The aim of this study was to evaluate the 8th edition of the American Joint Committee on Cancer Staging Manual: Head and Neck Section on oropharyngeal squamous cell cancer (OPSCC) and to clarify the relationship between p16 overexpression and the presence of human papillomavirus (HPV) DNA using fresh frozen samples.Samples from 100 OPSCC patients were analyzed using polymerase chain reaction (PCR) and p16 immunohistochemistry.n = 34), and p16-negative (n = 66) cases, respectively. OS tended to be better aligned with stage in the 8th edition than in the 7th edition. The 5-year OS was 96.0% in never or light smokers (< 40 pack-years), and 87.5% in heavy smokers (≥ 40 pack-years) in the p16-positive group, respectively (p = 0.027). HPV infection was found in 100% of p16-positive and 21.2% of p16-negative cases. The p16-positive cases had higher viral load and integrated physical status than the p16-negative cases. Although 1 case with p16 overexpression showed no PCR amplification using consensus primers, PCR amplification was detected using HPV 16 E6-specific primers.Five-year overall survival (OS) was 73.0%, 93.9%, and 62.2% in all, p16-positive (The 8th edition predicts OPSCC prognosis more accurately than the 7th edition and p16-overexpression is an excellent surrogate marker for detecting HPV infection. Although high-risk-type HPV infection was observed in p16-negative cases, it showed no significant effect in survival outcome. Human papilloma virus (HPV) infection has become an increasingly common cause of oropharyngeal squamous cell carcinoma (OPSCC) , 2. HPV-Comparing the 7th and 8th editions of the AJCC Staging Manual , 5 in teThe aim of this study was to evaluate the 8th edition in view of survival estimation according to HPV infection status and to clarify the relationship between p16 overexpression and the presence of HPV DNA using fresh frozen samples.18F-fluorodeoxyglucose-positron-emission tomography CT imaging.This study involved 100 treatment-naïve determined OPSCC patients without distant metastasis who gave written informed consent for study participation. All patients were diagnosed as having OPSCC by pathologic examination of biopsy samples and were treated at the Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, between January 2007 and December 2014. Classification of the TNM stage was performed according to both the 7th and 8th AJCC editions . Tissue This study was approved by the Institutional Review Board of the University of the Ryukyus and was carried out in accordance with the Helsinki Declaration of 1975. Informed consent was obtained from all OPSCC patients before enrollment.2 on day 1) and 5-fluorouracil . The regimen was given twice with a 4-week interval. When the primary tumor failed to show a partial response regardless of the neck lymph node response at 39.6 Gy irradiation, and patients underwent curative surgery for the primary lesion combined with neck dissection. Also, patients with T1 stage in the 7th edition underwent primary lesion removal with neck dissection in cases where there was no likelihood of severe surgical complications related to swallowing ability and quality of life.The principal treatment with curative intent for OPSCC was concurrent chemoradiotherapy regardless of the presence of HPV. All patients had CT-assisted three-dimensional radiation treatment planning in the treatment position with mask immobilization. The full treatment protocol was as reported previously . BrieflyAll tissue samples from primary lesions were analyzed with both polymerase chain reaction (PCR) using fresh frozen samples and p16 immunohistochemistry using formalin-fixed paraffin-embedded (FFPE) samples. The detection methods have been published in detail previously , 13.In brief, DNA was isolated from the tumor samples using the Gentra Purification Tissue kit . The presence and integrity of the DNA was verified by PCR β-globin gene amplification using the primers PC04 and GH20. The presence of HPV DNA was analyzed with PCR using the consensus primer sets GP5+/GP6 + and MY09/11 . DNA samE2 and E6 open reading frames were used. The primers and probes recognize the E2 hinge region, which is deleted on HPV-16 integration. Two standard curves for the E2 and E6 genes were created by amplification of tenfold serial dilutions  of the plasmid pB-actin carrying the complete HPV-16 early gene region , a gift from Karl Munger. Viral DNA load was assessed by calculating E6 copy numbers. An external standard curve was created using known serial dilutions  of human genomic placental DNA  for cellular DNA quantification and β-globin was amplified as described previously [E6 copy number in 50 ng cellular DNA was determined. Second, the integrated E6 was calculated by subtracting the E2 copy number  from the total E6 copy number . An E2/E6 ratio ≥ 1 indicates the predominance of the episomal form, whereas ratios of E2 copy number/total E6 of < 1 indicate the presence of both integrated and episomal forms (mixed form) [To evaluate the viral load and physical status of HPV-16, quantitative real-time PCR was performed as previously described . Brieflyeviously . The amoeviously . First, ed form) .For p16 immunohistochemistry, serial 4-µm-thick sections from FFPE samples were deparaffinized in xylene and hydrated in a graded series of alcohol . After eE6 and E2 on further analysis using the above-mentioned method.It is possible for HPV samples to show negative PCR results when consensus primers are used, particularly in cases where the L1 region is deleted during integration into the host genome. Thus, despite HPV not being detected by PCR using the consensus primer sets GP5+/GP6 + and MY09/11, the samples with p16 overexpression were found to have HPV p < 0.05 was considered statistically significant.Pearson’s chi-square test was used to compare the characteristics of OPSCC patients according to p16 status. Overall survival was calculated using the Kaplan–Meier method and was compared between the two groups using the log-rank test. All analyses were performed with SPSS Statistical Package . n = 100), p16-positive OPSCC patients (n = 34), and p16-negative OPSCC patients (n = 66) are shown in Tables p < 0.001, Fig. The clinical characteristics of all OPSCC patients n = 00, p16-pE6-specific primers. The patient in this case had concurrent chemoradiotherapy and survived without recurrence.There were no significant differences in sex, age, T category, N category, SCC differentiation, and tumor subsite between p16-positive and negative cases. There were significant differences in the rate of secondary primary cancer, tobacco consumption, alcohol consumption, and primary treatment between p16-positive and negative cases, as shown in Tables p = 0.027, Fig. p = 0.840) and related to smoking habit and drinking habit in the p16-negative group . Of the 100 OPSCC cases, 48.0% were HPV DNA positive from PCR. All p16-positive cases were found to harbor HPV DNA by PCR with consensus primers or HPV 16 E6-specific primers; there were also 14 (21.2%) p16-negative cases who harbored HPV DNA. There was substantial agreement between p16 immunohistochemistry and PCR for HPV DNA (kappa = 0.716). According to HPV DNA status, 5-year OS of HPV DNA-positive cases was 83.1%, and for HPV DNA-negative cases was 63.9% in all 100 OPSCC cases. The difference in OS between these two groups was marginal but did not reach significance .The 5-year OS was 96.0% in never or light smokers (< 40 pack-years), and 87.5% in heavy smokers (≥ 40 pack-years) in the p16-positive group, respectively .HPV-16 is the dominant HPV type, comprising 82.6% of all high-risk HPV types. The HPV-16 viral load in 25 cases in the p16-positive group and in 9 cases of p16-negative group could be measured with DNA of a sufficient quality. Marked significant differences in the median value of HPV-16 viral load were observed: 13.33 × 10Staging of the p16-positive OPSCC cases differed considerably between the 7th and 8th editions of the AJCC staging manual Tables , 5. Althp = 0.05). Since clinical stages in the p16-negative group were identical in both the 7th and 8th editions, there were no significant differences in OS in the p16-negative group based on both editions , and type of sample preparations used for analysis (fresh frozen or FFPE samples) , 16–18.  cancers . Contrar cancers . A recenpression . Furtherpression  might bepression . In the The p16-positivity criteria in the 8th edition states that the cutoff point for p16 overexpression is diffuse ≥ 75%) tumor expression with at least moderate (+ 2/3) staining intensity [% tumor eE6-specific primers. Although the number of p16-positive cases without HPV DNA was limited, the cause of p16 overexpression and survival outcome of these cases should be clarified in the near future to aid the design of a clinical trial.Interestingly, 1 out of the 100 OPSCC cases had no HPV DNA on PCR using consensus primers with p16 overexpression. Previous studies with a large sample size showed that approximately 3–4% of OPSCC had p16 overexpression without HPV infection , 22. TheSeveral studies have indicated that the 7th edition of the AJCC staging system has not properly described prognosis in HPV-mediated OPSCC , 23. TheIn conclusion, the 8th edition of the AJCC staging manual reflects the prognosis of OPSCC using p16 immunoreactivity for the detection of HPV-mediated OPSCC. However, a further study is needed to clarify the nature of cases that are p16 negative with HPV infection. The discordance between p16 overexpression and the presence of HPV DNA in relation to survival outcome should be clarified to design a de-escalation study.


