Pipe robots can perform inspection tasks to alleviate the damage caused by the pipe problems. Usually, the pipe robots carry batteries or use a power cable draining power from a vehicle that has many equipments for exploration. Nevertheless, the energy is limited for the whole inspection task and cannot keep the inspection time too long. In this paper, we use the total input energy as the cost function and a more accurate DC motor model to generate an optimal energy-efficient velocity control for a screw-drive pipe robot to make use of the limited energy in field environment. We also propose a velocity selection strategy that includes the actual velocity capacity of the motor, according to the velocity ratio  With the advancement of the robotics and industrial technology, many pipe robots have been developed to explore the pipes that have cracks or defects to avoid serious accidents –4.Up to now, there are more focus on the energy-efficient control of robots , 6. RoboThis paper proposes an energy-efficient solution for the control of a screw-type pipe robot by using an improved DC motor model and employing the total input energy as the performance index that reflect the whole system energy consumption. Straight pipe structure is the most common type; thus, this paper is limited to discuss the condition that the pipe robot is used in the straight horizontal pipe. Additionally, sinusoidal fashion control and the loss minimization control that only considers armature resistance are used as the comparison methods.The environment of the pipe is not invariable; thus, the pipe robot should possess the characteristics of multifunction, adaptability and efficiency. The screw-drive robots are not rare , 4, and Typical screw-drive robot is usually composed of a rotator, elastic support arms, rollers and a motor for driving. The rollers have a constant incline angle with respect to the cross section of the pipe. When the motor turns, the whole body moves forward. If the motor turns reversely, the body moves backward. To propel a screw-drive-type robot, one motor is enough for straight pipe and elbow. However, for the T-shape pipe, extra navigation mechanism is needed.A screw-type robot considered in this paper is shown in Fig. An armature-controlled DC motor is shown in Fig. The basic equations of circuit arelculated . \documeBy using the above equation, we can also calculate the value of As shown in Fig. From the above equations, the mechanical power generated by the motor is The above equations have shown the influence of mentclass2pt{minimv and D is the inner diameter of the pipe; The kinematics of the wheel-type robot is generally calculated byfound in , 4)10\don is the number of the driving arms. The static equilibrium equations can be derived from Fig. 5Let mentclasspt{minimaThus, the load torque term Now, Cruise start/stop mode: Cruise start mode is used to start the motor and the robot at a specified speed; then, the motor and the robot keep this speed moving to find the potential defect of the pipe. When the detecting camera finds the suspicious defect, the robot will stop and detect that area carefully; thus, stopping the robot is called cruise stop mode.Location mode: Sometimes, a segment of pipe need not to be checked; thus, the robot just passes by. The operator only inputs the displacement fined in .According to the task requirements, this robot has two modes:Under this mode, the final velocity The above problem is to find the optimal control Since many segments of the pipe have been checked and do not need to be checked again, the robot needs position to position control. Under the location mode, the operator inputs the desired displacement ted from . Let \doTrzynadlowski had discussed the optimal velocity by plotting a figure . We willment}ea,  yields36nt}a→+∞,  yields37mentclass2pt{minimIt is known that each motor has its maximum speed limit; moreover, it also has a maximum continuous working speed From the above discussion, we will get cated in , and we tAs for the condition After discussing the above working conditions of the robot, we can formulate the optimal velocity selection strategy, which considers the velocity constrains, under location modeWe have derived the energy-efficient control laws of the robot in cruise start/stop mode and location mode, respectively. In this section, simulations are performed to evaluate the proposed energy-efficient control law, and these results are compared with two benchmark methods. One is sinusoidal velocity function based on the computed torque control and is given byFigure In Fig. Figure entclass1pt{minimaFigure see Fig. . While trding to , which mentclass1pt{minimaThis paper considers a more accurate motor model and uses total input energy as the cost function to generate energy-efficient control laws for a pipe inspection robot. This pipe robot has two working modes: driving mode and detecting mode. Robot needs to keep a speed to move or move a distance to check the pipe; thus, we propose two types control: One is cruise start/stop control, and the other is location control. For the cruise mode and location mode, we have derived the optimal velocity and propose a velocity selection strategy according to the


Chlamydia trachomatis and Mycoplasma genitalium.It is well-established that male circumcision reduces acquisition of HIV, herpes simplex virus 2, chancroid, and syphilis. However, the effect on the acquisition of non-ulcerative sexually transmitted infections (STIs) remains unclear. We examined the relationship between circumcision and biological measures of three STIs: human papillomavirus (HPV), A probability sample survey of 15,162 men and women aged 16-74 years  was carried out in Britain between 2010 and 2012. Participants completed a computer-assisted personal interview, including a computer-assisted self-interview, which asked about experience of STI diagnoses, and circumcision. Additionally, 1,850 urine samples from sexually-experienced men aged 16-44 years were collected and tested for STIs. Multivariable logistic regression was used to calculate adjusted odds ratios (AOR) to quantify associations between circumcision and i) self-reporting any STI diagnosis and ii) presence of STIs in urine, in men aged 16-44 years, adjusting for key socio-demographic and sexual behavioural factors.The prevalence of circumcision in sexually-experienced men aged 16-44 years was 17.4% (95%CI 16.0-19.0). There was no association between circumcision and reporting any previous STI diagnoses, and specifically previous chlamydia or genital warts. However, circumcised men were less likely to have any HPV type  0.13-0.50) including high-risk HPV types   detected in urine.Circumcised men had reduced odds of HPV detection in urine. These findings have implications for improving the precision of models of STI transmission in populations with different circumcision prevalence and in designing interventions to reduce STI acquisition. It is well-established that male circumcision reduces HIV acquisition.–3 A metaHuman Papillomavirus (HPV) is highly prevalent. PersisteChlamydia trachomatis and Mycoplasma genitalium are bacterial STIs possibly associated with upper reproductive tract infection in women. A randomised trial of circumcision to prevent acquisition of HIV in Kenya found Chlamydia infection was reduced in the intervention arm, but this lacked significance .[M. genitalium , after controlling for behavioural risks.[C. trachomatis in circumcised participants .[32–1.00). However,C. trachomatis and M. genitalium in urine among men in the British general population.Our study examined the relationship between male circumcision and reporting previous STI diagnoses or detecting HPV, The third National Survey of Sexual Attitudes and Lifestyles  was a stratified probability sample survey of 15,162 men and women (6293 men) aged 16–74 years in Britain, who were interviewed during 2010–2012. The overall response rate was 57.7%. Participants were interviewed using computer-assisted face-to-face and self-interviews (CASI). The CASI included questions about male circumcision, sexual behaviour and history of STI diagnoses. Further methodological details have been described elsewhere.,12C. trachomatis and M. genitalium. STI test results were available from 1,850 men. HPV types were classified as high-risk, possible high-risk or HPV-6/11 using the IARC Monograph Working Group classification [C. trachomatis used the Aptima Combo 2 assay (Hologic Gen-Probe) as the initial screen and all positive or equivocal results were confirmed with the Aptima monospecific assay. An in-house Luminex-based genotyping assay was used to detect HPV types.[M. genitalium detection used an in-house Real-Time PCR assay which targets the adhesin protein (MgPa) gene, [After the interview, a sample of participants aged 16–44 years who reported at least one lifetime sexual partner were invited to provide urine for STI testing.,11 Samplfication  (see TabPV types. M. genita) gene,  with posStata (version 12.1) was used for all statistical analyses accounting for the stratification, clustering, and weighting of the sample, which was broadly representative of the British population. Analyseswww.natsal.ac.uk).All participants were given an information leaflet to read prior to participating in the survey and had the opportunity to discuss this with the interviewer. Verbal informed consent was obtained for participation in the interview and interviewers had to confirm that respondents had read the information leaflet in the computer programme before commencing the interview. Participants gave written informed consent for anonymised testing of urine samples, without the return of results, the ethical rationale for which has been previously described. The NatsThe prevalence of reported circumcision in sexually-experienced men aged 16–44 resident in Britain was 17.4% (95% CI 16.0–19.0). Prevalence increased with age, from 13.2% (95% CI 11.3–15.5) among men aged 16–24 to 18.4% (95% CI 15.5–21.6) among men aged 35–44 . PrevaleC. trachomatis, but not M. genitalium detected . Unadjusted and adjusted ORs for the association between circumcision and M. genitalium differed substantially due to confounding by ethnicity.Circumcision was not associated with reporting previous diagnosis/es of any STI, or specifically chlamydia or genital warts in 16–44 year old men . Resultsdetected . After adetected , circumcFindings from this large British population-based survey show that male circumcision is strongly associated with lower detection of any HPV and HR-HPV in urine, even after adjusting for key demographic and behavioural variables. However, circumcision was not associated with self-reported previous STI diagnosis/es in this population.C. trachomatis and M. genitalium),[C. trachomatis and M. genitalium are regarded as the ‘gold standard’ in men, but urine is considered a suboptimum specimen for detection of HPV. Hernandez et al assessed HPV prevalence at different sites and found that it was highest in the penile shaft (52% at this site) compared to just 10% in urine,[The strengths of this study are that the initial Natsal-3 sample size is large, and probability sampling gives data representative of the British general population. In contritalium), statistiin urine, suggestiin urine, Althoughin urine,,20,21 whin urine, suggestiC. trachomatis, consistent with some,[M. genitalium to the findings of other studies due to insufficient statistical power.The lack of association between male circumcision and self-reported previous STI diagnoses is in keeping with Natsal-2 findings, and the ith some, but not ith some, However,et al. observed that monogamous women whose partners had reported six or more sexual partners and were circumcised were at lower risk of cervical cancer than monogamous women whose partners were uncircumcised.[Data from a large national probability survey have shown that male circumcision is associated with lower odds of detecting oncogenic HPV types. Reduced prevalence of oncogenic HPV types among men may result in reduced transmission to sexual partners, with the consequent effect of reduced risk of cervical cancer. Castellasgue cumcised. Currentlcumcised. reductiocumcised. and the


