The newly gained knowledge of the viral etiology in cervical carcinogenesis has prompted industrial interests in developing virology-based tools for cervical cancer prevention. Due to the long incubation period from viral infection to developing an invasive cancer, a process whose outcome is influenced by numerous life-style and genetic factors, the true efficacy of the genotype-specific human papillomavirus (HPV) vaccines in cervical cancer prevention cannot be determined for another 30 years. Most HPV DNA test kits designed to replace the traditional Papanicolaou (Pap) smears for precancer detection lack the analytical sensitivity and specificity to comprehensively detect all potentially carcinogenic HPVs and to perform reliable genotyping. The authors implemented the classic nested PCR and Sanger DNA-sequencing technology for routine HPV testing. The results showed a true negative HPV PCR invariably indicates the absence of precancerous cells in the cytology samples. However, 80.5% of single positive HPV-16 tests and 97.3% of single positive HPV-18 tests were associated with a negative or a largely self-reversible Pap cytology. Routine sensitive and reliable HPV type-specific or perhaps even variant-specific methods are needed to address the issues of persistence of HPV infection if a virology-based primary cervical screen is used to replace the Pap cytology screening paradigm. Celopment .Cervical cancer is also a highly preventable disease if detected at its precancerous stages and treated by ablative procedures. Since there are no treatments to cure the persistent HPV infections, prevention of cervical cancer is accomplished by physical removal of the cancer-susceptible transformation zone of the uterine cervical epithelium when a biopsy shows the presence of high-grade cervical intraepithelial neoplasia (CIN3), representing pathologic changes with a high frequency of progressing to invasive cancer. In the United States, the widespread use of Papanicolaou (Pap) smear screening for detection followed by treatment of these precancerous lesions reduced the incidence of cervical cancer from 44 in 100,000 women in 1947 to 8.8 in 1970 . About 7i.e., HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 for cervical screening [i.e., HPV-16 and HPV-18, with the aim to prevent cervical cancer in the subjects.The discovery of DNA viruses as a major factor in causing most of the cervical squamous cancers has led to new hopes to use a virology test for cervical screening of precancerous lesions and to use an HPV vaccine for cancer prevention . A commecreening ,12. Sinc® (Merck) and those for HPV-16 and HPV-18 are used as the active ingredient of a bivalent HPV vaccine Cervarix® (GlaxoSmithKline). After being injected intramuscularly with an aluminum salt adjuvant, HPV VLPs are exceptionally effective in eliciting production of neutralizing antibodies against HPV-16 [The VLPs in the vaccines are irregularly shaped 30–50 nm structures composed of self-assembled HPV major capsid L1 protein pentamers manufactured by a DNA recombinant technology ,14. Genot HPV-16 ,16,17. It HPV-16 . Howevert HPV-16  and are t HPV-16 ,21,22, at HPV-16 , the trut HPV-16 . Sporadit HPV-16 , raisingt HPV-16 , suggestt HPV-16  and the t HPV-16  advise tt HPV-16 .At the present time, detection of persistent high-risk HPV infection and cytologic monitoring of the precancerous changes, if present, are the two screening tests which the practitioners rely on for patient management in cervical cancer prevention. The U.S. FDA has recently approved using a human papillomavirus test for primary cervical cancer screening for women of 25 or older .i.e., the E1, E2, E4, E5, E6 and E7 genes, are expressed as viral DNA replicates from the episomal DNA. The late genes, L1 and L2, are expressed in the cells of the upper epithelial layers to generate the L1 and L2 capsid proteins to encapsulate the viral genomes to form virions. The proteins E6 and E7 are known to inhibit the tumor suppressor genes p53 and pRb, and the protein E5 appears to have a weaker oncogenic property which results in increased activity of epidermal growth factor receptor (EGFR) [In a normal life cycle, when a high-risk HPV infects the basal epithelial cells of the cervical epithelium, the early HPV genes, r (EGFR) . HoweverThe proteins E2 and E1 play a leading role in control of transcription and replication, and the absence or loss of activity of these proteins leads to deregulation of E6 and E7 oncoproteins . If the HPV has a genome of about 8000 base pairs (bp) consisting of a covalently closed circular double-stranded DNA molecule. By definition, a genotype of HPV differs in the L1 gene DNA sequence, which is about 1600 bp in size and highly conserved, by at least 10% from every other known HPV type; HPV subtypes are those having DNA sequence similarities between 90% and 98% with a prototype in its L1 gene; and variants of an HPV genotype are those having a DNA sequence identity of over 98% of a prototype . Since sThe presence of HPV episomal DNA in the epithelial cells is often associated with koilocytotic atypical changes as the manifestation of HPV-induced cytopathology in cervical dysplasia , a reverAs much as 99.7% of the cervical cancers studied have been found HPV-positive in some reports . HoweverViral load is not a useful parameter to predict CIN3 or cancerous lesions  because Most commercially available HPV test kits have been shown to be suboptimal in analytical sensitivity and typing specificity. It has been reported that in a comparative study one of the approved HPV DNA test kits on the market was found to generate two to four times more positive results than the other . In a 20Using a nested PCR amplification for detection followed by automated DNA sequencing for genotyping as the standard for validation, the high-risk HC2 test (Digene/Qiagen) detected only 57.6% 388/674) of the high-risk HPV isolates in clinical specimens, mislabeled 46.8% (88/188) of the low-risk HPV isolates as high-risk genotypes, and misclassified 27.4% 180/657) of the negative samples as being infected by high-risk HPV . Based o7 of the 4 of the Recently, many studies worldwide have reported failures of detecting HPV DNA in some histologically confirmed cases of invasive cervical cancers, including squamous cell carcinoma. For example, 8% of patients with cervical squamous cell carcinoma were found to be HPV-negative within 30 months preceding the histological diagnosis of a cervical squamous cell carcinoma ; and 12.We have modified the protocol of primary PCR amplification with a pair of MY09 and MY11 degenerate primers followed by heminested PCR with a pair of consensus GP6 and MY11 primers at low stringency PCR condition for HPV detection and performed a routine Sanger DNA sequencing on all nested PCR amplicons for HPV genotyping. This nested PCR protocol is known to be able to amplify a single copy of the highly conserved HPV L1 gene DNA fragment with hypervariable base regions  and the Due to the diversity of the L1 gene DNA sequences between the individual HPV genotypes, the use of type-specific primer PCR to amplify all possible clinically relevant HPV genotypes individually on each clinical sample is impractical and uneconomical. For PCR detection of HPV DNA in clinical specimens, specificity is sacrificed for high sensitivity and efficiency to detect all possible clinically relevant genotypes, including the less frequently encountered variants or mutants, with one set of consensus general primers. The target DNA can be validated by DNA sequencing of the inter-primer segment of the PCR amplicon. For example, there are three subtypes of HPV-18, namely the European, the Asian-American and the African subtypes . All of Since there is a two-nucleotide-base difference in DNA sequence immediately downstream of the MY11 primer between the European/Asian-American subtype and the African subtype of HPV-18, a commercial HPV assay kit designed to target one subtype for hybridization may fail to detect the other , and thiAs previously reported, our routine work with DNA sequencing unveiled a novel HPV-39 variant with an A-to-C mutation, thus changing the 14th amino acid from leucine (TTA) to valine (GTA) in the L1 major capsid protein; and this mutant was repeatedly isolated from a patient with persistent HPV infection and a history of precancerous cytopathology . AnotherFrom 2007–2010, the department of pathology of Milford Hospital in Milford, Connecticut, USA received and processed 8461 alcohol-preserved liquid-based cervicovaginal Pap cytology specimens  collected by board-certified gynecologists in private practice with request for HPV testing according to the American College of Obstetricians and Gynecologists guidelines .The HPV tests were ordered as part of regular medical office women’s health care for patients aged 30 years and older as adjunctive screening to routine Pap cytology, and for patients below 30 years who had a cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS) or more severe cytological changes in most of the cases. This study was a retrospective analysis of laboratory data and the data analysis presented here did not influence the decision of patient management. Publication of these laboratory data with blinded patient identities was approved by the Milford Hospital Institutional Review Board as an ongoing research project .The Pap cytology screening was performed by an independent commercial laboratory , and all abnormal cytology slides classified as ASCUS, low grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL) were reviewed by a board-certified pathologist for final classification. Detection of HPV DNA by GP6/MY11 heminested PCR followed by direct automated DNA sequencing for genotyping was performed according to the method previously published ,66,67, aThe data presented in There were no LSIL or HSIL cytology results associated with a negative HPV PCR. Our findings support the conclusion made by others that HPV infection is a necessary factor for initiating the pathologic process which may lead to cervical cancer ,2,3,4. AOn the other hand, a positive high-risk HPV DNA test is not consistently associated with a precancerous or cancerous Pap cytology. A negative Pap cytology was found in a low 39.5% of HPV 31-positive samples to a high 71.0% of HPV 45-postitive samples. When HPV-16 was detected on single tests at the time of entrance in this study, only 19.5% (22/113) of the split samples were found to have a HSIL cytology, an indication for immediate colposcopic biopsy to rule out cancer or an advanced precancerous CIN3 lesion. The other HPV genotypes associated with a HSIL cytology were HPV-33 with 15.8% (3/19) HSIL, HPV-31 with 13.2% (5/38) HSIL, HPV-35 with 8.7% (2/23) HSIL, HPV-52 with 7.4% (4/54) HSIL, HPV-59 with 3.3% (1/30) HSIL, HPV-45 with 3.2% HSIL, and HPV-18 with 2.7% (1/37) HISL. An HSIL cytology was found in 4.8% (2/42) mixed HPV infections. This latter finding was not a surprise because genotype-specific PCR amplification followed by automated DNA sequencing for validation has confirmed that 82% of the cervicovaginal cell samples infected by multiple HPV genotypes found by nested PCR in fact contained an HPV-16 in addition to various other companion genotypes . In the The findings presented above indicate that a reliable negative HPV test can eliminate the need for a Pap cytology test. However, a single positive HPV test alone is not a good indication for colposcopic biopsy because 80.5% (91/113) of the HPV-16 positive samples and 97.3% (36/37) of the HPV-18 positive samples were associated with a negative Pap cytology or a largely self-reversible Pap cytology in the category of ASCUS or LSIL. Using an FDA-approved industry-standard test kit for triage of patients, more than 95% of referrals to colposcopic biopsies for diagnostic workup based on a single positive high-risk HPV DNA test result have been found to be unnecessary and potentially excessive [Recently, in an attempt to reduce the number of unnecessary and excessive 4-quadrant colposcopic biopsies the supporters of using commercial HPV DNA test kits for triage of patients to biopsy workup have introduced a pair of clinical sensitivity and clinical specificity targets as the “gold standard” for validation of novel HPV