II on a 4-valent vaccine was published . In the The longest follow-up period so far was 10 years in the P019-21 trial . The P01Several extension studies of V501-P020 ,60 were NCT00090285 trial  is an opIn P020-21, a follow-up study of V501-P020, a preventive effect against HPV 6/11/16/18-related anal cancer and its precursor lesion, condyloma acuminata, was observed for up to 11.5 years after the third inoculation . The V50Furthermore, in the V501-018-11 trial for men and women aged 9–15 years, no breakthrough disease in the forms of genital warts or cervical and/or genital precancers and cancers were observed in the 10-year follow-up period .The observation periods for the 9-valent vaccine was 5 years in a Latin American study , 6 yearsThe Latin American study included a randomized, double-blind, controlled trial in women aged 16–26 years and an immunogenicity study in girls and boys aged 9–15 years . The 9-vThe V503-001 trial was a randomized, international, multicenter, double-blind study in women aged 16–26 years . In the In the LTFU extension study of V503-002 ,56, the In the Scandinavian study, 1448 women aged 16–26 years were followed for the development of HPV16/18/31/33/45/52/58-related high-grade cervical dysplasia . The resTo the best of our knowledge, there are no reports on the long-term effects of the 9-valent vaccine in adult women.In the LTFU extension study of V503-002 ,63, in aThere are several reports on bivalent and 4-valent vaccines .The observation period of long-term effectiveness for the 4-valent vaccine was 9 years in an Australian study (2015) , 10 yearThe Australian study (2015)  comparedIn the Danish study , 19,951 The Australian study (2020)  investigp = 0.0018), and the vaccine effectiveness was 100%. Cross-protective-type HPV31/45/52 infection rate in the vaccinated group was significantly lower than that in the unvaccinated group (3.3% vs. 10.0%: p = 0.013).Recently, the long-term 9-year effectiveness of the bivalent vaccine against HPV16/18 and 31/45/52 infection was reported in Japanese women aged 25–26 years . HPV16/1p = 0.001), and its effectiveness was confirmed 9 years after vaccination. The authors stated that despite the low vaccination rate of 40.3% among women aged 25–35 years, the significant reduction in HPV prevalence may be due to the effect of strong herd protection.In the Australian study (2015), the 4-valent HPV vaccination program targeting girls aged 12–13 years commenced in 2007, with catch-up vaccination of women aged 14–26 years through 2009. In the study, HPV prevalence in women aged 18–24 and 25–35 years in 2015 was compared with that in 2005–2007 . The resThe observation period of long-term persistence of HPV vaccine-induced seropositivity for the bivalent vaccine was 4 years in HPV 032/063 , 6 yearsIn the HPV-001 , 007 3434, and 0The P014 trial  and its In healthy boys, the P011-NCT00309166 trial examined the efficacy of the bivalent vaccine in healthy boys . The antThe longest period of long-term persistence of HPV vaccine-induced seropositivity for the 4-valent vaccine was 14 years in FUTURE II   in women aged 16–26 years, the antibody positivity rates of HPV 6/11 (LR-HPV) and HPV 16/18 (HR-HPV) were 90.6%, 91.1%, 98.3%, and 52.4% at 14 years after the first inoculation, respectively .In the P020 trial for men aged 16–26 years, the antibody positivity rates for HPV 6/11/16/18 after 3 years were 86.9%, 78.0%, 92.3%, and 60.7%, respectively . FurtherThe V501-018-11  trial waThe longest period of long-term persistence of HPV vaccine-induced seropositivity for the 9-valent vaccine was 5 years in Extension V503-001  and the The V503-001 trial confirmed that the antibody response against the 9-valent HPV vaccine type persisted for at least 5 years in women aged 16–26 years, and the antibody positivity rate was ranged from 78% to 100% at 5 years after the third inoculation ,65. In tA Latin American study  confirmeIn the LTFU extension study , three dHPV is mainly transmitted through sexual activity and causes infections that can lead to HPV-associated cancers in both men and women. The preventive effect of HPV vaccines against anogenital and oropharyngeal cancers has been proven in both clinical trials and real-world data. The longest periods of preventive effect for the bivalent, 4-valent, and 9-valent vaccines were 11 years in the Costa Rica trial , 14 yearRegarding the bivalent vaccine, it is possible that research on the long-term follow-up trial was not planed due to the launch of the 4-valent and 9-valent vaccines. In some studies of the 9-valent vaccine, the results have not yet been published because of the short observation period ,55. The In this review, antibody titers were also mentioned. However, the correlation between the immunological efficacy of antibodies and the clinical efficacy of HPV infection prevention has not yet been established, and it has not been confirmed whether the preventive effect of the vaccine is due to sustained antibody titers. In addition, there is still no consensus on the minimum amount of antibodies needed to prevent HPV infection.In a national immunization program, most girls and boys are inoculated with HPV vaccine by the time puberty begins; thus, it is important to monitor the vaccine effect at least until the sexually active period in their 20s and 30s.


Moreover, the areas under ROC curve (AUC) was 0.712 in 55–64 years group which showed a strong contribution of age classification for HR-HPV infection. This study provided baseline data on the prevalence characteristics of HPV infection and the critical age group of HR-HPV prevalence rate was 55–64 y among the samples receiving physical examinations.This paper aimed to investigate the characteristics of female HPV infection in the Shangcheng District, Hangzhou city, China. The retrospective study was designed to analyze the HPV prevalence rate of 22,382 women receiving physical examinations from 2016 to 2020 in the Shangcheng District of Hangzhou city in China. A commercial kit was designed to detect the HPV genotypes. Trends were examined for age-specific groups . A receiver operating characteristic (ROC) analysis was used to assess the correlation of age classification in high risk HPV (HR-HPV) infection. 22.41%  of samples were HPV positive, 91.28% (4578/5015) of HPV positive women were infected by HR-HPV. The most prevalent HR-HPV genotypes were 16, 52, 18, 58, 56, and 51. The trend of HPV prevalence showed the significant differences in age-specific groups (χ More than 100 HPV genotypes have been identified, which are classified into high risk (HR) and low risk (LR) types on the basis of their correlation with cervical cancer11. HPV  and HPV  were clearly classified by International Agency for Research on Cancer (IARC) as common high risk types and low risk types13.Cervical cancer is one of the major malignant tumors threatening women all over the world. Persistent infection of high-risk human papillomavirus (HR-HPV) is a necessary factor for the occurrence of cervical cancerIt is difficult to establish an accurate database of the prevalence and genotype distribution for HR-HPV infection in China because of the immense geographical area and the largest population. There is still lack of comprehensive data among women receiving physical examinations on the prevalence and genotype distribution of HPV infection in Hangzhou. The objective of this study was to investigate the characteristics of the distribution of HPV genotypes among women with different age group living in Hangzhou and provide a theoretical basis for cervical cancer screening in this region.2 = 164.70, P < 0.001)  .In age-specific groups, HPV-52 was highest detected in the ≤ 30 y (4.51%), 45–54 y (3.29%) and ≥ 65 y (3.95%) age groups, HPV-16 was commonly detected in the 31–44 y (3.35%) and 55–64 y (5.86%) age groups , Guangdong (20.0%), Chongqing (27.3%), Jinan (25.7%), Shanghai (22.6%) and Nanchang (18.4%)19. The present study showed HPV positive rate among women in the Shangcheng District of Hangzhou city in China was 22.41% and the HR-HPV positive rate was 20.45% which might be lower or similar compared with the above report. However, different HPV prevalence rates may be a combination of different cultural diversity, survey periods, geographic locations, and economic levels. The cervical cancer screening practices are heterogeneous and inadequate implementation even in Europe33, and the population-based organized cervical cancer screening has been completed in only 9 of 28 European countries34 which maybe more