CCND1 showed to be an independent predictor for worse survival in OSCC. These results show that copy number aberrations, mainly of the 11q13 region, may be important predictors and prognosticators which allow for stratifying patients for personalized treatment of HNSCC.Current conventional treatment modalities in head and neck squamous cell carcinoma (HNSCC) are nonselective and have shown to cause serious side effects. Unraveling the molecular profiles of head and neck cancer may enable promising clinical applications that pave the road for personalized cancer treatment. We examined copy number status in 36 common oncogenes and tumor suppressor genes in a cohort of 191 oropharyngeal squamous cell carcinomas (OPSCC) and 164 oral cavity squamous cell carcinomas (OSCC) using multiplex ligation probe amplification. Copy number status was correlated with human papillomavirus (HPV) status in OPSCC, with occult lymph node status in OSCC and with patient survival. The 11q13 region showed gain or amplifications in 59% of HPV-negative OPSCC, whereas this amplification was almost absent in HPV-positive OPSCC. Additionally, in clinically lymph node-negative OSCC (Stage I–II), gain of the 11q13 region was significantly correlated with occult lymph node metastases with a negative predictive value of 81%. Multivariate survival analysis revealed a significantly decreased disease-free survival in both HPV-negative and HPV-positive OPSCC with a gain of Wnt-induced secreted protein-1. Gain of  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is characterized by a biologically highly heterogeneous group of tumors. Treatment is predominantly based on location and TNM-classification and comprises mainly conventional methods such as surgery, radiotherapy, chemotherapy, or a combination of these. Currently, no treatment modalities exist that rely on the tumor-specific biology of HNSCC. The 5-year overall survival remains relatively poor at ∼50–60% and has not changed significantly over the past decades Previous studies have shown an association between certain types of HNSCC and the presence of human papillomavirus (HPV). Molecular profiling may prove valuable to determine the exact role of HPV in oropharyngeal squamous cell carcinoma (OPSCC) and to the prediction of occult nodal metastasis in oral cavity squamous cell carcinoma (OSCC). Along with known risk factors such as alcohol and tobacco consumption, HPV infection has been identified to play an etiologic role in HNSCC, especially in OPSCC Constituting an important element in the causal chain to cancer initiation and progression, genetic imbalances could serve as predictive or prognostic biomarkers in the near future. Genomic copy number aberrations (CNA) are alterations of the DNA resulting in an abnormal copy number of a region within the DNA. To find potential relevant aberrations for clinical decision making, this study correlates CNAs of a panel of 36 common oncogenes and tumor suppressor genes in two major subsites of HNSCC, knowingly the oral cavity and the oropharynx, with both clinicopathological features and survival.The study population was described previously www.federa.org). Anonymous or coded use of leftover tissue for scientific purposes is part of the standard treatment agreement with patients in our center For OPSCC and OSCC, respectively, material from biopsies and resection specimen was used. Since we used leftover tissue from routine diagnostic procedures, no ethical approval was required according to the Dutch national ethical guidelines . In addition, specimens consisting of normal oral cavity mucosa of patients treated for an oral fibroma  with no history of head and neck cancer were used as controls in OSCC experiments. Normal oropharynx mucosa biopsies derived from patients with neck metastases from an unknown primary tumor in head and neck region were used as controls in OPSCC experiments.HPV type 16 positive tumors were determined by a validated test algorithm as described before μm paraffin blank slides using a scalpel. Tumor tissue was suspended in direct lysis buffer  and subsequently lysed by overnight incubation at 56°C in proteinase K , followed by heat inactivation at 98°C for 10 min and subsequently DNA extraction by means of centrifugation after which the supernatant was recovered.Hematoxylin and eosin-stained slides were reviewed by a dedicated head and neck pathologist (S. M. W.) to confirm the presence of malignancy. Samples with a tumor percentage of at least 30% were included in this study. After deparaffinization, corresponding tumor areas were scraped off from 5 μL of isolated DNA was used for MLPA analysis according to the manufacturer's instructions. A set of 36 genes for 12 different chromosomal locations  was investigated. For this kit, the genes were selected based on a thorough literature search (by S. S. and W. R.) for frequent CNAs in HNSCC in association with prognosis. After centrifugation, 5 All statistical analyses were performed using IBM  SPSS 20.0 statistical software. MLPA results were dichotomized as loss versus no loss (cut-off 0.70), gain versus no gain (cutoff 1.3), and nonamplified versus amplified (cut-off 2.0). The Pearson chi-square test (or Fisher's exact when appropriate) was used to compare baseline characteristics for categorical variables and frequencies of loss, gain, or amplification for individual genes and chromosomal arms between HPV-negative and HPV-positive OPSCC and between lymph node-positive and lymph node-negative OSCC. The analysis of variance test was used to compare continuous variables  between these groups in baseline characteristics. Backward logistic regression was performed to compare CNA between HPV-positive and HPV-negative OPSCC, taking into account differences in clinicopathological features between the two groups.P-value <0.05.Disease-free survival (DFS) was used for survival analysis. DFS was defined as survival after primary treatment without any signs or symptoms of recurrent or persistent disease. Both recurrence and death were recorded as events. Since over 95% of all HNSCC recurrences occur within 36 months after treatment and patients in our center are discharged from follow-up after a disease-free period of 60 months, analysis was cutoff at 60 months. Univariate analysis was demonstrated by Kaplan–Meier curves and statistical significance was determined using log rank tests. Multivariate analysis was performed using the Cox proportional hazard model. Clinicopathological characteristics both significantly related to survival as well as those acting as possible confounders  were included in the multivariate model. The level of significance was set at In 28 cases, the quality or quantity of DNA was insufficient for MLPA analysis which resulted in the availability of copy number data for 355/383 (92%) tumors  from our initial study population. Twenty-one percent (41 out of 191) of the OPSCCs were positive for high-risk HPV. All HPV-positive tumors contained HPV type 16, whereas two of 41 were co-infected with HPV 33 or HPV 52 as well. None of the OSCC were positive for high-risk HPV. Clinicopathological features of our study population are listed in TableP = 0.04) higher total number of CNAs compared to HPV-positive tumors. Gain of CCNL1 was independently associated with positive HPV status. Copy number gain of EGFR and both amplification and gain of genes located at 11q13  were significantly more frequent in HPV-negative tumors. After correction for baseline differences, multivariate analyses revealed that FADD, CTTN, CCND1, and FGF4 were independently associated with negative HPV status. No significant differences in frequencies of gene copy number losses were observed between HPV-positive and HPV-negative OPSCCs. Besides these observed differences, several genes showed frequent aberrations in both HPV-positive and HPV-negative OPSCC. The genes CCNL1, PIK3CA, TP63, MYC, MCCC1, and CDK6 showed a recurrent gain (>10% of cases) and RARB showed a recurrent loss (>10% of cases) in each group.Forty-one 21%) patients with an OPSCC showed an HPV-positive tumor. At baseline, patients with HPV-positive tumors had a significantly lower alcohol intake and smoked less than patients with HPV-negative tumors. Patients with HPV-negative tumors presented with larger tumors and were clinically less suspected to have lymph node metastases (LNM) . Differe% patientCCND1, FGF4, FADD, and CTTN) and loss (CSMD1) correlated significantly with LNMs. However, in the clinically relevant subgroup of clinically lymph node-negative OSCC , statistical significance of amplification in several of these biomarkers disappeared. In this clinically relevant subgroup, gain of chromosomal region 11q13 had the most diagnostic value for determining occult LNM (P = 0.002) with a NPV of 81% , see TableMCCC1 and MYC were commonly gained (>10%) in OSCC with and without LNMs.CNA of the 36 analyzed genes in lymph node-positive and negative OSCC are illustrated in Figure P = 0.00 with a NFADD and gain of wnt-induced secreted protein-1 (WISP1) correlated with a worse DFS. Multivariate analysis was performed to estimate the association of all analyzed factors with DFS. Gain of WISP1, age, advanced T stage (T3–T4), clinical nodal metastasis, and HPV-negative status were correlated independently with decreased DFS in OPSCC, see FigureFor survival analysis, only patients treated with curative intention were included. Hereto, 22 cases of OPSCC were excluded from survival analysis. DFS was defined as survival without recurrence of disease. The mean DFS oncologic follow-up of patients alive without recurrence was 40 months for OPSCC and 58 months for OSCC. In OPSCC, the baseline characteristics age, clinical nodal metastases N1–3), clinical advanced T classification (T3–T4), and HPV negativity were significantly correlated with a decreased DFS. From the 36-gene panel, amplification of , clinicaCCND1, FGF4, FADD, CTTN) correlated with a decreased DFS, with CCND1 gain acting as the strongest predictor . Multivariate survival analysis revealed a different effect of CCND1 gain on DFS pending on nodal status: in lymph node-positive tumors no correlation between gain and DFS was found, while lymph node-negative tumors with CCND1 gain had a significantly worse survival than lymph node-negative tumors without CCND1 gain, see FigureCCND1 gain was an independent predictor for worse DFS in this subgroup of OSCC, see TableIn OSCC, both gain and amplification of chromosomal region of 11q13 and its individual genes  and gain of EFGR occurred more frequently in the HPV-negative tumors. The relationship between CNA and HPV status in OPSCC has been shown in five previous studies FADD, CTTN, FGF4, and CCND1 are all located at chromosomal region 11q13. This region is the most frequently amplified region in HNSCC and is associated with unfavorable prognosis CCNL1, was significantly associated with HPV-presence, as stated in one previous study In our OPSCC cohort, 21% of the tumors were high-risk HPV-positive, which is in line with earlier reports of high-risk HPV prevalence in the Netherlands CCND1 amplification showed significant correlation  with LNM CCND1 amplification in Stage I–II OSCC with an NPV of 83%, which is similar to our results CCND1 amplification