Supplemental Digital Content is Available in the Text. Women with HIV face an increased risk of human papillomavirus (HPV) acquisition and persistence, cervical intraepithelial neoplasia, and invasive cervical cancer. Our objective was to determine the cost-effectiveness of different cervical cancer screening strategies among women with HIV in South Africa.We modified a mathematical model of HPV infection and cervical disease to reflect coinfection with HIV. The model was calibrated to epidemiologic data from HIV-infected women in South Africa. Clinical and economic data were drawn from in-country data sources. The model was used to project reductions in the lifetime risk of cervical cancer and incremental cost-effectiveness ratios (ICERs) of Pap and HPV DNA screening and management algorithms beginning at HIV diagnosis, at 1-, 2-, or 3-year intervals. Strategies with an ICER below South Africa's 2016 per capita gross domestic product (US$5270) were considered “cost-effective.”HPV testing followed by treatment (test-and-treat) at 2-year intervals was the most effective strategy that was also cost-effective, reducing lifetime cancer risk by 56.6% with an ICER of US$3010 per year of life saved. Other cost-effective strategies included Pap  at 1-, 2-, and 3-year intervals, and HPV test-and-treat at 3-year intervals. Pap (ASCUS+), HPV testing with 16/18 genotyping, and HPV testing with Pap or visual triage of HPV-positive women were less effective and more costly than alternatives.Considering per capita gross domestic product as the benchmark for cost-effectiveness, HPV test-and-treat is optimal in South Africa. At lower cost-effectiveness benchmarks, Pap (HSIL+) would be optimal. Recent guidelines from the American Society for Clinical Oncology recommend HPV testing at the time of HIV diagnosis and subsequently at an interval of every 2–3 years if resources are available; for lower-resource settings, screening for HIV-infected women is recommended twice as often as in the general population.9 In South Africa—where screening guidelines recommend cytology-based screening, with phasing in of HPV testing based on resource availability—the principles of equity, quality, efficiency, and sustainability have been adopted by the Department of Health.10 Yet data on the cost-effectiveness of different screening and management strategies in HIV-infected women are limited.The World Health Organization (WHO) recommends that women with HIV receive cervical cancer screening with HPV testing at least every 3 years if resources are available; for countries with fewer resources, visual inspection with acetic acid (VIA) is an acceptable screening alternative.To inform ongoing discussions about clinical guidelines, optimal resource use, and the integration of women's health interventions with HIV-related care in South Africa and other low-resource settings with a high burden of HIV, we evaluated the cost-effectiveness of different Pap and HPV screening and management algorithms among HIV-infected women in South Africa.12 to reflect the burden of HPV in HIV-infected women in South Africa . Women in the model are infected with HIV at the age of 20 years, approximating the peak HIV incidence in South Africa.13 Based on HIV progression estimates in the absence of ART15 and estimates of CD4+ cell count at HIV presentation,16 we optimistically assumed (as HIV testing and ART access increase) that women would be diagnosed with HIV around the age of 25 years, at a CD4+ cell count of approximately 350 cells/µL. Before HIV acquisition at the age of 20 years, background mortality rates for uninfected women were drawn from the THEMBISA model (version 2.5).13 On HIV infection at the age of 20 years, women face excess mortality rates dependent on CD4+ cell count and rate of HIV progression.14 On HIV detection at the age of 25 years, women are assumed to begin ART immediately and then face excess mortality from HIV based on a relative survival model that considers current age, age at ART initiation, CD4+ category at ART initiation, and duration on ART.7 Excess mortality due to cervical cancer was derived from estimates of 5-year survival in middle-income countries with a low burden of HIV17 multiplied by hazard ratios from cervical cancer mortality in HIV-infected versus uninfected women.18 Time-dependent survival by stage  was derived from cervical cancer survival estimates in HIV-infected women in Botswana.19 After 10 years following cancer treatment, we assumed women no longer faced excess mortality due to cervical cancer.Because of limited data on the interaction between HPV and HIV, we assumed a similar course of HIV infection and progression for all women in the model  and type-specific immunity. To reflect the greater risk of HPV infection and progression (to CIN and cancer) and the decreased likelihood of HPV clearance and CIN regression in HIV-infected women, as well as parameter uncertainty, we modified the previously described natural history model by setting plausible ranges for factors to apply to baseline transition probabilities derived from longitudinal studies, guided by previous work modeling HPV infection in the general population of women in several low- and middle-income countries with a low burden of HIV20 and published hazard and risk ratios in HIV-infected versus HIV-uninfected women.21 The model underwent repeated model simulations in the absence of any intervention, and for each simulation, single random values from a uniform distribution spanning the plausible range for each factor were selected and applied to the relevant baseline probability to create a unique natural history input parameter set. We then computed a goodness-of-fit score by summing the log likelihood of model-projected outcomes for each unique parameter set to represent the quality of fit to epidemiologic data  on age-specific prevalence of oncogenic HPV and the proportion of HPV type-specific infections in CIN3 and cervical cancer among women with HIV in South Africa.24 We selected the 50 top-fitting parameter sets for analyses and calculated the expected value, as well as the range of values, for all outcomes. Model fit to calibration targets is displayed in Figures 1 and 2, Supplemental Digital Content, http://links.lww.com/QAI/B185. Data sources used to inform the factor search space and the ranges for each transition probability value are provided in Table 1, Supplemental Digital Content, http://links.lww.com/QAI/B185. Model validations to CIN2/3 prevalence and cervical cancer incidence in South Africa are presented in Figures 3 and 4, Supplemental Digital Content, http://links.lww.com/QAI/B185.To inform transitions between HPV-related health states, baseline “prior” transition probabilities were estimated from large epidemiologic studies in predominantly HIV-uninfected populations, as previously documented.10 and WHO guidelines for HIV-infected women.8 Strategies included (1) liquid-based cytology with referral to colposcopy for atypical squamous cells of undetermined significance or worse (ASCUS+) [“Pap (ASCUS+)”]; (2) liquid-based cytology with referral to colposcopy for atypical squamous cells cannot rule out high-grade/high-grade squamous intraepithelial lesions or worse (HSIL+) [“Pap (HSIL+)”]; (3) HPV DNA testing with referral to treatment for all HPV-positive women (“HPV test-and-treat”); (4) HPV DNA testing with triage to VIA for all HPV-positive women, and treatment for all women who are both HPV-positive and VIA-positive (“HPV-VIA”); (5) HPV DNA testing with triage to Pap for all HPV-positive women, and referral to colposcopy for all women who are both HPV-positive and ASCUS+ (“HPV-Pap”); and (6) HPV DNA testing followed by HPV16/18 genotyping for HPV-positive women, with referral to treatment for 16/18-positive women and referral to colposcopy for women with other oncogenic types (“HPV16/18 genotyping”). Screening coverage was 70% of the target population. Of those screened, 85% were assumed to receive the strategy of interest , whereas 15% were assumed to receive a separate VIA strategy, to represent the potential availability of VIA followed by treatment for women without access to Pap or HPV-based strategies; we weighted cost and health outcomes accordingly. Screening and management algorithms are displayed in Figures 5–11, Supplemental Digital Content, http://links.lww.com/QAI/B185. All strategies were considered at 1-, 2-, and 3-year intervals.Screening and management algorithms were based on recently drafted guidelines for South Africa33 Compliance with each recommended clinical encounter was 85% relative to the previous visit. Values for screening and treatment variables are displayed in Table For HPV-testing strategies, we assumed provider-collection of samples. For HPV testing followed by either visual or Pap triage, women who were HPV-positive but triage test–negative were assumed to receive repeat HPV testing in 1 year. After colposcopy, women with normal or CIN grade 1 (CIN1) on histology were referred to repeat screening in 1 year; those with CIN2 or higher were referred to treatment. Treatment included cryotherapy for eligible women, and large loop excision of the transformation zone (LLETZ) for women who were determined to be ineligible for cryotherapy. Screening test performance and treatment effectiveness values were based on studies in HIV-infected women.34; costs were estimated from in-country data sources and included direct medical costs , direct nonmedical costs (women's transportation), and women's time  using consumer price indexes and average annual official exchange rates to report findings for global policy makers. Because HPV DNA and HPV genotyping tests have not yet been procured by the government of South Africa, list prices  did not necessarily reflect economies of scale from bulk purchasing. Each month, beginning at HIV diagnosis, women incurred the average monthly outpatient cost per adult on ART and average monthly inpatient cost for individuals on ART with a CD4 count above 350 cells/µL.40 Further details and data sources are provided in the Supplemental Digital Content, http://links.lww.com/QAI/B185.Consistent with guidelines for cost-effectiveness analysis, we adopted a modified societal perspectiveme Table .35–38 Co34 We then calculated incremental cost-effectiveness ratios (ICERs). An ICER is the additional cost of a strategy divided by its