difficult in China because of geographic and population diversity.HPV is associated with varied epithelial lesions, including cervical intraepithelial neoplasia and genital warts20. In China, HPV-16, HPV-58 and HPV-52 were the top three most common types in most regions22. Analysis of HPV prevalence showed that the positive rate of HPV-16 was still the highest in our research, accounting for 3.95% of the total population . The top six HR-HPV types were 16,52,18,58,56and 51, which were the same as those reported in several domestic studies25. Figure 14. These findings indicated that in addition to HPV-16 and HPV-18, an HPV vaccine in Hangzhou should include HPV-51, 52, 56 and 58 genotypes.HPV-16, HPV-18, HPV-31, HPV-58 and HPV-52 were the five most common HR-HPV types worldwide, HPV-16 and HPV-18 account for approximately 70% of cervical cancer cases27, however, two peaks of age-specific prevalence of HPV including younger and older women were also revealed in other reports30. Various conclusions of HPV infection were usually generated by heterogeneous study cohorts including inpatients or outpatients, indeed, women receiving physical examinations also play an important role in epidemiological investigation of HPV infection. In our study, chi-squared test showed the significant differences in HPV infection among age-specific groups and the highest HPV infection rate was found in age group of 55–64 y with the prevalence rate of 28.08% , and the test of HR-HPV infection was no significant differences among age-specific groups . Although the peak age of HPV infection in different studies was diverse, the peak occurred primarily in perimenopause women which may be due to physiologic and immunologic dysregulation31. Of all population receiving physical examinations, the prevalence of HPV-16 and 52 in the 55–64 y age groups (5.86% and 4.54%) was statistically higher than the others . The findings suggested that cervical cancer screening programs should pay more attention to the women cohorts of 55–64 y.The HPV infection rates in different age groups were also varied. Previous studies have showed that HPV prevalence decreased with the increasing age worldwideAge is an important factor associated with HR-HPV infection. The true positive rate and false positive rate of HR-HPV infection in age-specific groups were generated by ROC curve, and values of AUC were used to estimate the correlation between HR-HPV and ages. As showed in Fig. 32. Lack of individualized organized cervical cancer screening program for women receiving physical examinations including opportunistic cervical lesions is another limitation which will bring about the biased prevalence. Nevertheless, statistical methods were used to overcome these shortcomings.The limitation of current study is absent analysis of mix HPV infection. The reason is that whether HPV mix infections increase the risk of disease is not clear and antigens vary among different HPV types, cross-defense is not effective enoughIn conclusion, we conducted a study with population receiving physical examinations on the prevalence of HPV and age-related groups in the Shangcheng District of Hangzhou city. The HPV prevalence and distribution varied significantly in different female age groups especially in 55–64 y. Prevention and screening strategies should be adjusted according to the HPV characteristics of age-special groups in this region.The retrospective study was designed to analyze the results of cervical HPV test of 22,382 women who receiving physical examinations in the Shangcheng District of Hangzhou city from October 2016 to November 2020. The mean age of the population was 43 years ranging from 16 to 81 years and the data was divided into 5 age groups . The inclusion criteria for the study were as follows: women who were attending first-time cervical screening, living in the Shangcheng area, were not currently pregnant and had no history of total uterus or cervix resection. The exclusion criteria were the following: women who were in their second round of screening or more, receiving cervical physical therapy or had a history of any cancers. This study was approved by the Ethical Committee of Hangzhou Women’s Hospital. Informed consent was obtained from all participants including parents of 17 young people under the age of 18 years, and all methods were performed in accordance with the relevant guidelines and regulation.HPV detection and typing were performed on the collected specimens using the real-time polymerase chain reaction (RT-PCR) melt curve test by a commercial kit  which can identify 16 HR-HPV types  and 7 LR-HPV types . The RT-PCR platform possesses the advantages of convenience, high throughput, low time and cost, and low risk of false positive results due to cross-contamination of PCR. The experimental conditions for RT-PCR Test followed the guidelines of associated protocols. Briefly, 23 HPV specific fragments were amplified by this kit, and the products were hybridized with the designed molecular beacon probe to collect fluorescence signals which could determine the HPV types according to the melting temperature (Tm) values.The age classification was chosen for the estimated metric of HR-HPV infection in population receiving physical examinations. ROC curve analysis was used to assess the performance between age classification and HR-HPV infection. AUC with a value of 1 indicates an ideal result, whereas values lower than 0.5 means an insignificant result.All statistical analyses were performed using SPSS V.19.0 . Chi-squared tests were used to assess the statistical significance of any differences in HR-HPV prevalence. P < 0.05 was considered statistically significant.


Vaccine confidence reflects social, individual, and political factors indicating confidence in vaccines and associated health systems. In Japan, the government ceased proactive recommendation of the human papillomavirus (HPV) vaccine in June 2013, only several months after the recommendation had begun. Seven years later, as of October 2020, the suspension persists and vaccine coverage has precipitously declined, resulting in many young women being continually exposed to the risk of preventable HPV-related diseases. Accordingly, understanding stakeholder opinions on HPV vaccination issues is critical for informing strategies to improve HPV vaccine confidence and acceptance. In October 2019, we performed a nationwide, web-based survey of 1646 mothers of HPV-vaccination–eligible girls, 562 female adolescents aged 15–19 years, and 919 healthcare professionals (HCPs) in Japan. This survey captured key elements of vaccine confidence , awareness, and the willingness to receive (in HPV-vaccination–eligible girls) or recommend (in HCPs) the HPV vaccine, and the factors responsible for these decisions. HPV vaccine confidence was generally higher among HCPs than among mothers or female adolescents. Nearly half of all stakeholders were neutral regarding their willingness to receive/recommend the HPV vaccine. The seriousness of cervical cancer and the HPV vaccine’s effectiveness or safety were important deciding factors for receiving/recommending the HPV vaccine. Besides these factors, sufficient information and free vaccination were crucial. Our results suggest several factors that could help shape public policy and communication strategies to improve HPV vaccine confidence and acceptance in Japan. The suspension persists. HPV vaccine coverage has sharply declined from approximately 70% to less than 1%.8 Among Japanese women born between 1994 and 2007, an excess of 24,600–27,300 preventable cervical cancer cases and 5000–5700 cervical cancer deaths are projected to occur over their lifetime owing to the precipitous decline in HPV vaccination coverage recorded between 2013 and 2019.9Robust evidence supports the effectiveness and safety of HPV vaccines.11 Understanding and implementing effective approaches to restoring confidence in the HPV vaccine is critical to improving and sustaining vaccination acceptance to potentially avert adverse public health consequences.10 We therefore conducted a survey focused on understanding HPV vaccine confidence and the willingness to receive the HPV vaccine among Japanese mothers and female adolescents, and among healthcare professionals (HCPs) to recommend the HPV vaccine.Vaccine confidence is defined as trust in the importance, effectiveness, and safety of vaccines and the health system in which those are approved and delivered.This was a nationwide, cross-sectional, web-based survey of mothers