and LNM are the differences in the used detection method and cutoff values for amplification between these studies. Myo et al. used fluorescent in situ hybridization with ≥3 spots in >20% of 100 cells as cutoffs for amplification, which could be a less hard definition for amplification than a copy number change of >2 with MLPA in our study In early OSCC, appropriate management of the neck region is still topic of debate. Current strategies include elective neck dissection (END), sentinel node biopsy (SNB), irradiation, and watchful waiting. According to the decision tree analysis developed by Weis et al. in 1994, management that consists of observation only—as opposed to END—is an accepted treatment modality if the probability of occult LNM is less than 20% FADD amplification in OPSCC is not clear. In our study, 11q13 amplification showed to predict worse survival in univariate analysis. However, this predictive ability was confounded by the strong correlation between FADD amplification and HPV status; in a multivariate model FADD amplification did not appear to be an independent predictor. Furthermore, in a subgroup of HPV-negative OPSCC 11q13 amplification or gain showed no association with outcome, altogether suggesting that 11q13 copy number gain or amplification has no prognostic value in OPSCC. Only one other study similarly found 11q13 amplification in OPSCC to be associated with worse overall survival The exact prognostic value of WISP1 at 8q24.22 turned out to be a predictor for worse DFS in OPSCC, independent of HPV status. WISP1 is a member of the CCN family , which is a group of six secreted proteins that regulates adhesion and migration or functions as growth factors that modulate cell proliferation and differentiation WISP1 could also play an important role in the development of HNSCC and might predict a poorer prognosis. Moreover, because of the possible role of WISP1 in the development of radiation resistance, it is questionable to enroll HPV-positive tumors with WISP1 gain in de-escalating trials.Interestingly, gain of CCND1 as a predictor for worse DFS. The correlation between CCND1 gene aberrations and worse survival has been shown in other studies, though only Hanken et al. and Miyamoto et al. performed multivariate analyses CCND1 amplification being independent predictors for survival CCND1 amplification did not function as an independent predictor for survival in the study by Hanken et al. Survival analysis in OSCC revealed gain of CCND1 gain has no prognostic value in patients with proven histologic LNM in our cohort of OSCC, however it does correlate with worse overall survival in patients without LNM. The DFS of patients with CCND1 gain without LNM is comparable to patients with LNM, see FigureCCND1 gain without proven LNM, micrometastases could have been present which are known to be potentially missed by pathologists examining a neck dissection specimen CTTN, FADD, CCND1, and FGF4) in tumor growth and invasion  and staging of OSCC and OPSCC, no reliable comparison of CNA between these sites could be made. Although there seem to be differences between these sites, see FigureThis study was performed in a large consecutive cohort of OSCC and OPSCC patients. Nevertheless, some limitations require mentioning. First, although the OSCC data are derived from a prospective consecutive cohort, the OPSCC cohort has been gathered retrospectively and is nonconsecutive. Second, due to a limited registration of treatment response after radiotherapy DFS was used as a marker for treatment outcome in OPSCC. Therefore, it was not possible to correlate WISP1 gain correlates with decreased DFS in OPSCC independent of HPV status, potentially due to radiotherapy resistance. These findings could have implications for de-escalation trials in HPV-positive OPSCC. Finally, we showed that 11q13 gain is a promising biomarker for predicting occult LNM in patients with clinically Stage I–II OSCC. Consequently, CNA profiling increases our understanding of the specific biology of HNSCC and may prove of considerable value to personalizing future cancer therapy in these patients.In conclusion, we have identified CNA that are associated with HPV status in OPSCC and with prognosis in OSCC. Furthermore, we showed that


It is important to understand the specific HPV genotype distribution in screen-detected lesions. HPV Genotype is helpful for separating HPV-positive women at greater risk of cancer from those who can regress spontaneously and for preventing cervical cancer at early stage. The aim of this study was to investigate the high-risk HPV genotype distribution among cervical cytology abnormality in Pearl River Delta Region, Southern China5585 HPV-infected women were screened from 77069 women in Pearl River Delta Region. Information was obtained from 3226 screened subjects through questionnaires and personal interviews. Exfoliated cervical cells were collected by doctors for HPV test with MassARRAY  technique based on the matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS). The ThinPrep cytology test was performed to screen for cervical cancer. Unconditional logistic was used to determine the most common HPV carcinogenic types.Of the 3226 HPV-positive samples tested, 1744 (54.1%) with normal cervical cytology, 1482 (45.9%) with abnormal cytology. The five most common HPV types in this study were HPV16 (20.2%), HPV52 (17.1%), HPV58 (13.2%), HPV18 (9.5%), HPV6 (7.6%). Overall, HPV16 , HPV33 , HPV58 , HPV31 , multiple genotype infection , especially HPV16 and HPV33, increased the risk of cytology abnormalities.HPV16, HPV31, HPV33, HPV58, and multiple HPV genotype infection increased the risk of cytology abnormalities in Pearl River Delta Region and might be useful for the screening, preventing, treating, and monitoring of pre-cancer lesions in southern China. Cervical cancer is estimated to be the one of the leading causes of cancer among women worldwide; approximately 530000 new cases and 275000 resultant deaths are reported each year. About 85% of cervical cancer cases occur in developing countries. InfectiBesides HPV16 and HPV18, the next most common types in all world regions, other high-risk carcinogenic types, namely HPV31, HPV33, HPV45, HPV52, HPV58 and HPV35 have been reported with different distribution across world regions. Sub- Saharan Africa with equally HPV35 prevalence with HPV16, and the second most common types were HPV31 in Europe, HPV58 in South America, and HPV33 in Asia. Heterogeneity of HPV infection was significant across Asia, especially for HPV58.Notably, even high-risk HPV genotypes can contribute to different progression of cervical disease. A meta-There were about 58000 new cervical cases and approximately 20000 deaths in China. And the2 and with a population over 47 million. Notably, this area accounts for 10% of China’s gross domestic product (GDP) and over 80% of Guangdong’s GDP, which shows highly concentration of economy activity. Genotyping can be helpful for estimating the attribution of HPV genotype in this area[The Pearl River Delta Region (PRD) is located in mid-southern coastal part of Guangdong, Southern China, which with a total area over 56000 kmhis area. Thus, iThe study was conducted between May 2011 and November 2012 in Guangdong province, China. 5585 women were screened from 77069 women from nine cities, of these 2359 were excluded. Subjects were women between the ages of 18 and 68 years old (median age: 38.0 years) from nine cities in the Pearl River Delta region: Dongguan, Guangzhou, Huizhou, Jiangmen, Qingyuan, Shenzhen, Zhaoqing, Zhongshan, and Zhuhai . Total DNA from cervical cells was extracted using the commercial magnetic beads kit  according to the manufacturer’s instructions. Then 16 HPV genotypes were detected, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 ; and HPV6 and HPV11 (both LR HPV) with MassARRAY  technique based on the matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS). All theAnother cervical exfoliated cells sample was collected by cyto-brush  among women with HPV infection. ThinPrep 2000 (Hologic Inc.) and SurePath liquid-based Pap test  were used for the TCT test which was performed by Kingmed Center for Clinical Laboratory Corporation . Cytological slides were read by three cytopathologist. Cervical cell samples were categorized according to the Bethesda System (2001),19 as foχ2 test was used to compare the differences in HPV genotype representation for different cytological grades. Independent predictors for cervical lesions across different HPV genotype infection were evaluated via unconditional logistic regression with odds ratio (ORs) and 95% confidence intervals (CIs) (LR-HPV: HPV6 or HPV11 as reference). Data analysis was performed using SPSS13.0 . All statistical tests were two-sided; P values < 0.05 were considered statistically significant.The The study participants came from nine cities in Guangdong’s PRD, China, where is adjacent to Hong Kong, one of the most prosperous city in China Figure . Figure The most common HPV genotypes were HPV16 (20.2%), followed by HPV52 (17.1%), HPV58 13.2%), HPV18 (9.5%), HPV6 (7.6%), HPV45 (7.1%), HPV51 (6.7%), HPV66 (6.7%), and HPV39 (6.4%). Across all cytology grades, HPV16 was the most prevalent type than that of any other HR-HPV genotype. HPV6, HPV11, HPV45, and HPV66 were prone to be concentrated in normal cytology than HSIL. Similar percentages were detected for each HPV type between ASCUS and LSIL except for HPV51 and HPV56 in LSIL  Table . Women wIt is necessary to know the predominant HPV genotypes associated with cervical lesions with the aim of preventing cervical cancer. Our study presented the distribution of HPV genotypes and the association with cytological characteristics in a population consisted of 3226 women with HPV positive and an age range between 18 and 68 years in PDR, Southern China.Findings from our analysis show that HPV16 was the most predominant genotype in HPV positive women in PRD, which is similar with the most part of the world. Besides HPV16, HPV52 and HPV58 were also common in this study. These results are consistent with studies confined within part of the same geographical area,20. AlsoPrevious studies showed that HPV52 was one of the predominant genotypes in our study region and also in other parts of China,27,28, bThe distribution of HPV genotype and risk of cytology abnormalities in the present study are different from those in previous studies, which may be the variations in the study design, specimen types, screening methods. Our stuFurthermore, determining the causal attribution of carcinogenic HPV genotypes to cervical disease is important in the application of HPV genotypes included in future screening assays, and in the management and developing prophylactic vaccines. HPV-based primary screening is not only more sensitive for earlier detection of clinically high-grade cervical lesions than cytology, but alsThe design of our study should be considered when interpreting our results. Our data are from a cross-sectional study which was designed to identify detriments of early-stage cervical disease, so all of the women were HPV-infected. Indeed, detection of cytology abnormalities was more common among the participants with HPV infection in our study compared to these samples randomly obtained from women in PRD. We do not believe, however, that study design solely accounts for the findings observed in our study. Although future study is needed to confirm the findings from this study, our data provide valuable initial insight into the distribution of HPV among PRD women and suggest that some specific HR-HPV may help explain the existing cervical cancer disparities in PRD, furthermore, our results not only give support to the use of