additional health benefit, compared with the next most costly strategy after eliminating strategies that are dominated . Across the 50 top-fitting input parameter sets, the ICER was calculated as the ratio of the mean cost divided by the mean health effect. We considered the following possible cost-effectiveness thresholds as a benchmark to indicate strategies that provide good value for money: (1) South Africa's per capita gross domestic product (GDP) (2016 US$5270)41; (2) 50% of South Africa's per capita GDP (2016 US$2640); and (3) the cost per disability-adjusted life-year averted for extending ART eligibility from adult patients with CD4 counts of 350 cells/µL or less to patients with CD4 counts of 500 cells/µL or less—an approximation of the opportunity cost of HIV care in South Africa—as estimated by a recent cost-effectiveness analysis using 12 mathematical models (high estimate for South African models: 2016 US$1190).42Model outcomes included the absolute lifetime risk of cervical cancer incidence and mortality, life expectancy, and lifetime costs; costs and life-years were discounted at an annual rate of 3%.Scenario analyses assessed the impact of alternate parameter values and assumptions on cost-effectiveness results Table .The impact on cervical cancer incidence and mortality, lifetime cost, life expectancy, and ICER for each screening strategy is presented in Table Pap (HSIL+) every 3 years had the lowest ICER [$340 per year of life saved (YLS)], followed by Pap (HSIL+) every 2 years ($900 per YLS), but these strategies also were the least effective. More effective strategies that were also efficient  included HPV test-and-treat every 3 years ($1780 per YLS), Pap (HSIL+) every year ($2420 per YLS), HPV test-and-treat every 2 years ($3010 per YLS), and HPV test-and-treat every year ($8020 per YLS) , Pap testing (HSIL+ threshold) every 1–2 years would be considered optimal but would yield much lower reductions in cervical cancer risk . Optimal strategies under various cost-effectiveness benchmarks were robust across many scenario analyses. However, when the high direct medical cost of the HPV test in the base case was reduced to $35, HPV test-and-treat at 2- or 3-year intervals was the optimal strategy for all benchmarks considered. Furthermore, because of the lower number of required visits for this strategy compared with Pap-based strategies, less favorable assumptions regarding visit compliance improved the relative attractiveness of HPV test-and-treat.41” Yet recent analyses suggest that accepting interventions according to per capita GDP may displace interventions yielding substantial health benefits with less effective interventions.43 In the absence of a formal threshold based on empirical data from South Africa, we considered alternative thresholds, including 50% of per capita GDP and $1190, which represents the ICER (from a recent modeling study in South Africa) of expanding ART coverage from all adults with CD4 counts below 350 cells/µL to those with CD4 counts below 500 cells/µL. In addition to consideration of value for money, it is critical to consider health equity in determining what services should be available.To determine whether an intervention should be included in a package of services, a benchmark of health opportunity costs, or cost-effectiveness threshold, must be established. The WHO Commission on Macroeconomics and Health suggests that interventions with ICERs less than per capita GDP are “very cost-effective” and less than 3 times per capita GDP are “cost-effective.10 The effectiveness of treatment in HIV-infected women is uncertain. One recent randomized trial found that HIV-infected women with CIN2/3 were more likely to experience recurrence after cryotherapy than after LEEP at 12 and 24 months after treatment, as measured by HSIL+ on cytology; another trial in South Africa found that more women had recurrent CIN2+ after cryotherapy than LEEP at 6 months, but by 12 months, the difference was not significant.44 When we assumed all women were treated with LLETZ instead of cryotherapy, HPV test-and-treat every 3 years was optimal at cost-effectiveness thresholds of per capita GDP and 50% of per capita GDP; less frequent screening was needed because of the assumed greater effectiveness of treatment. Although our analysis included the costs associated with potential overtreatment of screen-positive women in HPV test-and-treat strategies, we did not consider the impact of potential adverse reproductive outcomes. Guidelines will need to weigh safety, effectiveness, and logistical considerations  in recommending surgical versus ablative treatments for women with HIV.In South Africa, access to cryotherapy is limited, but recently drafted guidelines rely heavily on this form of treatment, and our analysis assumed that most eligible women would receive cryotherapy.45 found that HPV testing every 1–2 years followed by Pap triage of HPV-positive women was cost-effective in Brazil but did not consider HPV testing with treatment for all HPV-positive women. Lince-Deroche et al35 estimated the cost per case of CIN2+ detected associated with screening in South Africa and found that HPV testing followed by colposcopy of HPV-positive women had the highest cost per case detected, whereas VIA had the lowest; however, this analysis did not consider the cost offsets of prevented cancers.Because of the complexities of modeling HIV and HPV coinfection, few studies have evaluated the cost-effectiveness of cervical cancer screening in HIV-infected women in a low- or middle-income country. Vanni et al46 Although we modeled screening strategies based on WHO recommendations and recently drafted guidelines in South Africa, HPV testing is not widely available in South Africa at this time. Although there are plans for scale-up as resources become available, the delivery of HPV testing and management may need to be refined based on system resources or improved for more efficient delivery. We did not consider reduced screening intervals after a specific number of negative screening tests, nor did we model switching from Pap-based to HPV-based screening or cotesting after the age of 30 years, as US guidelines suggest.47 We did not consider the future impact of HPV vaccination of girls, which was introduced in South Africa in 2014.There are several limitations to this analysis. We did not have data to inform HPV transitions stratified by CD4 count, viral load, or ART status and thus assumed a common trajectory of HIV disease in the modeled cohort. Further research on the time-dependent impact of ART on HPV and cervical cancer transitions will inform future modeling efforts. Furthermore, our calibration to oncogenic HPV prevalence relied on data from the VICAR study of HIV-infected women in Johannesburg, and we thus could not calibrate to the prevalence of HPV for women younger than 20 years before HIV infection, nor can we assume generalizability to HIV-infected women in other parts of the country. However, by achieving good fit to HPV prevalence beginning at the age of 20 years, when screening does not begin until the age of 25 years, we believe the model reflects appropriate clearance versus persistence of earlier HPV infections. Furthermore, our validation exercises examining model-projected cervical cancer incidence relative to registry data for the general population are consistent with hazard ratios gleaned from studies in HIV-infected versus uninfected women.4; however, tremendous disparities in access to screening remain. Our model-based analysis demonstrates that cervical cancer screening is likely to be both effective and very cost-effective in HIV-infected women in South Africa. If sufficient resources are available, HPV testing at 2-year intervals can achieve greater reductions in cancer risk than Pap-based strategies in South Africa through increased detection of precancer and fewer visits per screening episode. As the demand for HPV testing rises and test costs decline, a shift toward HPV-based test-and-treat strategies in South Africa and other low-resource settings with a high burden of HIV may improve access and save lives. Health services research will be necessary to integrate cervical cancer prevention with HIV-related services through community outreach, task-shifting to nonphysicians, and strengthening information systems to track patients48; integrating and improving service delivery for high-risk women is a tremendous opportunity to transform the health care system and reduce health disparities. We present optimal strategies under a range of benchmarks for cost-effectiveness to stimulate action from international donors, advance policy discussions, and inform implementation efforts—so that women who are saved from HIV will not die of cervical cancer.As women live longer due to improved access to life-saving ART, they will likely continue to face an elevated risk of cervical cancer. In South Africa, expanded access to organized cervical cancer screening with cytology has reduced cervical cancer incidence in HIV-infected women