of HPV vaccination–eligible girls, female adolescents, and HCPs in Japan.Mothers and female adolescents were recruited from a database managed by INTAGE Healthcare, Inc. . HCPs were recruited from a database managed by PLAMED, Inc. . Inclusion criteria included mothers who have at least one daughter aged 12–16 years, female adolescents aged 15–19 years, and HCPs who are gynecologists (obstetricians/gynecologists), pediatricians, or internists who had seen a female adolescent aged 12–19 years in clinical practice within the year prior to study participation. Although the recommended age for HPV vaccination in Japan starts at 12 years, girls aged 12–14 years were excluded because of the respondent panel’s age restriction. Outcomes were assessed among (i) mothers who have daughters unvaccinated against HPV, (ii) female adolescents who have never received the HPV vaccine, and (iii) HCPs who currently do not recommend HPV vaccines to their patients aged 12–16 years. Factors associated with receiving or recommending the HPV vaccine were also assessed among mothers who have daughters vaccinated against HPV, female adolescents who have received the HPV vaccine, and HCPs who currently recommend HPV vaccines to their patients aged 12–16 years.The primary outcome was measures of HPV vaccine confidence  and awareness of cervical cancer and the HPV vaccine among mothers, female adolescents, and HCPs. Secondary outcomes included HPV vaccination practices and the willingness to receive, have their daughters receive (for mothers), or (for HCPs) recommend the HPV vaccine, and factors associated with receiving or recommending the HPV vaccine, including timing and factors associated with acceptance of the HPV vaccine.11 Other questions for this study were developed based on previous studies and modified for the situation in Japan.13 Question wording was finalized after a pilot study conducted from August 28 to 30, 2019, to ensure question accuracy and comprehensibility.Potentially eligible respondents were randomly sampled and sent e-mail invitations with a web-link to participate in the study. A maximum of three reminders were sent to survey participants. An eligibility check was performed. The web-link included in the e-mail invitation directed participants to a webpage providing the following: (i) relevant study information, (ii) the option to provide informed consent for study participation and data utilization for possible publication, and (iii) screening questions . Only participants who provided informed consent and met the inclusion/exclusion criteria were able to proceed and respond to the web-based study questionnaire. Respondents were allowed to respond to the questionnaire only once. Mothers with more than one daughter were requested to answer questions only for the daughter whose age was closest to 13 years. All respondents were asked basic demographic questions. The total number of items per respondent was approximately 30. The survey questionnaires were developed based on items in the Vaccine Confidence Index™ developed by the Vaccine Confidence Project as a tool for estimating vaccine confidence, against a mix of socio-demographic variables, and using a five-point Likert scale: Strongly agree, Tend to agree, Do not know, Tend to disagree, and Strongly disagree, for three domains of confidence.To prevent missing or out-of-range numerical values, restrictions were implemented on the online response entries. Upon completing the entire questionnaire, respondents received points that could be exchanged for various items, such as cash and gift cards.Vaccine confidence was defined as a composite of answers to questions about the HPV vaccine, assessing vaccine importance, effectiveness, and safety. Other questions assessed levels of vaccine confidence regarding views on the importance, effectiveness, and safety of non–HPV vaccines, trust in the government to protect citizens from health risks, awareness of cervical cancer and the HPV vaccine, and factors associated with vaccination/recommendation. For mothers and adolescents, adolescents’ HPV vaccination history was queried, as was HCPs’ current status for HPV vaccine recommendation.Respondents’ consent for study participation and data utilization was obtained electronically by having them click a checkbox. The study protocol was approved by the ethics committee of Kitamachi Clinic , and the study was conducted in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects and the Act on the Protection of Personal Information in Japan. Under this act, surveys were anonymized to prevent personal information from being traceable by the sponsor to identify a specific individual.P-value of <0.05 considered as statistically significant.The analysis was primarily descriptive in nature; thus, no formal hypothesis tests were conducted. For continuous variables, descriptive statistics  were calculated. For categorical variables, the number and proportion of respondents within each corresponding category were calculated. All statistical analyses were performed using SAS version 9.4 (SAS Institute) and R version 3.6.2 . The proportions for each outcome were summarized descriptively in subgroups by each covariate. Pearson’s chi-square test was used to analyze statistical differences in the proportions for any comparisons between groups, with a The survey was conducted in October 2019, with 1646 mothers, 562 female adolescents, and 919 HCPs included in the analysis populations. We describe vaccine confidence, awareness, and willingness to receive/recommend the HPV vaccine among 1591 mothers with daughters unvaccinated against HPV, 466 female adolescents who had never received the HPV vaccine, and 548 HCPs who at the time of the survey did not recommend the HPV vaccine to their patients. For all respondents, the factors that determined their decisions were assessed . Tables P< .001). Mothers (46%–54%) and female adolescents (36%–40%) most commonly selected “Do not know” for questions related to the three factors of vaccine confidence  on the importance, effectiveness, and safety of the HPV vaccine was reported by 24%, 25%, and 11% of mothers; 49%, 46%, and 34% of female adolescents; and 83%, 84%, and 52% of HCPs, respectively . Views on vaccines and vaccination in infants/toddlers and adolescents are shown in Supplementary Figure 2. The levels of confidence in non–HPV vaccines were higher than those in the HPV vaccine.Ten percent of mothers and 18% each of female adolescents and HCPs agreed with the statement, “The information about HPV vaccines from the government is sufficient” .P<.001 for both items). Approximately one-third of female adolescents were not aware of the HPV vaccine  to the awareness of cervical cancer and the HPV vaccine was reported by 61% and 44% of mothers, 27% and 16% of female adolescents, and 90% and 86% of HCPs, respectively  gynecologists and 71% of hospital physician (HP) gynecologists—more than twice the proportion of any other specialists—answered that they knew about the HPV vaccine in detail. Eleven percent of HP internists, 58% of GP pediatricians, and 32%–55% of other specialists knew that the HPV vaccine is included in Japan’s NIP.P<.001). However, neutral willingness (answer “Do not know”) was the largest proportion in all groups .Among mothers and female adolescents, 9% and 23%, respectively, were willing to receive the HPV vaccine, whereas 43% of HCPs were willing to recommend the HPV vaccine (l groups . RespondThe main reasons why mothers chose to have their daughters vaccinated were because they thought HPV vaccines are effective (45%) and cervical cancer is dangerous . The mother’s recommendation was the main reason for female adolescents having received the HPV vaccine ; SupplemSixty percent each of HCPs who currently recommend the HPV vaccine and HCPs who do not but stated their willingness to do so reported that they would recommend the HPV vaccine based on patient inquiry . SupplemThis is the first nationwide, comprehensive survey on vaccine confidence related to HPV vaccination in Japan conducted since the 2013 suspension of the government’s proactive HPV vaccine recommendation. Collectively, the findings could help inform the development of effective public health communications, public engagement, and advocacy campaigns that could lead to greater awareness of, confidence in, and improved uptake of the HPV vaccine in Japan.P<.001). Among mothers, HPV vaccine confidence regarding safety was slightly lower than that for effectiveness and importance. Overall, respondents