HPV testing, especially including HPV16, HPV58, HPV31, and HPV33 testing for women aged 30 years and older but also provided valuable fundamental data to the future study with the aim of reducing the burden of cervical cancer, last but not least, these results give us a hint that may there is great potential use of these HPV genotypes in improving risk stratification of women with HPV infection in cervical screening programmes, and in improving performance and cost-effectiveness of programs while reducing over treatment and patient anxiety.There are some limitations in our study. Since we analyzed a population based on HPV-positive results from a large cross-sectional study in Guangdong, women without HPV infection that cause cytological abnormalities are underrepresented in our population. Furthermore, the accuracy of the information about their sexual and reproductive history might impact the authenticity of this survey.In summary, the results of this study provide clear indication of the HPV infections among cytology abnormal women in the Pearl River Delta region of Guangdong Province of China, and only a few high-risk HPV genotypes might be more useful for the screening and monitoring of pre-cancer lesions.HPV16, HPV31, HPV33, HPV58, and multiple HPV genotype infection increased the risk of cytology abnormalities in Pearl River Delta Region and might be useful for the screening, preventing, treating, and monitoring of pre-cancer lesions in southern China.All authors declare that they have no competing interests.LPJ and XMZ wrote the manuscript. XCW, GY and CXJ conceived the study, assisted in drafting the article for important intellectual content. WHH, YL and MW performed the surveys and analyzed the data collected. ZLM, XPZ and JZ performed the HPV test and analyzed the results. BWZ, CDC and XML performed the cytology study and interpreted the results. All authors participated to the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/388/prepuba.Distribution of HPV genotypes among different cytology grades stratified by ageClick here for file


Cervical cancer is the most common female malignancy in the developing nations and the third most common cancer in women globally. An effective, inexpensive and self-applied topical treatment would be an ideal solution for treatment of screen-detected, pre-invasive cervical disease in low resource settings.Between 01/03/2013 and 01/08/2013, women attending Kenyatta National Hospital's Family Planning and Gynaecology Outpatients clinics were tested for HIV, HPV (Cervista®) and liquid based cervical cytology (LBC -ThinPrep®). HIV negative women diagnosed as high-risk HPV positive with high grade squamous intraepithelial lesions (HSIL) were examined by colposcopy and given a 2 week course of 1 capsule of Lopimune (CIPLA) twice daily, to be self-applied as a vaginal pessary. Colposcopy, HPV testing and LBC were repeated at 4 and 12 weeks post-start of treatment with a final punch biopsy at 3 months for histology. Primary outcome measures were acceptability of treatment with efficacy as a secondary consideration.A total of 23 women with HSIL were treated with Lopimune during which time no adverse reactions were reported. A maximum concentration of 10 ng/ml of lopinavir was detected in patient plasma 1 week after starting treatment. HPV was no longer detected in 12/23 . Post-treatment cytology at 12 weeks on women with HSIL, showed 14/22  had no dysplasia and 4/22  were now low grade demonstrating a combined positive response in 81.8% of women of which 77.8% was confirmed by histology. These data are supported by colposcopic images, which show regression of cervical lesions.These results demonstrate the potential of Lopimune as a self-applied therapy for HPV infection and related cervical lesions. Since there were no serious adverse events or detectable post-treatment morbidity, this study indicates that further trials are clearly justified to define optimal regimes and the overall benefit of this therapy.48776874ISRCTN Registry  Infection with high-risk types of HPV has now been established as the main aetiological agent for invasive cervical cancer (ICC),2,3 and The development of ICC can take 10–20 years and is preceded by HPV related pre-invasive pathology which are characterised as either low-grade (CIN1) or high-grade cervical intraepithelial neoplasia (CIN2/3).The reduction in ICC related mortality in the developed world has been largely dependent on organised cytology screening and similar trends in cervical cancer mortality have been achieved by organised single screen and treatment in low resource settings . HoweverCurrent treatment options in clinical practice are either by ablative (destructive) or excisional modalities which have similar success rates but have different morbidities . In the There are a variety of locally-applied, non-surgical approaches which have been evaluated for the treatment of cervical dysplasia  may have activity against HPV related cervical disease and identified lopinavir as the most effective  althoughBased on our pre-clinical studies it was concluded that direct application of lopinavir to the cervix as a vaginal pessary or gel, might achieve the concentration required for activity against HPV related cervical dysplasia. Since we already had research collaborations in place with clinicians at Kenyatta National Hospital/University of Nairobi in Kenya, we built on these to conduct a trial of self-applied lopinavir (in the form of Lopimune) in this setting with the aim of developing an effective, inexpensive, non-surgical, self-applied treatment with minimal side effects. We now report the results of a small, single-arm study to evaluate the safety, acceptability and ability of Lopimune to induce clearance of HPV infection, and related dysplasia, in a group of 23 women diagnosed with HSIL.This was a collaborative proof-of-concept clinical trial between the University of Manchester and the University of Nairobi/Kenyatta National Hospital (KNH), Kenya. Clinical and basic diagnostic tests were done at KNH while scientific aspects of the study that required specialized equipment were carried out at the Viral Oncology Laboratory, St Marys Hospital in Manchester, UK. KNH and the University of Nairobi Ethics and Research Committee (KNH/UoN-ERC) approved the trial on the 25/11/2011, an extension was granted on the 07/01/2013 and the trial was conducted between 01/03/2013 and 01/11/2013. Patients were recruited from women attending the Family Planning and Gynaecology Outpatient clinic at KNH for routine follow up. Registration was carried out retrospectively (ISRCTN48776874) since the original intention was to recruit a small number of patients to investigate acceptability of treatment.TM  and if positive was confirmed by UnigoldTM  as per Kenya HIV testing algorithm. All patients were then given a speculum examination during which two cervical cyto-brush samples were taken. The first of these was used for LBC ThinPrep® and HPV testing and samples were immediately sent to Manchester. The second was used for a conventional smear test as per standard Pap smear screening procedures, which was examined and reported in Nairobi by the Kenyan study pathologist. Patients were then reviewed after one month at which time those that were HIV negative and HPV positive with abnormal cervical cytology, were enrolled into the Lopimune trial. It was decided this should be pragmatic in nature and biopsies were not taken at this point since it is well known this can affect treatment outcomes [The study protocol is illustrated in outcomes . FurtherAt enrolment a pelvic examination was carried out which included baseline colposcopy. Cervical morphology was initially visualised with acetic acid (VIA) followed by Lugol’s Iodine (VILI) using x5 magnification . This was carried out according to standard clinical practices and images recorded . Blood wAll ThinPrep® LBC samples sent to Manchester were analysed by staff trained and certified by Hologic . HPV testing of LBC samples was carried out using the FDA-approved Cervista HPV HR test  in conjuUltrapure DNA isolated from each ThinPrep® LBC sample which was used for the Cervista® test, was then further analysed by PCR to test for which HPV genotypes were present in each respective Cervista® mastermix positive sample. A novel hot-start, touch-down multiplex method was used which simultaneously detects the L1, E6 and E7 ORFs of HPV type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70 as previously described . Each asLBC slide preparation was carried out using the ThinPrep-2000 system (Hologic) automated slide preparation unit, according to the manufactures directions and Pap stained (Hologic) at the Regional Cytology Laboratories . Reporting of the LBC samples was done using a Dual Review Imaging System (Hologic).Final punch biopsy samples of the cervical transformation zone and VILI positive lesions were transported to Manchester, wax embedded and 3–4μm sections cut at 3 levels according to standard pathology laboratory practices. The sections were then stained with haematoxylin and eosin and reported separately by two pathologists Independent review of cases where there was disagreement was carried out by a third pathologist (Dr Bianca Da Gama Rose).Lopinavir was extracted from plasma using a method based on that of Djerada et al . MSMS anAgreements between the conventional and ThinPrep cytology and the two study histopathologists were analysed by the McNemar test using R program version 3.1.2. All 95% confidence intervals were computed using the R binom package and a p value < 0.05 was considered statistically significant.All baseline bloods and health checks were normal for the study participants.The original intention was to block randomise Lopimune treatment starting at the higher dose. However, due to the nature of recruitment being few patients spaced by long time intervals, it was noted that the twice-daily dose produced excellent cervical lesion clearance with no significant adverse effects. This prompted the decision to adopt an adaptive design and abandon the planned lower dose arm since no patient had been put on the lower dose at that time. The study, therefore, became a single-arm non-randomized trial.st month of taking Lopimune as described.In the 4 weeks post start-of-treatment there were no serious adverse reactions or drug events such as death, life-threatening events, hospitalization or disability observed. Additionally, none of the study subjects reported, vaginal dryness, bleeding/spotting, painful intercourse, palpitations, unusual weakness, vomiting, decreased appetite, diarrhoea, pale stools, itchy skin, skin rashes, increased thirst, increased frequency of urination, visual disturbance, chest pains, or jaundice. However, there were some minor side-effects  but onlyInformation on drug adherence was self-reporting and 10 (43.5%) of patients completed their treatment on schedule without skipping a dose. Out of the remaining 13, 7 (53.8%) missed once, 2 (15.4%) missed twice, 1 (7.6%) missed three times, 2 (15.4%) missed for times and one subject missed 5 consecutive doses. The commonest reason for skipping doses was interruption by menstrual flow (6/46.2%), forgot (5/38.5%) and the inconvenience of carrying refrigerated Lopimune whilst away from home (2/15.4%). Significantly, none of the subjects skipped due to side-effects, problems inserting pessaries or because their partner wanted intercourse. Indeed, despite being advised to abstain during the treatment period, 5 (21.7%) of patients still reported as having had intercourse at least once. Neither omitted doses nor engaging in sexual intercourse had any statistically significant effect on the observed treatment outcomes.The