To elucidate oncogenic human papilloma virus (HPV) types in Japan, HPV genotyping was performed in 1526 cervical intraepithelial neoplasia (CIN) and 371 invasive cervical cancer (ICC) patients with the novel Genosearch-31+5 HPV test. The HPV-positive rates were 89.3% and 90.8% in CIN and ICC. Regarding single-type infections, 13 internationally recognized high-risk (13HR) types excluding HPV 35, and probably HR HPV 53, 67, 69, and 70 were identified in ICC, suggesting that all these types may be oncogenic. HPV16 and 18 were identified in both SCC and adenocarcinoma (ADC). HPV HPV52, 31 and 58  were predominantly detected in SCC, whereas HPV 18, 45, 39 and 59  were in ADC. The prevalence of HPV 18 in SCC significantly decreased with increasing age of patients, whereas the opposite trend was observed in the other HR types. HPV18 is likely to induce SCC rapidly. All ICC cases aged 20–29 were positive for HPV 16 or 18, suggesting that present HPV 16, 18 vaccines may be quite effective to prevent ICC in young women. The sensitivity of the Genosearch HPV31 was confirmed to be equivalent (maximumly six time difference) for all target HPV types, with no cross-reactivity with each other type (19). Using this platform with a detection system for an additional 5 HPV types (Genosearch HPV31+5), we determined the HPV genotypes in pooled samples of 1526 cases of cervical intraepithelial neoplasia and 371 cases of invasive cervical cancer to elucidate probable high-risk HPV types in Japanese women.22.1This cross-sectional epidemiological study used liquid-based cytology (LBC) samples from cervical intraepithelial neoplasia (CIN), and LBC and tissue samples from invasive cervical cancer (ICC) obtained from Japanese women whose diagnoses were histologically confirmed. Written informed consent was obtained from all women for use of their HPV typing data, diagnoses, and ages. LBC samples were collected in the first visit in the outpatient clinics, and tissue samples were obtained from women at initial surgeries or at colposcopy examinations. Tiny tissue specimens were excised from cancer specimens by an experienced gynecological oncologist, and the remaining tissues were used for pathological diagnosis. Two clinical pathologists made diagnoses independently, and the final diagnosis was determined by consensus.Details of the pooled samples are as follows. LBC samples of 936 CIN cases and 9 ICC cases were collected from multiple institutions from 2011 to 2012 (Japanese Human Papillomavirus Disease Education Research Survey [JHERS] study groups) (19), and of 621 CIN and 19 ICC cases were collected in outpatient clinic of Kanazawa Medical University Hospital from 2009 to 2017. Fresh tissue fragment samples from 211 ICC patients were collected from Hospitals of Kanazawa Medical University, Kanazawa University, and Osaka International Cancer Institute from 2002 to 2017, and formalin-fixed and paraffin-embedded tissue (FFPE) specimens from 141 ICC patients were collected from Kanazawa Medical University Hospital from 1990 to 2017. The pellet of 1 ml of LBC samples spin down by centrifugation, and a small fragment of tissue of cervical cancer specimen was stored at −80 °C until use.DNA was extracted from the LBC samples and frozen tissue of ICC using the Smitest EX&D , and from FFPE specimen using the pinpoint slide DNA isolation system . DNA extracted from FFPE was immediately used for analysis.A total of 31 CIN and 7 ICC samples were omitted due to poor quality for DNA. Ultimately, 1526 cases of CIN, including CIN1 (597 cases), CIN2 (526 cases), and CIN3 (403 cases), AIS (12 cases), and 371 ICC cases were examined . ICC sam2.2HPV genotyping was performed in a commercial laboratory . In brief, 10 ng of DNA was used in all analyses, and the beta-globin gene was used as internal control. Genosearch HPV31+ Genosearch HPV5  was used with the PCR-SSOP-Luminex method. The GS-31 is a genotyping method that can detect 31 different HPV types, including 12 low-risk types (LRs), i.e., HPV 6, 11, 42, 44, 54, 55, 61, 62, 71, 84, 89, and 90; six probable high-risk types (Probable HRs), i.e., HPV 26, 53, 66, 70, 73, and 82; and the 13 internationally recognized high-risk types (13HRs), i.e., HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The GS-5 was designed to detect an additional 5 probable HR types, i.e., HPV 30, 34, 67, 69, and 85. The Roche Linear Array detects 37 HPV types including 16 LRs, i.e., HPV 6, 11, 40, 42, 54, 55, 61, 62, 64, 71, 72, 81, 83, 84, IS39, and 89; eight probable HRs, i.e., HPV 26, 53, 66, 67, 69, 70, 73, and 82; and the 13HRs, i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.The Japanese Society of Obstetrics and Gynecology recommends dividing the 13HR types into two groups: relatively higher risk types (Relatively HR) and relatively lower risk types (Relatively LR). The former group includes HPV 16, 18, 31, 33, 35, 45, 52, and 58, and the latter includes HPV 39, 51, 56, 59, and 68. Based on this recommendation, the HR HPV types were further divided into four groups in the present study: HPV 16; HPV 18; high-grade HRs (HG-HR) HPV 31, 33, 35, 45, 52, and 58; and low-grade HRs (LG-HR) HPV 39, 51, 56, 59, and 68.We ascertained that no LRs were identified as single types in high-grade squamous intraepithelial lesions (CIN3) and invasive cancer. Therefore, 10 ICC cases that showed infection with multiple HPV types, i.e., one HR and one or more LR HPV types, were conventionally classified as single-type infections of the HR type.2.3Chi-squared tests for trends were performed using GraphPad Prism ver. 6.05 .33.1P < 0.0001), whereas that of multiple-type infection showed the opposite trend . Thus, single-type infection was observed in 54.8% and 66.7% of CIN3 and adenocarcinoma in situ [AIS] cases, respectively, whereas it was observed in 36.3% of CIN1 cases. The single HPV types identified in CIN3 were 11 of the 13HRs, excluding HPV 45 and 59, and Probable HRs HPV 26, 66, 67, and 82 , 1363 of 1526 (89.3%) cases were positive for HPV, and single- and multiple-type infections were detected in 43.8% (N = 668) and 44.8% (N = 684) of cases, respectively . The pre, and 82 . The top, and 82 .Table 2HHPV types were examined in 371 ICC cases comprising 279 squamous cell carcinoma (SCC) cases, 20 adeno-squamous cell carcinoma (ADSCC) cases, 59 adenocarcinoma (ADC) cases, and 13 other histological type carcinoma (OTC) cases. A total of 90.8% of ICC, 95.3% of SCC, 95.0% of ADSCC, 71.2% of ADC, and 76.9% of OTC cases were positive for any type of HPV . When on3.2When only single HPV type infections were evaluated, HR HPV types were detected in 78.0% (170/218) of CIN1, 82.2% (189/230) of CIN2, and 92.2% (213/231) of CIN3 cases. In contrast, only 16.1% of CIN1, 4.8% of CIN2, and none of CIN3 cases were positive for LR types.P < 0.0001) increased with increasing grade of CIN (P = 0.0013) and LR types  significantly decreased with increasing CIN grade ; HPV 31, 33, 35, 45, 52, and 58, and low-grade high-risk types (LG-HRs); HPV 39, 51, 56, 59, and 68. When only the cases with single-type HPV infection were evaluated, the prevalence of HR types (data not shown) and HPV 16 , whereas Probable HR showed the opposite trend   . No such 0.0042) . No singP = 0.0098) and HPV16  decreased with increasing patient age (P = 0.0007). Such tendencies were not seen in LG-HR, or Probable HR HPV types. The same analysis was done for SCC, ADSCC, and ADC cases, and only SCC exhibited a similar trend to ICC. It should be noted that all patients with SCC and ICC aged 20–29 years were positive for either HPV 16 or HPV 18  HPV types in Japanese women, we identified HPV types infecting HSIL (CIN2 and 3) and ICC cases using a new HPV genotyping test that can detect 36 HPV types including 13 h, 11 Probable HRs, and 16 LRs. The present results demonstrated that 11 of the 13HRs (excluding HPV 45 and 59) and probable HR types such as HPV 26, 66, 67, and 82, were detected as single HPV types in CIN3, and 12 of 13 h HPV types (excluding HPV 35) and Probable HR types such as HPV 53, 67, 69, and 70 were identified in ICC. It is noted that HRs HPV 39 and 59 and the Probable HRs HPV 53 and 67 were detected as single-type infections at the same prevalence in ICC, and HPV 35 and HPV 26, 66, 67, and 82 had the same prevalence in CIN3. These results suggest that HPV 26, 53, 66, 67, 69, 70, and 82 are likely to be oncogenic types. In 2012, the IARC working group defined 12 high-risk types (group 1A) and the following additional types as probable (group 2A), or possible (group 2B) 4.2When only single-type infection was evaluated, the present study demonstrated that LR types were observed in CIN1 and CIN2, whereas they were not identified in CIN3 and ICC. In contrast, HR types and Probably HR types were identified in CIN3 and ICC. An evaluation of single type infections estimates the risk of each HPV type for cancer.The same analysis showed that HPV 31, 33, 52, 58 , 51 , and 56  were identified in SCC, whereas these types were not identified in adenocarcinoma (ADC). In contrast, HPV 18 and 45  were more common in ADC than in SCC. The former types were predominantly the alpha-9, and the latter types were the alpha-7. Moreover, HPV 45 was not identified as a single type at all in CIN3, whereas it was identified in AIS. These data suggest that HPV types in the alpha-9 predominantly induce SCC, whereas HPV types belonging to the alpha-7 induce ADC. However, HPV 16  and 18  appear to induce both types of cancer. Previous studies demonstrated that HPV 16, 18, 45, and 52 are common HPV types in ADC 4.3Many reports suggest that HPV 16 and 18 are the most malignant HPV types, as their progression from premalignant to malignant lesions is earlier than that of other types In the analysis of age-dependent prevalence in SCC, HPV 18 significantly decreased with increased age of patients. Such trend for HPV16 was not significant. In contrast, the opposite trend was observed in HG-HR types . HPV18 appeared to induce SCC more quickly thande novo pathway, since the cancer appears to be rapidly developed by HPV18 than other high-risk HPV types. Some studies have suggested that CIN3 may be developed directly from normal epithelium de novo carcinogenesis by HPV18 warrants further investigation in the future study.other HR types did if the infecting age was the same. These results may support the previous finding that HPV 16 and 18 are the most aggressive in promoting malignant progression in squamous cells 4.4Although Genosearch-31 was used in the previous epidemiological study (19), Genosearch-31 plus 5 was first used in the present study. The limitation of this study may be lack of the data of comparative study with the gold standard methods such as Roche LINEAR ARRAY and the INNO-Lipa.5We elucidated the prevalent HPV types in CIN3 and ICC in Japanese women using a new HPV genotyping method in evaluation of single-type HPV infections. Probable HRs such as HPV 53, 67, 69, 70, and 82 were also identified as single-type infections as equivalent prevalence as the internationally established HR HPV types in CIN3 and ICC. This suggests that these types are likely to be HR types in cervical cancer in Japanese women. HPV 16 and 18 can induce both SCC and ADC. Age or CIN grade -dependent distribution suggest that HPV18 induces cervical cancer rapidly, and the progression might be through de novo pathway. HPV 31, 52, and 58  predominantly induced high-grade squamous lesions and SCC, whereas HPV 39, 45, and 59  induced ADC. All ICC cases aged 20–29 were positive for HPV 16 or 18, suggesting that currently available prophylactic HPV vaccines may be quite effective on prevention of ICC in young Japanese women.These authors declare no potential conflicts of interest.