with high HPV vaccine confidence were more willing to receive/recommend the HPV vaccine. The seriousness of cervical cancer, the HPV vaccine’s effectiveness and safety, sufficient information, and free HPV vaccination were important factors for respondents’ decisions to receive/recommend the HPV vaccine. Sixty percent of HCPs who currently recommend the HPV vaccine or who were willing to do so each reported that they would recommend it based on patient inquiry. As patients rarely ask their physicians about the HPV vaccine, this result indicates that HCPs are not necessarily recommending the HPV vaccine proactively or that often.Key findings are that HPV vaccine confidence and willingness to receive/recommend the HPV vaccine were higher among HCPs than among mothers and female adolescents  recommend the HPV vaccine are crucial to better understand behavioral motives and the dynamics of the interaction and communication between them that could in turn be addressed by tailored interventions.Previous surveysPublic trust and confidence in the HPV vaccine and its providers, as well as in the vaccination process, development policies, and recommendation, are at the core of increasing vaccine acceptance and implementing successful immunization programs. Our results reveal the current state of HPV vaccine confidence in Japan and its possible determinants. Such evidence could help shape public policy and communication strategies to improve HPV vaccine confidence and acceptance in Japan.We found that the level of awareness/understanding and type and source of information shape perception of the HPV vaccine. HCPs recommending the HPV vaccine or who were willing to do so relied on background scientific knowledge more than other sources of information. Moreover, HCPs who referred to scientific information showed high acceptance of the HPV vaccine. By contrast, HCPs, as well as mothers and female adolescents who were negative about the HPV vaccine, were more likely to be influenced by media reporting. Both mothers and female adolescents selected “TV news or health-related programs” as the most frequently used source of information. Mothers willing to recommend the HPV vaccine were influenced by information from the local government and HCPs.A high proportion of the HCPs we surveyed were not even aware that the HPV vaccine is included in the NIP. Based on these results, better public communication is needed around the availability of the HPV vaccine in the NIP. HCPs who were asked about the reasons for recommending the HPV vaccine reported that safety, sufficient information, routine (free) vaccination, and care and support after vaccination were the most important.9 Including the HPV vaccine along with routine adolescent vaccinations has been shown in the United States to be a key determinant of HPV vaccination rates.25 We found that many mothers, female adolescents, and HCPs have higher confidence in vaccines for infants and toddlers compared with the HPV vaccine.Since mothers’ and female adolescents’ reliance on information about the safety and effectiveness of the HPV vaccine can influence willingness to receive the HPV vaccine, all stakeholders should be given sufficient and appropriate information about the HPV vaccine. Findings from this study could help shape public policy and communication strategies to improve future HPV vaccine confidence and acceptance in Japan and ultimately increase HPV vaccination rates to avert the rising trend of cervical cancer.26 (as of October 2018) consisted of approximately 9,850,000 individuals in Japan and that of PLAMED, Inc.27 (as of April 2016) consisted of approximately 45,400 doctors from 37 medical departments in Japan. The analysis population included 34 female adolescents who reported that they had received the HPV vaccine at the age of 9 years or 10 years—ages ineligible for the routine vaccination program in Japan. Results excluding these respondents were similar to those that included them (data not shown).Because of the differences between Japan and other countries regarding HPV vaccine policy, some of our results may not be widely generalizable. Within Japan, however, both INTAGE and PLAMED databases have similar patterns of distribution of age, gender, geographic locations, and clinical departments as nationwide data. The selected database populations are not expected to greatly differ from the target Japanese populations because the database from INTAGE Healthcare, Inc.Several limitations should be noted. Respondents’ characteristics might be different from those of the general population because people without internet access were not included, nor were females aged 12–14 years. Mothers, females aged 16–19 years, and HCPs were randomly sampled given the large number of such individuals registered in the database. In contrast, all registered females aged 15 years received an e-mail invitation because of the relatively small number of such individuals in the database. Response bias may have occurred in this study as it is likely that individuals who responded to the questionnaire had an interest in the research area compared with those who did not, which could distort the samples’ representativeness. The causal relationships between factors associated with the intentions of the respondents cannot be determined because of the cross-sectional nature of this study. Additionally, the intended willingness to receive or recommend the HPV vaccine may not reflect actual behavior. An important clinical question revealed by the current study is what can bridge the gap between willingness and behavior.In conclusion, levels of HPV vaccine confidence were higher among HCPs than among mothers and female adolescents. The seriousness of cervical cancer and perceptions of HPV vaccine effectiveness and safety were the most important factors among all stakeholders for the decision to receive/recommend the HPV vaccine. Mothers and female adolescents were more likely to be influenced by media reporting, but some of them indicated the desire to get information from HCPs. Given that patients rarely ask their physicians about the HPV vaccine, this represents an impediment to HCPs recommending the HPV vaccine in the future. This is because HCPs stated that their willingness to recommend the HPV vaccine would be based on patient inquiry. This thus represents another gap that could be addressed through future interventions. Overall, our survey results suggest the existence of several critical factors that could change the perception and behavior around HPV vaccination. These in particular include resumption of a proactive recommendation by the MHLW and HCP recommendation.Click here for additional data file.


The risk of HIV acquisition is low on a per-contact basis but increased by transmission co-factors such as other sexually transmitted infections (STIs). Human papillomavirus (HPV) is a prevalent STI that most individuals will acquire HPV in their lifetime. Current HPV vaccines can prevent newly acquired infections, but are largely ineffective against established HPV, complicating worldwide eradication efforts. In addition to being the causative agent of cervical cancer, accumulating evidence suggests that HPV infection and/or accompanying cervical inflammation increase the risk of HIV infection in men and women. The fact that immunological features observed during HPV infection overlap with cellular and molecular pathways known to enhance HIV susceptibility underscore the potential interplay between these two viral infections that fuel their mutual spread. Here we review current insights into how HPV infection and the generation of anti-HPV immunity contribute to higher HIV transmission rates, and the impact of HPV on mucosal inflammation, immune cell trafficking, and epithelial barrier function. HPV is the most common viral infection of reproductive tracts and the vast majority of sexually active individuals will acquire this virus at some point in their lives . At 4 yeDespite availability of several effective, prophylactic HPV vaccines, the burden of HPV-associated pathology and cancer remains high . The twoPapillomaviridae family and there are over 200 genotypes of HPV which are divided into low-risk HPV, causing anogenital and cutaneous warts or no obvious disease, and high-risk HPV, which in some cases lead to oropharyngeal and anogenital cancers  is exposed through epithelial trauma, HPV L1 major capsid protein binds the heparin sulfate proteoglycans (HSPGs) on BM . This bi+ T cells are of particular importance, exemplified by patients with regressed HPV+ lesions containing significantly higher numbers of HPV16 E6 and E7-specific CD4+ T cells  and tissue resident macrophages and function to recruit CD4f lesion . As partf lesion . HPV oncf lesion . E6 and f lesion . Lastly,f lesion , limitinf lesion . TLR9 lef lesion , suggestf lesion Figure 2f lesion .via tumor necrosis factor (TNF) (+ lesions are characterized by high infiltration of predominantly M2-polarized tumor-associated macrophages (TAMs)   or antibor (TNF) . High grs (TAMs)  and are s (TAMs) . M2 TAMss (TAMs) . It is ss (TAMs) . Additioof IL-10 .