results of repeat ThinPrep® LBC and Cervista® HPV tests carried out at 4 and 12 weeks post-start of treatment are summarised in A total of 22 patients were reported at 4 and 12 weeks since E19 had inadequate cellularity for HPV testing and cytology at 4 weeks whereas E15 was adequate for HPV testing but not cytology at 12 weeks. After 4 weeks  14/22 6 14/22  had no detectable dysplasia and 4/22  had CIN1, which equates to a 77.3% positive response to treatment. Only 4/22  had CIN2 or CIN3 and 1/22  had hCGIN. A total of 22 patients were reported for pathology since the biopsies obtained from E19 at 12 weeks were inadequate and thus excluded from the analysis. McNemar test demonstrated a p-value of 0.025 which indicated that there was a statistically significant difference in reporting by the two study pathologists which was resolved by referral to a third independent pathologist.Extracted ion chromatograms of research grade lopinavir showed this to have a mass/charge ratio of 629.37 m/z eluting at 5.1 min. Extraction of compound from control plasma was shown to be >90% efficient when compared to the same amount of drug added directly to solvent prior to analysis and could detect lopinavir to a level of ~6.00 ng/ml when added to plasma. Out of 23 samples tested, the maximum level of drug detected in patient plasma, after one week of vaginal delivery, was approximately 10 ng/ml although most samples were lower than this.When used for oral HIV therapy, lopinavir is normally co-adminstered in a 4:1 ratio with ritonavir . Due to cost and supply issues, reformulation of lopinavir as a single agent was not possible so evaluation of the orally-administered Lopimune gelatine capsule formulation was undertaken with the axiom this might serve as a vaginal pessary for local delivery to the cervix. Clearly the local concentration of drug achieved by this mode of delivery will be much higher than the 74–215 ng/ml previously reported in cervico-vaginal fluid as a result of normal oral dosing for HIV therapy . AnalysiThe primary outcome was to assess tolerability and any adverse events associated with local application of this treatment to the cervix. Out of 23 women treated, no significant serious adverse indications were reported. Compliance was good and Lopimune was well tolerated with few side-effects and only nausea achieving statistical significance. Acceptability was also good, given that none of the subjects omitted a dose because of side-effects or because their partner wanted sexual intercourse. Moreover, 5 patients had intercourse during the treatment period and yet this did not appear to affect treatment outcome which suggests this may not be need to restricted. However, further study is clearly required to investigate this possibility.A potential criticism of this study was the reliance on cytology for the pre-treatment and 4 week follow up diagnosis of underlying cervical dysplasia since it is well known that there is considerable variation (30–80%) in the accuracy of cytology between different centres throughout the world . The ManAnother potential criticism is the lack of a placebo control arm since CIN2/3 lesions can spontaneously regress if left untreated although there is a paucity of data regarding natural regression rates over a 3 month period with the majority of published work analysing this over 6 to 12 months or longer. However, a study, conducted by Alvarez et al, did report over 3 months and found that ~30% of CIN2/3 lesions regressed in this period  which waComparison of histopathology with cytology at 12 weeks post-treatment was mostly consistent although there were some differences. However, pathology still showed that ~60% of women had no detectable neoplasia at 12 weeks and others had reduced severity of disease (CIN1) indicating a combined positive response in ~77% of women. Most significantly these rates of regression are generally higher than would be predicted for untreated cases of high-risk HPV positive CIN2/3 .Regarding the different HPV genotypes present, there was no significant correlation with response to treatment for any of those identified nor was there any obvious correlation with other socio-demographic factors such as age, type of contraception used, number of lifetime sexual partners or parity. Notably, none of the women in this study smoked.How does the current treatment with Lopimune compare to other non-surgical treatments for cervical dysplasia? PDT has been extensively evaluated for this purpose with a large range of response rates ranging from 0–100% for CIN and 53·4–80% eradication of HPV . DisadvaThe current study illustrates the potential of Lopimune to serve as an effective alternative to surgery for the treatment of HPV related cervical dysplasia although it is clear that the dose and regimen used in our study were sub-optimal and need further refinement. Evidence for this was that some women showed a transient improvement in HPV and/or cytology at 4 weeks but then went on to develop HPV positive abnormal cytology/pathology at 12 weeks Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOC)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.S5 Text(PDF)Click here for additional data file.


Apocrine acrosyringeal keratosis is a rare skin lesion showing a unique benign keratotic lesion associated with syringocystoadenoma papilliferum. It is characterized by an exophytic proliferation of the epidermis with two distinct keratinocytic structures: a) columns of hyperkeratotic mass surrounded by acanthotic epidermis and b) papillated and/or cystic invaginations typical of syringocystoadenoma papilliferum. No causative agents were reported.The present report describes a typical case of apocrine acrosyringeal keratosis localized in the right retro-auricular area of 57-year-old man in which the presence of HPV was evaluated. PCR analysis and direct sequencing revealed the presence of HPV 89. The presence of this low risk mucosal HPV in a skin localization was never reported as well as in association with this rare tumor. Furthermore rolling circle amplification, RT-PCR and in situ hybridization confirmed the presence of a transcriptionally active HPV 89.Taken together our results suggest that HPV89 plays a role in apocrine acrosyringeal keratosis with syringocystoadenoma papilliferum, in consideration of the documented biological activity of the virus. The association of low risk mucosal HPV infection with this skin lesion opens new perspectives in its clinical management. Further studies on samples from other patients are needed to confirm this association. Alphapapillomavirus , mainly isolated from genital lesions; Betapapillomavirus (beta-HPV) and Gammapapillomavirus (gamma-HPV), the latter two genera predominantly isolated from skin [Human papillomaviruses (HPVs) can be considered etiological agents of invasive cervical, anogenital, and oropharyngeal, in particular tonsil, cancers , 2. Currrom skin .The tissue tropism of a group of viruses is an important characteristic to understand how viruses evolve in ecological niches and induce pathogenic consequences in their hosts. In line with this assumption persistent infections by mucosal high-risk oncogenic alpha HPV types such as HPV type 16 are associated with some oral and/or oropharyngeal cancers , 4. HoweOn the contrary, mucosal types have been detected in skin lesions and in particular HPV 16 was consistently detected in periungueal carcinoma with Bowenoid features .This study was approved by the local Ethical Committee (Prot.n. CE/312/05). A 57-year-old man was showing a lesion in the right retro-auricular area that was diagnosed as apocrine acrosyringeal keratosis. Anything relevant to this pathology was in the clinical history of the patient and in particular no evidence of immunosuppression. The lesion was exophytic and revealed two different components: a well-developed syringocystoadenoma papilliferum (SCAP) with free floating papillae and a more verrucous proliferative area resembling a wart Fig. . This waSensitive PCR with degenerate primers followed by direct sequencing was utilized to detect a broad spectrum of HPV types , 8. RollPCR analysis demonstrated the presence of an amplified band that upon direct sequencing revealed a nucleotide sequence with 98 % homology to that of HPV 89 Fig. . RCA shoHPV mRNA expression was detected by reverse transcriptase–PCR Fig. . Total RSince no cloned HPV89 was available, viral load was measured in real time-PCR respect to two different standard curves: SiHa cells with one integrated copy of HPV16 and a plasmid carrying HPV16 full genome . Briefly, real time PCR was performed with Kapa SYBR Green Master Mix  in i-Cycler apparatus  according to manufacturer's instructions. Amplifications of L1 and β-globin gene were performed in a 20 μl volume containing 2× Kapa SYBR Fast qPCR Master Mix, 10 μM of each primer and 20 ng of genomic DNA, using the following protocol for 40 cycles: 3 s denaturation at 95 °C, annealing and extension at 60 °C for 30 s, with the initial denaturation at 95 °C for 3 min. Viral load was normalized by β-globin gene and calculated as 0.73 copy/genome equivalent. Finally, ISH revealed a HPV positive nuclear signal confirming data of RCA about the presence of episomal HPV 89 Fig. .In the present report, we bring evidence that transcriptionally active low risk HPV 89 was present in apocrine acrosyringeal keratosis, a unique benign keratotic lesion associated with syringocystoadenoma papilliferum described on 2000 by Khishimoto et al. .It is characterized by an exophytic proliferation of the epidermis with two distinct keratinocytic structures: a) columns of hyperkeratotic mass with swollen corneocytes containing keratohyalin granules surrounded by acanthotic epidermis and b) papillated and/or cystic invaginations, typical of SCAP. This tumor is rarely observed and, at the best of our knowledge, only one case is reported in the English literature. Khishimoto et al. made the hypothesis of HPV involvement but failed to detect any HPV by immunohistochemistry . It is tOn the contrary, Carlson and co-workers reported the presence of alpha and beta HPV DNA in cases of syringocystadenoma papilliferum associated with nevus sebaceus, a complex hamartoma of the skin with epithelial and dermal components, often associated with secondary neoplasms as trichoblastoma, basal cell carcinoma and syringocystadenoma papilliferum .The presence of this low risk mucosal HPV in a skin localization was never reported as well as in association with this rare tumor. HPV 89 was recently associated with a long duration of viral persistence in limited case studies suggesting some peculiar characteristic of this HPV . Taken tIn alternative, the virus presence, even if transcriptionally active, may represent only a reactivation of latent infection during the lesion development.Nevertheless, the ascertained presence of mucosal papillomavirus in skin locations reinforces the hypothesis that a single type of HPV may have a different tropism in comparison with other members of the same species suggesting that genotyping classification may not always reflect the biological behavior of the HPVs. The role of symbiotic HPV infections, their relationship with the host  and the emergence of their pathogenic potential remain to be better understood.In conclusion, apocrine acrosyringeal keratosis with syringocystoadenoma papilliferum can be associated with HPV infection. Although conclusive data cannot extrapolated from a single case, nevertheless the possibility of an association of this rare skin pathology with an Alfa papillomavirus exists in consideration of the documented activity of the virus. Further studies on samples from other patients are needed to confirm this association.