Empetrum nigrum L.) represents an important source of flavonols  and anthocyanins. The fruit components could be utilised as natural colourants or as a part of functional foods and, because of the high antioxidant activity, the berries of black crowberry can be used in the treatment of diseases accompanied with inflammation, or as an effective antibacterial and antifungal remedy. Moreover, the reduction of lipid accumulation and total cholesterol as well as an improvement of postprandial hyperglycaemia have been proven. This review summarizes for the first time the main antioxidants (flavonoids) of black crowberry fruits, with a focus on their health promoting activity.Nowadays, much research attention is focused on underutilized berry crops due to the high antioxidant activity of fruits. Black crowberry ( Empetrum nigrum, Vaccinium ulinosum, Vaccinium vitis ideae, Aronia melanocarpa, Vaccinium oxycoccus, and Sorbus aucuparia display higher antioxidant activity in comparison with well-known and distributed berry crops  [Recently, the attention of researchers has been focused on lesser-known and underutilized species of edible berries, such as honeyberry, lingonberry, rowanberry, black crowberry, and others with respect to their health benefits ,2,3,4. Wspberry) . Moreovespberry) . It is aspberry) .E. nigrum (crowberry or black crowberry) and its tetraploid subspecies E. nigrum ssp. hermaphroditum [E. nigrum could also be used as ingredients in functional foods [Black crowberry includes two forms—hroditum . Black chroditum ,10, the hroditum , but thehroditum . Anotherhroditum . The heahroditum —especialhroditum ,15. Desphroditum . As menthroditum  and theyhroditum . Powdereal foods .A summary of the main antioxidants—flavonoids—of black crowberry fruit together with the utilization of health promoting compounds has not yet been published. Moreover, this review brings a comparison of these bioactive compounds with components of other underutilized plant species. This could therefore help to clarify the position of black crowberry fruit with respect to anthocyanin content, as these are the most important flavonoids in the fruit.Empetrum nigrum fruit, and contribute to the high antioxidant and health promoting activities of plants [Polyphenols  represent the main group of bioactive compounds of f plants ,4. Polyphenols are the most significant bioactive compounds in black crowberry fruit. The total polyphenol content (TPC) of black crowberry fruit represents 4.3 ± 0.09 mg GAE/g DW  in fresh fruit and 7.51 ± 0.17 mg GAE/g DW in dried fruit , althougEmpetrum fruits was 2.46 ± 0.01 and 3.94 ± 0.106 mg CE/g DW, respectively [p-coumaric acid and caffeic acid in black crowberry fruit from northern Finland using high performance liquid chromatography (HPLC) method with DAD (diode array) detector. The total sum of phenolic acids reported by Dudone [p-coumaric acid as the major phenolic acid (8.33 ± 0.63 mg/100 g FW), and other forms of acid were present in lower amounts . Furthermore, they also detected p-hydroxybenzoic acid (0.47 ± 0.03 mg/100 g FW), ferulic acid (0.21 ± 0.01 mg/100 g FW), caffeic acid (0.18 ± 0.01 mg/100 g FW), and protocatechuic acid (0.19 ± 0.03 mg/100 g FW) [The fruit of black crowberry also represents a valuable source of anthocyanins (460 mg/100 g FW fruit) . The conectively . Crowberectively ,20,21. Aectively  identifiectively  detectedy Dudone , analyse00 g FW) .Most research concerning black crowberry polyphenol composition is performed in relation to another lesser known fruit species. Dudonne et al.  providedHowever, the measurement of polyphenols and their composition could be varied depending on the climate. The content of polyphenols and their composition was also investigated in fruits ripened under tundra conditions. The concentration of stilbens increased under light grazing and warming. On the other hand, under the mentioned conditions, some parameters decreased, such as the concentration of flavonols and condensed tannins, with no change in total phenolic content. Under heavy grazing, a weak but consistent decrease among the different phenolic compound groups was evident, resulting in a decrease in total phenolics ,25,26. TEmpetrum nigrum flavonoids is given in Flavonoids represent the predominant group of phenolic compounds. Flavonols, flavan-3-ols, and anthocyanins are the most important flavonoids in crowberry fruit. A summary of Empetrum nigrum fruits. A more detailed spectrum of black crowberry flavonoids was provided by HPLC-DAD analyses by Laakson et al. [O-galactoside and quercetin-3-O-glucoside were determined as main flavonols.Flavonols are the most important group of flavonoids in black crowberry fruit. Ogawa et al.  identifin et al. . AccordiAccording to the results of the flavonoids of crowberry fruit originating from Korea , determiThe total content of crowberry flavonols—quercetin, myricetin, and kaempferol—reached up to higher levels in comparison with commonly consumed fruits or vegetables such as onion, kale, and broccoli . Mlcek eIn black crowberry fruit, flavan-3-ols represent the second most abundant group of flavonoids. Among flavan-3-ols in black crowberry fruit, epicatechin predominated, in quantities of 7.09 ± 0.41 mg/100 g FW. That is a higher concentration compared to the amount found in Saskatoon berry, chokeberry, or honeysuckle . ProanthBlack crowberry is a unique and exceptional source of anthocyanins. Crowberry fruit has high levels of anthocyanin pigments in the skin, which have potential as natural food colourants and food additives .Empetrum nigrum subsp. hermaphroditum (Hagerup Böcker) were extracted by ethanol and acetic acid and purified by extraction on Ionex. Separation and analyses of aglycones were provided by HPLC method, and glycoside units were analysed by GLC-MS (gas liquid chromatography tamdem mass spectrometry). Researchers identified 12 acetylated monosides: glucosides, galactosides, arabinosides of delphinidin, petunidin, cyanidin, peonidine, and malvidine. The three main anthocyanins of Empetrum nigrum fruit were galactosides of cyanidin, delphinidin, and malvidin, which formed about 60% of total anthocyanins. More detailed identification and quantification of individual anthocyanidins was provided by Ogawa et al., using 80% methanol with 0.5% acetic acid for extraction [The first attempt to quantify anthocyanins comes from Linko et al. and Käppa et al. ,19. The traction . They idtraction  and Heymtraction ), with tGenerally, anthocyanins are present in the form of glycosides in the majority of berry crops . The preThere are interesting comparative studies of anthocyanin quantification and identification among berry crops. The fruits of black crowberry together with bilberry  present Anthocyanins in black crowberry, chokeberry, and blackcurrant juices are often analysed by high performance liquid chromatography coupled with mass spectrometry. The experimental results showed that crowberry juice had a more variable anthocyanin profile compared to chokeberry and blackcurrant juices. In fruit juices, the total anthocyanin content varied from 44.8 to 128 mg/100 mL, with the highest value in crowberry juice. Generally, cyanidin-3-galactoside was the most abundant in lingonberry, alpine bearberry, Saskatoon berry, chokeberry, and black crowberry . One of 3+ and Al3+ ions. Authors found out that the half-life (t1/2) of pure untreated crowberry juice was 4–6 weeks. When 90% of the oxygen was removed, the stability was improved by a factor of 3 to 4. Glucosides and galactosides had a longer degradation half-life than arabinosides. The presence of Fe3+ and Al3+ ions increased the stability of anthocyanins, but significantly decreased the colour shade of the juice. In the dark-coloured crowberry juice, the total anthocyanin content constitutes about 45%–50% of the fruit juice. Due to the relatively bland taste of the juice it may have future use as a natural food colourant [Anthocyanins are highly unstable and very susceptible to degradation. The stability of crowberry anthocyanins was examined by Kallio et al.  in relatolourant ,35. 1/2 for total anthocyanins = 6.7 weeks at 21 °C; 23.8 weeks at 9 °C; and 32.5 weeks at 4 °C) whilst in blackcurrant it is t1/2 = 3.0 weeks at 21 °C; 11.5 at 9 °C; and 20.3 weeks at 4 °C, respectively, and in crowberry juice is t1/2 = 2.2 weeks at 21 °C; 7.3 at 9 °C; and 12.3 weeks at 4 °C, respectively. Hellstrom et al.  comparedCrowberry fruit can be utilised as a natural colour enhancer; a summary of the main anthocyanins in the fruit of black crowberry is depicted in The evaluation of the antioxidant activity of fruits is a way of expressing the biological activity of fruit that should be coupled with the study of the major biologically active substances ,37,38,392O2-induced cytotoxicity using fruit extracts of black crowberry from Korea. The fruit extract displayed a strong antioxidative effect in the free radical scavenging and superoxide dismutase (SOD)-like enzyme. The black crowberry fruit extract also inhibited lipid peroxidation in human LDL, detected as a reduction in malondialdehyde production [The extract of crowberry fruits restores the activity of cellular antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase-1 , and theoduction ,38.Empetrum hermaphroditum) (9.63 mmol/L) was reported to be higher than in blackcurrant (5.49 mmol/L), blueberry (7.57 mmol/L), strawberry (7.01 mmol/L), and raspberry (4.01 mmol/100 mL) [The total antioxidant activity in the crowberry (/100 mL) . All rad/100 mL) . Hakkinen et al.  found thr2 = 0.921 and 0.890, respectively, with significance at p < 0.001) [Polyphenolic compounds are a major group of secondary metabolites that mostly contribute to the antioxidant activity of crowberry. Black crowberry fruit showed a good correlation of free radical scavenging activity with TPC and TFC  (< 0.001) ,30.Berries of black crowberry—in relation to their high flavonoid content—are reported to have potential health-promoting effects, such as antioxidative, anti-inflammatory, and anticarcinogenic effects . In folk medicine, the fruits of black crowberry are utilized for the treatment of epileptic and paralytic states. Crowberry extract is a part of an homeopathic drug for the treatment of epilepsy in Russia (Empetrin). It also displays astringent and diuretic properties . Crowberry has been used in folk medicine to treat inflammatory diseases . Hyun etThe most-studied activity in black crowberry fruit is antimicrobial (AM) and antifungal activity, firstly published by and Maatsuura et al. . The antBacillus cereus and weak inhibition against Campylobacter jejuni. Another similar study by Rauha et al. [Staphylococcus aureus, Staphylococcus epidermis, and Escherichia coli, moderate antimicrobial activity (3–4 mm) against Bacillus subtilis, and clear antimicrobial activity (4–10 mm) against Micrococcus luteus. The juice of black crowberry is also effective against pneumococcal infections. Rauha et al.  studied a et al.  proved tStaphylococcus aureus, Candida albicans, and E. coli within 51 species of higher plants collected from the Oymyakon region of the Republic of Sakha (Yakutia) and Russia. The results were evaluated according to the size of the inhibition zone. The findings showed that only two species of higher plants—Empetrum nigrum and Cassiope tetragona—displayed very strong AM activity against S. aureus (inhibition zone: ≥20 mm) and moderate AM activity against C. albicans (inhibition zone: 10 mm).Paudel et al.  studied E. nigrum and E. hermaphroditum L.) juice fractions against Streptococcus pneumoniae. Human bronchial cells  were used as an adhesion model. All studied berry juices showed remarkable antiadhesive and AM activity. Pneumococcal growth was totally inhibited at a concentration of ~86 mg/g polyphenolic fraction of Empetrum nigrum juice. The strongest effect was displayed by the cranberry juice fraction. Gordien et al. [Mycobacterium aurum and M. tuberculosis H(37)Rv. The results of the experiment showed that the highest activity against M. tuberculosis was noticed for extracts of C. arbuscula, E. nigrum, and J. communis roots, Calluna vulgaris aerial parts, and Myrica gale roots and stems (93% to 99% inhibition at 100 μg/mL). The fruit of black crowberry also contains 2′-methoxy-4′-hydroxy-α- and β–dihydrochalcone with antimicrobial activity [50 values of 23.8 and 32.8 μM, respectively) in comparison to mammalian (HEK 293) cells , which was confirmed by a study by Li et al. [Huttunen et al.  comparedn et al.  studied activity . Anti-myi et al. .Empetrum nigrum fruit is based on protective actions against UVB radiation in human HaCaT keratinocytes  [E. nigrum’s activity such as absorbing UVB radiation and scavenging UVB-induced intracellular reactive oxygen species (ROS) in HaCaT keratinocytes. Moreover, E. nigrum protected HaCaT keratinocytes in the level of cellular components  following UVB irradiation and against UVB-induced apoptotic cell death [E. nigrum recovered cell viability by inhibiting apoptosis after cells were exposed to radiation. E. nigrum treatment also reduced γ-radiation-induced Bax proteins (apoptosis regulator), and caspase 9 and 3 expression in irradiated cells. The mechanism of the anti-apoptotic effect of E. nigrum is given by the inhibition of mitogen-activated protein kinase-4 (MKK4/SEK1)-c-Jun N-terminal kinase (JNK) cascades induced by γ-radiation [The anticancer activity of ethyl acetate extract of an skin) . Severalll death . In addiadiation .The black crowberry can be utilised in food processing as well. Wines made from a mixture of crowberry and blackcurrants have slightly higher antioxidant activity compared to red grape wines . The stuBlack crowberry fruit also seems to be an effective antidiabetic agent in fortified preparations of functional drinks. Torronen et al.  studied Proanthocyanidin-enriched extracts of alpine blueberry and black crowberry were found to reduce lipid accumulation in mouse adipocytes, in a positive correlation with total Proanthocyanidin content .Human studies of the antioxidant effect of black crowberry fruit were provided by Park et al. . They evCrowberries are good dietary sources of natural antioxidants with a dominant composition of flavonoids, α-glucosidase inhibitory, and anti-inflammatory components, suggesting that crowberry consumption can be a potential natural way to improve human health .There is a current preference for the use of lesser-known fruits like black crowberry in fruit production. This is because this species contains great amounts of biologically active substances, especially flavonoids (flavonols and anthocyanins), that display high antioxidant activity. For this reason, crowberry has a promising potential for future utilization as an alternative herbal drug displaying antidiabetic and antibiotic effect. Moreover, this species displays anti–inflammatory and anticancer activity, and has a positive effect on lipid metabolism. Crowberry extract can also find use as a nutraceutical component or for functional foods.