test kits ,82. One The FDA-approved Roche Cobas 4800 HPV kit used for detection of ≥CIN 2 and ≥CIN 3 in an ASCUS population was reported to have a clinical sensitivity 90.0% and a clinical specificity 70.5% for ≥CIN 2 lesions, and a clinical sensitivity 93.5% and clinical specificity 69.3% for ≥CIN 3 lesions . These pAccording to the existent guidelines of practice, patients with a HSIL cytology  were advThe data in Based on data generated by laboratories using various test kits, the overall HPV prevalence in women with normal cervical cytology worldwide was estimated to be 10.4% , compareIn a head-to-head comparative study performed on split clinical samples, using nested PCR for detection followed by direct DNA sequencing for HPV genotyping as the standard for comparison, an FDA-approved high-risk HPV test was found to correctly detect 57.6% of the high-risk HPV isolates in clinical specimens, mislabel 46.8% of the low-risk HPV isolates as high-risk genotypes, and classify 27.4% of the “true-negative” samples as being infected by high-risk HPV . TherefoIn this series, there were 229 patients who had at least one additional positive HPV test showing the same genotype in the second sample taken between 6 months and 3 years after the first sample was tested, thus considered having a persistent HPV infection, and at least one follow-up cytology test associated with the second HPV assay. Comparing the subsequent cytology with the first, these patients were placed into three classes, namely no change (NC), regress to lower class (REG) or progress to higher class (PRO) in Pap cytology. These follow-up results in cytology were tabulated  against As shown in In the 229 patients with a persistent HPV infection and a Pap cytology <HSIL, 166 (72.5%) showed no changes or a regression in the follow-up Pap cytology while 63 (27.5%) showed cytologic progression from a lower class to a higher class. Colposcopic biopsies confirmed that the cervical lesion in 15 patients with persistent infection by HPV-16, -31, -45 or -52 progressed to CIN3. These biopsies were performed because the cytology was found to progress to HSIL on a follow-up Pap test or because detection of the same high-risk HPV genotype associated with an unresolved LSIL or ASCUS cytology on repeat tests. Of the 63 patients with a persistent HPV-16 infection, 23 (36.5%) showed Pap cytology progression from a lower class to a higher class (negative > ASCUS > LSIL > HSIL), 6 (9.5%) regressed from a higher class to a lower class (negative < ASCUS < LSIL), and 34 (54%) showed no class changes in the follow-up cytology. The corresponding numbers in these cytologic change categories for the patients with HPV-31 persistent infection were 6 (66.7%), 1 (11.1%) and 2 (22.2%); those with HPV-45 were 6 (50%), 0 (0%) and 6 (50%); and those with HPV-52 were 7 (38.9%), 0 (0%) and 11 (61.1%), respectively. Patients with persistent infections by HPV-18, -35, -39, -53, -54, -58,-59, -59, -66, -70, and -73 did not develop CIN3 or more severe lesions although 16 patients with persistent infections caused by these HPV genotypes had colposcopic biopsies during follow-ups which were proven to be below CIN3. While 12.7% (8/63) of the patients with persistent HPV-16 infection developed a biopsy-proven CIN3 lesion, 16.7% (3/18) of the patients with HPV-52 persistent infection developed a CIN3 lesion during this observation period. HPV-52 has been reported to be a leading cancer-causing genotype among certain populations ,92, but an FDA-approved HPV test that detects the same genotypes”, or validated by “PCR followed by sequencing of the amplicon” [According to the U.S. FDA guidance, a commercial test kit for HPV detection and genotyping may be validated against “mplicon” . Practicmplicon” ,99,100. mplicon” ,101 and mplicon” , all othmplicon” ,99,100. mplicon” . As a remplicon” .In this series, 372 of the 770 patients who were positive for HPV at entrance  were fouAlthough HPV persistence is consistently and strongly associated with CIN2-3/HSIL+ lesions, the magnitude of association may vary by duration of persistence, testing interval and HPV genotypes. Precise definition and standardization of HPV testing, sampling procedure, and test interval are needed for reliable clinical testing . There aAs stated above, the 8461 alcohol-preserved liquid-based cervicovaginal Pap cytology specimens were collected by board-certified gynecologists in private practice in the town of Milford for HPV testing according to the American College of Obstetricians and Gynecologists guidelines .The HPV tests were ordered as part of regular medical office women’s health care for 6011 patients aged 30 years and older  as adjunctive screening to routine Pap cytology, and for 2450 patients below 30 years  as adjunctive screening to Pap cytology or after the patients were found to have a cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS) or more severe cytological changes. Since most of the patients below 30 were known Pap cytology-positive before the HPV testing as a result of insurance payment policy, the HPV-positive rate in this younger age group was higher than those patients aged 30 and older. This study was a retrospective analysis of laboratory data and the data analysis presented here did not influence the decision of patient management. The lifestyle and genetic makeups of these private patients may be different from those participating in other research projects, and may influence the data presented in this article. As previously reported, Milford has a rural and suburban population of over 50,000, and 93.6% are non-Hispanic white residents, and publication of these laboratory data with blinded patient identities was approved by the Milford Hospital Institutional Review Board as an ongoing research project .The laboratory performing the diagnostic HPV tests on these clinical samples is certified by the State of Connecticut Department of Public Health under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) for human papillomavirus (HPV) by PCR and DNA sequencing based on methods previously published ,65,66,67in situ in the practice of gynecologic pathology). All CIN1, CIN2 and CIN3 lesions are considered to be precancerous, not a life-threatening disease unless progressing further to invasive cancer.After a specimen was received in the laboratory, about 1 mL (5%) of the ThinPrep, or 0.5 mL (5%) of the SurePath cell suspension, was transferred to a 1.5 mL plastic tube for HPV testing. The remaining liquid-based specimen was forwarded to an independent commercial laboratory  for Pap cytology screening. The abnormal cytology was classified and reviewed by a board-certified pathologist as atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesion (LSIL), or high grade squamous intraepithelial lesion (HSIL). The 4-quadrant colposcopic cervical biopsies, if performed, were processed in the department of pathology, Milford Hospital and the histologic sections were interpreted by the hospital pathologist, and the cases were classified as normal, cervical squamous intraepithelial neoplasia 1 (CIN1), cervical squamous intraepithelial neoplasia 2 (CIN2), or cervical squamous intraepithelial neoplasia 3  centrifugation for 5 min in a Microcentrifuge 5424 w/rotor 022620444 , and washed first in reagent grade water, then in 1 mL buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, 0.5% Tween 20, pH 8.1. The washed cell pellet was re-suspended and digested at 45–55 °C overnight in 100 μL of 0.1 mg/mL proteinase K  dissolved in the same washing buffer. After denaturing the proteins in the cell digestate in a metal block heated to 95 °C for 10 min and a final centrifugation of the digestate at 13,000 rpm for 5 min, the supernatant was used for HPV DNA PCR amplification without further purification.® DNA polymerase ready-to-use mix  which contained all the components needed for low temperature PCR, including dNTPs, Mg++, buffer, HiFi® DNA polymerases, dsDNA melting agents and dNTP preservatives, to reach a final 25 μL reaction volume. For thermocycling, the temperature steps of a TC-412 Thermal Cycler  were programmed for an initial heating at 85 °C for 10 min, followed by 30 cycles at 85 °C for 30 s, 40 °C for 30 s, and 65 °C for 1 min. The final extension was 65 °C for 10 min.For primary PCR amplification, 1 μL of the crude proteinase K digestate, 1 μL of 10 μmolar MY09 primer (5'-CGTCCMARRGGAWACTGATC-3'), 1 μL of 10 μmolar MY11 primer (5'-GCMCAGGGWCATAAYAATGG-3') and 2 μL of water were added to a PCR tube containing 20 μL of LoTemp™ HiFi® DNA polymerase ready-to-use mix, 1 μL of 10 μmolar GP6 primer (5'-GAAAAATAAACTGTAAATCA-3'), 1 μL of 10 μmolar MY11 primer and 3 μL of water to generate a ~190 bp nested PCR product for detection by standard gel electrophoresis. The thermocycling steps for heminested PCR were identical to those used for primary PCR.For heminested PCR, a “trace” of the MY09/MY11 PCR products was transferred by a micro-glass rod of about 1.5 mm in diameter with clean wettable surface to a second PCR tube containing 25 μL of complete nested PCR reaction mixture consisting of 20 μL of LoTemp™ HiFiThe positive heminested PCR products were subjected to direct automated DNA sequencing without further purification. Briefly, a trace of the nested PCR products was transferred from the PCR tube with a calibrated micro-glass rod described above into a reaction mixture containing 1 μL of GP6 sequencing primer, 1 μL of BigDye Terminator , 3.5 μL 5× buffer, and 14.5 μL of water in a total volume of 20 μL. This reaction mixture was subjected to 20 enzymatic primer extension/termination reaction cycles, according to the protocol supplied by the manufacturer (Applied Biosystems). The final reaction mixture was loaded in an automated ABI 3130 four-capillary genetic analyzer for sequence analysis. A computer-generated base calling electropherogram usually shows an unambiguous sequence of about 70 bases  for accuOne μL of each digestate sample was placed in a separate PCR tube with a β-globin primer pair for human genomic DNA amplification as a control of specimen adequacy. Specimens with no β-globin gene amplification were excluded as insufficient. All HPV-negative samples had a concomitant positive β-globin gene amplification.The purified full-length plasmid DNAs of HPV types-6B, -11, -16, and -18 purchased from American Type Culture Collection (ATCC) were used as HPV DNA standards. The heminested PCR products generated of these standard genotypes by the primers used were confirmed by direct automated DNA sequencing to be those of the L1 gene DNA of the expected HPV genotype. This preliminary experiment was to demonstrate that the methodology used in this study was capable of generating genotype-specific template suitable for DNA sequencing.The purified HPV type-16 DNA was used as the routine positive control and molecular grade pure water instead of DNA extract was used as negative control for each PCR run. To avoid cross contamination, three separate rooms with no air re-circulation were dedicated to the nucleic acid amplification tests. Two of the rooms were each equipped with a 32” PCR workstation . All pre-amplification procedures were performed in PCR station I. All post-PCR procedures were carried out in PCR station II, including preparations for the nested PCR and sequencing reaction. Gel electrophoresis and DNA sequencing were performed in the third isolation room. No post-PCR materials or any items potentially contaminated by amplicons or equipment used in the post-PCR rooms were allowed to enter the pre-PCR working space. Transferring of PCR products from one test tube to another to initiate a new chemical reaction was always accomplished by a micro-glass rod to avoid cross contamination due to PCR amplicon aerosol which may occur during micro-pipetting.i.e., highly trained medical doctors. It cannot be replaced by a virology test in spite of the ASCCP guidelines which recommended referral to colposcopy of HPV-16/HPV-18+ women with negative cytology [In spite of our newly gained knowledge on the viral etiology in cervical carcinogenesis, a proposed policy depending on prophylactic vaccination of adolescents against carcinogenic HPV infections as a primary means to prevent cervical cancer in developed countries  is beingcytology . To avoi