Diadema are known to occur around the Japanese Archipelago. Three species  are widely distributed in the Indo-Pacific Ocean. The fourth species was detected by DNA analysis among samples originally collected as D. savignyi or D. setosum in Japan and the Marshall Islands and tentatively designated as Diadema -sp, remaining an undescribed species. We analyzed nucleotide sequences of the cytochrome oxidase I (COI) gene in the “D. savignyi-like” samples, and found all 17 individuals collected in the mainland of Japan (Sagami Bay and Kyushu) to be Diadema-sp, but all nine in the Ryukyu Archipelago (Okinawa and Ishigaki Islands) to be D. savignyi, with large nucleotide sequence difference between them (11.0%±1.7 SE). Diadema-sp and D. savignyi shared Y-shaped blue lines of iridophores along the interambulacrals, but individuals of Diadema-sp typically exhibited a conspicuous white streak at the fork of the Y-shaped blue iridophore lines, while this feature was absent in D. savignyi. Also, the central axis of the Y-shaped blue lines of iridophores was approximately twice as long as the V-component in D. savignyi whereas it was of similar length in Diadema-sp. Two parallel lines were observed to constitute the central axis of the Y-shaped blue lines in both species, but these were considerably narrower in Diadema-sp. Despite marked morphological and genetic differences, it appears that Diadema-sp has been mis-identified as D. savignyi for more than half a century.Four long-spined sea urchin species in the genus  Diadema Gray, 1825 are widespread species found in tropical and sub-tropical regions Diadema species are reported from the southern part of the Japanese Archipelago D. savignyi  and D. setosum  are the most abundant and well known, both having the widest geographic range in the Indo-Pacific Ocean of any species in the genus. The Hawaiian Islands and South Pacific D. paucispinum A. Agassiz, 1864, and the species is suspected to inhabit other parts of the tropical Indo-Pacific Diadema was found among samples collected from Japanese and Marshall Island samples initially identified as D. savignyi or D. setosum and tentatively designated as Diadema-sp Diadema-sp has received almost no attention from researchers Long-spined sea urchins in the genus Diadema species from the Arasaki area of Sagami Bay, Japan, in order to investigate species abundance and the annual reproductive cycle, in which two types of Diadema morphologically corresponding to D. savignyi and D. setosum were observed. However, preliminary nucleotide sequence analysis of mtDNA cytochrome oxidase I gene (COI) from these samples indicated that our “D. savignyi” collected in Sagami Bay was the previously reported Diadema-sp Diadema-sp, among samples originally identified as D. savignyi collected in waters of mainland and the southern islands of Japan.We have undertaken monthly sampling of Diadema sampled in the present study were not endangered in Japan and verbal permits for collecting Diadema species were obtained from the above organizations.All field surveys were approved by Nagai-machi Fishermen's Cooperative Association , Gonoura-cho Fishermen's Cooperative Association , Yaeyama Fishermen's Cooperative Association , and Sesoko Station of the University of the Ryukyus . The species of the genus D. savignyi-like” and D. setosum were sampled from four locations between December 2011 and May 2013  with a conspicuous orange (or red) ring, and five conspicuous white spots on the median naked interambulacral areas. In comparison, “D. savignyi-like” individuals did not typically project a periproctal cone, and when a cone was observed, a prominent orange ring was not present. In addition, “D. savignyi-like” individuals exhibited five blue lines of iridophores along the interambulacrals, and a pentamerous ring around the periproctal region. All individuals were photographed underwater in situ or alive in aquaria following collection and transfer to the laboratory.Individuals of “May 2013 , Table 1Tube feet or muscle tissue surrounding the Aristotle's lantern was collected from each individual for genomic DNA extraction. The remaining body was immersed in bleach, primarily consisting of sodium hypochlorite to remove spines and other soft tissues to examine test morphology. Primer sequences used for PCR and nucleotide sequencing were as follows: COI120F (5′TTCTTCATGGTAATGCCAAT 3′) and COI1300R (5′ATTCCGGCTARACCTAAGAA3′). PCR was performed in a 20 µl reaction mixture containing 2 µl 10x buffer, 1 mM each dNTPs, 0.4 µM each primer, 0.8 units EX Taq polymerase , and DNA template. The reaction mixtures were preheated at 94°C for 4 min, followed by 30 amplification cycles , with a final extension at 72°C for 7 min. PCR products were treated with ExoSAP-IT (Amersham Biosciences) to remove primers. Sequences were generated on an automated sequencer  using the ABI Big-dye Ready Reaction kit (Applied Biosystems) following standard cycle sequencing protocol.D. setosum-a (AY012747 and AY012746), D. setosum-b (AY012732 and AY012733), D. savignyi (AY012742 and AY012743), and Diadema-sp (AY012744 and AY012745) sequences previously reported Sequence chromatograms were manually checked using ChromasPro v1.42 (Technelysium Pty Ltd), and aligned by MEGA v6 Underwater photographic images of live individuals were compared. Diameter and height of naked tests were measured, and primary tubercle arrangements on the interambulacral coronal plates were recorded.D. savignyi-like” and D. setosum were observed, each of which was further divided into two sub-clades  collected in Sagami Bay, and nine (DSV9-17) in Iki Island . All nine “D. savignyi-like” individuals (DSV18-26) collected in the Ryukyu Archipelago . On the other hand, average sequence divergence between Diadema-sp and D. savignyi was much larger (11.0%±1.7 SE). All 10 individuals of our D. setosum (DST1-10) were clustered in the D. setosum-a sub-clade, and nucleotide sequence divergence among individuals was small (0.5%±0.2 SE), while the divergence between D. setosum-a and D. setosum-b was 7.3%±1.3 SE. The largest divergence (13.9–18.7%) was observed among individuals representing D. setosum and “D. savignyi-like” clades.Of the 758 to 984 nucleotide sequences resolved in 36 individuals, a 526 bp region was shared with previously reported sequence data b-clades . All clai Island  were cluhipelago  were cluD. setosum, D. savignyi, Diadema-sp). Compared to D. setosum, D. savignyi and Diadema-sp lacked conspicuous white spots on the interambulacral area, and the periproctal cone projection was also usually absent. Even when the periproctal cone was observed, it was dark in color and lacked an orange ring  from Ishigaki Island were immersed in 50% bleach for several hours with occasional gentle shaking. Naked test proportion (height/diameter) results were as follows: D. savignyi (n = 7), 0.482±0.032 SD; Diadema-sp (n = 29), 0.479±0.027 SD; and D. setosum (n = 20), 0.508±0.035 SD. A slight but significant difference was observed between D. setosum and Diadema-sp in the naked test proportion (height/diameter) among the three species examined , but the proportion appeared not to be diagnostic. The apical system did not always remain in the original position, even after short bleach treatments, therefore the following sample sizes were measured for primary tubercle arrangements in naked tests; four of seven D. savignyi collected at Ishigaki Island, 19 of 29 Diadema-sp, and 17 of 20 D. setosum additionally collected in the Arasaki area. From these representative images of primary tubercles on the interambulacral area for each species were prepared  coronal plate, and the inner series on the 6th (rarely 5th) plate  coronal plate, and inner series on the 6th plate  coronal plates, and inner series on the 7th or 8th (rarely 6th)  plate . In D. sth plate . In D. sely 6th) . TherefoDiadema-sp. A previous phylogeographic study that examined population structure and speciation in Diadema species throughout global tropical regions indicated support for a new Diadema species Diadema species (D. savignyi and D. setosum) had been reported in Japanese waters D. savignyi and included the absence of the orange ring on the periproctal cone and blue lines of iridophores on the apical system and interambulacral midline Diadema-sp, however, cannot be distinguished from D. savignyi based on these morphological traits. In a survey of sea urchins from Sagami Bay, Shigei D. savignyi”, which resembled Diadema-sp based on prominent white streaks and a short central axis of the Y-shaped blue lines of iridophores. Diadema species observed in the Oki Islands, Sea of Japan, were reported as D. savignyiDiadema-sp. “D. savignyi” photographs provided in several Japanese guide books Diadema-sp. Furthermore, allozyme electrophoresis patterns were compared between D. savignyi and D. setosum collected in mainland of Japan, and allelic differences were observed between the two species at G6P and AAT loci D. savignyi and D. setosum shared the same alleles at these loci D. savignyi” collected from the mainland of Japan and used for allozyme analyses Diadema-sp. Our mtDNA COI results revealed that the “D. savignyi-like” specimens collected in Sagami Bay and Iki Island were Diadema-sp, but those collected in the Ryukyu Archipelago were D. savignyi. A previous study indicated an greater number of Diadema-sp versus D. savignyi occurred along the coast of mainland of Japan, while Diadema-sp were not observed in the Okinawa Island area Diadema-sp appears to be within mainland waters of Japan, and the taxon has been mis-identified as D. savignyi for more than half a century. D. savignyi appears to be much less abundant in mainland Japanese waters and the distribution appears to be restricted in the southern islands. Diadema-sp has only been reported from mainland Japan and Marshall Island region to date D. savignyi” taken in Australia .html and http://bie.ala.org.au/species/urn:lsid:biodiversity.org.au:afd.taxon:386810d3-25a5-4952-be0a-f396fe5cab50) and Singapore (http://www.flickr.com/photos/wildsingapore/2440065733/) represent characteristics of Diadema-sp, suggesting that the species may have much wider distribution and has been mis-identified as D. savignyi in many occasions.The present study is the first to report morphological attributes characterizing the as yet undescribed Diadema individuals from Sagami Bay, and suggested that the populations might represent an undescribed species. Following several mainland Japan collections, Ikeda Diadema clarki after Clark D. clarki was supposed to be a synonym of D. setosumDiadema-sp might be one of Clark's undescribed Diadema species D. clarki by Ikeda Diadema specimen exhibited only eight or nine coronal plates in each column Diadema species examined in the present study. On the other hand, qualitative affinities were observed between Diadema-sp and D. clarki. Ikeda D. clarki; the inner series of interambulacral primary tubercles began on the 5th or 6th coronal plates, and conspicuous white streaks were present in the median naked interambulacral area instead of white spots observed in D. setosum. Although Ikeda D. clarki showed that the outer series of the interambulacral primary tubercles begun on the 2nd or 3rd coronal plates. However, Ikeda D. clarki to be orange as in D. setosum, suggesting that Diadema-sp is unlikely to be D. clarki. Consequently, the relationship between Diadema-sp and D. clarki is equivocal, so that further investigation on the distribution and intraspecific morphological variation in Diadema-sp is required prior to formal species description.In the early 20th century, Clark