Persistent infections with high-risk genital Human papillomavirus (HPV) especially types 16 and 18, are associated with cervical cancer. However, distribution of HPV types varies greatly across geographical regions and the available vaccines target only few types. This study was designed to determine the HPV types circulating in Southwestern Nigeria, thereby providing necessary information for effective control of the virus.Endocervical swab samples were collected from a total of 295 consenting women attending routine cervical cancer screening, STI clinics and community-based outreach programme. Viral DNA was extracted from the samples and the consensus region of the HPV DNA was amplified by PCR using GP-E6/E7 primers. Type-specific nested multiplex PCR and Sanger sequencing were used to genotype the HPV isolates.In this study, 51 (17.3%) individuals were positive for HPV DNA using consensus primers that target the E6/E7 genes but only 48 (16.3%) were genotyped. A total of 15 HPV types  were detected, with HPV-31 being the most predominant (32.8%), followed by HPV-35 (17.2%) and HPV-16 (15.5%). Two rare HPV types; 74 and 86 were also detected. The HPV-74 isolate had three nucleotide (CCT) insertions at E7 gene that translated into amino acid proline. Highest nucleotide substitutions (n = 32) were found in HPV-44 genotype. Among positive individuals, 20.8% had dual infections and 86.2% had High-risk HPV types.Multiple Human papillomavirus types co-circulated in the study. Most of the circulating Human papillomavirus are high-risk type with type 31 being the most predominant. Although the implication of HPV-74 with proline insertion detected for the first time is unknown, it may have effect on the transformation potential of the virus. Polyvalent HPV vaccine will be more effective for the infection control in Nigeria. Genital Human papillomavirus (HPV) infection is the most frequent sexually transmitted infection globally and most sexually active individuals will be infected with HPV at some periods in their lives , 2. YounPapillomaviridae family  as previously described by Sotlar et al. . The cycet al. . The NMPF and twoSamples that were not typeable by PCR were sequenced and further analysed for genotype identification. The PCR products were purified with a commercially available PCR purification kit  according to the manufacturer’s instructions. The sequencing was performed commercially  on ABI Prism 3130 genetic analyzer (Applied Bio Systems) using the same consensus primers used for the PCR. However, only one of the reverse primers (either GP-E7-5B or GP-E7-6B) was used together with the forward primer (GP-E6-3F) to sequence the DNA samples.https://www.hpvcenter.se/human_reference_clones/) were downloaded and aligned with sequences of the sequenced isolates for phylogenetic analysis. Multiple sequence alignment of the isolates and the reference sequences was carried out using the MUSCLE algorithm on MEGA 6.0.6 software [Reference sequences of 179 HPV types available on the International Human Papillomavirus Reference Centre site (software . Evolutisoftware . MultiplThe socio-demographic characteristics of the study participants have been previously described . Out of p = 0.001). The six most commonly detected types were HPV-31, 16, 35, 18, 33 and 86. The HPV isolates included 8 high risk types  and 5 low risk types . However, two rare HPV types (HPV-74 and 86) with unclassified risk were also identified. High-risk HPV types were detected more frequently (86.2%) among HPV positive individuals than low-risk types (8.6%).Overall, 15 HPV types were detected at various frequencies in 58 infections . Some inp = 0.518). High-risk HPV  were found among both groups of participants. All the detected low risk types were found among symptomatic individuals (p = 0.007).Nine HPV types  were detected among 26 apparently healthy individuals. However, 11 HPV types  were detected among 22 participants with clinical symptoms of STI . There wFourteen HPV types  were identified by sequence analysis from 19 sequenced samples. Each HPV type has unique nucleotide and amino acid differences (Tables (CCT) at the E7 region between A512 and G513 position of the reference sequence (P) between L28 and D29 position. The phylogenetic trees of sequences of the study isolates with their reference sequences are shown . The HPV-74 sequence had an insertion of a triplet codon sequence  that traThis study is a cross-sectional study of Human papillomavirus in Nigeria. Fifteen HPV types  were detected in this study indicating co-circulation of multiple HPV types in Southwest Nigeria. The prevalence of high-risk HPV (HR-HPV) type was significantly higher than the low-risk type. The high prevalence of high-risk type in this study is a cause for concern because persistent infections with these types have been recognised as a necessary cause for cervical cancer. Similar results of higher prevalence of HR-HPV have been previously reported in several studies in Nigeria –29. RelaThere were significantly more single infections (79.2%) in this study than dual infections (20.8%) which corroborates the findings of past studies , 30. NonThe predominant HPV type in this study is HPV-31, followed by HPV-35, 16, 18, 33 and 86. HPV-31 has been reported to be very common in Europe and Latin America , 37. SimSeveral studies in Nigeria have shown disparities in the prevalent HPV types. In Lagos, HPV type 31 also predominated albeit among HIV positive individuals, followed by types 52, 53 and 35 while among HIV negative group, type 18, 16, 52 and 56 were the commonest . The mosHigh-risk HPVs found both among apparently healthy individuals and symptomatic participants further buttress the fact that most infections with HPV do not manifest clinically. Routine HPV testing among general population would therefore be important for diagnosis. Further studies on the rare types (HPV-74 and 86) detected in this study would be essential to access their distribution and determine their association with cervical carcinoma. All the low-risk HPV types detected were found among symptomatic individuals, although, only one individual out of those with genital wart had low-risk HPV (type 81). This could imply that HPV types other than low-risk types may also be associated with genital warts. Hence, regular HPV testing and not the mere presence of a wart will be required to detect HPV types in an infected individual.The phylogenetic analysis of the 19 sequenced isolates showed that the E6 and E7 sequences of HPV types are conserved within the types but varies between types. The overall mean distance between the isolates being 0.659 showed a high level of diversity among the isolates in the E6/E7 region. To the best of our knowledge; this seems to be the first study that describes the sequences of HPV types by E6/E7 genes among Nigerian women.The analysis of HPV E6 and E7 sequences has been used to determine the variants of some HPV types –12. AlthThe two HPV-18 sequenced in this study have 3 silent mutations at their E6 regions as previously found , 60. Isoet al. [The HPV-44 sequence with the highest nucleotide substitutions had only two of the E6 substitutions (C183T and C396T) reported by Maver et al. . The isoet al. , 8. The et al. , 66, 59 (CCT) insertion that translated to amino acid Proline (P) at the E7 region. Based on information available in the literature, this is the first report of the insertion. Although the implication of the insertion is unknown, it may have effect on the oncogenic potential of the virus.HPV-86 and 74 sequenced in this study are very rare. Their prevalence has been reported by only limited studies –73. The The presence of HPV-74 and 86 among participants in this study could not be linked to any factor. However, the genome of HPV-74 was first identified from an immunosuppressed woman with persisting low-grade vaginal intraepithelial neoplasia . Future In this study, 3 (5.9%) out of the 51 positive samples could not be successfully genotyped. The three samples had unreadable sequences due to mixed peaks shown on their sequenced data which means that it is still possible to have some more HPV types in the samples. Hence, the genotypic result of this study might not be representative of the overall circulating genotypes in Southwest Nigeria. This limitation however can be tackled in future studies by the use of Next Generation Sequencing technology.In conclusion, the high prevalence of high-risk HPV types detected in this study even in multiple infections reveals the burden of HPV infection in the country. Infections with high-risk types have the tendency of progressing to malignancy; thus an increased surveillance to determine the women at risk of cervical cancer is advocated. Further studies into detecting HPV-74 and 86 (rare types) will be important to determining their association with cancer. Multiple HPV types detected with non-HPV 16 and non-HPV 18 dominating indicated that HPV-16 and 18 might not be the major circulating types in Nigeria and the available vaccines in the country may be less effective in controlling the infection. However, 7  out the 15 circulating types, detected in 40 infections (69.0%) are vaccine types in Gardasil-9. It is therefore necessary for policy makers to consider a more protective polyvalent vaccine like Gardasil-9 for effective control of HPV infection, and the vaccine should be made available at a subsidised rate to the target population in Nigeria.