High oxidative stress and chronic inflammation can contribute to the pathogenesis of coronary artery disease (CAD). Coenzyme Q10 is an endogenous lipid-soluble antioxidant. Statins therapy can reduce the biosynthesis of coenzyme Q10. The purpose of this study was to investigate the effects of a coenzyme Q10 supplement  on antioxidation and anti-inflammation in patients who have CAD during statins therapy.Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery and who were treated with statins for at least one month were enrolled in this study. The subjects (n = 51) were randomly assigned to the placebo (n = 24) and coenzyme Q10 groups . The intervention was administered for 12 weeks. The concentrations of coenzyme Q10, vitamin E, antioxidant enzymes activities , and inflammatory markers  were measured in the 42 subjects  who completed the study.P < 0.001) and antioxidant enzymes activities (P < 0.05) were significantly higher after coenzyme Q10 supplementation. The levels of inflammatory markers  were significantly lower after coenzyme Q10 supplementation. The subjects in the Q10-300 group had significantly higher vitamin E (P = 0.043) and the antioxidant enzymes activities (P < 0.05) than the placebo group at week 12. The level of plasma coenzyme Q10 was significantly positively correlated with vitamin E (P = 0.008) and antioxidant enzymes activities (P < 0.05) and was negatively correlated with TNF-α (P = 0.034) and IL-6 (P = 0.027) after coenzyme Q10 supplementation.The levels of the plasma coenzyme Q10 (Coenzyme Q10 supplementation at 300 mg/d significantly enhances antioxidant enzymes activities and lowers inflammation in patients who have CAD during statins therapy.NCT01424761.Clinical Trials.gov Identifier:  Cardiovascular disease is the leading cause of death worldwide . HyperliHigher levels of oxidative stress and inflammation play a role in the development of CAD ,11. CoenThe circulating levels of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) are positively correlated with the risk of primary and recurrent myocardial infarction and death -18. AdipThis study was designed as a single blinded, randomized, parallel, placebo-controlled study. CAD patients were recruited from the cardiology clinic of Taichung Veterans General Hospital, which is a teaching hospital in central Taiwan. CAD was identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery or receiving percutaneous transluminal coronary angioplasty (PTCA). The subjects in this study were treated with statins therapy for at least 1 month. The subjects with diabetes, liver, or renal diseases, or who currently use vitamin supplements were excluded. Informed consent was obtained from each subject. This study was approved by the Institutional Review Board of Taichung Veterans General Hospital, Taiwan.With a sample size calculation, we expected that the change in the levels of antioxidant enzymes activities would be 5.0 ± 7.0 U/mg of protein after coenzyme Q10 supplementation; therefore, the desired power was set at 0.8 to detect a true fact and at an α value equal to 0.05 with a minimal sample of 18 in each intervention group. We enrolled 51 CAD patients in this study and used random numbers table to random assign the subjects to the placebo (n = 24) or to the coenzyme Q10  groups. The female subjects in this study were postmenopausal women who were not receiving hormone therapy. The coenzyme Q10 and placebo (starch) capsules were commercially available preparations . The intervention was administered for 12 weeks. The subjects were instructed to take two capsules daily . To monitor compliance, the researchers reminded subjects to check the capsules bag every 4 weeks to confirm that the bag was empty. The researcher also asked some questions to the subjects, such as ①When did you take the supplements? ; ②How many times did you take the supplements every day? (We advised subjects to take the supplements two times per day); ③How many capsules did you take of the supplements each time? (We advised the subjects to take one capsule of supplement each time); and ④How did you feel after taking the supplements? (for monitoring adverse effects), and then we checked the capsule bags. We placed 56 capsules in each bag, and the bag should be emptied after 4 weeks of taking the supplements. If the subjects took the empty bag back and answered the questions correctly, the researcher then gave them another bag of capsules. In addition, we also measured the concentration of coenzyme Q10 every 4 weeks supplementation for monitor the compliance of the subjects. The age, blood pressures, and smoking, drinking, and exercise habits of all of the subjects were recorded. The body weight and height were measured; the body mass index (BMI) was then calculated.Fasting venous blood samples (15 mL) were obtained to estimate the hematological and vitamin status. Blood specimens were collected in Vacutainer tubes  that contained EDTA as an anticoagulant or that contained no anticoagulant as required. Serum and plasma were prepared after centrifugation  and were then stored at -80°C until analysis. Hematological entities  were measured by an automated biochemical analyzer . The level of CRP was quantified by particle-enhanced immunonephelometry with an Image analyzer . Plasma TNF-α , IL-6 , and adiponectin  levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available kits and according to the instructions made available from the suppliers.Plasma coenzyme Q10 and vitamin E were measured by high-performance liquid chromatography (HPLC) and were detected by a UV detector at 275 nm and 292 nm, respectively ,23. The p < 0.05. The values presented in the text are means ± standard deviation (SD).The data were analyzed using SigmaPlot software . The normality of the distribution of the variables was evaluated using the Shapiro-Wilk test, and the normally distributed variables were age, waist circumference, the ratio of waist to hip, BMI, TC, LDL-C, HDL-C, the frequency of smoking and exercise, the ratio of vitamin E to TC, SOD, and IL-6. The multiple linear regression analyses were performed to examine the time and the interventional effects on the variables . We set the dummy variables for the time  and the intervention . If the variables had significantly effects by the time or the intervention, then the intergroup differences (intervention effect) between the placebo and the Q10-300 groups were evaluated by the Student’s t-test or the Mann-Whitney rank sum test; within a group (time effect), the paired t-test or the Wilcoxon signed rank tests were used. When comparing the changed levels of coenzyme Q10, vitamin E, antioxidant enzymes activities and inflammatory markers between the placebo and the Q10-300 groups, the Student’s t-test or the Mann-Whitney rank sum test was used. For categorical response variables, the differences between two groups were assessed by the Chi-square test (parametric method) or the Fisher’s exact test (non-parametric method). To examine the relationships of coenzyme Q10 concentration with the level of vitamin E, antioxidant enzymes activities, and inflammatory markers after the supplementation, the Pearson product moment correlations were used in the parametric data and the Spearman rank order correlations were used in the non-parametric data. The results were considered to be statistically significant at The sampling and trial profiles are summarized in Figure P < 0.001) and the ratio of coenzyme Q10 to TC (P < 0.001) were significantly increased after coenzyme Q10 supplementation. The subjects in the Q10-300 group had significantly higher levels of coenzyme Q10 (P < 0.001), ratio of coenzyme Q10 to TC (P < 0.001), and vitamin E (P = 0.043), and ratio of vitamin E to TC (P = 0.025) than those in the placebo group at week 12.The effects of coenzyme Q10 supplementation on the levels of coenzyme Q10 and vitamin E are shown in Figure P = 0.001), CAT (P = 0.009), and GPx (P = 0.021) were significantly increased after coenzyme Q10 supplementation. The subjects in the Q10-300 group had significantly higher activities of SOD (P = 0.005), CAT (P = 0.025), and GPx (P = 0.040) than those in the placebo group at week 12.The effects of coenzyme Q10 supplementation on the antioxidant enzymes activities are shown in Figure P = 0.039). There were no effects on the level of CRP, IL-6, and adiponectin after 12 weeks of supplementation.The effects of coenzyme Q10 supplementation on the levels of inflammatory markers are shown in Figure P < 0.001), the ratios of coenzyme Q10 to TC (P < 0.001), vitamin E (P = 0.046), the ratios of vitamin E to TC (P = 0.032), antioxidant enzymes activities , and inflammatory markers  were significantly different between the placebo and the Q10-300 groups.The changed levels of coenzyme Q10, vitamin E, antioxidant enzymes activities, and inflammatory markers after the supplementation are shown in Table r = 0.41, P = 0.008), antioxidant enzymes activities , and the inflammatory markers  at week 12.The correlations between coenzyme Q10, vitamin E, antioxidant enzymes activities, and inflammatory markers after coenzyme Q10 supplementation are shown in Table In this clinical trial, we have demonstrated that coenzyme Q10 at the dose of 300 mg/d for 12 weeks increases the antioxidant enzymes activities  and decreases inflammation in patients with CAD during statins therapy. Tiano et al.  administBoth groups of statins-treated CAD patients in this study had a low level of coenzyme Q10 at baseline Figure , and theCAD is considered to be a chronic inflammation status . In the Regarding the safety of statins therapy and its combination with placebo or coenzyme Q10 in the present study, there were no clinically significant changes in the subjects’ vital signs, serum chemical values, or hematological values, and there were no serious adverse events, no complaints of myalgia or muscle weakness, and no withdrawals due to adverse events. Thus, coenzyme Q10 at a dose of 300 mg/d is safe for co-administration with statins therapy.Two limitations of the present study should be mentioned. First, the number of participants was small, although we did recruit more subjects than expected. Second, a few of the subjects had higher inflammation in the present study. Only 10% of the subjects had a high inflammation according to the level of CRP (≥ 3.0 mg/L), and this finding might have contributed to observing the null effect on CRP after the supplementation. Large studies are needed to establish the beneficial effect of coenzyme Q10 supplementation on inflammation, especially in subjects who have high inflammation status.In conclusion, we have demonstrated that coenzyme Q10 supplementation at a dose of 300 mg/d significantly increased antioxidant enzymes activities and reduced the levels of inflammatory markers (TNF-α and IL-6) in CAD patients during statins therapy. CAD patients might benefit from using coenzyme Q10 supplements to increase their antioxidation and anti-inflammation capacity during statins therapy.The authors have no conflict of interest.BJL carried out the study, performed the data analyses, and drafted the manuscript. YFT and CHY carried out the study and sample analyses. PTL conceived of the study, participated in its design, and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