There are variations in the proportions of head and neck cancers caused by the human papillomavirus (HPV) between countries and regions. It is unclear if these are true variations or due to different study designs and assays.We tested formalin‐fixed paraffin‐embedded diagnostic biopsies for p16 immunohistochemistry and HPV‐DNA (by polymerase chain reaction [PCR] and in situ hybridization [ISH]) using validated protocols on samples from 801 patients with head and neck cancer recruited prospectively between 2006 and 2011 in 4 randomized controlled trials (RCTs).p < .0001) and between Western Europe and Asia . Other independent determinants of HPV positivity were tumor site and smoking.Twenty‐one percent of patients (170 of 801) showed both HPV‐DNA and p16‐positivity, detected almost exclusively in oropharyngeal cancer ; and only 1% of the patients (5 of 499) with nonoropharyngeal cancer were HPV positive. HPV‐positive oropharyngeal cancer differed between Western and Eastern Europe (37%, 155 of 422 vs 6%, 8 of 144; Head Neck38: E1863–E1869, 2016This is the first study to establish geographic variability as an independent risk factor in HPV‐positive oropharyngeal cancer prevalence, with higher prevalence in Western Europe. © 2016 The Authors Head & Neck Published by Wiley Periodicals, Inc.  The proportion of head and neck cancers caused by the human papillomavirus (HPV) seems to have increased considerably over the past decade in several countries.It has become important to ascertain the regional differences in HPV‐positive head and neck cancer to understand the current trends and relative burden of HPV‐positive and HPV‐negative head and neck cancer. This will enable local clinicians and health commissioners to plan for the provision of health care, rehabilitative, and social support services for patients who are HPV‐positive who enjoy higher survival rates but have to deal with the considerable sequelae of their intensive treatment.The variations in reported regional prevalence rates may be a reflection of regional differences in the profiles of established risk factors, such as primary site, smoking, or sexual habits,In this study, our purposes were to overcome these potential deficiencies, to provide a recent and more accurate representation of the proportion of HPV‐positive head and neck cancer in different geographic regions, and to assess the role of region as a risk factor for HPV‐positive head and neck cancer. We compared the proportion of patients with HPV‐positive head and neck cancers and analyzed possible risk factors in a multinational cohort of patients with head and neck cancer who were all recruited prospectively in randomized controlled trials (RCTs), within a recent time frame. All samples were assessed for high‐risk HPV‐DNA using the same validated protocols in 2 quality‐assured laboratories.We examined formalin‐fixed paraffin‐embedded diagnostic biopsies from a total of 1049 patients with head and neck cancer who gave informed consent and were recruited to 4 multicenter RCTs: PET NECK trial (187 subjects), GSK EGF104334 (107 subjects), GSK EGF105884 (67 subjects), and GSK EGF102988 (688 subjects). Most subjects included in our study were recruited between 2007 and 2010.Ethical approval for PET NECK trial (reference: 07/Q1604/35) was granted by the Oxfordshire Research Ethics Committee, May 2007. GSK EGF Ethical approval (reference: 06/MRE12/82) was granted by the Thames Valley Research Ethics Committee, March 2007.The procedures followed were in accordance with the ethical standards of the committees on human experimentation and in accord with the Helsinki Declaration of 1975, as revised in 1983.Analysis was undertaken on tissue sections (4 μm) or on tissue microarrays (TMAs) with up to four 0.6‐mm diameter cores from each case. We took 10‐μm thick tissue curls from available blocks for DNA extraction and downstream polymerase chain reaction (PCR) analysis. All samples were analyzed for p16 expression by immunohistochemistry using a proprietary kit  on a Benchmark Autostainer . Two pathologists (H.W. and M.R.) scored the test results independently, after undergoing calibration. Samples were assessed for high‐risk HPV‐DNA using a protocol based on that described and validated by Smeets et al.For all samples in this study, cases that showed evidence of HPV‐DNA (either by ISH or consensus PCR) and showed high p16 expression (HPV‐positive/p16‐positive) were defined as “harboring biologically relevant oncogenic HPV infection.”All subjects who were enrolled into the studies but who had not yet provided tumor tissue for biomarker testing were included in a summary of baseline and disease characteristics, but excluded from all other analyses. Subjects who had provided tumor tissue that was inadequate for testing were not included in the patient characteristics summaries. The latter are based on the subset of subjects who had valid results for both HPV and p16.Subjects with at least one positive result for HPV and one positive result for p16 for any of the cores and sections were considered to be positive overall for that biomarker. If the patient had no positive cores or sections, then they were considered negative, otherwise their result was treated as unknown, and they were not included in the analysis. Subjects who tested negative for p16 and who had an inconclusive result for HPV were considered as HPV‐negative overall.The analysis includes only subjects with valid results for both biomarkers (HPV‐DNA and p16). The demographic and baseline disease characteristics, including age, sex, primary tumor type, disease stage, smoking status, and geographic region, were summarized for subjects who had valid results for biomarkers, as well as those who did not provide tumor tissue for testing or who had insufficient tissue. The frequency and proportion of subjects testing HPV‐positive, p16‐positive, and those with combined positivity (HPV‐DNA‐positive and p16‐positive) were also reported by these characteristics. Where possible, odds ratios for combined positivity (HPV‐DNA‐positive and p16‐positive) were calculated for each characteristic, with suitable dichotomization where necessary. A logistic regression analysis was used to model the probability of testing positive for both HPV‐DNA and p16, adjusting for the risk factors listed above. Combined test results were grouped into 4 categories: (1) HPV‐positive/p16‐positive; (2) HPV‐positive/p16‐negative; (3) HPV‐negative/p16‐positive; and (4) p16‐negative/HPV‐negative, and summarized for patients with oropharyngeal cancer and nonoropharyngeal cancer tumors, as well as by geographic region.A total of 1049 patients submitted samples, constituting 81% of total subjects recruited in all 4 studies at the time of the analysis. Eight hundred one patients had valid results for both biomarkers. The primary tumors included were squamous cell carcinomas originating from the oral cavity in 241 patients (30%), the oropharynx in 302 patients (38%), the larynx in 159 patients (20%), and the hypopharynx in 99 patients (12%). Compared to those who were included in the analysis, the cases that were not included had very similar baseline characteristics  showed HPV‐DNA (by ISH or PCR), 218 (27%) tested positive for p16, and 170 (21%) showed both HPV‐DNA and p16‐positivity .There was a large difference in the distribution of HPV and p16‐positivity between tumor sites. Biologically relevant, oncogenic high‐risk HPV infection (HPV‐DNA‐positive/p16‐positive) was detected almost exclusively in oropharyngeal cancer , whereas 1% (5 of 499) of nonoropharyngeal head and neck cancers were positive  and Eastern Europe  compared to Western Europe  and 9% (2 of 23) in Asia  in oropharyngeal cancer also differed significantly by geographic location: 63% of the patients 152 of 242) in Western Europe, compared to 24% (8 of 33) in Eastern Europe , and laryngeal cancers  and for former smokers relative to nonsmokers . Subjects with stage IV disease were more likely to have HPV‐positive cancer  compared to those with stage II disease. Importantly, subjects from Western Europe had a significantly higher risk of being HPV‐positive compared to those from Eastern Europe  compared to Eastern Europe, but this was not statistically significant. There was no association with age or sex , for former smokers relative to nonsmokers  and significantly higher for subjects from Western Europe  compared to those from Eastern Europe , or sex , or disease stage , and second using stepwise selection. Only primary site, smoking status, and geographic region were found to have a significant influence on the probability of biologically relevant oncogenic HPV infection (HPV‐DNA and p16‐positivity) in head and neck cancer (Table p = .69).When examining risk factors for oropharyngeal cancer alone, multivariate analysis revealed only smoking status and geographic region to be significantly associated with combined HPV‐positivity and p16‐positivity . This was the same whether the factors were introduced together or in a stepwise fashion. There was no statistically significant interaction between the risk factors (This study demonstrated large, significant differences in the proportion of HPV‐positive oropharyngeal cancer among Western Europe, Eastern Europe, and Asia, with HPV‐associated head and neck cancer being predominantly a disease of the oropharynx in Western Europe. Geographic location seems to have a strong association with HPV‐positivity in oropharyngeal cancer, regardless of smoking status, which is a well‐established risk factor. To the best of our knowledge, this is the first time that a study firmly has established geographic variability in the prevalence of HPV‐positive oropharyngeal cancer. The study highlights the need for ascertainment of local rates before deciding on local health policies and strategies related to oropharyngeal cancer. The results also demonstrate the very low, relative prevalences of HPV in nonoropharyngeal cancer tumors, and emphasize that, despite the current interest in HPV‐related disease, the prevalence of HPV‐negative head and neck cancer in many countries remains considerably higher than that of HPV‐positive disease.By collating one of the largest cohorts ever collected, using prospective multinational trials, and analyzing them using the same assay and methodology, this study has been able to overcome some of the main limitations of previous studies. After accounting for tumor site and smoking status, geographic region has a significant influence on the probability of testing positive for HPV. The reasons for these geographic variations remain speculative. A likely factor may be related to differences in the prevalence rates of oral HPV infection.Some hypothesize that smoking may protect against the development of HPV‐positive oropharyngeal cancer.This study had some limitations. Only 76% of eligible subjects had evaluable samples. Although this is high when compared to similar studies, it may have still introduced bias to our results. The subjects included had been recruited within trials with eligibility criteria, including having treatment for curative intent. This introduces the possibility of bias compared to population‐based estimates, which may also include subjects with very early disease or those undergoing palliative treatment, who are more likely to have HPV‐negative oropharyngeal cancer.Our study also enabled the quantification of the relative prevalence of HPV in nonoropharyngeal head and neck cancer compared to oropharyngeal cancer, reports of which have varied widely in the literature.Our findings have implications for the planning of future clinical trials. It is clear that HPV‐positive and HPV‐negative head and neck cancers have different characteristics and survival rates, and are thus different disease entities.The findings of this study also have significant implications for local health care policies. In regions where HPV‐positive disease is prevalent, it will be necessary to consider and plan for the long‐term support and survivorship needs of these younger subjects, who are much more likely to survive their cancer, but who will also have to deal with the long‐term effects on them and their families of their disease and treatment.Over three fourths of the subjects recruited in our study had HPV‐negative head and neck cancer Table . AlthougFinally, the significant differences in the prevalence of HPV‐positive head and neck cancer have implications for preventative public health measures, especially in resource‐limited settings. The cost‐effectiveness of the HPV vaccination for prepubescent boys is highly dependent on the prevalence of the disease, among other factors. In areas of low HPV prevalence, especially in countries with low health care expenditure, resource and efforts may be best directed toward smoking cessation policies.Supporting InformationClick here for additional data file.