+ T cells infiltrate HPV-associated tumors, they do not seem to impair tumor growth, indicative of sub-optimal priming  that reside in the epidermis normally migrate to secondary lymphoid organs and prime the adaptive immune response. HPV has been shown to impair trafficking of APCs to the lymph node, delaying the onset of the immune response. Low infiltration of LCs into HPV-associated cancerous tumors is associated with disease severity . This is priming .+ cervical lesions express high levels of IDO1 (+ DCs may drive cancer progression by generating Tregs and/or inducing T cell anergy.Maturation of APCs is also impacted in HPV-infected tissues, as expression of MHC and co-stimulatory molecules CD80 and CD86 are downregulated . Many ty of IDO1 , suggest+ T cell populations in the genital epithelium and sub-mucosa, the prime targets of HIV during mucosal exposure (+ T cell numbers found within the stroma and epithelium are a marker for HPV lesion regression (+ T cells (The per-exposure risk of HIV acquisition ranges from 0.08% for receptive vaginal sex  to 1.4% exposure . Meta-anexposure . A 2.4-fexposure . While bgression  and thergression Table 1 gression . Overlapgression . Increasgression . Increasgression , and indgression . However T cells . Finally T cells , but NK  T cells .in vivo would be an invaluable tool to address some of these outstanding questions (While HPV infection can cause cervical cancer in women, it is also a strong risk factor for HIV acquisition. This may have a biological basis; HPV infection induces genital inflammation, vaginal barrier disruption, and influx of activated target T cells, which have all been implicated in HIV risk. However, a clear mechanistic understanding of the factors that directly facilitate HIV acquisition is lacking. The fact that HPV can establish a latent state and re-establish infection complicates HIV prevention strategies in high-risk individuals, prompting the need for a better understanding of the cellular and molecular basis of anti-HPV immune control. Recent characterization of a mouse papillomavirus to study mucosal disease progression, host immune control and cervical cancer development uestions . Murine Conceptualization, writing and editing, RZ, TM, and LM. Figures, RZ. Supervision, TM and LM. Funding acquisition, TM and LM. All authors contributed to the article and approved the submitted version.This work was supported by the CIHR grant MRT-CDAA-159236 to TM and a studentship from Research Manitoba and University of Manitoba Student Fellowship to RZ. LM is supported by CIHR New Investigator Award.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


HPV-B19感染后网织红细胞及血红蛋白变化：9例患儿以贫血伴网织红细胞下降为主要特点，HPV-B19感染后患儿网织红细胞下降幅度、速度明显大于血红蛋白。例1移植后网织红细胞持续偏低，其余8例患儿均出现网织红细胞比例和绝对计数快速、明显下降，网织红细胞开始下降后7 d内网织红细胞比例、绝对计数降幅中位数分别为90.4％（24.7％～98.7％）、90.7％（18.6％～99.0％）。相应时间段内，患儿血红蛋白下降不明显或尚未开始出现下降（2. HPV-B19感染后血象及骨髓细胞形态学变化：除常见红系造血受抑表现外，此类患儿还具有非红系受抑的血液学改变。9例患儿中3例外周血出现红系、粒系及血小板三系下降，2例出现红系、粒系下降，2例表现为单纯红系下降，2例出现红系、血小板下降（而骨髓细胞形态学改变具有多样性。例1红系、巨核系受抑，与外周血表现一致，其余8例患儿外周血改变与骨髓不完全一致。5例患儿外周血中性粒细胞下降，但骨髓涂片均未见粒系增生受抑；6例患儿骨髓巨核系增生减低，其中5例出现外周血血小板减少。全部9例患儿均出现贫血，但仅5例患儿骨髓红系增生减低，其余4例患儿中红系增生正常、偏高各2例。仅1例患儿骨髓涂片中见到巨大原始红细胞。详见−1·d−1，中位疗程为18（9～35）d。在IVIg治疗后9例患儿均在短期内获得网织红细胞及血红蛋白恢复。以开始IVIg治疗为起点，7例患儿网织红细胞比例恢复至≥1％、绝对值恢复至≥0.024×1012/L的中位时间均为7（6～16）d，8例患儿网织红细胞比例、绝对值恢复到HPV-B19感染前水平的中位时间分别为17（6～35）d、16.5（6～35）d。9例患儿血红蛋白升高≥10 g/L中位时间为6（5～28）d，≥20 g/L中位时间为14（6～133）d。3. HPV-B19感染治疗后血象及骨髓细胞形态学改变：9例患儿IVIg中位剂量为0.30（0.09～0.40）g·kg8例患儿中，7例骨髓红系比例由治疗前的8.0（0～51.5％）增至治疗后的42.0％（12.5％～55.0％）、巨核细胞数由治疗前的3（0～60）个/全片增加至20（3～101）个/全片，2例患儿发生HPV-B19感染复发，再次IVIg治疗仍有效。详见P＝0.04）1975年，Cossart等成人HSCT患者合并HPV-B19感染多发生于移植后3个月或以后以往研究显示，在免疫功能正常和免疫功能缺陷人群，HPV-B19感染均可引起外周血三系细胞减少allo-HSCT造血重建后HPV-B19感染患儿的骨髓同样具有非红系受抑的表现。本研究中，6例患儿骨髓巨核系增生减低，提示HPV-B19感染除累及红系外，巨核系同样易受累及。既往报道中，接受自体造血干细胞移植（auto-HSCT）的多发性骨髓瘤患者HPV-B19感染以血小板减少为主要表现本研究中，5例患儿骨髓涂片见红系增生减低，但2例患儿红系增生正常，还有2例患儿红系增生偏高。Crook等治疗后网织红细胞比例及绝对值恢复到正常水平的中位时间为7 d，这与既往免疫缺陷患者治疗反应的报道一致综上，本组病例结果显示，儿童血液病患者allo-HSCT造血重建后合并HPV-B19感染的血液学表现具有一定异质性且以贫血伴网织红细胞下降为主要特点，快速、明显的网织红细胞下降对HPV-B19感染的筛查可能具有重要意义。


Human papillomavirus screen in female cervical cells has demonstrated values in clinical diagnosis of precancerous lesions and cervical cancers. Human papillomavirus tests of cervical cells by utilizing Polymerase Chain Reaction (PCR) method provides human papillomavirus infection status however no further virus in situ information. Although it is well known that the tests of human papillomavirus E6/E7 RNA location in infected cervical cells and cell internal malignancy molecular will provide clues for gynecologists to evaluate disease progression, there are technique difficulties to preserve RNAs in cervical scraped cells for in situ hybridization.In current study, after developing a cervical cell collection and preparation method for RNA in situ hybridization, we captured the chance to screen 98 patient cervical cell samples and detected human papillomavirus E6/E7 mRNAs of high-risk subtypes, low-risk subtypes and long non-coding RNA (lncRNA) TERC in the cells.There were 69 samples exhibited consistence between human papillomavirus PCR and human papillomavirus RNA in situ hybridization results in cervical collected cells. Among them, 23 were both positive and 46 were both negative. In the rest 29 samples, 8 were HPV RNAscope positive, either high risk or low risk subtypes, however HPV PCR negative. Another 9 samples were HPV PCR results positive whereas RNAscope negative. The last 12 samples were HPV positive detected by both RNAscope and PCR methods, however inconsistent between high-risk and low-risk subtypes. In RNAscope positive samples, viral E6/E7 mRNAs were observed to distribute in cervical scraped cell nucleus and cytoplasm. Moreover, HPV viral RNA gathered clusters were observed outside of cells through human papillomavirus RNA in situ hybridization detection. Varied numbers of human papillomavirus infective cells were detected by RNAscope assay in different patients even though they were all human papillomavirus high-risk subtype positive discovered by human papillomavirus PCR results. A cell malignancy related long non-coding RNA, TERC, has been detected in seven patient samples. The patient follow-up information was further analyzed with RNAscope results which indicated a combination of RNAscope positive signals of TERC and human papillomavirus high risk signals in more than 10 cells (cytoplasm or nucleus) may connect with cervical lesion fast progression which deserves further studies in the future.CTaken together, current study has provided an observable clue for gynecologists to evaluate human papillomavirus infection stage and cell malignancy status which may contribute for assessment of cervical disease progression.The online version contains supplementary material available at 10.1186/s12985-021-01696-9. Human papillomavirus (HPV) is a group of more than 200 related viruses, which are widely spread through vaginal, anal or oral sex. It has been proven that HPV can cause multiple types of cancers including cervical cancers and head and neck cancers. More than 9 of every 10 cases of cervical cancer are caused by HPV which brings the fact that monitor HPV status benefiting the diagnosis of precancers and cervical cancers. Given the fact that cervical cancer is once the leading cause of cancer deaths among women worldwide, the HPV detection becomes a key test for cervical diseases [Among eight genes HPVs encode, E6 and E7 are best-known for their transforming properties . E6 and i.g. cervical biopsy samples or surgery collected tissues, to support clinical diagnosis [In the past several years, studies have focused on HPV E6/E7 mRNA features in cervical pathological samples, iagnosis . In the iagnosis , 16.This study was conducted with the approval of the Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital, Institutional Review Board (IRB) Committee on Human Research in the Medical Sciences (CHRMS). A written informed