Military veterans often experience physical health problems in later life; however, it remains unclear whether these problems are due to military service or are a feature of the ageing process. This study aimed to explore veteran and non-veteran perceptions of the impact of their occupation on their physical well-being later in life.Semi-structured qualitative interviews analysed using thematic analysis.Interviews were conducted face-to-face in participants’ homes or via telephone.35 veterans (≥65 years), 25 non-veterans (≥65 years) were recruited, as well as a close companion of all participants for triangulation (n=60).Most veterans reported good physical health later in life which they attributed to the fitness they developed during military service. However, several veterans described challenges in maintaining their desired level of physically activity due to new commitments and limited sports facilities when they left service. Fewer non-veterans had experienced work-related fitness activities or exercise in their civilian jobs. Ongoing physical health difficulties, such as deafness, were perceived to be due to exposure to workplace hazards and appeared more common in veterans compared with non-veterans. Veterans also described greater reluctance than non-veterans to seek medical treatment for physical health difficulties, which could be challenging for close companions who had to provide informal care.Military service was largely perceived to be beneficial for physical well-being; although when occupation-related physical health problems were experienced, many veterans were unwilling to seek treatment. These findings may inform clinicians of the needs of older veterans and highlight potential barriers to care. Large-scale qualitative study (n=120) which recruited veterans and non-veterans aged over 65 years and a close companion of each participant.Semi-structured, qualitative interviews were conducted face-to-face in participant’s homes or via telephone.Inclusion of the views of a close companion  to triangulate the study findings.The UK Armed Forces (AF) veteran population is becoming increasingly elderly, with approximately 64% of UK AF veterans over the age of 65.The limited available evidence indicates that UK AF veterans may be more likely than the general population to experience physical health conditions that affect their daily functioning. For example, working age UK AF veterans have higher rates of hearing and musculoskeletal problems compared with non-veterans.A deeper understanding of the perceived long-term impact of AF service on physical health may inform clinical practice, identify whether there is a need to alter the way in which services for this population are provided and highlight areas for occupational policy change. We conducted in-depth, qualitative interviews with older veterans, older non-veterans and a close companion  of each veteran/non-veteran participant. We aimed to explore perceptions of the impact of military versus non-military occupation on physical well-being later in life.This was a qualitative study using in-depth interviews. The qualitative approach utilised allowed for the exploration of issues that were most salient to participants and the subjective meaning attached to their career and well-being. The present study was nested within a larger programme of researchFour subsamples of participants were recruited: (1) 25 older veterans  not known to have mental health problems, (2) 25 older non-veterans not known to have mental health problems , (3) 10 older veterans with self-reported experiences of mental health issues  and (4) a close companion of all participants . The 25 veterans without mental health diagnoses and 25 non-veterans were age (+/-5 years) and gender matched. Exclusion criteria included: brain damage or cognitive impairment, inability to speak English or current self-injurious behaviour/suicidal intent.Veteran and non-veteran physical health and quality of life were measured using the 12-item Short Form Health Survey (SF-12)The semi-structured interview questions focused on participants perceptions of their career, the impact of their career on their physical health and their experiences of informal care for physical health problems (see online 10.1136/bmjopen-2018-028189.supp1Supplementary dataThe majority of interviews were conducted face-to-face (n=82), with 38 conducted via telephone. All participants gave written (if interview conducted in person) or audio-recorded verbal (if interview conducted via telephone) informed consent for their participation. Interviews were conducted by study researchers who had training and experience in qualitative methods. Interviews lasted for an average of 67 min . All interviews were digitally recorded and transcribed verbatim. Interviews were completed by 120 participants to represent a wide range in occupations and ages to reflect the possible diversity of opinions. Thematic saturation was achieved, and this was determined by the research team when no additional themes were found from the reviewing of successive data.14Purposive sampling was used. As previous research has highlighted the relationship between poor mental health and physical health problems,A total of 86 veteran/non-veteran participants were approached to take part in the study, 60 of which were recruited (69.8% recruitment rate). Eligible individuals who did not participate were largely not contactable or had no close companion to take part. All participants were recruited between March and December 2017. All participants were given a £20 voucher as a thank you for their time.Data were entered and analysed using the statistical analysis software package SPSS (V.24). Missing data on the EQ-5D VAS and SF-12 were excluded from the analysis . T-tests were used to determine whether statistically significant differences between groups existed, with p values <0.05 used to indicate statistical significance.All transcripts were entered into NVivo V.11 . Data were analysed using thematic analysis using the steps described by Braun and ClarkeThe study involved patients in shaping the study design and outcome measures as the study materials were piloted with veterans (n=3) and non-veterans (n=2). Patients were also involved in the study recruitment as recommendations for potential recruitment sites were sought from patient participation group meetings. The findings were disseminated to participants via a study newsletter.Participant demographic information can be found in Differences in scores on the SF-12 and EQ-5D were not statistically significant between veteran, veterans with mental health diagnoses and non-veteran samples , this activity was considered to have kept them in good health at the time but was often seen as having no long-term benefits.Nonetheless, several veterans stated they had chronic arthritis and other musculoskeletal health complaints in older age which were thought to be due to the physically demanding activities required in the AF . These health problems were less commonly described by non-veterans and were not thought to be related to occupation.On leaving the AF, a minority of veterans experienced difficulties continuing physical activity, either due to the long working hours of their civilian job, being employed in a more sedentary civilian role , limited access to sports facilities or due to a military-related physical injury. Veteran difficulties continuing exercise were also discussed by their close companions.Participants in both veteran and non-veteran samples reported sustaining physical injuries in the workplace, with the majority of injuries viewed as being due to a lack of protective equipment or poor health and safety regulations .Veterans more frequently reported long-term health difficulties due to a lack of protective equipment compared with non-veterans. Veterans often described experiencing hearing loss due to a lack of ear defenders in loud engine rooms or on shooting ranges, as well as skin cancers believed to be caused by having no sun protection during training activities or deployment.For non-veterans, those in physically demanding jobs often reported strain injuries due to heavy lifting or spending several hours standing. Physically demanding jobs were thought to exacerbate existing physical health conditions . Some non-veterans in healthcare roles or jobs requiring foreign travel  also described that they had contracted serious illnesses . Such injuries/illnesses were often felt to have long-lasting effects, with physical pain or disability experienced into retirement.It reportedly became more challenging for close companions to cope with veteran and non-veteran’s occupation-related health problems later in life, particularly in cases of veteran/non-veteran hearing loss or debilitating illness. These close companions described feeling lonely as well as frustrated that their plans to exercise or travel as a family in retirement were now not feasible.The health of close companions or other family members could also reportedly be affected by veteran and non-veteran’s exposure to workplace hazards. For example, some close companions discussed being exposed to asbestos on veteran/non-veteran’s work clothes. This indirect exposure was thought to cause serious illness, and family members reportedly experienced cancer, infertility, breathing problems and other conditions as a result. When family members became unwell in such circumstances, veterans and non-veterans disclosed experiencing significant guilt and anxiety for their family members’ well-being, often taking considerable steps to address the perceived harm they had caused .Veterans with and without diagnoses of mental health problems described experiencing similar self-reported physical health problems . Veterans without diagnoses of mental health difficulties more often considered that their physical health conditions related to AF service compared with veterans with mental health diagnoses. Few other differences in terms of physical health problems were described between the two subsamples.Reluctance to seek medical treatment for physical health problems both during and following military service was commonly reported in veterans. This reluctance was largely not described by non-veterans. Unwillingness to admit physical health problems and access treatment was reportedly due to a sense of self-sufficiency instilled by the AF and a need to feel and be seen as ‘tough’.Close companions detailed how they play a central role in encouraging veterans to access medical treatment, often accompanying them to appointments and ensuring medication was taken. In cases of serious illness/disability, many close companions of veterans described that veterans were extremely reluctant to accept informal care, such as assistance with getting dressed, daily hygiene, etc, often due to embarrassment or pride, and unwillingness to accept care was less common in non-veterans.Providing support and care to unwilling veterans could be a distressing and frustrating experience for close companions who often felt their efforts were not appreciated. To cope, close companions positively reframed their experience, for example describing that the veteran would provide them with the same care if needed or seeking support from other family members and friends. Close companions of non-veterans described facing less resistance to their provision of support.The aim of this study was to explore the perceived impact of military and non-military occupations on physical health later in life. Our findings illustrate the perceived considerable benefits of occupation on well-being, particularly for those who served in the AF, such as long-term good physical health as a result of physical exercise. At the same time, these results also highlight the significant long-term implications of workplace practices on well-being, including chronic physical health problems due to a lack of protective equipment and beliefs regarding the wider impact of such workplace hazards on an employee’s family.Our findings delineate the positive influences of occupation on health, including the perceived long-lasting benefits of physical exercise in the AF. Physical exercise is associated with lower risk of health problems later in life, including dementiaPrevious research into the impact of military service on physical health has also highlighted that, compared with the general population, working-age veterans are more likely to experience hearing difficulties, musculoskeletal problems and arthritis on leaving service.Another key theme emerged in relation to treatment seeking as veterans appeared to be more reluctant than non-veterans to seek medical treatment for physical health problems or accept informal care - reportedly due to embarrassment or an AF instilled need to be ‘tough’. As delays to necessary medical care can adversely impact well-being,The current study provided a perspective on an important, yet understudied, group in examining the impact of military service on well-being in older UK AF veterans. We recruited a diverse sample of veterans and non-veterans from multiple professions and took steps to ensure the data was robust by triangulation with close companions. At the same time, there are limitations that must be considered. First, this study largely included male veterans/non-veterans, although this is broadly consistent with the gender distribution of the UK AF. Second, this study included only those veterans and non-veterans who agreed to contact by the research team or chose to self-refer in response to study advertisements. This may mean that participants had particularly salient occupation-related issues they wished to discuss. Third, while the key outcome measure of the study was the qualitative interview, a small proportion of participants did not fully complete questionnaire assessments resulting in missing data which must be taken into consideration. Furthermore, the inclusion of non-veterans with diagnoses of mental health problems in future studies would further our understanding of the potential relationship between occupation, physical and mental health. Finally, given the qualitative nature of the study, the findings reflect the lived experiences of a sample of veterans, non-veterans and close companions and large-scale quantitative investigations would be useful in determining the generalisability of the results.Despite these limitations, this study adds to the very limited research into the long-term physical health implications of military service on older UK veterans in several ways. First, by directly comparing the experiences of veterans and non-veterans, our results suggest that military service may have considerable beneficial effects on physical health later in life due to greater opportunities for exercise. Second, these findings demonstrate that poor health and safety practices, particularly in the AF, may lead to physical health problems which can impact long-term functioning. Finally, this research highlights that additional advice and support may be beneficial to veterans and their families to facilitate veteran access to medical treatment which could ultimately improve their well-being and reduce familial stress.