HPV infections are detected in sexually naive children. This has raised the question about the role of early HPV infections in either protecting or predisposing to further HPV infections. HPV16-specific cell-mediated immunity (CMI) was studied in 10 case-children born to mothers with an incident cervical intraepithelial neoplasia (CIN) diagnosed during their 14-year follow-up (FU), and in 21 children born to mothers, who remained constantly HPV-negative (controls). The mean age of children was 12.3 years.Peripheral blood mononuclear cells were isolated from blood and stimulated with peptide pools covering HPV16 E2, E6 and E7. Proliferation of lymphocytes, their secretion of cytokines, and the frequency of regulatory T-cells were determined. The results were correlated with the HPV status and analyzed in a nested case–control setting.All children, except two controls, displayed CMI against HPV16 E2, E6 and/or E7 peptides associated with type 1 and 2 cytokine secretion. Only two statistically significant differences were found in the nested case–control setting; (1) case-children had a higher TNF-α response to HPV16 E2 (p = 0.004) than controls and (2) controls had no response to HPV16 E7.2 peptide pool while 3/10 case-children had (p = 0.013). Totally, 50 and 57 % of the cases and controls, respectively, had HPV positive oral samples at some FU-visit. In addition, the children without any HPV antibodies before the age of 6 months showed proliferative responses of PBMC after HPV16 exposure more frequently than other children (p = 0.045).HPV16-specific CMI is common in young, sexually inexperienced children. This suggests that oral HPV infections occur frequently in children. Our results might also explain the previous findings that half of healthy adults demonstrate HPV-specific CMI irrespective of their partner/sexual status.The online version of this article (doi:10.1186/s12967-015-0733-4) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) is the main cause of cervical cancer (CC) and also important in the etiology of other cancers, such as anal and head and neck cancers. HPV is also causing benign epithelial growths in mucosa i.e. papillomas and condylomas. The traditional concept of HPV infection as an exclusively sexually transmitted disease has strongly focused the research mainly to HPV infections in adults. However, the recent meta-analysis provided evidence that HPV infection affect also children under the age of sexual debut, as well as newborns at perinatal period : HPV infAll these data support the view that HPV infection can be acquired early in life. This has raised the question about the significance of early infections for later encounters with HPV in the view of immunity. It has been speculated that if a child acquires the first infection before the maturation of her/his immune system, the outcome of infection could eventually be either HPV-specific immunological tolerance or immune response with antibody production and cell-mediated immunity , 6–9. HoTo further elucidate the dynamics of HPV-specific cell-mediated immunity (CMI) we compared the responses of these 10 children born to the mothers who developed incident CIN, to those 21 children whose mothers remained constantly HPV-negative at cervix during the 6 year follow-up. The mononuclear cells isolated from the venous blood of the children were stimulated with HPV16 peptide pools and the following CMI response was determined by measuring the proliferation of lymphocytes, their secretion of cytokines, and the frequency of regulatory T-cells.The Finnish HPV Family Study is a longitudinal cohort study conducted at the Department of Oral Pathology, Institute of Dentistry, University of Turku and the Department of Obstetrics and Gynecology, Turku University Central Hospital. The study plan was approved by the Research Ethics Committee of Turku University Hospital (#3/1998 and 45/180/2010). Originally, 329 pregnant women at their 3rd trimester of pregnancy and all their newborns  were enrolled in the study between 1998 and 2001, as described previously , 12. As The authors acknowledge the concept of the Minimal Information About T-cell Assays (MIATA) framework, which was recently published , 17. The® assay  as described earlier -Thymidine incorporation assay. The cut-off value for counts per minute (CPM) values was determined by the average plus 3× SD of the eight medium-only control wells. Stimulation index (SI) was calculated as the average of tested eight wells divided by the average of the medium-only control wells. The proliferative response was defined positive if the CPM values of at least six of the eight wells were above the cut-off value and if the SI was ≥3.LST was performed as described previously , 23, 24.The supernatants collected from eight replicative wells of LST at day 6 of culturing were pooled and analyzed using Cytometric Bead Array (CBA) human enhanced sensitivity flex set system  according to the manufacturer’s instructions. In this array, the levels of IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-17A were determined, as previously described . The detRegulatory T-cell identification was performed as previously described , 21, 25.®  software package . Frequency tables were analysed using the χ2-test, with the likelihood ratio or Fisher’s exact test for categorical variables. Differences in the means of continuous variables were analysed using non-parametric (Mann–Whitney) tests for two independent samples. All statistical tests were two-sided and declared significant at p value ≤0.05.All statistical analyses were run using IBM SPSSThe laboratory of the Oral Pathology at the Institute of Dentistry, Faculty of Medicine, University of Turku, Turku, Finland, is a research laboratory where the T-cell assays are performed according to SOPs, including the predefined criteria for positive responses.All children of this study were followed from birth until the age of 6-years. HPV DNA status of the oral mucosa was determined at birth, at the age of 3 days, and 1, 2, 6, 12, 24, 36 and 72 months. HPV serology to genotypes 6, 11, 16, 18, and 45 was determined at same time points starting from the age of 1–36 months. Table The HPV16-specific proliferative responses of PBMC in both groups are shown in Fig. In addition to mother HPV and CIN status, the LST results of all 31 children of case and control groups were also analyzed according to the presence of maternal HPV antibodies in their sera as infants. The children were grouped accordingly: (1) children (n = 13) with HPV antibodies before the age of 6 months and (2) children without any detectable HPV antibodies before the age of 6 months. This grouping resulted in a statistically significant difference in the proliferative responses against HPV16 peptide pools. The children without any HPV antibodies before the age of 6 months showed proliferative responses of PBMC after HPV16 exposure more frequently than other children (p = 0.045).The cytokine levels of IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-17A after stimulation of PBMC with HPV16E2, E6 and E7 peptides are summarized in Fig. Figure The frequency of CD4 + CD25 + Foxp3 + regulatory T-cells (Tregs) after 7-day stimulation of PBMCs was analyzed with flow cytometry Fig. b. In theTwo children (ID13 and ID20) in the control group had positive Treg responses against peptide pool E2.2., and child ID20 also against peptide pools E2.1, E6.1, E6.4, and E7.2. Child ID13 had also response against the MRM but not the child ID 20.Since the cell-mediated immune response (CMI) has been regarded as the paramount player in the resolution of the HPV infection and diseases induced by HPV, the present HPV-specific T-cell memory of individual might provide information on both, the encountered infections and possible reactivity for infections to come. Recently, we published the first work showing that HPV-specific CMI exists already in children naïve to any sexual contacts or HPV vaccinations . As all Altogether, proliferative responses against HPV16 E2 peptides were the most predominant in both groups; all 10 children of the case group and 18 of the 21 children of the control group displayed E2-specific immunity, especially against the peptide pool E2.1. (amino acids 1–195 of HPV16 E2 protein). Only one child in the control group reacted only with the peptide pool E2.2. but not with pool E2.1.The HPV16 E6 peptides induced proliferative responses in 5 of 10 children in case group and 10 of 21 children in control group. These observations are congruent with several studies, where the HPV16 E2 and E6-specific immune responses are prevalent among healthy adults , 24, 26.As HPV E2 is abundant in infected cells at the stage of viral amplification, the reactivity to HPV16 E2 peptides may be a sign of a previous replicative HPV16 infection . This raThe relatively high HPV18 seropositivity found among the control-children is surprising as the prevalence of oral HPV18 is low. Furthermore, HPV 18 genotype has a strong association with cancer or cancer development. The levels of HPV18 L1antibodies in the sera of were low, but still exceeding the cut-off value of 200MFI. Only two children (ID 182 and 228) had titers higher than the 600MFI. The HPV serology was analyzed in the reference laboratory .In the present study, the HPV16-specific proliferative T-cells secreted higher levels of cytokines than the cells with no detectable proliferation response. The levels of cytokine TNF-α showed difference between the two groups in that the children in the case group had higher levels. Altogether, the T helper type 1 cytokine IFN-γ and T helper type 2 cytokine IL-5 secretion, characteristic to the HPV16-specific immune response in healthy population , were frFurthermore, we detected Foxp3+ T regulatory-cell activation in four children. Two children in the case group and two children in the control group had Foxp3+ Treg responses against peptide pools of HPV16 E2. In addition, two of them also reacted against peptide pools of E6 and, one against peptide pool E7.2. Since Foxp3+ T regulatory-cells are known to suppress immune responses and several studies suggest that association between increased CD4+ Foxp3+ frequency and persistent HPV infection and/or progression of cervical dysplasia and other malignancies –35, our To conclude, the HPV16-specific immunity seems to be common in young, sexually inexperienced children. Though, the HPV infection status or HPV related disease of the mother did not show any effect on the HPV-specific immunity of her child. Our data also supports the view that in many cases, the first HPV infection is acquired at early infancy, before the onset of sexual life. In fact, these results show that oral HPV infections occur much more frequently in young children than expected before. The HPV-specific humoral and cellular immunity induced by these early infections could explain previous observations showing T-cell immunity in about half of healthy adults irrespective of their partner/sexual status. Even though the number of the study subjects was limited, the present well characteristic follow- up cohort is unique in its design, and gives a strong evidence of the existence of HPV immunity in children. However, the higher number of study subjects would be needed for the discrimination of the possible effects of the mother’s HPV status or special characteristics of the child to the HPV immunity.These data may also give rise to the discussion about the significance of these early HPV infections to the effectiveness of prophylactic HPV vaccines. Thus, the virus like particles in HPV vaccines are shown to induce neutralizing antibodies against HPV L1 remarkably more efficiently than natural infections and be very efficacious in reduction of infections by vaccine related HPV types –38. HoweAnother issue to be resolved is that despite the protection resulting from natural infection and/or HPV vaccination, the sexual behavior still remains a risk factor for cervical cancer development . A possi