patient consent was signed by each patient before joining this study project. All agrees to provide specimens and their data to be further published as part of the study results.115 adult female outpatients ranging in age from 23 to 71 years were included in this study. The cervical samples were collected between Dec 2018 and March 2019 followed the procedures described below. After exposure cervical entrance, its surface was scratched two circles to collect cervical scraped cells using two different Thinprep Cytologic Test (TCT) sample collection brushes, respectively. For most patients, first circle of scraped cells was sent for HPV PCR tests, whereas the second circle of scraped cells was tested for RNAscope assays. For the latter collected ones, the TCT sample collection brush was cut and the tip with cells were kept in a 50 ml tube with 10% neutral buffered formalin (NBF). The tubes were kept at 4 °C overnight then the cell samples on the brush tips were physically scraped down from the brush into 10% NBF. The tubes were centrifuged at 800rmp for 10 min to collect cells. Cell pellet of each sample were transferred into 2 ml EP tube and washed by PBS once. 1.5 ml 70% ethanol was used to resuspend the cells and stored at room temperature (RT) for 2 h. The cells in 2 ml EP tubes were then centrifuged at 8000–10,000 rpm for 5 min and the supernatant was discarded. The cell pellets were regarded as a chunk and went through 70%, 80%, 95% and 100% ethanol, respectively, at RT for 10 min. After 100% ethanol, the cell pellet chunk floated and were transferred into a filter paper to totally try. Melt CellGel  was dropped onto hydrophobic paper (parafilm paper). The dried cell pellet chunk was embedded in the CellGel, solidified with the CellGel and became a bigger block. The latter one was transferred into tissue processing histology cassette and went through 85% ethanol for 45 min; 95% ethanol for 30 min, 100% ethanol I for 30 min, 100% ethanol II for 30 min, 100% ethanol III for 45 min, xylene I for 30 min, xylene II for 30 min, xylene III for 45 min, Wax I for 30 min, Wax II for 30 min, Wax III for 30 min and Wax IV for 30 min, then embedded in paraffin to become Formalin-fixed paraffin-embedding (FFPE) blocks. Each FFPE block carried a patient cervical cell pellet were sectioned of 5 μm for RNA in situ hybridization tests.RNA in situ hybridization was performed on FFPE cell pellet sections (5 μm) using the RNAscope 2.5 HD assay-Red  and the RNAscope Probes  including HPV-HR18 , HPV-LR6  , 14 and Human Papillomavirus Polymerase Chain Reaction HR-HPV PCR was performed using the 23 HPV Genotyping Real-time PCR Kit  containing 17 high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and 6 low risk HPV types: 6, 11, 42, 43, 44 and 81.i.e. big amount of dot signals with high-density in limited area, above one or more cells which were recorded as well. For TERC results, RNA signals were only discovered in nucleus which were recorded.RNAscope stained FFPE cell sections were scanned using Leica AT2 scanner . Whole sections were examined at 40× magnification. RNAscope results of HPV were recorded based on signal location and positive cells. For probe-HPV-HR and probe-HPV-LR results, the signal locations of cells and the positive cell numbers in each sample, < 3, ≥3≤10 or > 10, were recorded. Besides the classic RNAscope dot signals detected in cytoplasm or nucleus, there were HPV RNA signals gathered as clusters, Two gynecologists (Z. H. and H. Y.) evaluated the scanned sections independently. If a disagreement occurred during RNAscope assay result recording, they reviewed the case together and reached a final agreement. The interpretation was generally straightforward; therefore, no significant disagreements led to incompatibility.In current study, totally 115 patient cervical cell samples have been collected. 101 samples have been successfully prepared into FFPE blocks and passed RNAscope positive control tests using the probe of Hs-UBC. Three of 101 samples failed RNAscope negative control tests with background of probe DapB staining. All 98 QC passed samples were studied using RNAscope HPV high-risk probe (V-HPV-HR18) and HPV low-risk probe (V-HPV-LR6). Hs-TERC probe targeting on cell malignance has also been detected in the samples.Among 98 analyzed patient samples, 69 showed consistent results between RNAscope and PCR from patient cervical scraped cells. 46 of 69 patient samples were PCR and RNA-scope both negative  was popular for such assays with 100% specificity reported of individual HPV subtype RNAscope probe from the pooled ones compared to HPV subtype PCR results [High-risk subtypes of HPV infection are known to have chance to lead cervical cells into transforming stages and may have more chance to develop into high squamous intraepithelial lesion (HSIL) . HPV PCR results . HPV RNA results –30, whicCurrent study is the first report trying to estimate cervical lesion progression by using RNAscope HPV in situ hybridization in cervical scraped cells as we have known. By developing a new method which solved the main technique difficulties of cell detachment and RNA quality issue of cervical scraped cells, it makes RNA in situ hybridization of HPV E6/E7 become feasible in such sample type. The results uncovered a window to study active HPV infection status and how the viral locally interacts with cervical cells which provides observable clues for disease progression assessment. TERC is a long non-coding RNA associated with high grade squamous intraepithelial neoplasia (HSIL) and progression of invasive carcinoma . The amp98 patient samples which passed RNAscope QC have been examined by using HPV high-risk probes, RNAscope HPV low-risk probes and TERC probe. RNAscope results have been compared to PCR assay data for further analysis. In most cases, cervical scraped cells were collected twice, the former set was used for PCR assays and the latter set was used for RNAscope assays. In several cases, PCR used cell samples were collected at different date based on patients’ situation. The collection time variations may partly lead to the 29 inconsistence of HPV results between RNAscope and PCR assays. HPV DNA results detected by PCR study were compared to active HPV transcribed E6/E7 mRNAs captured by RNAscope assays, which may also lead to varied results if HPV virus infection happened previously however with inactive status. In current study, three RNAscope HPV positive cells observed in one sample and ten RNAscope HPV positive cells in one sample were selected to separate HPV infection status as low, medium and high, which helps to predict disease progression combined with RNAscope TERC results. The cut off numbers, three and ten, may be modified followed by a bigger patient sample pool collected in the future studies. There is software available for RNAscope quantification data analysis. RNAscope Red Assay has provided clear red RNA signal dots for positive cell identification which is straightforward for scoring by gynecologists. Since there is no disagreement on result interpretation by two recorders to raise a final decision, no software quantification was further performed. RNAscope assay results demonstrated two types of HPV signals. One is typical RNA signal dots located in cell nucleus and cytoplasm. The other type of signals exhibited big clusters, many RNA dot signals detected with high-density, located above one or several cells, which looks like “out of cell” signals. The latter signal phenotype has been observed in both high-risk and low-risk HPV probe detected samples which demonstrated viral secreting status. In the patients whose sample were RNAscope positive whereas PCR negative, 4 were after-LEEP or CIN I-III stage. RNAscope positive signals in the samples were most out of cells, with only 4 patient samples showed HPV positive signals in nucleus (< 10). In nine RNAscope negative whereas PCR positive samples, seven were HPV High-risk subtypes. RNAscope results negative may due to sample and tested cell variations, inactive virus status or other sample preparation caused unknown reasons.Five of ninety-eight samples  exhibited active HPV E6/E7 mRNA signals in more than 10 cells  may connect with cervical lesion fast progression which deserves highly attention.Additional file 1. Supplemental Table 1. RNAscope and PCR results in patient cervical scraped cells. Both negative. Supplemental Table 2. RNAscope result summary of patients with high HPV signal dots and/or TERC positive signals, and the disease follow-up records.