Cervical lesions caused by integrated human papillomavirus (HPV) infection are highly dangerous because they can quickly develop into invasive cancers. However, clinicians are currently hampered by the lack of a quick, convenient and precise technique to detect integrated/mixed infections of various genotypes of HPVs in the cervix. This study aimed to develop a practical tool to determine the physical status of different HPVs and evaluate its clinical significance.The target population comprised 1162 women with an HPV infection history of > six months and an abnormal cervical cytological finding. The multiple E1-L1/E6E7 ratio analysis, a novel technique, was developed based on determining the ratios of E1/E6E7, E2/E6E7, E4E5/E6E7, L2/E6E7 and L1/E6E7 within the viral genome. Any imbalanced ratios indicate integration. Its diagnostic and predictive performances were compared with those of E2/E6E7 ratio analysis. The detection accuracy of both techniques was evaluated using the gold-standard technique “detection of integrated papillomavirus sequences” (DIPS). To realize a multigenotypic detection goal, a primer and probe library was established.The integration rate of a particular genotype of HPV was correlated with its tumorigenic potential and women with higher lesion grades often carried lower viral loads. The E1-L1/E6E7 ratio analysis achieved 92.7% sensitivity and 99.0% specificity in detecting HPV integration, while the E2/E6E7 ratio analysis showed a much lower sensitivity (75.6%) and a similar specificity (99.3%). Interference due to episomal copies was observed in both techniques, leading to false-negative results. However, some positive results of E1-L1/E6E7 ratio analysis were missed by DIPS due to its stochastic detection nature. The E1-L1/E6E7 ratio analysis is more efficient than E2/E6E7 ratio analysis and DIPS in predicting precancerous/cancerous lesions, in which both positive predictive values (36.7%-82.3%) and negative predictive values (75.9%-100%) were highest (based on the results of three rounds of biopsies).The multiple E1-L1/E6E7 ratio analysis is more sensitive and predictive than E2/E6E7 ratio analysis as a triage test for detecting HPV integration. It can effectively narrow the range of candidates for colposcopic examination and cervical biopsy, thereby lowering the expense of cervical cancer prevention.The online version of this article (doi:10.1186/s12967-014-0282-2) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) is a sexually transmitted pathogen that has been linked to more than 90% of cervical cancer events in women of childbearing age . The genHPV genomic integration is a form of persistent viral infection . A seriePrevious in vitro experiments have confirmed that in precancerous cervical epithelial cells that originally harbored only episomal HPV copies, the frequency of HPV integration into the human genome increases with cell division and with the canceration degree that these cells exhibit . TherefoIn this study, we developed a novel multiple E1-L1/E6E7 gene copy number ratio-based technique  to detect integrated HPV infection in the cervix. This technique is derived from an already existing but less sensitive E2/E6E7 gene copy number ratio-based polymerase chain reaction (PCR) technique  ,15. To iFrom January 1, 2011 through December 31, 2012, cervical cytological samples that were diagnosed with pathological abnormalities  in Pap −8 μM  were considered qualified samples. The consensus HPV L1 primer pair MY09/11 was adopted to amplify viral genomic DNA. The resultant PCR product was hybridized with a panel of 45 HPV-genotypic probes on a positively charged nylon membrane  based on the method described by Oh et al. [For each patient, the total DNA was isolated from a 15-mL cervical cytological sample  using a QIAamp DNA Mini Kit  and maintained in phosphate-buffered saline at −20°C). The quality of the DNA samples was tested by measuring the β-actin (ACTB) copies. An ACTB primer pair .For each HPV genotype, a pair of E6E7-specific primers  was used, and p < 0.05 was defined as statistically significant.A total of 1162 women with single-genotypic HPV infections in the cervix were enrolled. All of the cervical biopsies were performed due to abnormal findings in the cervical cytological examinations  I, 189 cases of CIN II, 36 cases of CIN III and 3 cases of invasive cancer. The first-round biopsy detected 477 (92.8%) cases; the second-round biopsy detected 26 (5.1%) cases; and the third-round biopsy detected 11 (2.2%) cases. The age-related distribution pattern of biopsy-diagnosed precancerous/cancerous cases is shown in Figure 2 test). The age-related distribution patterns of the DIPS-confirmed  and DIPS-denied  cases are shown in Figure Based on the DIPS analysis, HPV integration events (including the integrated and mixed HPV infection) were detected in 312  patients with precancerous/cancerous lesions and in 45  patients without such lesions. The cervical lesion-related HPV integration rates are shown in Figure Table To confirm whether the viral load affects the detection consistency between the copy number ratio-based techniques and DIPS, we divided HPV-infected cases into tertiles based on the E6E7/ACTB ratios. In the highest (first) tertile, relative to the results of DIPS, both the numbers and percentages of false-negative cases in the two copy number ratio-based analyses peaked   of cervical precancerous/cancerous lesions, we compared the efficacies of the three techniques, namely, DIPS, E2/E6E7 ratio analysis and multiple E1-L1/E6E7 ratio analysis, in predicting CIN or invasive cancer cases among the enrolled women. As shown in Figure We examined the detection reproducibility of the three techniques one month after a first-round test. Following two additional rounds of analyses on the remaining cervical samples preserved at −20°C, the coefficients of variation (CVs) of each copy number ratio analysis were obtained, which were 5.1%-7.8% (E1/E6E7), 3.9%-5.6% , 2.3%-6.9% E4E5/E6E7), 2.9%-8.2% (L2/E6E7) and 3.1%-7.1% (L1/E6E7), respectively. Using CV = 10% as a threshold, the quantification stability of the two techniques was acceptable. Moreover, the detection consistency  of the three rounds of tests was 100% for both of the techniques. However, 79  of the integrated/mixed infection cases that were identified during the first-round DIPS failed to be detected again during the second- or third-round DIPS. Meanwhile, two of the so-called false-positive cases  for their physical statuses. The number of HPV genotypes that were evaluated, to the best of our knowledge, has reached the maximum among known studies concerning integrated HPV infection. In previous studies, only two to five genotypes of HPVs have been evaluated via three major techniques, namely, APOT, DIPS and the E2/E6E7 ratio analysis. However, such a narrow genotype range does not satisfy the clinical demands, which could be associated with a high misdiagnosis rate in women that are infected with other HPV genotypes. In a nine-country collaborative international study, Muñoz et al. detected 30 genotypes among the 1998 cases of cervical HPV-infected women  [It is noteworthy that we only adopted the primer and probe library but not the traditional degenerate  primer strategy for adapting the three techniques, i.e., DIPS, the E2/E6E7 ratio analysis and the E1-L1/E6E7 ratio analysis, to detect multigenotypic HPV -21. MoreBased on the three compared techniques, i.e., the DIPS analysis and the two copy number ratio-based analyses, the importance of applying HPV integration tests as tools to prevent cervical cancer was consistently demonstrated by a panel of mutually corroborative lesion-, genotype- and age-related distributional characteristics of integrated/mixed HPV infections in the enrolled women. Our results indicate that, with increasing lesion grades, the percentages of integrated/mixed infection cases increased gradually Figure B. This fMultigenotypic DIPS - a common reference platform was established in our study for comparing the respective diagnostic accuracies of the E2/E6E7 ratio analysis and the E1-L1/E6E7 ratio analysis in the enrolled women. The original version of DIPS was designed for two genotypes of HPVs, namely, HPV 16 and 18 . The opeIn the detection of integrated/mixed HPV infection, the two copy number ratio-based techniques were being confronted with the same problem: the complete viral genome of episomal HPV copies can cover up a few viral sequence deletions that are caused by the integration events, leading to an indifferent copy number ratio to that of episomal infection and, thereafter, to false-negative cases ,31. In tThe enrolled population of this study consisted of women with HPV infection > six months who had positive findings in their cervical cytological examinations. Previous studies indicated that 30%-60% of these women would be eventually diagnosed as normal or cervicitis cases ,38. TherCervical biopsy is a key method to acquire direct evidence of precancerous/cancerous lesions. This process provides reliable guidance to develop a proper treatment scenario. However, we and other groups have demonstrated a rate of missed biopsy, especially in women with no visible lesions in the cervix -44. In tThe clinically collected samples usually need to be preserved for a period of time until they can be examined or re-examined under necessary conditions. A clinically applicable test, therefore, is expected to be able to maintain its detection ability as the sample quality begins to decrease. We re-examined the DNA extractions of cervical samples after one-month of preservation at −20°C. A lowered technical reproducibility was observed only among the results of triplicate DIPS Table , while bOur study of women with single-genotypic HPV infections has its limitations. Previous studies indicated that women with multi-genotypic HPV infections can account for 10% to 20% of the total cervical HPV-positive cases ,27. ThisIn conclusion, our study demonstrates that the multiple E1-L1/E6E7 ratio analysis is more sensitive and predictive than its prototype the E2/E6E7 ratio analysis, and it is also a more convenient and stable technique if the gold-standard technique, DIPS, is used as a reference. This technique is capable of meeting the growing demands for detecting integrated/mixed HPV infection cases in the current clinic. Meanwhile, regarding identifying the high-risk women of cervical cancer and selecting candidates for colposcopy and cervical biopsy, this technique possesses a substantial value as a triage test, which can effectively reduce the range of the target population, thereby concentrating limited medical resources to those in the most need and ameliorating unnecessary stress in low-risk women. At last, as a novel, extensible and broad-spectrum technique, the multiple E1-L1/E6E7 ratio analysis could be used by more HPV integration-related studies to explore the epidemiological mechanisms of cervical cancer that is induced by a score of HPV genotypes.