HPV genotypes have distinct distributions among various ethnic populations worldwide. In December 2013, 237 and 159 cervical samples were collected from Hani and Han ethnic women, respectively, in Mojiang, a rural county in southern Yunnan. The overall HPV infection rate (21.1%) among the Hani women was significantly higher than that among the Han women (12.6%). The high-risk (HR) and low-risk (LR) HPV and single- and multiple-genotype infection rates among the Hani women were 11.0%, 4.6%, 15.6%, and 5.5%, respectively. HPV-16 (3.8%) was the most prevalent genotype among the Hani women, followed by HPV-52 (1.7%), HPV-31 (0.8%), and HPV-33 (0.8%). Comparatively, the Han women had lower infection rates of high-risk (8.2%), low-risk (1.2%), single-genotype (9.4%), and multiple-genotype HPV infections (3.1%). HPV-16 (3.1%) was also the predominant genotype among the Han women, followed by HPV-52 (1.3%), HPV-33 (0.6%), HPV-44 (0.6%), and HPV-54 (0.6%). The area background, number of children, and past history of STIs were recognized as potential risk factors for HPV infection. Rural background, age, education level, number of children, and illness history were significantly associated with HPV infection among the Hani women. These findings highlight the urgent need for HPV prevention and control strategies in Yunnan, particularly for the Hani ethnic women. Human papillomavirus (HPV) is one of the most common sexually transmitted pathogens of the genital system and plays a vital role in the development of cervical cancer , 2. It iEpidemiologically, the HPV prevalence rates and genotype distributions vary among different geographical regions. The highest HPV prevalence in women without cervical abnormalities has been reported in Africa (24%), followed by Eastern Europe (21%), Latin America (16%), and Southeast Asia (14%) . The HPVCervical cancer cases are increasing in China, and due to distinct topographical variations, HPV prevalence varies among different regions of China . The highttp://www.hxen.com/word/xinwen/2011-04-28/142164.html), the size of the Hani ethnic population living in Yunnan is 1.63 M, making it the third largest local population. The majority of individuals in the Hani population are illiterate, have a high fertility rate, mainly work in agricultural farming, and live in remote mountainous areas with poor sanitary conditions [Hani group is a Chinese certified ethnic minority group that mainly inhabits southeast China . AdditioIn June and July of 2014, this cross-sectional study was collaboratively conducted at Mojiang by the Department of Gynaecology, the First People's Hospital of Yunnan Province, and Kunming University of Science and Technology. Interested women were requested to visit a local hospital in Mojiang County, Yunnan, and written consent was obtained. A standardized questionnaire was used to collect information about ethnic background, age, education, marital status, illness history, sexual activity, and profession. After completion of the questionnaire, a qualified gynaecologist performed pelvic examination and sample collection. Women who met the following criteria were recruited into the current study: those who (a) were not a gynaecological outpatient with symptoms related to genital tract disease (commonly cervicitis and/or vulvar discomfort); (b) were not currently pregnant; and (c) had not undergone a total uterus or cervical resection. Finally, a total of 396 participants from general population, including 237 Hani and 159 Han women, were considered qualified and were recruited for this study. The protocol was followed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Kunming University of Science and Technology and the Center for Disease Control and Prevention (CDC) in Yunnan Province, China.A qualified gynaecologist collected exfoliated cervical cells from each participant using a cytobrush (Hybribio). Each sample of exfoliated cervical cells was inserted into a vial with preservation solution  and vigorously swirled 10 times. The vial contents were sent to Yunnan First People's Hospital for cytological analysis. All cytological slides were individually prepared by two qualified technicians. All of the cytology specimens were classified according to the Bethesda classification system . All smeCervical samples were collected with a cervix brush, and cells were placed into a vial containing preserving media. The samples were transported at −20°C to a central laboratory. A QIAamp DNA Mini Kit  was used to extract DNA from the cervical samples according to the manufacturer's instructions.β-globin gene was performed to evaluate DNA quality [β-globin positivity were further analyzed by PCR amplification of HPV DNA. HPV-positive samples were confirmed by PCR with universal L1 primers using the MY09/11 and GP5/6 systems [PCR amplification of the housekeeping  quality . Samples systems . DNA extμL containing 4 μL of the DNA extract, 2 μL of 10 μmolar MY09 primer, 2 μL of 10 μmolar MY11 primer and 25 μL of the provided master mixture, and 17 μL dH2O. The consensus primer products were separated by electrophoresis on a 2% agarose gel and purified with Tiangel PCR purification kit. The DNA was then directly sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction  on an ABI 310 DNA analyzer. The nucleotide sequences were aligned and compared with those of known HPV genotypes available through Genbank by using the BLAST 2.0 software server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sample was identified as a particular HPV genotype if the sequence was 95% homologous with the reference standard.HPV genotypes were determined using an HPV GenoArray Test Kit  according to the manufacturer's instructions. The GenoArray test is an L1 consensus primer-based PCR assay that is capable of amplifying 21 HPV genotypes, including 15 HR-HPV genotypes  and 6 low-risk HPV genotypes (LR-HPV)  . The assP value of 0.05 was considered statistically significant. The results of all manual statistical analyses were confirmed with SPSS software package (version 20.0).The HPV prevalence rates, including the total rates, and the rates for single- and multiple-genotype infections, were compared using the Chi-square test. Corresponding 95% confidence intervals (CIs) were estimated by binomial distribution analysis. The effects of age-related variables on HPV infection in women were assessed using the logistic regression model, with the grouping of age in 10-year intervals , and odds ratios and their 95% CIs were calculated. Possible risk factors for HPV infection acquisition were evaluated by univariate analysis and multivariate logistic regression analyses, and odds ratios and their 95% CIs were calculated. A P value of 0.048 . Of them, 52 (13.1%) were positive for an HPV single-genotype infection, while 18 (4.5%) had a multiple-genotype infection. A total of 23 genotypes were identified, with the most frequently detected HR-HPV genotypes being HPV-16 , HPV-52 , HPV-33 , HPV-58 , and HPV-18 . Comparatively, the most common low-risk HPV genotypes were HPV-54 , HPV-11 , HPV-55 , and HPV-42 . The prevalence of HR-HPV  was significantly higher than that of low-risk human papillomavirus (LR-HPV) , with a of 0.048 .Coinfection of multiple HPV genotypes did not occur frequently. In total, 4.5% (18/396) of the patients had a multiple-genotype HPV infection. Of them, 3.5% (14/396) had a double infection , 0.8% (3/396) had a triple infection , and only 0.2% were found to be infected with four genotypes (HPV-16/33/11/40) . Among tP value of 0.032. As the primary infection form, the HPV single-genotype infection rate among the Hani women  was significantly higher (P = 0.016) than that among the Han women . The multiple infection rate among the Hani women  was also higher than that among the Han women . Furthermore, the average percentage of samples containing at least one HR-HPV genotype was higher among the Hani women (11.0%) than among the Han women (8.2%), although this difference was not significant (P = 0.35). The LR-HPV prevalence among the Hani women was 4.6% higher than that among the Han women .Among the 237 recruited Hani women, 50  were found to be HPV DNA positive. In contrast, HPV DNA was detected in only 12.6% (20/159) of the Han women. The HPV prevalence observed among the Hani women (21.1%) was significantly higher than that among the Han women (12.6%), with a A total of 23 HPV genotypes were identified in this study . Of themP = 0.67). Correlation analysis revealed that HPV prevalence was more complex in the younger and older participants compared with the middle age group. The highest overall HPV prevalence was observed in the <35-year age group, and it declined thereafter with increasing age. A less pronounced second peak in prevalence was observed for the Han women in the older age group (>46 years). However, high-risk and multiple-genotype infections among the Hani women also exhibited two peaks in prevalence, with the first peak of 12.3% and 7.7%, respectively, occurring at <36 years and the second peak of 11.2% and 6.2% occurring at >46 years. The prevalence of single-genotype infections among the Hani women peaked in the middle age group and declined thereafter as age increased  and median of 41 years. There was no significant age difference between the Hani (41.53 years (median = 41), SD = 8.79, 95% CI, 40.41–42.66) and Han ethnic groups (41.86 years (median = 41), SD = 4.99, 95% CI, 41.08–42.64)  had a significantly higher risk of being infected with HPV .Cervical malignancy is the 8th most common cancer among Chinese females . It is wP < 0.05) higher than that among the Han women (12.6%). The prevalence of HPV among Hani is lower than results among Tibetan women (27.4%) from Northwestern Yunnan and is higher compared with Naxi (11.9%) and Han (17.2%) cases [In this study, the overall HPV infection rate of 17.6% was higher than the documented rates in neighbouring countries  , 28. Howith Naxi .9% and HHR-HPV prevalence has been examined in previous investigations due to its vital role in the development of cervical cancer . This prAn understanding of HPV genotype distributions in different areas or populations is crucial for HPV prevention and vaccine development. In this study, HPV-16 (3.5%) was detected as the most frequent genotype among the women in both ethnic groups, but it was more common among the Hani women (3.8%) than the Han women (3.1%). However, coinfection with HPV-18, which is considered a highly carcinogenic genotype, and HPV-16 was not frequently detected in this study , 40. HPVAge is another important factor affecting HPV prevalence due to the various physiological developmental stages. A cross-sectional study of women from Hong Kong has shown that an initial infection peak occurs at young ages, with a second peak occurring in women over 60 years of age, while a single peak in HPV prevalence has been detected in 20- to 29-year-old Guangzhou women. Thereafter, the prevalence has been shown to decrease with increase in age . AnotherWomen residing in a rural area were found to be at high risk of HPV infection. Most of the Hani women were from rural areas, where they lived in concentrated villages. The sanitary conditions in their houses were found to be the worst. Further, medical hospitals in these areas are lacking, and if inhabitants become sick, they prefer to be treated at home with their traditional medicine. A possible interpretation of these findings is that women living in villages without health care facilities may be at high risk of HPV infection and the development of cervical cancer. It is therefore crucial to identify these populations and to stress the importance of cervical screening. Various reproductive characteristics are linked to the acquisition of HPV infection. In this study, Hani women with a past history of sexually transmitted diseases and those with multiple children were at significantly higher risk of HPV infection. This finding suggests that sexually transmitted diseases may also be a risk factor for HPV infection in other populations , 46. ThuWe have identified different HPV prevalence rates and genotype distributions among the Hani and Han women and have described the roles of different potential risk factors in the acquisition of HPV infection. Further, we have determined the age-specific HPV infection rates among these two groups; however, there are still some limitations to this study. For example, the number of participants is low, particularly the Han women . Thus, the results generated from the two groups are unreliable, which limits the generalizability of the findings. In view of the findings of previous studies indicating that the older age group is at high risk of acquiring HPV infection due to their compromised immune systems, it is crucial to perform further investigations to clarify our results.To our knowledge, this is the first Hani ethnic population-based study conducted in Yunnan, China. Significantly higher prevalence rates of both general HPV and single HPV genotype infection were discovered among the Hani women compared with the Han women. A young age was found to be a risk factor for acquiring HPV infection. In addition, residing in a rural area, a past history of STIs, and a history of multiple pregnancies were found to be associated with acquisition of HPV infection. These findings contribute to the understanding of the epidemiological characteristics of HPV among women in different ethnic groups and will facilitate the design of effective strategies for the prevention and control of HPV.