This study presents the clinical assessment of the Onclarity HPV Assay (Becton Dickinson) on the novel COR high-throughput instrument (Becton Dickinson) using the international guidelines in a routine setting.Screening samples collected in BD SurePath from women aged 30 years and older were used in this validation. Noninferiority of the Onclarity HPV Assay on the COR instrument (Onclarity-COR) was assessed with the comparator assay glycoprotein 5–positive (GP5+)/6+ enzyme immunoassay (GP-EIA) for clinical sensitivity on 122 cervical intraepithelial neoplasia 2 and greater samples. Specificity was assessed using 887 samples with twice-normal cytology. Inter- and intralaboratory reproducibility analysis was assessed using 525 samples. Finally, a time-and-motion study was performed to evaluate COR instrument performance characteristics.P = .0016) and specificity (P < .0001). The intralaboratory reproducibility was 98.3% (κ = 0.96), and interlaboratory agreement was 98.5 % (κ = 0.96). The daily hands-on time for the COR instrument was 58 minutes, and walk-away time was 7 hours, 2 minutes per 8-hour day shift.The Onclarity-COR was noninferior to the GP-EIA for both sensitivity (The Onclarity-COR instrument fulfills international validation criteria on sensitivity, specificity, and laboratory reproducibility. The Onclarity assay’s extended genotyping capability, together with its high-throughput characteristics, makes the COR instrument an excellent candidate for use in human papillomavirus primary cervical cancer screening. Key PointsWe evaluated the BD COR high-throughput platform combined with the BD Onclarity HPV assay.Successful clinical validation was compliant with the international guidelines for human papillomavirus (HPV) assays for cervical cancer screening.The BD COR high-throughput platform allows for unprecedented laboratory HPV test production with limited operator interaction requirements.1 A trend in cervical cancer screening programs is a continued consolidation process toward larger, centralized laboratories with unprecedented high throughput. This shift necessitates validation of existing HPV assays and novel high-throughput HPV test instruments using well-defined clinical assay performance specifications compliant with the international criteria for HPV tests used in cervical cancer screening.2 The migration of an already-validated HPV test to a new high-throughput instrument platform can result in changed test characteristics. Validation of the new combination of assay and instrument is therefore as important as the original validation of the assay itself, especially if the new high-throughput platform entails different sample processing from the previous instrument. Operator interaction (hands-on time), workflow, and system maintenance requirements are also important determinants for the clinical performance of fully automated high-throughput HPV instruments, and these elements can differ considerably between automated systems.4Cervical cancer screening is transitioning worldwide from cytology toward primary human papillomavirus (HPV) screening. At the same time, there is a paradigm shift toward risk-based guidelines using clinical action thresholds.4-6 the Alinity instrument from Abbott,8 and the COR HPV instrument from BD Diagnostics are all branded on sample turnaround numbers surpassing their predecessors: the Roche Diagnostics cobas 4800,9 the Abbott RealTime High Risk HPV Assay,10 and the BD Viper LT system,12 respectively.Commercially available high-throughput HPV instruments are only now being introduced and validated. The cobas HPV Test on the cobas 6800/8800 instruments from Roche Diagnostics,13 Here, HPV genotyping plays a role in the management of index screening samples but also, when combined over time with subsequent follow-up samples or multiple screening rounds, enables clinicians to assess HPV persistence vs new infections as an integrated element in the evaluation of risk of disease and subsequent management.Besides the move toward larger instruments for existing assays, a change toward the use of HPV genotype information in management algorithms is defining cervical cancer screening. The 2019 ASCCP guidelines describe a paradigm shift to risk-based guidelines using clinical action thresholds.14-16 It is US Food and Drug Administration (FDA) approved for clinical cervical cancer screening using the Viper LT instrument.17-19 The newly developed, fully automated high-throughput BD COR instrument runs the Onclarity HPV Assay (Onclarity-COR) with identical chemistry, aspiration, and transfer volumes as on the well-validated Viper LT instrument (Onclarity-Viper). The COR system received CE Mark approval in 2019, and a premarket approval supplement was submitted to the FDA in 2020. The COR instrument consists of 2 interconnected units: a processing unit (PX) and an analytical unit (GX). SurePath or ThinPrep vials are loaded directly into the COR system, which handles the scanning, aliquoting, preheat treatment, DNA extraction, and real-time polymerase chain reaction (RT-PCR) analysis in a fully automated workflow that performs all preanalytic and analytic assay testing in a single system.The BD Onclarity HPV Assay is an extended genotyping assay that can detect 6 individual HPV genotypes and 8 genotypes in 3 groups . The Onclarity HPV Assay has been validated according to the international guidelines for samples collected in both ThinPrep and SurePath liquid-based cytology media in several studies.21 where clinical sensitivity and specificity were evaluated against glycoprotein 5–positive (GP5+)/6+ enzyme immunoassay (GP-EIA), accepted as a standard comparator assay. Furthermore, the validation panel was complemented with an independently collected reproducibility panel in concordance with international validation guidelines.2 A detailed genotype concordance analysis between Onclarity-COR and Onclarity-Viper was performed as well. Finally, a time-and-motion study was conducted to assess the laboratory performance of the COR instrument in a routine 8-hour day setting.Here, we validate the Onclarity HPV assay after migration from the Viper LT instrument to the COR high-throughput HPV instrument using SurePath screening samples from women aged 30 years and older who participated in the organized cervical cancer screening program in Denmark. The validation is performed using the samples from the fourth installment of the VALGENT validation study,20 In short, the VALGENT4 panel was collected at the Department of Pathology, Hvidovre Hospital, Denmark (parent laboratory) in 2016 and consisted of 2 populations: 998 consecutive screening samples from routinely screened women  consisting of 947 samples negative for intraepithelial lesions or malignancy (NILM); 6 with abnormal squamous cells of undetermined significance (ASCUS); 21 with low-grade squamous intraepithelial lesions (LSILs); and 24 with high-grade SILs (HSILs), atypical glandular cells, atypical cells—cannot exclude HSIL, and adenocarcinoma in situ 14Sample collection, processing, cytology, and histology procedures have previously been described in detail.2—in total, 169 positives and 355 negatives. In total, 525 samples were tested twice for intralaboratory agreement at the parent laboratory in Copenhagen. The original SurePath vial was tested on the COR instrument twice on 2 separate runs . For interlaboratory agreement, an aliquot of the samples, preprocessed at the parent laboratory from the original vial to instrument-compliant molecular (M) tubes, was shipped to Østfold Hospital Trust, Norway, and tested once. Only samples valid on all 3 runs were included in the analysis.The reproducibility agreement panel contained 525 samples collected from routine samples tested with the Onclarity HPV assay on the Viper LT instrument from Danish women going for screening, with a predefined split of 32% HPV-positive and 68% HPV-negative samples, as stipulated by the international guidelines22 The COR system has a flexible, modular design and consists of a centralized preanalytical PX module that performs all primary specimen conversions, including uncapping/capping, vortexing, aliquoting, heating (as needed), specimen holding (as needed), and control rehydration.The Onclarity assay is an RT-PCR assay with extended genotyping for 9 genotype readouts . The Onclarity assay on the COR instrument uses the same chemistry and sample aspiration and transfer volumes as the Onclarity assay on the Viper LT instrument.The processed specimens and controls are then sent to the analytic GX module, which is equipped with full assay automation (without user intervention). Depending on the particular needs of the laboratory, the preanalytical PX module can be connected with up to 2 independent GX modules, thus offering flexible options dependent on capacity needs. We evaluated the performance of the PX module paired with a single GX unit. Onclarity testing on the COR instrument was performed in 2019, with mean time from sample reception at the laboratory to testing of 1,185 days .20The GP-EIA was used as the standard comparator for clinical accuracy of the sensitivity detection of ≥CIN2 or ≥CIN3 and the specificity of <CIN1 using the Onclarity assay performed on the COR instrument. The high-risk HPV (hrHPV) GP-EIA assay has pooled detection of 14 hrHPV types , and the GP-EIA testing was performed as part of the VALGENT4 study described previously.14 Testing was performed in 2016, with mean time from sample reception at the laboratory to testing of 28 days . The samples were stored refrigerated during this period and were subsequently stored at −20°C before COR testing.Onclarity testing was performed on the Viper LT instrument according to the