HPV vaccination with the bivalent vaccine is efficacious against HPV16 and 18 infections and cross-protection against non-vaccine HPV types has been demonstrated. Here, we assessed (cross-) protective effects of the bivalent HPV16/18 vaccine on incident and persistent infections and viral load (VL) of fifteen HPV types in an observational cohort study monitoring HPV vaccine effects.Vaginal samples were obtained annually. Type-specific VL assays were developed for HPV6,11,31 33,35,39,45,51,52,56,58,59 and 66 and used in addition to existing HPV16 and 18 assays. Rate differences of incident clearing and persistent infections were correlated with differences in VL and vaccination status.HPV16/18 vaccination resulted in significantly lower incidence of HPV16/18 infections and significantly lower VL in breakthrough HPV16 (p<0.01) and 18 infections (p<0.01). The effects of vaccination on non-vaccine type VL were ambiguous. Incidence and/or persistence rates of HPV31, 33, 35 and 45 were reduced in the vaccinated group. However, no significant type specific VL effects were found against HPV31, 33, 45, 52 in the vaccinated group. For HPV 6, 59 and 66 no significant reductions in numbers of incident and persistent infections were found, however borderline) VL reductions following vaccination were observed for HPV6 (p = 0.01), 59 (p = 0.10) and 66 (p = 0.03), suggesting a minor effect of the vaccine on the VL level of these HPV types. Overall, vaccination resulted in infections with slightly lower VL, irrespective of HPV type.In conclusion, vaccination with the bivalent HPV16/18 vaccine results in significantly reduced numbers of HPV16 and 18 incidence rates and reduced VL in breakthrough infections. Significant reductions in incident and/or persistent HPV31, 33, 35 and 45 infections were found, but no significant effect was observed on the VL for infections with these types. For the other non-vaccine HPV types no reduction in incident and/or persistent infections were found, but overall the VL tended to be somewhat lower in vaccinated women. Cervical intraepithelial lesions (CIN) and cervical cancer are caused by persistent high-risk human papillomavirus (hrHPV) infections . ProgresO-desacyl-4′-monophosphoryl lipid A adsorbed on aluminum salt), which appears to induce enhanced and longer-lasting antibody titers than the aluminum hydroxyphosphate sulfate adjuvant used in the quadri- and nonavalent vaccines [To prevent HPV-associated malignancies, three highly efficacious prophylactic recombinant vaccines are currently registered and commercially available. The bivalent vaccine protects against HPV16 and 18, quadrivalent adds HPV6 and 11, and nonavalent further adds HPV31, 33, 45, 52 and 58 –15. The vaccines . Vaccinavaccines , 22.The protective effect against persistent HPV16/18 and some non-vaccine type HPV infections and associated CIN2+ lesions –24, combIn 2009, bivalent HPV vaccination was introduced in the Netherlands. At the same time, the HPV Amongst Vaccinated and Non-vaccinated Adolescents (HAVANA) cohort study was started to monitor vaccine effectiveness , 27, 28.HPV Amongst Vaccinated and Non-vaccinated Adolescents” (HAVANA) observational cohort study, which has been described previously [Vaginal self-samples were obtained annually from participants of the “eviously . In shoreviously . Informe25 platform [Total DNA was isolated from 200μl of self-samples using the MagNAPure96 platform (Roche Diagnostics) and eluted in 100μl elution buffer. A phocine herpesvirus-1 spike was added to each sample as an internal control for DNA isolation. Isolated DNA was genotyped using the analytically sensitive SPF10-DEIA-LiPAplatform –31.E. coli (High Efficiency) (New England Biolabs) according to the manufacturer’s protocol and the cell suspension was subsequently plated on MH agar plates containing ampicillin. Plates were incubated overnight at 37°C and single colonies were transferred to liquid LB medium containing ampicillin. Cultures were grown while shaking to OD600 = 2–3, and then centrifuged at 5000g for ten minutes. Plasmid was isolated from cell pellets using GeneJet Plasmid Midiprep kit (Thermo Scientific) according to the manufacturer’s protocol. Isolated plasmid was eluted in 40μl elution buffer and quantitated in triplicate in three individual experiments to minimize assay variation using PicoGreen dsDNA quantification assay (ThermoFisher).Ampicillin resistant plasmids for HPV6, 11, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 were kindly provided by the HPV Reference Center, Karolinska Institutet, Sweden. Plasmids were transformed into heat-competent NEB 5-alpha 7 genome equivalents (GEq)/reaction). To assess possible over- and underscoring of assays, nine dilutions from each calibration curve were tested in the presence and absence of high concentrations (>1.7x105 GEq/reaction) of the eight most prevalent hrHPV types in this study; HPV16, 18, 31, 33, 45, 51, 52 and 58. All measurements were performed in singleplex for optimal sensitivity and normalized by measuring cellular content via beta-actin qPCR [−5 copies/cell [New quantitative VL assays for HPV6, 11, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 were developed in line with HPV16/18 assays described previously . Primer tin qPCR . Samplesde facto exclusion for other HPV types.To be included in the analysis of any HPV type, study participants had to be genotyping negative at baseline for that specific HPV type. HPV (baseline) positivity was assessed on a per type basis. Study participants could be included independently in multiple TS analyses. An exclusion for any one HPV type did not result in Infection definitions are explained in Incidence rates (IR) were calculated for incident clearing (IRI) and persistent (IRP) infections to compare with VL measurements. Person-time for any infection was only counted when at least two subsequent measurements were available after the first HPV TS positive sample. As the exact moment of infection could not be determined, interval censoring was applied for incident clearing infections after the first TS HPV positive measurement. For persistent infections one more sample after the initial TS HPV positive sample was included after which the participant was censored. IRI and IRP were adjusted for factors associated with vaccination status, as reported previously: age, urbanization degree, ever smoked, ever used contraception, ever had sex . AdjusteParticipants supplied samples annually. Participants were excluded from a type-specific (TS) analysis if positive at baseline for that human papillomavirus (HPV) type (A). Following a negative TS HPV baseline measurement, participants were either negative, incident clearing or incident persistent for that HPV type . Intermittent infections (G) were excluded due to uncertainty about true incidence or latent persistence. Infections meeting definitions for both clearing and persistent infections were only included based on the first infection identified for that HPV type .et al. [Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software) and SAS 9.4 (SAS Institute Inc.). Two-tailed p-values <0.05 were considered statistically significant. Differences in study populations for the present analysis and the study population as published by Donken et al.  were asset al. [From a total of 1832 HAVANA study participants, 115 women were excluded due to a missing sample at baseline or incomplete or unknown vaccination status. 1717 non-vaccinated n = 769; 44.8%) or three-dose vaccinated  girls were included in this analysis. Characteristics of the selected study population are listed in et al. . In tota4.8% or tThe present analysis was compared to a previous epidemiological description of this cohort study  and diffet al. [et al. [Incidence rates and ratios were calculated per thousand observed person years for vaccinated and non-vaccinated individuals and adjusted according to Donken et al. , as show [et al. , we findFor non-vaccine types, a reduced incidence of incident clearing infections was found in the vaccinated group for HPV31 , HPV33 , HPV35  and HPV45 . Additionally, less HPV31 persistent infections were found among vaccinated study participants . Interestingly, more HPV59 persistent infections were found in the vaccinated group . For other HPV types, no (significant) differences were observed.7 copies of different HPV types. Sensitivity was <5 GEq/reaction for each individual assay. Reproducibility of VL measurements was assessed by measuring duplicates of a subset of HPV16 (n = 62) and HPV18 (n = 22) samples. Duplicates were found to be on average <0.1 ct values apart . VL measurements were performed based on genotyping results. Figs HPV6, 11, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 TS VL assays were specific in the presence of at least 10VL measurements correlated well with observed reductions in incident/persistent HPV16/18 infections. Median HPV16 VL was significantly lower in vaccine recipients for the total study analysis p<0.01, , as wellDespite finding some cross-protection of the bivalent vaccine against HPV31, 33, 35 and 45  based vaccines) primarily leads to sterilizing immunity against HPV16/18 via type-restricted neutralizing antibodies, preventing the majority of incident and persistent infections . We repoet al. , 40, whiet al. , 26.While we are unable to clearly associate vaccine effects on incidence of infections with VL, we do observe a general trend where median VL for any HPV type was lower for vaccine recipients than non-vaccinated individuals. If these findings are indeed caused by the vaccine, they might help alleviate or prevent long-term disease effects by dampening infection development through reduced viral load.Several limitations need to be addressed for this study. First, we included participants based on baseline TS HPV DNA negative samples, since serology was only available for a subset of our cohort. While baseline DNA positivity is informative for the present sample, it does not inform about any past exposure to HPV and any possible effects on VL. Fortunately, due to low sexual activity at inclusion for this study , we beliCombined, our results suggest that vaccination against HPV16/18 affects VL in breakthrough infections when compared to non-vaccinated women. Although some effects are observed against non-vaccine types, this study is insufficiently powered to suggest a clear correlation between vaccination and effects on VL of non-vaccine HPV types. Further population-based studies are required to identify which effects are truly causal to and maintained by the vaccine.S1 Table(DOCX)Click here for additional data file.