The Iran-Iraq imposed war lasted eight years and was one of the longest wars of the last century. Twenty-three years have passed since the war ended, but little has been discussed about the long-term results of war amputations in the literature.In this long-term study, we have evaluated the activities of daily living among veterans with hip or hemipelvis amputations.A cross-sectional study was performed on Iran-Iraq war veterans with hip or hemipelvis amputations in Iran. Eighty-four (96.5%) veterans out of 87 registered veterans with hip or hemipelvis amputations participated in the study. The degree of independence for activities of daily living (ADL) was assessed by the Barthel index. The degree of independence for instrumental activities of daily living (IADL) was assessed by the Lawton-Brody scale.The average follow-up time was 26.6 ± 3.7 years. The average age of veterans was 44.1±7 years old. Of 84 amputees, 57 (67.85%) had limitations in at least one domain of the ADL. The most common single item that affected the patients was ascending and descending stairs seen in 45 (78.9%) veterans, followed by eating seen in 4 (7.01%) veterans. In addition, 70 (83.33%) had limitations in at least one domain of the IADL. The most common single item that affected the veterans was shopping seen in 56 (80%), followed by responsibility for own medications seen in 13 (18.57%) veterans. Spearman correlation coefficient of the sum scores of ADL and IADL showed an intermediate to strong correlation (r = 0.58).Increasing dependency in ADL is accompanied by increasing dependency in IADL. In the past, the duty of health care providers was saving the life of veterans due to injuries while at present, because these injuries occurred in young and healthy individuals, the need for increased function is being highlighted. The Iran-Iraq imposed war lasted eight years and was one of the longest wars of the last century in which many healthy, young people took part and were left with different injuries. Presently, 20801 veterans suffer from various types of limb amputations and 12981 veterans underwent lower limb amputations  veterans out of 87 registered veterans with hip or hemipelvis amputations participated in this study, which was held in Mashhad, Iran, in 2011. The study team included an orthopedic surgeon, psychiatrist, physical medicine and rehabilitation, and prosthetic specialists. All participants signed an informed consent form before taking part in the study.Basic characteristics and clinical data were collected. The degree of independence for activities of daily living (ADL) and independence for instrumental activities of daily living (IADL) were assessed by the Barthel index and the Lawton-Brody scale, respectively. Data were evaluated by an epidemiologist and community medicine specialist and were analyzed by SPSS 12 .The ADL was evaluated by the Barthel Index , which cIADL score was evaluated by the Lawton-Brody scale , which cEighty-four veterans with hip or hemipelvis amputations participated in this survey. The average follow-up time was 26.6 ± 3.7 years ranging from 23 to 32 years. The average age of veterans was 44.1 ± 7 years old with a minimum and maximum age of 36 and 72 years old, respectively. Fifty-nine (70.2%) amputees were single before being injured, but 79 (94%) amputees were married at the time of study. The average number of veterans' children was 3.1 ± 1.7 . Forty-fThe participants had the following ambulatory conditions: normal ambulation without any aid in 4 (4.7%) amputees, abnormal ambulation without any aid in 9 (10.7%), ambulation using a single crutch in 12 (16%), ambulation using double crutches in 50 (59.5%), ambulation using a walker in 1 (1.3%) and mobilization by wheelchair in 7 (9.3%). Among these veterans, 50 (59.5%) patients complained of decreased ambulation velocity in comparison with the previous year.Among these 84 hip or hemipelvis amputees, 66 (78.5%) veterans were dissatisfied with their prosthesis and 41 (48.8%) veterans abandoned their prosthesis. It was owing to its heaviness in 16 (19%), pain stimulation in 6 (15%), skin disorders in 6 (7.1%), fatigue in 8 (9.5%), need for frequent repair of prosthesis in 7 (17.5%), need for frequent prosthesis adjustment in 5 (12.5%), need for prosthesis replacement in 7 (17.5%), stump ulcer in 2 (2.6%), phantom pain in 1 (1.3%), and other reasons in 8 (20%) patients. Furthermore, an accompanying amputation included finger amputation in 1 (1.1%), below elbow amputation in 2 (2.2%), and above-elbow amputation in 2 (2.2%) patients.Of 84 amputees in this study, 57 (67.85%) had limitations in at least one domain of the ADL. The most common single item that affected the patients was ascending and descending stairs in 45 (78.9%) veterans, followed by eating in 4 (7.01%) veterans .In the 84 amputees, 70 (83.33%) had limitations in at least one domain of the IADL. The most common single item that affected the veterans was shopping in 56 (80%), followed by responsibility for own medications in 13 (18.57%) veterans . We calculated the correlation between the sum score of ADL and IADL. Spearman correlation coefficient of sum scores of ADL and IADL was 0.58 (P = 0.001) that showed an intermediate correlation.The eight-year Iran-Iraq war, which was initiated by the former Iraqi dictator Saddam-Husain in 1980, left more than 200,000 casualties and more than 400,000 veterans in Iran , followed by eating (7.01%). Therefore, 85.91% of the patients had limitations in ascending and descending stairs in combination with eating. The results of our study were similar to a study by Roehrig, in which the greatest dependency was in "ascending and descending stairs" and "shopping" .Amini et al. evaluated Iranian blind veterans by the Barthel index  and showIn the current study, IADL was assessed by the Lawton-Brody scale and due to the high proportion of male veterans, five domains out of eight were assessed. The greatest dependency among these 84 veterans with hip or hemipelvis amputation was shopping (80%) followed by responsibility for own medications (18.57%). Therefore, 98.57% of the patients had limitations in shopping in combination with responsibility for their own medications. According to the current literature, the Lawton-Brody scale was used in studies for other ailments; however, it had not been used for veterans and amputees. Therefore, our study can be used as a basis for future studies.In our study, the calculation of the correlation coefficient between the sum score of ADL and IADL was 0.58. A correlation coefficient of more than 0.4 is a good correlation, which shows a linear correlation between dependency in ADL and IADL. Hence, increasing dependency in ADL was accompanied by increasing dependency in IADL. Roehrig et al. also achieved the similar results and suggested that an abbreviated form of six items instead of a total of 18 items of the ADL and IADL could be used for screening .Due to the inversion of mortal factors to disabling factors, the average age and the number of senile people in this population is increasing and therefore, the need for rehabilitation services is increasing as well , 22. MorIn the past, the duty of health care providers was saving the life of veterans due to injuries, but at the present time, because these injuries have occurred in young and healthy individuals, the need for increasing their function is being highlighted . Therefo


Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity. Papillomaviruses are small circular double-stranded DNA viruses that preferentially infect the basal layer of skin and mucosal epithelia. They are highly species-specific and have been identified in a broad variety of animal species, including reptiles, birds, and mammals . To datei.e., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HP45, HPV51, HPV52, HPV56, HPV58, HPV59) have been classified as oncogenic types because of the experimental and/or clinical evidence of their etiological role in the development of cervical cancer and of a subset of anogenital and head and neck squamous cell carcinomas [Differences in tissue tropism and ability to promote cell proliferation and transformation have been demonstrated among papillomavirus types. As for HPV types, 12 . In addition, clinical and experimental studies have shown that the presence of nucleotide changes in some HPV intra-type variants may be associated with increased oncogenic activity and probability to induce cancer ,9,10,11.While further studies are required to confirm the significance of HPV intra-type genetic variants in conferring an increased risk of cancer development, especially for non-HPV16/18 oncogenic types . So far,In this study, we designed and validated a new method for the characterization of intra-type variants of HR-HPVs based on deep sequencing of the whole E6/E7 region. Potential applications of this method include epidemiological studies on the distribution of HPV lineages, investigation of HPV-related cancers to identify HPV variants associated with increased oncogenic potential, and risk-stratification in patients with HR-HPV infection.A bioinformatics pipeline was implemented with the aim of generating a set of PCR primers able to amplify the whole E6/E7 region of all HPV types classified as oncogenic groups 1 and 2A by the International Agency for Research on Cancer (IARC)  in orderThe setup of the pipeline developed for this purpose allowed to two distinct tasks to be accomplished: (1) the possibility to detect and amplify all HR-HPV types in the same reaction and (2) the robustness of the design to the effect of HPV genome variability, at least for all known variants that are deposited in the Nucleotide Database.To this aim, all nucleotide sequences belonging to HR-HPV types (IARC group 1 and 2A) were downloaded from GenBank . For eacStarting from these haplotype sequences, the PCR primers were generated with Hyden , a tool The following samples were analyzed in order to evaluate distinct features of the multiplex PCR-NGS-based system for identification of HR-HPV types and subtypes.To assess the sensitivity of designed PCR primer set, HPV16 and HPV18 DNA-positive controls were prepared with DNA purified from CaSki and HeLa cells  and added at ten-fold dilutions in the DNA carrier obtained from the HPV-negative HEK293 cells, according to the WHO Human Papillomavirus Laboratory Manual . These sTo evaluate the ability of the E6/E7 primer set to identify HR-HPV intra-type variants in clinical samples, we analyzed leftover anogenital swab specimens collected in Specimen Transport Medium  at Padova University Hospital from patients with a positive result by the INNO-LiPA HPV Genotyping Extra kit . Specimens included in this study, which were stored at −80 °C until use, were selected from consecutive samples in order to include a representative series of all HR-HPV types. HPV DNA-negative samples were also included as negative controls.The primer set reported in this study was designed for broad applications, including Sanger sequencing and NGS protocols. Thus, the performance of the primer set in the identification of HR-HPV intra-type variants was validated by both Sanger sequencing in 36 samples with a single HPV type infection detected by LiPA and by a NGS-based deep sequencing protocol in eight samples with multiple