manufacturer’s recommendations and as described previously.A time-and-motion study of the COR instrument workflow was performed. For 1 working week, 330 samples were loaded into the COR daily for a single-shift workflow. Time points were assessed on each of the 5 days for (1) system startup, (2) daily maintenance, (3) weekly maintenance, (4) reagent loading, and (5) sample loading. Hands-on time and time to result for each day were registered.Two daily interactions were required with the instrument during the 8-hour shift: At startup, we loaded all the SurePath vials to be processed that day together with the required consumables and reagents , and a second interaction in the afternoon was necessary to replenish pipette tips, extraction trays, M tubes, and PCR plates. The BD COR system has 6 extraction drawers (capacity = 30 patient samples and 2 QCs), 5 of which are replenished in the afternoon to allow the instrument to continue processing unattended, for a combined total of 11 × 30 = 330 specimens. Weekly maintenance was not included in the daily contact time but consisted of cleaning selected touch points on the GX and PX units with 1% bleach, which took approximately 30 minutes.23A sample was considered Onclarity positive (for both instruments) if the cycle threshold value was 34.2 or less for HPV-18, 31, 45, 51, 52, 33/58, 35/39/68, and 56/59/66 and 38.3 or less for HPV-16. The GP-EIA detects all 14 HPV genotypes detected by the Onclarity assay as a pooled hrHPV positive result. Between the Onclarity assay on the Viper LT and COR instruments, the level of genotype agreement was determined by the percentage of overall agreement and κ statistics, as was the reproducibility element. The following categories were distinguished based on κ statistics: 0.00-0.20 = poor, 0.21-0.40 = fair, 0.41-0.60 = moderate, 0.61-0.80 = good, and 0.81-1.00 = excellent.24 The reproducibility element was evaluated by using the predefined setup with an 87% or greater lower confidence bound and a κ value above 0.6, as defined by the international criterion.2The accuracy of the GP-EIA assay was used as a comparator for clinical validation of the Onclarity assay on the COR instrument. Noninferiority assessment of the Onclarity assay on the COR instrument compared with the GP-EIA assay was according to the international guidelines using the preset 90% and 98% benchmarks for relative sensitivity and specificity, respectively.Sample collection and data retrieval for the VALGENT4 study were approved by the Danish Data Inspection Agency J. No. AHH-2017-024, I-Suite: 05356.All collected samples were cross-referenced and found eligible with the Danish register on collection, storage, and use of human biological material in health research projects (Vævsanvendelsesregistret).The overall HPV positivity of Onclarity-Viper and Onclarity-COR was similar, as was the concordance of the 9 individually reported genotypes P = .0016) and ≥CIN3 (P = .0005) sensitivity. The absolute specificity was 92.6% for Onclarity-COR and 89.3% for the comparator assay; relative specificity was 1.04 . Onclarity-COR was found to be noninferior to the comparator assay for specificity (P < .0001).The clinical accuracy of Onclarity-COR and the comparator assay (GP-EIA) is shown in The intralaboratory reproducibility and interlaboratory agreement were assessed using 525 cervical cancer screening samples. The intralaboratory reproducibility of Onclarity-COR was 98.3% , with a κ of 0.96 When looking at the individual genotype concordance The time-and-motion study was performed to assess the sample turnaround time and overall single-shift production capacity of the COR instrument 14This study presents our validation of the Onclarity HPV assay on the novel high-throughput COR instrument using SurePath collected screening samples from Danish routine cervical cancer screening. Onclarity-COR clinical and analytical performance was similar to Onclarity-Viper on all parameters evaluated  12 the interlaboratory agreement was 96.8% (κ = 0.92), and the intralaboratory reproducibility was 97.4% (κ = 0.93). It should be noted, however, that Viper LT and COR testing was performed on 2 different reproducibility panels.Also, the intralaboratory reproducibility and interlaboratory agreement fulfilled the international validation criteria Reproducibility among the individual genotype groups showed excellent concordance for all 9 genotype groups for both intralaboratory reproducibility 6 and the intra- and interlaboratory reproducibility of the Alinity HPV test7 was 96.7% (κ = 0.92) and 98.7% (κ = 0.97), respectively.Comparing COR performance with similar high-throughput instruments—the Roche cobas 6800/8800 and the Abbott Alinity instruments—inter- and intralaboratory analysis showed equally good results, at more than 98% reproducibility for cobas in the 2 separate studies,22The time-and-motion study evaluated a workflow of 330 samples per day and a production of 1,650 test results in a regular week. In comparison, the Viper LT instrument processed 90 to 120 samples per day per instrument.The average daily hands-on time for a full COR workflow was 58 minutes, with 2 interactions . The daily walk-away time was 7 hours, 2 minutes 4 where initialization, time to first results, time to last result, and total hands-on time amounted to 24 minutes; 2 hours, 28 minutes; 7 hours, 7 minutes; and 59 minutes, respectively. In contrast to our study, the cobas 6800 study did not include preanalytical processing before testing on the cobas 6800 instrument; a recent study estimated that the combined preanalytical (cobas p480) and analytical throughput of the cobas 6800 instrument was 384 samples in an 8-hour shift. To the best of our knowledge, similar time-and-motion studies have not been published on the cobas 8800 or Alinity instruments. Hands-on time, maintenance, and cleaning operations on a high-throughput HPV instrument can greatly affect the daily workload of a laboratory.25A similar approach evaluating the cobas HPV assay on the cobas 6800 instrument showed comparable test turnaround figures,26-28 and distinguishing new from persistent infections.29 The cobas HPV assay maintains the 2010 assay design on all cobas instruments, with individual reporting of HPV-16 and HPV-18 and reporting of the remaining 12 hrHPV genotypes as 1 group.9 The Abbott RealTime High Risk HPV Assay on the Alinity instrument is the newest design of the 3 and individually reports HPV genotypes 16, 18, and 45, whereas HPV-31/33/52/58 and HPV-35/39/51/56/59/66/68 are reported in 2 groups.10The 3 high-throughput HPV test instruments from Abbott, Becton Dickinson, and Roche, respectively, rely on widely different strategies with respect to reporting HPV-positive findings. HPV genotyping can play a major role in risk management of HPV-positive women, allocating women to risk tiers by HPV genotype30 The use of extended genotyping is gaining ground, however, and several studies have shown the 12 non–HPV-16/HPV-18 hr genotypes to carry distinct and markedly different risk of cervical disease.31-33 Consequently, more detailed genotyping than just HPV-16 and HPV-18 could be important in near-future triage algorithms and risk management. Of special note for the Onclarity and Alinity assays, the joint probe for HPV-33 and HPV-58 may result in overreferrals because the risk of ≥CIN2 is significantly greater for HPV-33 compared with HPV-58,34-36 and a separation of those 2 genotypes could be beneficial in improving assay specificity.In comparison, the Onclarity assay allows for extended genotyping on every positive sample, with individual typing of 6 genotypes  and the remaining 8 genotypes in 3 groups . In the United States, Australia, many European Union countries, and other countries, partial genotyping for HPV-16 and HPV-18 is used to risk-stratify women for follow-up.2 In comparison, the novel cobas 6800/8800 HPV assay on the cobas 6800 instrument was validated against the cobas 4800 assay.6 The argument for this deviation was that the 2 internationally recognized comparator assays—GP-EIA and Hybrid Capture 2 (Qiagen)—have been more or less discontinued in clinical routine worldwide. We acknowledged the merit of this argument, and the guidelines are expected to extend the number of standard comparator tests to reflect this reality.A major strength of our study is that the Onclarity assay on the COR instrument was validated against the assay on the already-validated Viper LT instrument as well as against an internationally recognized comparator assay, the GP-EIA assay.Another strength of our study is that the COR testing was done using the same panel of samples as previously used to validate the Onclarity assay on the Viper LT instrument. This approach makes the results directly comparable yet also confides a weakness in that the test rounds are 3 years apart. If storage had any negative impact, however, we would have expected performance differences. Such was not observed, and the Onclarity-COR concordance to the comparator assay remained high and well within validation criteria acceptance.The Onclarity assay was successfully migrated from the medium-throughput BD Viper LT HPV instrument to the high-throughput BD COR instrument. The Onclarity assay on the novel COR instrument was found to be noninferior to the comparator assay for clinical sensitivity and specificity. The inter- and intralaboratory reproducibility was high. The Onclarity assay allows for extended genotyping, which, together with the COR instrument’s capacity to run a high number of samples, makes the COR instrument an excellent candidate for use in HPV primary cervical cancer screening in laboratories that use screening algorithms, including genotyping.