HPV type infection detected by LiPA.v/v, 3 mM MgCl2, dNTPs 0.8 mM each, oligonucleotide primers 0.03 μM each and two units of AmpliTaq Gold® DNA Polymerase . The amplification was carried out with an initial denaturation step of 10 min at 95 °C, followed by 40 cycles of amplification  and with a final elongation step  in a MyCycler thermal cycler .Total DNA was purified from specimens by using the MagNA Pure 96 Viral NA Small Volume Kit on a MagNA Pure 96™ instrument . For PCR amplification, two primer pools composed by forward and reverse primers respectively were prepared, with each primer at 5 µM concentration. A multiplex PCR reaction was performed in a final reaction volume of 50 μL with reaction buffer 10% Sanger sequencing was performed by using the single forward or reverse oligonucleotide primers specific for the HR-HPV type identified by LiPA at a 0.3 μM concentration and a BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher) on an ABI PRISM 3130xl Genetic Analyzer System (ThermoFisher). Electropherograms were visualized through the Chromas Lite 2.1.1 software  and aligned to reference sequences from PaVE  using JaDeep sequencing was performed on a Roche 454 GS FLX+ instrument (Roche 454 Life Sciences) according to the following Roche GS FLX+ protocols: Amplicon Library Preparation Manual (2014); emPCR Amplification Manual Lib-A-SV (2013); Sequencing Method Manual (2013). Due to the insertion of two distinct barcodes in primer sequences, four pools were generated by mixing equimolar amounts of DNA, each one consisting of two samples. The pools were loaded in separate lanes of the sequencing plate.Sequences obtained by 454 deep sequencing were demultiplexed and primers removed from the 5′ and (if present) 3′ end. The quality control step was performed with USEARCH : sequencFor each sample, HPV typing was performed through BLAST  alignmenAll the steps not explicitly performed with third-party tools were carried out with custom-made Perl scripts that are available upon request.The phylogenetic analysis of E6/E7 consensus sequences obtained from the samples, together with the reference sequences for the different genotypes and variants, was performed with the Maximum Likelihood method based on different models for the different species datasets, as reported in figure legends. The analyses were conducted in MEGA6 .Finally, a score based on SNP data was implemented in order to infer viral variants within HPV types. Briefly, SNPs detected in the E6/E7 region of the HR types and associated to each variant were extracted from previous reports ,46,47,48High-risk HPV intra-type variant lineages and sublineages were named according to the alphanumeric nomenclature .By using the bioinformatics pipeline described in the Methods section, a set of primer sequences, including 13 forward and 16 reverse primers, were designed in order to target all HR-HPV types and all their nucleotide variants. Nucleotide sequences and identified targets of the primer set are reported in i.e., CaSki and HeLa cells, respectively. The LoD for HPV16 and HPV18 was estimated as 900 and 700 genome equivalents in 200 µL STM, respectively. These LoD values were lower than the LoD of LiPA, which was estimated as five genome equivalents per 5 µL of purified nucleic acids for both HPV16 and HPV18, as demonstrated by participation to external quality assurance evaluations promoted by WHO Global HPV DNA typing proficiency studies [The sensitivity of the DNA amplification-sequencing method was assessed by testing replicates of serial dilutions of HPV16 and HPV18 DNA-positive cell lines,  studies ,50.The sequences obtained by Sanger sequencing and the reads obtained by deep sequencing and assigned to each sample were genotyped by means of a BLAST search against a custom database of HPV genome sequences. The multiplex approach of the method, which is designed to target the E6/E7 region of 13 HR-HPV genotypes in the same reaction, allowed detection of the presence of single or multiple infections in all samples.A complete agreement was observed between the HPV typing results obtained by LiPA and PCR-Sanger sequencing, as shown in Since Sanger sequencing was performed by using only forward and reverse primers specific to the HR-HPV types identified by LiPA as definitely present, HR-HPV types defined as possibly present by LiPA  a phylogenetic analysis and (2) a scoring system based on SNPs.As regards phylogeny, distinct phylogenetic trees were generated for HPV species α-7, α-9, and α-5/α-6 by using the Maximum Likelihood method. The phylogenetic trees included reference sequences for all known variant lineages/sublineages and the HPV sequences obtained from clinical samples . In the In almost all cases, sample sequences were able to cluster with the reference sequence of a single variant and with a reliable bootstrap value greater than 0.7, as reported in In order to strengthen and validate the results of HPV subtype classification, we developed an alternative method based on a scoring system of SNPs associated with the different HPV variants. In this method, each nucleotide position in the E6/E7 region presenting variability within an HPV type was evaluated to build a ranking of variants, as detailed in the Methods section. The results are presented in vs. C and C vs. B comparisons are not symmetrical, because B is a superset of C in this particular region of the genome and, inevitably, C reaches the same score when compared to itself and B. Similarly, in some clinical samples, HPV sequences did not obtain the maximum score for any variants, e.g., HPV16 in sample 004. In these cases, possible sources of bias might be considered, such as the incompleteness of available information about lineage variability or the misclassification of the sequences used to build the scoring matrix.As shown in i.e., phylogenetic analysis and SNP scoring system) showed an agreement in 52 out of 54 samples . The two exceptions included sample 004, which was assigned to HPV16 variant D by phylogeny while the SNP score was unable to discriminate among lineages, and sample 541 which was equally assigned to HPV68 variants A and B by both methods. In the first case, both classifications are quite uncertain, since the bootstrap value for the clade containing sample 004 and HPV16 variant D1 is 0.7 while the SNP score is similar among all HPV16 variants, with no clear separation between the highest score and the others; in the second case, variants A and B of HPV68 are clustered together by phylogeny and obtain a comparable SNP score, suggesting that the E6/E7 region does not provide enough information to discriminate between them.The comparison of the results obtained by the two methods for the classification of HPV variants  to detect variants of all HR-HPV types in a single reaction; (ii) to detect variants of HR-HPV types in clinical specimens with multiple infections; (iii) being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.a priori knowledge of variable sites within the E6/E7 region and their association with HPV intra-type variants. In our evaluation, in some cases, the SNP score method was not able to unambiguously discriminate among HR-HPV intra-type variants. Nonetheless, with the growing availability of sequence data in public databases, an improvement in the discriminating power of the method is expected, helping to overcome the limitations observed for few specific variants. In alternative, an approach based on the detection of haplotypes, instead of independent variable positions, could be adopted.The novel HPV subtyping algorithm based on the SNP score implemented in this study could be used as an alternative to phylogeny for variant classification, since it showed comparable performance. This algorithm can be provided as an online platform, as it only requires In the literature, methods for HPV variant detection have been generally designed for testing single HPV types ,56,57,58The region targeted in HPV genome for variant detection included the full sequence of the E6 and E7 oncogenes. The most accurate method for the identification of HPV variants/lineages and sublineages is obviously based on full genome sequences ,59, althet al. [Sanger sequencing, which still represents the gold standard sequencing method, was used in the present study to benchmark the performance of the E6/E7 multiplex primer set in amplifying different HR-HPV types from clinical specimens and identifying their lineages/sublineages. Classification of HR-HPV lineages and sublineages was performed following the nomenclature and the reference HPV genomes proposed by Burk et al. . At varii.e., approximately 50,000 reads per sample) that allowed identification of HPV types that were not identified by LiPA probably because of their relatively low concentration in comparison with co-infecting HPV types. A good sensitivity of deep sequencing in the detection of HPV types representing a small proportion  in multiple infections was also observed in our previous studies with different primer sets targeting the L1 region [After validation of the multiplex PCR protocol by Sanger sequencing, the method was implemented in a 454 deep sequencing protocol. A proof-of-principle sequencing run, performed with eight clinical specimens containing multiple HR-HPV types, showed that the NGS method could correctly identify HPV types and characterize intra-type variants in multiple infections. The inclusion of MID sequences in PCR primers allowed pooling of samples in the sequencing run, thus leading to a reduction of sequencing costs per sample. In our proof-of-principle run, we used a high depth of coverage  different consensus sequences can be obtained from the reads belonging to a certain genotype within a sample. We did not observe such event in the present series of samples, while we detected some cases with multiple infection of variants of the same HPV type in our previous study in a larger series of patients .In conclusion, this study reports the design and validation of a NGS-based method for characterization of HR-HPV intra-type variants in clinical samples. Notably, the primer set can be adapted to Sanger sequencing and different NGS platforms. This method could be used both for epidemiological studies on the distribution of HPV lineages and for diagnostic purposes. Diagnostic applications may include second line testing in patients with HR-HPV infection, to identify infections associated with increased risk of persistence and disease progression ,13. This


This study was performed to assess attribution of high grade cervical intraepithelial neoplasia (HG-CIN) and invasive cervical cancer (ICC) to human papillomavirus (HPV) genotypes and secondarily to assess reproducibility of HG-CIN/ICC diagnosis obtained in Poland. Formaldehyde fixed, paraffin embedded blocks of HG-CIN/ICC from two distant institutions were sent to a central laboratory together with original histological diagnoses. Central/expert review of histopathological specimens was performed and agreement between local and central/expert diagnoses was calculated. HPV detection and genotyping in the samples was carried out with the use of SPF10-LiPA25 technology. Results were analyzed for 205 HG-CIN and 193 ICC cases with centrally confirmed diagnoses. Kappa coefficients and 95 % confidence intervals for HG-CIN and ICC diagnoses were: 0.13  and 0.19  respectively. Cohen’s kappa coefficients for lesions with representative number of samples ranged from 0.01 for cervical intraepithelial neoplasia grade 2 to 0.75 for adenocarcinoma. HPV DNA was detected in 96.1 and 91.2 % of the confirmed HG-CIN and ICC specimens respectively. HPV positive HG-CIN was most commonly attributed to HPV types: 16 (62.8), 33 (7.8), 31 (6.6), 52 (3.7), 45 (2.6) and 58 (2.6 %). HPV positive ICC was most commonly attributed to HPV types: 16 (72.1), 18 (10.8), 33 (5.7), 45 (3.4) and 31 (1.7 %). Reproducibility of histological diagnosis of HG-CIN/ICC obtained in Poland generally increases with the severity of lesion and is lowest for cervical intraepithelial neoplasia grade 2 and highest for adenocarcinoma. Over 80 % of ICC cases are vaccine-preventable in Poland. Despite the long term opportunistic screening and initiation of active organized cytological screening in 2006 , cervicaIdentification of high risk types of human papillomaviruses (HR HPVs) as a necessary etiological factor of CC has led to elaboration and implementation of effective vaccines against HPV in many countries around the world, however primary prevention of CC through HPV vaccination is still not available in the Polish reimbursed immunization program . InformaThe current study is a part of a large project which assessed the HPV-type distribution in HG-CIN encompassing CIN2, CIN2/3, CIN3 and adenocarcinoma in situ (AIS) and also in invasive cervical cancer (ICC) in selected European countries for which data were incomplete . Our stuOne of the factors influencing the effectiveness of CC prevention programs and also crucial for clinical decision-making is the quality of histological diagnosis of cervical neoplasia. To our knowledge, data on this issue have not been published for Poland yet. For this reason we decided to use data collected in the international project  and perfIn comparison to the primary manuscript , our worOur study is based on the material collected in HERACLES and SCALE studies published recently  which weSince this study focuses on HG-CIN/ICC diagnosed in Poland, the material used was collected at two distant sites in Poland: the First Department of Gynecology and Oncologic Gynecology of Medical University of Lublin and the Department of Gynecology and Oncology of the Jagiellonian University Medical College of Gynecology and Obstetrics in Krakow. The two sites participate in both organized and opportunistic cervical screening, perform triage of abnormal pap results, carry out treatment and follow-up of women with symptomatic and asymptomatic cervical neoplasia. Therefore tissue samples archived at these sites should be representative of the local population of women with cervical disease.10-DEIA/LiPA25-polymerase chain reaction (PCR) system  [10-DEIA. In positive samples the HPV genotype was identified by reverse hybridization probe assay (SPF10-LiPA25), which detects 14 high-risk (HR)  and 11 low-risk (LR) HPV types . If the sample was HPV DNA negative, ten-fold DNA dilutions were retested. All HPV-negative and a random of HG-CIN and ICC samples were again reviewed by up to three expert pathologists blinded to HPV status. Cases were classified according to 2003 WHO classification [To assure the presence of lesions in the material used for HPV genotyping, a “sandwich” method of material sectioning was applied. The first and the last of the four 4 μm thick sections was stained with haematoxilin and eosin (H&E) and reviewed by an experienced gynecological pathologist blinded to the original local diagnosis. Only if the presence and the type of the lesion was confirmed in both H&E sections, the two middle sections were used for DNA analysis. The highest grade of lesion present in the sample was taken into account in the analysis. The sectioning was performed with strict measures to avoid contamination . Eligiblhnology) . Total DStatistical methods used in this study were different to those published in the primary manuscript  since noAgreement levels for each diagnosis category of cervical lesions were calculated in percent. Simple and weighted kappa coefficients were calculated separately for HG-CIN and ICC cases because study procedures and analyses were done separately and cases were not transferred between the two groups . The samThe flowcharts of study subjects with HG-CIN and ICC and their samples included in the analysis are presented on Fig. The agreement levels, simple and weighted kappa coefficients for local and central diagnoses of HG-CIN and ICC are presented in Tables HPV DNA was detected in 197 (96.1 %) of the 205 eligible HG-CIN and in 176 (91.2 %) of the 193 eligible ICC specimens. HPV DNA was detected in 86.0 % of CIN2, 96.9 % of CIN2/3, 99.2 % of CIN3, 94.2 % of SCC, 83.3 % of ADC, 75.0 % of ASC and in 80.0 % of specimens with other histological types of ICC. The two cases of AIS and AIS + CIN3 were HPV-positive.Among HG-CIN and ICC, there were 80.5 and 87.6 % samples with single HPV type respectively. In 12.7 % of HG-CIN and in 2.1 % of ICC multiple HPV types were detected. Undetermined HPV types were detected in 2.9 % of HG-CIN and 1.6 % of ICC. Minimal, proportional and maximal attributions of lesions to certain HPV genotypes are presented in Table Median ages (inter-quartile ranges) of patients at the time of histological specimen collection for women with HG-CIN and ICC positive for DNA of HPV: 16, 18, 31, 33, 45, other combined types and multiple types are presented at Fig. To our knowledge this is the first comprehensive report on HPV prevalence and genotype distribution in almost 400 HG-CIN and ICC tissue specimens collected consecutively at two distant institutions in Poland and representative for local population of women. HPV DNA was detected in 96.1 % of HG-CIN and 91.2 % of ICC. HPV 16, 33, 31, 52 and 45 were found to be most commonly causally related to the development of HG-CIN and approximately 84 % of these lesions in Poland can be attributed to the five listed HPV genotypes. Low Risk HPVs  are responsible for less than 10 % of HG-CIN in Poland. In our series they were detected only in three CIN2 cases but absent in CIN2/3, CIN3 and ICC samples as single infections. The most frequent genotypes in ICC were: HPV 16, 18, 33, 45, 31 and over 80 % of ICC in Poland can be attributed to the first two genotypes. ICC cases were exclusively related to HR HPVs.HPV DNA positivity rates of HG-CIN and ICC in Poland are very similar to rates observed for HG-CIN in our analysis of pooled European countries  and to rHPV 16 and 33 were the most commonly found among polish HG-CIN samples both in our study and in a previous report by Kędzia et. al  but the HPV 16 and 18 are universally the two most common genotypes among ICC in our Polish, European  series oRestrictive analysis incorporating only minimal attribution of ICC to HPV 16 and 18 obtained in our study reveals that theoretically 80.7 % of ICC cases are vaccine preventable in Poland. In the most optimistic scenario assuming maximal attribution of ICC to HPV 16 and 18 obtained in our study and a 50 % cross-protection of vaccines against HPV 31, 33 and 45, the rate of potentially vaccine preventable ICC cases in Poland would be close to 90 %. The data collected in this study are necessary for input into existing pharmacoeconomic models and analyses before decisions of health technology assessment regulatory agency in Poland on recommendations of reimbursement of HPV vaccination from public funds. Very high contribution of HPV 16 and 18 to the development of ICC in Poland is an opportunity for effective primary prevention of the great majority of invasive lesions. Unfortunately HPV vaccines are not yet reimbursed and immunization coverage is low.In the light of our results indicating very frequent prevalence of HPV 16 and 18 among patients with ICC compared to HG-CIN in Poland, the newest recommendations on the use of HPV16/18 genotyping in the triage of women with atypical squamous cells of undetermined significance (ASC-US) or no intraepithelial lesions or malignancy (NILM) but a positive cocktail HPV test  are of gTo our knowledge, this is the first literature report of the reproducibility of histological diagnosis of cervical neoplasia in Poland. The quality of CIN and ICC diagnosis is important for effective screening and treatment of cervical disease. Histological overdiagnosis of cervical lesions may result in overtreatment with subsequent higher rate of complications and adverse outcomes in the reproductive health . On the vs central diagnosis was fairly concordant.CIN2/3, according to WHO International Statistical Classification of the Diseases and Health Problems 10th edition , is not Despite very low reproducibility of CIN2 and low-to-moderate reproducibility of CIN3 between local and central diagnosis in our study, invasive lesions were found by central/expert pathologists only in 1 (0.6 %) case of CIN2 and 6 (2.9 %) of CIN3 cases. 64.5 % of CIN2, 60 % of CIN2/3 and 42.2 % of CIN3 lesions were downgraded by central review which suggests a tendency to overdiagnosis and possible risk of overtreatment of a number of patients treated at our centers. However the most alarming finding is downgrading of almost a quarter of local site diagnosis of SCC. It is possible that extensive treatment protocols, overtreatment and adverse outcomes in some patients occurred. Blinding of the tissue blocks sent for study procedures to the central laboratory required by the central study protocol precludes identification of the over-treated patients. Strikingly, our results indicating possible overtreatment of CIN and ICC as a result of histological overdiagnosis are in contrast with epidemiological data of relatively high mortality and case-fatality rate of women with cervical cancer in Poland . Low covOur results indirectly indicate that time of progression of HG-CIN to ICC may vary according to HPV genotype causing the lesion Fig. . HoweverThe strengths of the part of our study devoted to HPV detection and genotyping include the use of a standardized study protocol, consecutive collection of histological samples representative for local populations at two distant gynecologic oncology centers participating in all steps of national active and opportunistic cervical screening and providing comprehensive diagnosis and treatment of CIN and ICC. Central expert histopathological review of histological material from 2003 to 2008 and the use of a highly sensitive HPV genotyping method provide an up-to-date picture of the attribution of HG-CIN and ICC to specific HPV genotypes in Poland.The apparent drawback of our analysis, which should be addressed in future studies, is the lack of analysis of reproducibility of our local histological diagnosis of non-neoplastic epithelium  in cases suspected of cervical neoplasia (e.g. based on cytology/colposcopy/HPV testing results) and estimation of falls-negativity rate of our local histology. This information could have profound clinical implications however assessment of the quality of pathological work-up was not among the end-points of the main protocol  and we wNevertheless, further personnel training, improvement of quality control measures and possibly a wider use of biomarkers such as p16 (INK4a) and MIB-1 –22 could